Resonance Raman spectroscopy of squid and bovine visual pigments: the primary photochemistry in visual transduction. Resonance Raman spectra of squid rhodopsin have been obtained under a variety of temperature and illumination conditions. The data have been characterized in terms of spectral contributions from squid rhodopsin, isorhodopsin, bathorhodopsin, lumirhodopsin, mesorhodopsin, P-465, and acid metarhodopsin. The results are compared with the spectral features obtained from bovine rhodopsin, isorhodopsin, and bathorhodopsin. The data support a proposed structure for the chromophore in bathorhodopsin which is not all trans, 11-cis, or 9-cis. This structure can be generated from either rhodopsin or isorhodopsin by a similar motion (simultaneously rotating chromophore carbon atoms 10 and 11 out-of-plane). Furthermore, we detect the same distinct bathorhodopsin vibrational modes when rhodopsin is illuminated between 4 and 100 K. This demonstrates that under steady-state illumination the light-induced chromophore structural alterations occurring at 4 K are very similar to those occurring at higher temperatures. Finally, our data indicate that bathorhodopsin is generated not only by structural transitions in the chromophore but also alterations in the opsin conformation as has recently been proposed[Lewis, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 549].


Resonance Raman spectroscopy of the retinylidene chromophore in bacteriorhodopsin (bR570), bR560, M421, and other intermediates: structural conclusions based on kinetics, analogues, models, and isotopically labeled membranes. Resonance Raman spectra of various intermediates in the bacteriorhodopsin proton pumping cycle have been obtained at physiological and low temperatures. To interpret these data, spectra of modél compounds, bacteriorhodopsin analogues, and isotopically labeled membranes have been measured. These results demonstrate that a protein group interacts with the Schiff base proton and, thus, the chromophore in protonated bacteriorhodopsin species is not a simple protonated Schiff base. This accounts for the abnormally low frequency of the C=N+H vibrational mode in bacteriorhodopsin and other failures to model the chromophore in bR570 with a simple butylamine protonated Schiff base of all-trans-retinal. To obtain the resonance Raman spectrum of M412 at physiological pH and temperatures, a dual beam kinetic technique was developed. We demonstrate that in the fingerprint region of the resonance Raman spectrum M412 is modeled accurately by a simple unprotonated butylamine Schiff base of all-trans-retinal. Spectral resolution and the solution environment of the membrane suspensions play important roles in this conclusion. Kinetic resonance Raman techniques are also used to monitor the time evolution of the M412 species and the intermediates which precede it. We find spectral features in our kinetic data which can be assigned to L550, and we present evidence for a new unprotonated species (X) which occurs before M412. Single pass flow resonance Raman spectra of bR560 also have been obtained, and, although bR570 and M412 appear to have all-trans chromophores, there are 13-cis-like features in the spectra of bR560, L550, and X.


A circular dichroism study of the interaction of chlorpromazine with mouse brain tubulin. A circular dichroism study with subsidiary ultracentrifuge measurements on the interaction of chlorpromazine with mouse brain tubulin establishes the previous inference from binding studies that the drug induces a change in the structure of the protein. Binding of the first mole of chlorpromazine causes an alteration in secondary structure, which is reversible with respect to drug concentration, without detected change in tertiary structure or significant change in the state of association of the protein. The conformationally altered tubulin binds additional chlorpromazine molecules without further change in secondary structure.


Platelet 5-hydroxytryptamine transport, an electroneutral mechanism coupled to potassium. Transport of 5-hydroxytryptamine into plasma membrane vesicles isolated from porcine blood platelets is stimulated when a potassium gradient (in greater than out) is imposed across the vesicle membrane. This stimulation occurs in the absence of measurable electrical potential across the membrane. Addition of valinomycin induces a membrane potential of approximately 50 mV (interior negative) as estimated by uptake of the lipophilic cation triphenylmethylphosphonium, but has surprisingly little effect on 5-hydroxytryptamine transport. Addition of 2,4-dinitrophenol dissipates the valinomycin-induced membrane potential. In the absence of valinomycin, 2,4-dinitrophenol has no effect on 5-hydroxytryptamine transport but valinomycin and 2,4-dinitrophenol together inhibit transport, probably by dissipation of the K+ gradient. These results are consistent with an electroneutral mechanism in which 5-hydroxytryptamine influx is directly coupled to potassium ion efflux and argue against an electrogenic mechanism in which there is a net influx of positive charge with 5-hydroxytryptamine.


Primary structure of human erythrocyte glycophorin A. Isolation and characterization of peptides and complete amino acid sequence. Peptides of glycophorin AMN were prepared by cyanogen bromide cleavage and by chymotryptic and tryptic digestion. Cyanogen bromide cleavage produces three fragments which account for the entire polypeptide chain. Trypsin and chymotrypsin cleave completely at several sites, but incompletely at sites within the glycosylated segment of the polypeptide chain. Some of the latter sites become accessible to proteolysis after desialation in addition to exposure of new sites for cleavage. The amino acid sequence of glycophorin AMN has been determined by manual Edman degradation, using both the direct Edman and the dansyl-Edman procedures simultaneously for determination of glycosylated amino acid residues. The automated procedure was used for sequence determination of a hydrophobic peptide. Glycophorin A is a polypeptide chain of 131 amino acid residues and contains 16 oligosaccharide units attached to the amino-terminal third of the molecule. Fifteen oligosaccharides are linked O-glycosidically to either threonine or serine residues and one complex oligosaccharide unit is attached N-glycosidically to an asparagine residue. Amino-terminal sequences are different for glycophorin AM and AN, the two forms of the glycophorin A molecule coded for by genes at the MN locus. The differences in sensitivity to proteases of various sites on glycophorin A seem to be due to heterogeneity in the carbohydrate components and not to differences in the primary structure of the polypeptide chains. This work contains a number of revisions and corrections of earlier preliminary reports [Segrest, J.P., Jackson, R. chem. Biophys. Res. Commun, 49, 964-969; Tomita, M., & Marchesi, V.T. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2964-2968].


Circular dichroism and magnetic circular dichroism of iron-sulfur proteins. Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra are reported for the 2-Fe ferredoxins from Pseudomonas putida and Spirulina maxima, Chromatium HIPIP, the 4-Fe ferredoxin from Bacillus stearothermophilus, and the 8-Fe ferredoxin from Clostridium pasteurianum. The spectral range spans the near-infrared, visible, and near ultraviolet. In all cases except oxidized 2-Fe ferredoxins, electronic absorption is observed continuously from less than 5000 cm-1 to above 30,000 cm-1. The CD spectra of the two 2-Fe ferredoxins are similar. In contrast, the CD of the 4-Fe and 8-Fe proteins, for a given 4-Fe cluster oxidation level, varies considerable with protein. MCD is less sensitive to protein environment than is CD. In the 2-Fe proteins, MCD at 5 T is appreciably smaller than the CD; in the 4-Fe and 8-Fe proteins, MCD and CD are comparable in magnitude. Both CD and MCD are more highly structured than the corresponding absorption spectra. The CD and MCD spectra reported provide a broader base than heretofore available for the characterization of iron-sulfur proteins containing 2-Fe and 4-Fe clusters and for the evaluation of electronic structural models for these clusters.


Synthesis of peptides of arginine chloromethyl ketone. Selective inactivation of human plasma kallikrein. Synthetic procedures have been developed for the preparation of peptides of arginine chloromethyl ketone and applied in the preparation of affinity labels which correspond to the -Pro-Phe-Arg- C terminus of bradykinin, a physiological cleavage site of kallikrein in kininogen. Two such reagents, Ala-Phe-ArgCH2C1 and Pro-Phe-ArgCH2C1, proved to be highly effective as well as selective affinity labels for human plasma kallikrein. For example, Pro-Phe-ArgCH2C1 inactivates plasma kallikrein 50% in 24 min at a concentration of 2 x 10(-8)M, while other trypsin-like proteases are less susceptible in inactivation than kallikrein, differing by a factor of 48 for plasmin and factors of 10(2)-10(5) for factor Xa, thrombin, and urokinase. The affinity of human plasma kallikrein for Ala-Phe-ArgCH2C1 (Ki = 0.078 micron) is about 60 times that for Ala-Phe-LysCH2C1(Ki = 4.9 micron), whereas human plasmin exhibits about the same affinity for the former affinity label (Ki = 1.3 micron) as for the latter (Ki = 0.83 micron). The rate constants for the irreversible step of the affinity labeling reaction, k2, are similar for affinity labels tested with the individual proteases: 0.35 min-1 for plasma kallikrein and 0.18 min-1 for plasmin.


Importance of C4a- and N5-covalent adducts in the flavin oxidation of carbanions. The reaction of nitroalkanes, furoin, and phenacyl alcohol with 7- and 8-cyano-3,10-dimethylisoalloxazine (7-CNFlox, 8-CNFlox), 5-ethyl-3-methyllumiflavinium ion (Flox+Et), and 2.6-dichloroindophenol (DCI) have been studied. Nitromethane anion forms an adduct on reaction with Flox +Et. The condensation reaction has been shown (nuclear magnetic resonance) to occur through addition of the methylene carbon of the nitromethane anion to the 4a position of Flox+Et. This adduct undergoes spontaneous dissociation to its components in acid. It does not undergo conversion in base to reduced flavin (1,5-F1EtH), NO2-, and CH2O. For this reason one may conclude that nitroalkane anion oxidation by flavin does not involve the intermediacy of a 4a-covalent intermediate. Oxidation of nitroalkane anion through formation of an N5-flavin adduct is discussed in terms of the peculiar requirements of the substrate when compared with carbanions derived from normal flavoenzyme substrates. It is concluded that the mechanism of nitroalkane oxidation by flavin must bear no relationship to the flavin oxidation of normal substrates. Furoin and phenacyl alcohol (compounds containing the-CH(OH)--CO-functionality) are oxidized by 8-CNFlOX, 7-CNFlOX, DCI and by the N5-blocked flavin Flox+Et. These reactions are found to be zero order in oxidant at the concentrations of oxidants (approximately 1.0 X 10(-5) M) and reductant (approximately 10(-3) to 10(-4) M) employed. This finding, and that of an 1H/2H kinetic isotope effect of 3.5, as well as certain equalities of the determined rate constants establish the rate-determining step in all these oxidations to be carbanion formation. That the N5-blocked flavin (Flox+Et) serves just as well as an oxidant as do substituted and unsubstituted isoalloxazines provides evidence that the flavin oxidation of carbanions of general structure (-C-(OH)--CO-) does not require the formation of an intermediate N5-flavin adduct. This conclusion when taken with the fact that the N5-flavin adduct formed on reaction of dihydroflavin with -C(=O)--CO-is not an intermediate in the reduction of -C(=O)--CO-to -CH(OH)--CO- by dihydroflavin shows that N5 adducts do not arise as intermediates in these oxidations. Remaining mechanisms (4a addition and radical pair formation) are discussed. 4a addition of carbanions to Flox+Et is shown to be subject to considerable steric strain by the instability of the nitroethane adduct and the inability to observe the formation of the 2-nitropropane adduct.


T4 RNA Ligase joins 2'-deoxyribonucleoside 3',5'-bisphosphates to oligodeoxyribonucleotides. T4 RNA ligase catalyzes the ATP-dependent addition of a single 2'-deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of an oligodeoxyribonucleotide. The bisphosphate is joined to the deoxyoligomer by a 3' leads to 5' phosphodiester bond and the product, which is terminated by a 3'-phosphate, is one nucleotide longer than the substrate. Bisphosphates of dAdo, dCyd, dGuo, dThd, and dUrd are donors and oligodeoxyribonucleotides with dA, dC, dG, dT, or dU 3' termini act as acceptors. The preferred residue for both donor and acceptor is dCyd. Deoxyoligomers from 3 to 12 residues in length are active as acceptors. To obtain good yields, high concentration of enzyme, long incubation time at low temperature, and manganous rather than Mg(II) ion are required. Under optimal conditions, yields calculated with respect to deoxyoligomer converted to product vary from 40 to greater than 95%. The turnover number of the enzyme for DNA joining is extremely low but, because the preparation is nearly free of DNases, there is less than 3% degradation of substrate or product after 6 days of reaction. We anticipate that this reaction will serve as the basis for a method for the stepwise enzymatic synthesis of DNA of defined sequence.


Complete amino acid sequence of the precursor region of rat prolactin. Rat preprolactin was translated in a wheat germ in vitro synthetic system in the presence of 3H-labeled amino acids. The amino-terminal sequence of the radiolabeled precursor was quantitatively identified by high pressure liquid chromatography. The 29 residue precursor sequence is: Met-Asn-Ser-Gln-Val-Ser-Ala-Arg-Lys-Ala-Gly-Thr-Leu-Leu-Leu-Leu-Met-Met-Ser-Asn-Leu-Phe-Cys-Gln-Asn-Val-Gln-Thr-. A discussion of the structure-function relationships of the precursor region and the microsequencing methods used to determine the sequence is presented. The amino-terminal 30 residue sequence of the secreted form of rat prolactin was determined and found to differ at positions 7 and 8 (-Gly-Asp-) from a previously reported rat prolactin sequence (Parlow, A.F., & Shome, B. (1.76) Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 219).


Mechanism of the membrane potential sensitivity of the fluorescent membrane probe merocyanine 540. The fluorescence and optical absorption of the membrane-staining dye merocyanine 540 (M-540) have been widely used to measure cellular transmembrane potentials. We have studied the molecular mechanisms of these optical changes by measuring the fluorescence polarization of M-540 and its response to membrane potential changes in hemispherical lipid bilayer membranes. The fluorescence responds to a potential step in two distinct time scales: a fast response with a rise time less than the instrumental capability of 6 micromilligram and a slow response with a time constant around 10(-1) s. Both response amplitudes are proportional to the amplitude of the membrane potential change and both require an asymmetrical distribution of M-540 across the membrane. The slow response is ascribed to a net change of the dye concentration in the membrane. The fast response appears to be dominated by a change in the distribution of orientations of the dye molecules in the membrane, with a concomitant perturbation of a monomer-dimer equilibrium, due to interaction of the applied electric field with the permanent molecular dipol moment of M-540. The amplitude of the fast fluorescence response is concentration dependent and can be modeled by including membrane saturation effects and the presence of a nonfluorescent dimer species in the membrane at high dye concentrations. Absorbance changes reported by other investigators are consistent with this model mechanism.


Possible roles of bile lipids and colipase in lipase adsorption. The adsorption isotherms of bile salts, phospholipids, and cholesterol were determined with siliconized glass beads. It was observed that the molar fractions of cholesterol, phospholipid, and bile polypeptide fractions increased simultaneously and considerably on the surface of the beads in comparison to the corresponding fractions found in bile. The composition of the adsorbed film is approximately 1 cholesterol: 2 phospholipid: 3 bile salt molecules. The performed complex of lipase, colipase, and bile lipids behaves as an entity which determines lipase adsorption. The modification of the interface quality of a lipid substrate by a detergent is not perse the reason for the lack of lipase adsorption. A model is proposed according to which lipolysis under physiological conditions would occur in two steps requiring two cofactors. Colipase would be necessary for the formation of the lipase-bile lipoprotein complex, and bile lipids would be required to direct the adsorption of this lipolytic entity toward the emulsified substrate.


Reconstitution of 5-hydroxytryptamine transport from cholate-disrupted platelet plasma membrane vesicles. The bile acid cholate disrupts platelet plasma membrane vesicles and inactivates 5-hydroxytryptamine transport activity. The transporter appears not to be solubilized by this procedure, but remains associated with a high molecular weight aggregate. Merely removing cholate does not restore the vesicular structure or transport activity, but in the presence of added soybean phospholipids, cholate removal generates proteoliposomes which accumulate 5-hydroxytryptamine. This reconstituted transport activity is similar in its ionic requirements and inhibitor sensitivity to transport in native membrane vesicles and intact platelets.


A vicinal dithiol containing an essential cysteine in phosphoenolpyruvate carboxykinase (guanosine triphosphate) from cytosol of rat liver. The highly purified form of phosphoenolpyruvate carboxykinase (PEPCK) contained 13 thiols (all in the reduced state) per 72 000 daltons. Modification of the enzyme with equimolar 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) caused rapid formation of a cystine disulfide bridge and an even more rapid loss of enzymatic activity. Formation of the cystine bridge proceeded about 25 times faster than formation of the analogous intramolecular disulfide of dithiothreitol induced by Nbs2. o-Iodosobenzoate, Cd2+, and the 2,3-dimercapto-1-propanol complex of arsenite were potent, time-dependent, irreversible inhibitors of PEPCK. The inactivation by arsenite-2,3-dimercapto-1-propanol and o-iodosobenzoate was first order with respect to both time and inhibitor concentration. The sum of these data indicates the existence in PEPCK of a critical cysteine that is in a vicinal dithiol grouping with a second cysteine. PEP protected against cystine bridge formation induced by equimolar Nbs2 but not against the extent of inactivation. In the presence of PEP, the modification by Nbs2 of one cysteine/mol of enzyme (k = 1.2 X 10(6) M-1 min-1 at pH 7.2) caused nearly complete inactivation. Replacing the bulky 5-thio-2-nitrobenzoate moiety with cyanide did not result in any reactivation. This critical, cyanylated cysteine was determined to be 44% of the distance from the amino terminus.


On the light-stimulated coupling between rhodopsin and its disk membrane environment. Disks from bovine ROS undergo a rapid shrinkage when flash illuminated with green light (Uhl, R., et al. (1977) Biochim. Biophys. Acta 469. 113-122). This can be monitored as a light scattering transient, referred to as the P signal. In this paper the P signal is studied at various temperatures and pH. The temperature dependence of the kinetics reveals that "P" consists of two sequential reaction steps. Both appear to occur within the receptor molecule rhodopsin. The actually observed event, the shrinkage of the disk, is therefore not rate limiting under the tested conditions. Both steps of "P" take place while there is only one spectroscopically detectable reaction of the rhodopsin molecule, the metarhodopsin I-metarhodopsin II transition. This implies that there are intermediates of the rhodopsin photolytic cycle which are not evident as spectroscopically separate species. The amplitude of "P", i.e., the extent of the disk shrinkage, is independent of the state of the equilibrium between the two photoproducts absorbing at 478 and 380 nm respectively and called MI and MII. A scheme is suggested in which the irreversible decay of MI (478) triggers the disk shrinkage (and maybe transduction), and in which there is an equilibrium between MII (380) and a proposed isochromic photoproduct MI' (478).


Specificity of the retinal binding site of bacteriorhodopsin: chemical and stereochemical requirements for the binding of retinol and retinal. The complexes formed from bacteriopsin and various retinyl compounds were analyzed by fluorescence and absorption spectroscopy. The binding of retinol occurs in two steps. In the first reaction the molecule is fixed in the retinal binding site of the protein. In this state, energy transfer from aromatic amino acid residues to the retinyl moiety is observed. all-trans-Retinal and the 13-, 11-, and 9-cis-retinols are bound in the chromophoric site. In the second reaction the cyclohexene ring and the side chain of the retinyl moiety are forced into a planar conformation. This reaction is mediated by a base (B1) with a pK of 3.8 and requires the oxygen atom but not the free hydroxyl group of retinol, indicating interaction with a group AH (pK greater than or equal to 10.5). The ring-chain planarization reaction is blocked for the 9-cis isomer of retinol. Binding studies with bacterioopsin and retinal isomers reveal that, as in the case of the corresponding retinols, B1 mediates ring-chain planarization in the case of the all-trans, 13-cis, and 11-cis isomers but not with the 9-cis isomer. Reconstitution of the purple complex from the intermediate 430-460-nm chromophore requires the presence of a second base (B2) with a pK of 4.6. This reaction is exclusive for all-trans- and 13-cis-retinal


Correlations between subunit distribution, microheterogeneity, and iron content of human liver ferritin. Subunit heterogeneity of human liver ferritin was investigated by two-dimensional electrophoretic methods. The protein which ordinarily remains assembled in 10 M urea solution was dissociated into subunits in acid-urea or sodium dodecyl sulfate solutions. In agreement with earlier studies, the subunits migrated as two bands in sodium dodecyl sulfate or acid-urea gel electrophoresis systems or in two-dimensional combinations of these systems. Isoelectric focusing methods, however, resolved four major subunit bands and three to five minor bands. Each of these components migrated as either a 22 000 or a 19 000 molecular weight component in sodium dodecyl sulfate gel electrophoresis in the second dimension. The multiple subunit model, which is contrary to currently accepted representations of ferritin structure, is compatible with certain inherent properties of the protein. Thus, ferritin was fractionated on the basis of iron content to show that the relative amounts of individual subunit types were directly dependent upon the iron composition of the protein. Iron-loaded molecules were deficient in the most basic subunit types, and apoferritin was enriched in these components. Aspects of microheterogeneity of assembled ferritin molecules were correlated to subunit heterogeneity, and discrete differences in subunit populations among purified isoferritin components were demonstrated.


Inhibition of sickle hemoglobin gelation by amino acids and related compounds. The effects of amino acids, several aromatic compounds, and peptides on the gelation and solubility of deoxyhemoglobin S have been studied. The aromatic amino acids (tryptophan, phenylalanine, and possibly tyrosine) significantly inhibited the rate of gel formation and increased solubility. The dipeptide L-Thr-L-Phe, the tripeptide L-Lys-L-Phe-L-Phe, and various phenylalanine analogues (hydrocinnamic acid, phenethylamine, benzamine, and amphetamine) also inhibited gelation. However, aromaticity is not a sufficient condition for inhibiting gelation as shown by the fact that several aromatic compounds (acetylsalicylic acid, salicyclic acid, aniline, and phenol) enhanced gelation. Surprisingly, several oligopeptides (betaS1--12, betaS4--8, betaS3--13, and betaS4--10) also enhanced gelation. All of these additives follow the supersaturation relationship that the delay time for gelation is proportional to the ratio of the total hemoglobin concentration to the solubility of deoxyhemoglobin S to the nth power (n approximately 35). A possible mechanism for the action of these inhibitors is considered in terms of a specific site of interaction on the hemoglobin molecule. Although none of these compounds may prove to be efficacious in treatment of sickle cell anemia, they should yield information about the structure and process of formation of the deoxyhemoglobin S gel.


Expression of functionality of alpha-chymotrypsin. Effects of guanidine hydrochloride and urea in the onset of denaturation. Crystals of alpha-chymotrypsin (CHT) at equilibrium in solutions of 2.0 M guanidine hydrochloride and 3.0 M urea at pH 3.6 were prepared, three-dimensional X-ray intensities were measured, and difference electron-density maps were calculated and examined. The guanidine hydrochloride derivative displayed changes occurring exclusively on the surface of the protein. The difference peaks represented mostly small changes in various protein surface groups and in the adjacent solvent regions, and some displayed convincing evidence of binding of the guanidinium ion to the protein. The urea difference map likewise showed that changes had occurred on the surface of the protein, but also that numerous changes in the structure occurred in the hydrophobic interior of the CHT molecule. Further, the urea difference map contained evidence for two kinds of interactions of urea with protein groups. There are examples of bound urea either causing or accompanying structural changes and examples of urea binding with no accompanying changes to the protein. Examples of both kinds of binding were observed in both the surface regions and in the hydrophobic interior of the molecule. From an examination of these two derivatives, it is clear that guanidine hydrochloride and urea unfold proteins by different mechanisms.


Acetylcholine receptor and ionic channel of Torpedo electroplax: binding of perhydrohistrionicotoxin to membrane and solubilized preparations. The electric organ of the ray, Torpedo ocellata, can serve as a source for both the acetylcholine (ACh) receptor and its ionic channel. The two entities were identified by their specific binding of [3H]ACh and [3H]perhydrohistrionicotoxin ([3H]H12-HTX), respectively. Binding of [3H]H12-HTX was inhibited by certain drugs and toxins, e.g., histrionicotoxin (HTX), amantadine, and tetraethylammonium (TEA) ions at concentrations that did not inhibit [3H]ACh binding. However, the specific carbamoylcholine-induced 22Na efflux from microsacs from the electric organ membranes was blocked by inhibitors of either the receptor or its ionic channel. The ionic channel had the properties of a protein as judged by heat sensitivity and the inhibition of [3H]H12-HTX binding, after incubation of the electric organ membranes with protein reagents such as p-chloromercuribenzenesulfonic acid (PCMBS) or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The "binding" of [3H]H12-HTX at 4 X 10(-8) M to lipids in the microsacs was 12% of the total binding to intact microsacs and was nonsaturable and insensitive to heat or specific drugs. After solubilization with cholate, the [3H]H12-HTX binding subunits retained the same affinities for toxins and drugs. The Kd for [3H]H12-HTX was 3 X 10(-7) M. The majority of the ionic channel could be separated from the ACh receptors in the cholate extract by incubation with ACh-receptor affinity gel and ACh-receptor antibodies. The ACh receptor purified by this affinity gel contained only a few active ionic channel units as judged by low levels of high affinity binding of [3H]H12-HTX. On the other hand, after solubilization with Triton X-100, all the ionic channel molecules were either separated or denatured so that the purified ACh receptor did not exhibit high affinity binding for [3H]H12-HTX.


Lysine-rich histones and the selective digestion of the globin gene in avian red blood cells. Deoxyribonuclease I (DNase I) is known to preferentially digest the adult globin gene sequences in avian red blood cells. We have investigated the contribution of histones H1 and H5 in maintaining the nuclease-sensitive structure about the globin genes. When the lysine-rich histones H1 and H5 were selectively removed from avian red blood cell nuclei, the rate of digestion with DNase I increased several fold. However, the globin genes in H1-and H5-depleted nuclei were still selectively digested. Since histone H1 is necessary for the higher order folding of the nucleosomes, these data suggest that DNase I recognizes an aspect of structural heterogeneity within each core particle rather than some higher order packaging of the nucleosome cores.


Thallium-205 and proton nuclear magnetic resonance investigation of the complexation of thallium by the ionophores monensin and nigericin. A thallium-205 and proton nuclear magnetic resonance study of the ionophores monensin and nigericin is reported. Evidence is presented for the simultaneous existence of two forms of monensin free acid in chloroform solution, one containing a water molecule in the central cavity and one with a water molecule on the periphery, linking the ends of the monensin molecule by hydrogen bonding. There appear to be three structurally different monensin complexes of thallium, two in the salt form and one in the acid form. In free nigericin acid, both terminal hydroxyl moieties hydrogen bond to the terminal carboxylic acid oxygens; in the Na+ salt, the binding of one of the carboxylate oxygens to the metal ion allows but one of the terminal OH's to hydrogen bond strongly. Thallium-205 chemical shifts and spin-lattice relaxation times in the title complexes are discussed and compared to those in ionophores studied previously.


Oxidation of heme proteins by alkyl halides: a probe for axial inner sphere redox capacity in solution and in whole cells. Iron(II) porphyrins in homogeneous solution, in heme proteins, and in intact human erythrocytes and lysed cells are oxidized by certain alkyl halides to the corresponding iron(III) complexes at room temperature. The mechanism established for the oxidation of hemes in homogeneous solution operates at all levels of biological integrity. It is an axial inner sphere process. Deoxyhemoglobin has about the same reactivity within and without cells. The speed of the reaction with the proteins is primarily governed by the steric accessibility to iron. The reactivity of an array of iron(II) proteins accords well with theoretical prediction. In contrast the reactivity of cytochrome b5 does not. An examination of the oxidation and reduction of this protein was additional mechanistically defined reagents (trinitrobenzene and hydroquinone) shows it to be in the G rather than C conformation. The unusual redox characteristics of this protein can be rationalized on this basis.


Inactivation of ribulosebisphosphate carboxylase by modification of arginyl residues with phenylglyoxal. Phenylglyoxal rapidly and completely inactivates spinach and Rhodospirillum rubrum ribulosebisphosphate carboxylases. Inactivation exhibits pseudo-first-order kinetics and a reaction order of approximately one for both enzymes, suggesting that modification of a single residue per protomeric unit suffices for inactivation. Loss of enzymic activity is directly proportional to incorporation of [14C]phenylglyoxal until only 30% of the initial activity remains. For both enzymes, extrapolation of incorporation to 100% inactivation yields 4-5 mol of [14C]phenylglyoxal per mol protomer. Amino acid analyses confirm the expected 2:1 stoichiometry between phenylglyoxal incorporation and arginyl modification and suggest that other kinds of amino acid residues are not modified. (Thus, inactivation correlates with modification of 2-3 arginyl residues per protomer). The substrate ribulose bis-phosphate and some competitive inhibitors reduce the rates of inactivation of the carboxylases and prevent modification of about 0.5-1.0 arginyl residue per protomer. Inactivation is therefore a consequence of modification of a small number of residues out of the 35 and 29 total arginyl residues per protomer in spinach and R. rubrum carboxylases, respectively.


Human alpha-1-antichymotrypsin: interaction with chymotrypsin-like proteinases. The interaction of human plasma alpha-1-antichymotrypsin with serine proteinases from different tissues has been investigated. The protein was found to form stable complexes with pancreatic chymotrypsin, leukocyte cathepsin G, and mast cell chymotrypsin. No inhibition of pancreatic trypsin or leukocyte elastase could be demonstrated. With mixtures containing both alpha-1-antichymotrypsin and alpha-1-proteinase inhibitor, it was found that the former preferentially inactivated leukocyte cathepsin G, while the latter showed a strong preference for pancreatic chymotrypsin. However, leukocyte elastase was specifically inactivated by alpha-1-proteinase inhibitor even in 1:1 mixtures with chymotrypsin. All of these results taken together suggest that one of the primary functions of alpha-1-antichymotrypsin is to inactivate leukocyte cathepsin G, while alpha-1-proteinase inhibitor controls the activity of other serine proteinases, particularly leukocyte elastase.


Sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase including the sites of covalent and allosteric control. The sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase (EC 2.4.1.1) has been determined. Limited proteolysis of native phosphorylase b (841 residues, subunit molecular weight 97 412) by subtilisin BPN', Streptomyces alkaline protease, or elastase yielded two large segments (light and heavy). The light segment isolated from the subtilisin digest was cleaved at methionyl bonds with cyanogen bromide to yield eight major fragments and two minor overlapping fragments. The alignment of the major fragments was obtained by analysis of the two minor fragments, of five tryptic peptides containing methionine and of one large fragment generated by cleavage of an aspartylproline bond. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule identified the sites susceptible to limited proteolysis and the overlap between the light and the heavy segments. Serine-14 and tyrosine-155 were identified as the residues involved in the covalent and allosteric controls of the enzyme, respectively. Residues 108 and 142 were identified as the cysteine residues reported to be involved in the aggregation of subunits.


Amino acid sequence of two cyanogen bromide fragments of glycogen phosphorylase. This communication presents the strategy and experimental details to prove the amino acid sequence of two large fragments of rabbit muscle glycogen phosphorylase generated by cleavage with cyanogen bromide. These fragments, CB18 and CB15, represent 241 of the 841 residues in the whole molecule. In addition to applying methods of automated liquid phase Edman degradation, techniques of selective immobilization and solid phase Edman degradation are used. One of the two cyanogen bromide fragments (CB15) contains two of the sites of cleavage with hydroxylamine which have proved to be important in the overall strategy of determining the complete sequence of this molecule. Together with the accompanying reports by Koide, A., et al., and Titani, K., et al. ((1978) Biochemistry 17 (first and third papers, respectively, in a series in this issue)), the present communication completes the proof of the amino acid sequence of phosphorylase and provides the basis for examining the relationship between its structure and function.


Sequence of the carboxyl-terminal 492 residues of rabbit muscle glycogen phosphorylase including the pyridoxal 5'-phosphate binding site. This communication presents the strategy and experimental details which establish the amino acid sequence of the carboxyl-terminal 492 residues (residues 350 through 841) of rabbit muscle glycogen phosphorylase (EC 2.4.1.1). The heavy segment (Hs), derived from the native enzyme by limited proteolysis with subtilisin, was cleaved with cyanogen bromide to yield 15 fragments. The amino acid sequences of 12 of these are described herein. The sequence of 3 other fragments (CB17C, CB18, and CB15) is described in accompanying reports by Koide, A., et al., and Hermann, j., et al. ((1978) Biochemistry 17 (first and second papers, respectively, in a series in this issue)). These 15 fragments were aligned by analysis of three others generated by cleavage of the heavy segment Hs at asparaginylglycine bonds with hydroxylamine and of four more generated by acid cleavage of aspartylproline bonds. Lysine-679 was identified as the binding site of the essential cofactor pyridoxal 5'-phosphate. These data, together with those reported in the accompanying papers (vide supra), establish the complete sequence of the 841 amino acid residues in glycogen phosphorylase. They provide a chemical basis on which the relationship between structure and function of the enzyme can be examined.


Histone-histone interactions in a lower eukaryote, Tetrahymena thermophila. The six pairwise interactions of Tetrahymena macronuclear histones H2A (formerly HX), H2B, H3, and H4 have been studied using the techniques of circular dichroism and continuous variation. Parallel experiments have been performed with calf thymus histones as controls, and the 12 possible interspecies pairs have also been examined. The behavior of three of the ciliate histones, H2A, H2B, and H3 is virtually identical with that of their vertebrate counterparts. Tetrahymena H4 exhibits a pattern of interactions identical with that of calf thymus H4, but displays significant quantitative differences in the strength of those interactions. Although we have not entirely eliminated the possibility that these differences may be artifactual, we suggest that they reflect the unique primary structure of Tetrahymena H4. These results extend the pattern of histone-histone interactions defined for plants, animals, and fungi to include the protists as well and imply the strong evolutionary conservatism of this pattern in spite of the electrophoretic and compositional differences which exist between homologous histones of different organisms. In addition, they demonstrate conclusively that histone HX should be redefined as Tetrahymena H2A.


Covalent structure of collagen: amino acid sequence of chick skin collagen alpha1(1)-CB6B. The amino acid sequence of the peptide alpha1(I)-CB6B, the carboxyl-terminal peptide containing 91 residues of alpha1(I) chain of chick skin collagen, is described. The amino acid sequence was determined by automated Edman degradation of intact peptide plus its tryptic and thermolytic peptides. The comparison of this sequence with the corresponding segment of previously published alpha1(I)-CB6 of calf skin collagen showed a sequence identity of 90%.


Thyroxine-induced conformational changes in prealbumin. The effects of thyroxine binding on the conformation of human prealbumin and bovine serum albumin have been examined. A blue shift in protein absorption was observed with prealbumin, whereas a red shift was observed with bovine serum albumin. In the case of prealbumin, where the two binding sites are identical, the total absorption change was confined to the binding of the first ligand and has been interpreted as resulting from a conformational change. A blue shift observed in the absorption spectrum of thyroxine, however, was the same for the first and second bound molecules. These data have been interpreted in terms of two identical and interacting sites on prealbumin and explain the origin of the difference in binding affinities between the first and second sites. Fluorescence quenching by thyroxine and thyroxine effects on tryptic hydrolysis of prealbumin are in accord with the above interpretation.


Sphingomyelin multiple phase behavior as revealed by multinuclear magnetic resonance spectroscopy. 31P and 13C nuclear magnetic resonance (NMR) spectra are employed to study the phase behavior of bovine brain sphingomyelin as a function of temperature. The 31P NMR data suggest that, while at low temperatures sphingomyelin can form a lamellar phase, at physiological temperatures and higher the lamellar phase is unstable, and a new phase, best described as a hexagonal phase, is formed. Egg phosphatidylcholine is suggested to play an important role in stabilizing bilayers in natural membranes. Cholesterol also exhibits a sphingomyelin bilayer-stabilizing ability. The 13C NMR spectra suggest a gelling of the hydrocarbon chains of sphingomyelin at low temperature. Thus, bovine brain sphingomyelin undergoes both a gel to liquid-crystalline phase transition and a lamellar to nonlamellar transition.


DNA sequence organization in the pea genome. The reassociation kinetics of pea (Pisum sativum L.) DNA fragments (300 nucleotides) were measured with hydroxylapatite. The most slowly reassociating fragments do so with a rate constant of 2 X 10(-4) L mol-1s-1, as determined from experiments with total DNA as well as with a tracer enriched for slowly renaturing sequences. This rate is about 1000 times slower than that observed for Escherichia coli DNA included as an internal kinetic standard, indicating a kinetic complexity of 4.5 X 10(9) nucleotide pairs or 4.6 pg of DNA per haploid nucleus. This estimate is in good agreement with previous chemical and cytophotometric measurements. The majority (85%) of the 300 nucleotide fragments contain repetitive sequences. While the reassociation of repetitive DNA could be modeled with two theoretical second-order components, the data did not specify a unique solution. The reassociation kinetics of isolated high- and low-frequency fractions indicate that repetitive sequence families in pea DNA probably cover a broad range of frequencies ranging from 100 to 10 000 or more copies per haploid genome. Single-copy sequences account for about 30% of the DNA, but because of extensive interpersion of repetitive sequences only about 15% of 300 nucleotide fragments reassociate with single-copy kinetics. From studies of hydroxylapatite binding as a function of fragment length, we conclude that the major class of single-copy sequences has a modal length of about 300 nucleotides. Long tracer reassociation kinetics indicate that sequences with an apparent repetition frequency of about 10 000 copies are interspersed at intervals of less than 1300 nucleotides throughout 75% of the genome. At a detection limit of about 3%, we find no single-copy sequences longer than 1000 nucleotides.


Gene enrichment using antibodies to DNA/RNA hybrids: purification and mapping of Dictyostelium discoideum rDNA. Antibodies, shown to be specific for DNA/RNA hybrids, have been covalently attached to CNBr-activated Sepharose. The resultant affinity resin specifically binds DNA/RNA hybrids and has been used to enrich for the DNA which codes for rRNA in the slime mold Dictyostelium discoideum. By utilizing the technique of R-loop formation, DNA molecules containing the rRNA genes were isolated from total nuclear DNA in a double-stranded form. These rDNA molecules, which were recovered by high salt elution from the affinity resin, were typically 15-40 kbp in length, and thus contained DNA sequences adjacent to the selected sequences coding for the 17S and 26S rRNAs. In addition, evidence has been obtained concerning the structure of Dictyostelium rDNA which agrees with the finding (Taylor et al. (1977) ICN-UCLA Symp. Mol. Cell. Biol. 8, 309-313) that the rDNA molecules are not covalently attached to the chromosomes of this organism.


Stereodynamics of dimer segments of RNA in aqueous solution. Arguments are presented which show that conformations II and III proposed by Lee and Tinoco [Lee, C.H., and Tinoco, I., Jr. (1977), Biochemistry 16, 5403] for ribodinucleoside monophosphates in aqueous solution are untenable. It has been shown that ribodinucleoside monophosphates exist in aqueous solution as an equilibrium blend of the classically recognized right-handed stack (g-g-), loop stack (g+g+), skewed (g+t), and extended arrays. In order to determine the effect of epsilonA base on the conformer distribution in the equilibrium blend, detailed ring-current calculations were performed and the isoshielding curves for epsilonA were derived. Use of these curves vis-a-vis dimerization shift data indicates that introduction of epsilonA perturbs the equilibrium blend which causes an increase in the population of skewed (g+t) arrays.


Resonance Raman studies of hepatic microsomal cytochromes P-450: evidence for strong pi basicity of the fifth ligand in the reduced and carbonyl complex forms. Resonance Raman spectra have been measured for cytochromes P-450 purified from liver microsomes of phenobarbital-treated rabbits (PB P-450 and PB P-448) and of 3-methylcholanthrene-treated rabbits (MC P-448). In the reduced state, all three cytochromes P-450 exhibit Raman spectra of ferrous high-spin type but show the so-called "oxidation state marker" (band IV) at unusually low frequencies, indicating extensive delocalization of electrons from the iron dpi orbital to the porphyrin II (eg) orbital and, consequently, the strong pi basicity of the fifth ligand of the heme iron. The reduced CO complexes of the cytochromes P-450 also exhibit band IV at markedly lower frequencies than CO complexes of hemoglobin and myoglobin. These anomalies observed for the reduced form and CO complex disappear upon conversion of the cytochromes to the catalytically inactive form called cytochrome P-420. Oxidized PB P-450 shows a Raman spectrum which is characteristic of typical ferric low-spin heme compounds, whereas those of PB P-448 and MC P-448 are of the ferric high-spin type. PB P-450 is also clearly distinguishable from the two P-448 preparations in the reduced state. The reduced form of cytochrome P-420, produced by laser illumination, exhibits two sets of Raman lines and, therefore, seems to be a mixture of both high- and low-spin species.


Implication of a tyrosyl residue at the active site of mitochondrial L-malate:NAD+ oxidoreductase. The tyrosyl residues of porcine mitochondrial L-malate:NAD+ oxidoreductase (EC 1.1.1.37) have been studied spectrophotometrically and using selective chemical modification with iodine and tetranitromethane. CNBr hydrolysis and Sephadex G-25, G-50 and G-75 chromatography produced a peptide which contained two tyrosines in the native and the nitrated molecules when the nitration took place in an NAD+-oxaloacetate solution. Nitration in the absence of the substrates caused the tyrosyl residues to disappear. Spectrophotometric titrations indicate that one of the 10 tyrosyl residues in mitochondrial L-malate:NAD+ oxidoreductase titrate abnormally, while iodination experiments suggest that two fast-reacting tyrosines are not involved in activity. Nitration and iodination experiments, in conjunction with CNBr-mapping, suggest that two of the four nitrated tyrosyl residues are necessary for biological action. Titration of the sulfhydryl groups with 4,4-bis-dimethylaminodiphenyl carbinol before and after nitration indicate that none of the cysteinyl residues were oxidized by the tetranitromethane, thus ruling out the loss of enzyme activity due to the thiol oxidation.


Uricase of Bacillus fastidiosus. Properties and regulation of synthesis. Uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) of Bacillus fastidiosus was purified to homogeneity in a two-step procedure and was crystallized. The native molecule had a molecular weight of 145 000--150 000 and was composed of subunits of two kinds (Mr = 36 000 and 39 000) in a 1 : 1 ratio. The quaternary structure of the enzyme was reversibly altered, with concomitant loss of activity, at temperatures between 40 and 60 degrees C. No evidence was found for the involvement of metal ions or coenzymes in the uricase reaction. The enzyme was inhibited by various metal ions and by cyanide. The isoelectric point of the enzyme was 4.3, and pH optimum 9.5 and the optimal temperature 30--35 degrees C. Only uric acid was oxidized by the enzyme and 9-methyluric acid, xanthine, 8-azaxanthine and oxonic acid were competitive inhibitors. Uricase synthesis was repressed by allantoin and allantoate, even in the presence of uric acid, which induced synthesis of the enzyme. Molecular oxygen was an important environmental factor in the control of uricase synthesis, probably due to its effect, as cosubstrate in the uricase reaction, in assessing the cytoplasmic concentration of allantoin. The highest amounts of uricase, up to half of the intracellular soluble protein content, was found in cells growing under limited oxygen supply in media containing uric acid as the main substrate.


Nitrogenase: properties of the catalytically inactive complex between the Azotobacter vinelandii MoFe protein and the Clostridium pasteurianum Fe protein. The catalytically inactive complex generated by the combination of the Azotobacter vinelandii MoFe protein (Av1) and the Clostridium pasteurianum Fe protein (Cp2) inhibits N2 reduction, C2H3 reduction, H+ reduction and ATP hydrolysis catalyzed by the homologous nitrogenases. Kinetic data indicate that the inactive complex consists of two molecules of Cp2 to one molecule of Av1, with values for the inhibitor constant in the range of 1--10 nM. Inhibition of C. pasteurianum nitrogenase by Av1 produces a lag phase in acetylene reduction that increases with increasing Av1. The lag phase is found only at levels of Av1 sufficient to keep the ratio of Cp2 : Cp1 lower than 2. Gel filtration of a mixture of Av1 and Cp2 provides evidence for complex formation and indicates that each Av1 molecule binds more than one Cp2 molecule. The Av1-Cp2 complex binds two molecules of MgATP per molecule of Cp2. MgATP is not required for complex formation, but complex formation lowers the MgATP-Cp2 dissociation constant approx. 3-fold. Av1 protects the iron-sulfur center in Cp2 completely against the MgATP-induced reaction with chelators. This provides additional evidence for formation of the Av1-Cp2 complex and together with the results of the MgATP-binding studies suggests that the two binding sites for MgATP are some distance away from the iron-sulfur site on Cp2.


Properties of a non-specific nucleotidase in the membrane of rabbit red cells. A non-specific nucleotidase was found in rabbit red blood cell membrane and, using 5'-AMP as substrate, several kinetic parameters for the enzyme were determined. Rabbit red cell ghosts catalyzed the hydrolysis of a wide spectrum of nucleoside 5'-, 3'- and 2'-monophosphates and a limited number of non-nucleotide substrates. All these activities were heat inactivated at the same rate, suggesting that they are the result of catalysis by the same enzyme. The nucleotidase was not dependent on K+ or Mg2+ and was also insensitive to ouabain. Its specific activity and other kinetic parameters were identical in preparation of membranes from both reticulocytes and the mature erythrocytes.


Fibrinogen-fibrin transformations characterized during the course of reaction by their intermediate structures. A light scattering study in dilute solution under physiological conditions. Intermediate structures of human fibrin formed under physiological conditions were investigated by means of light scattering in the course of the polymer/network formation. Very low fibrinogen concentrations (c = 0.03--0.13 mg/ml) were used to lower the polymerization rate, and thrombin at five concentrations (0.0085--0.04 N.I.H./ml) was used for initiation. The light scattering data were evaluated from (i) a Zimm plot, (ii) a Holtzer plot, i.e., hRtheta/Kc vs. h2, and (iii) a Kratky plot, i.e., h2Rtheta/KC vs. h2. In the beginning of the polymerization process rod-like structures are formed. The dimensions of the rod-like monomeric unit in the fibrin polymer are 112 X 3.9 nm and agree with the dimensions of fibrinogen, which also was found to be a thin rod of 105 +/- 10 nm length and 3.9 nm diameter. The mass per unit length, obtained from the asymptote in the Holtzer plot, initially increases only slightly but for high thrombin concentrations increases steeply when a critical length of 1000 nm is exceeded. At this point also the total scattering behaviour changes considerably. The upturn in the Zimm plot and the occurrence of a maximum in the Kratky plot are clear indications for the onset of branching. At low thrombin concentrations the kink in the curve of Mw/Lw against Mw becomes smoothed out because of nonspecific side-by-side aggregation of fibrin strands. The results are discussed and compared with earlier findings by others, and lead to the following conclusions. (i) Fibrinogen is a polymer with some flexibility and can exist in conformations of a stretched rod 105 nm in length, a folded rod of 45 nm in length, and a banana-like conformation of 94 nm circumference. (ii) Under the conditions of the present work, fibrinogen has the thin stretched rod conformation, and has the same dimensions as the repeating unit in the fibrin polymer. (iii) After approx. 10--12 units, end-to-end aggregated monomer branching occurs. (iv) The end-to-end aggregation is promoted by the cleavage of A peptides, branching is caused by the cleavage of B peptides while side-by-side aggregation of strands is caused by nonspecific van der Waals interaction.


The complete amino acid sequence of the alpha-subunit of pea lectin, Pisum sativum. The complete primary structure of the alpha-subunit of the lectin from the pea (Pisum sativum) has been determined using a combination of tryptic and staphylococcal protease digestion, purification using Sephadex gel filtration and high-voltage electrophoresis followed by either manual or automated Edman degradation. The molecular weight of the alpha-subunit from sequence data and gel filtration in guanidine-HCl is close to 5800, which is lower than that determined by sedimentation equilibrium techniques. The sequence reveals considerable homology to concanavalin A and near identity to the alpha-subunit of the lentil lectin (Lens culenaris). As in the case of the lentil alpha-subunit, the alpha-methyl glucose binding site(s) are not present in this region, nor are the S1 and S2 metal ion binding sites as judged by homology consideration, though the residues for the S3 lanthanide binding (Glu 87 and Asp 136) are conserved from the available data on the alpha- and beta-subunits. Preliminary metal exchange experimens on the intact pea lectin indicate some differnces in the metal exchange properties of this lectin compared to concanavalin A, and therefore possible ligand variations in this region of the beta-subunit.


The primary structure of tubulin. Sequences of the carboxyl terminus and seven other cyanogen bromide peptides from the alpha-chain. The alpha-chain of calf brain tubulin was fragmented by treatment with cyanogen bromide and eight peptides together accounting for 108 residues were purified and sequenced. The COOH-terminal peptide contains a fractional amount (about 0.3 residues) of tyrosine at its COOH-terminus; this presumably represents tyrosine that is added post-translationally to alpha-tubulin. The beta-chain can be phosphorylated, and the probable site of this modification is identified also in the COOH-terminal peptide. Comparison of the sequences described here with the sequence of actin reveals no homology between actin and tubulin.


A contribution to the debate about hydrophobic effects. Based on available measurements of the concentration dependence of the volume and the heat capacity of aqueous solutions of low molecular weight compounds it is suggested that non-polar molecules or non-polar groups in aqueous solution fluctuate between solvated and non-solvated states. The experimental data indicate that the change in standard free energy of the hydrophobic solvation is positive and so large that the solvation is incomplete even in the most dilute solutions studied. It is tentatively suggested that the hydrophobic solvation enhances the solubility in water of non-polar substances, and that the reason why non-polar groups in protein solutions are buried in the interior of the protein conformations is a low degree of solvation of these groups in the solvent exposed state.


Thermodynamics of the isothermal interaction of human immunoglobulin G with guanidinium chloride. A thermodynamic study of the isothermal interaction of human immunoglobulin G with guanidinium chloride, a strong denaturant, has been performed. Free energies of interaction were calculated using preferential binding data obtained by measuring densities at constant chemical potential and constant composition, respectively. Enthalpies of interaction were determined calorimetrically. The values of both thermodynamic parameters as well as those of entropies of interaction have been found to depend crucially on the extent of denaturant binding.


Pressure effects on water-swollen elastin. A model for hydrophobic interactions in proteins. The effect of pressure on the swelling of elastin in pure water was investigated. Because elastin is a very non-polar protein, and because the swelling of elastin can be directly related to changes in the strength of hydrophobic interactions, we have used elastin as a model to study the effect of pressure on hydrophobic interactions in proteins. The elastin swelling model is particularly useful because it is based on a macromolecular system very similar to a globular protein and not on dilute aqueous solutions of small non-polar compounds. Increased pressure causes elastin to increase its swollen volume, and the observed swelling changes were analyzed in terms of the Flory-Rehner theory (Flory, P.J. and Rehner, Jr., J (1943) J. Chem. Phys. 11, 521--526) for the swelling of kinetically free, random polymer networks. Our calculations provide a measure of the volume change for the process of transferring 1 mol of an average non-polar amino acid side chain from a region where the side chains are surrounded by other non-polar groups and have no contact with water (i.e. a hydrophobic region) into contact with water. The results indicate that there is a small, negative volume change associated with this process, and quantitative estimates indicate that the volume change is of the order of --6 ml/mol side chain. The results support the hypothesis that the free energy required to transfer a non-polar side chain from a hydrophobic region into water becomes less positive (i.e. the hydrophobic interaction becomes weaker) as hydrostatic pressure is increased.


Cobalt-concanavalin A. An index of inactive and active conformational states. Studies on the circular dichroic spectrum of cobalt-substituted concanavalin A have been continued in particular with respect to calcium- and saccharide-induced spectral pertubations reported previously (Kalb, A.J. and Pecht, I. (1973) Biochim. Biophys. Acta 303, 264-268; Richardson, C.E. and Behnke, W.D. (1976) J. Mol. Biol. 102, 441-451). We find that the addition of calcium or cadmium to (CO2+)-concanavalin A induces slow time-dependent alterations in the extrinsic cotton effects. Moreover, one equivalent of calcium is sufficient to cause maximal changes in the cobalt spectrum provided sufficient time is allowed for the effect to be observed. The addition of mono, di-and trisaccharides, specific for concanavalin A, have no resolvable effect upon the cobalt spectrum of concanavalin A (Richardson, C.E. and Behnke, W.D. (1976) J. Mol. Biol. 102, 441-451). The data presented here suggest that these time-dependent processes are conformationally mediated and occur subsequent to S2 occupancy. Evidence is presented that a particular calcium-cobalt-concanavalin A conformer exists which is responsible for the generation of activity in a light-scattering assay system.


Three classes of sulfhydryl group in bovine alpha-crystallin according to reactivity to various reagents. alpha-Crystallin from calf eye lens is found to have 30 sulfhydryl groups per 800 000 daltons by modification with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 4,4'-dithiopyridine in 6 M urea. These -SH groups can be divided into three different classes in native alpha-crystallin from their reactivity with DTNB, 4,4'-dithiopyridine, iodoacetamide and ethylenimine. Results obtained with these reagents point to the presence of 7 +/- 1 thiol groups (Class I) which are likely to be surface exposed, with a concomitant 40--45% quenching of tryptophan fluorescence in DTNB-modified alpha-crystallin. Another 10 +/- 1 thiol groups (Class II) must be in a hydrophobic environment since they react only with the hydrophobic reagents, causing a further decrease in fluorescence intensity. Class III, 13 +/- 2 thiol groups, is inaccesible to any of these reagents. Introduction of up to 15 negatively charged thionitrobenzoate groups or seven positively charged aminoethyl groups in the alpha-crystallin molecule at pH 7.5--8.0 did not change the state of aggregation as judged from the sedimentation coefficients.


Protein modifications during the keratinization of normal and psoriatic human epidermis. Studies on the polypeptide chain compositions of human stratum corneum and callus have shown that characteristic modifications of the epidermal fibrous protein occur during normal eqidermal keratinization. The polypeptide chain composition of psoriatic scale differs from that of both normal stratum corneum and callus. A major constituent, the alpha-chain (Mr = 70 000) of the epidermal fibrous protein is either absent or greatly decreased in amount in psoriatic sacle. This chain is, however, present in normal amounts in the uninvolved epidermis of psoriatics and, during effective treatment, returns to normal levels in the previously involved areas. It is concluded that this is a major, reversible difference between involved psoriatic epidermis and uninvolved psoriatic or normal epidermis and that it most probably results from a defect in the synthesis of the alpha-chain.


The subunit composition of two high molecular weight extrinsic proteins from human erythrocyte membranes. Purified hollow cylinder (22.5 S) and torus protein (9.0 S) from human erythrocyte membranes, together with the intact membranes, have been dissociated using 2% sodium dodecyl sulphate and electrophoresed in the presence of 0.1% sodium dodecyl sulphate. The torus protein gives rise to a single subunit migrating slightly ahead of band 8 of the polypeptide profile of the intact membranes (Mr approximately 20 000) and the hollow cylinder gives rise to two main subunits, which migrate slightly behind that of the torus protein. It is clearly shown that neither protein is related to erythrocyte membrane spectrin (bands 1 + 2) or actin (band 5).


A low-sulfated chondroitin sulfate in rat blood: an acidic glycosaminoglycan with a high metabolic rate. The rate of metabolism of low-sulfated chondroitin 4-sulfate, a predominant glycosaminoglycan in blood, has been studied by administering intraperitoneally radioactive hexosamine and/or sulfate to rats. The biological half-life of the material was estimated to be 10--12 h, suggesting that the metabolic process of blood low-sulfated chondroitin sulfate is different from that of glycosaminoglycans in the tissue.


Low-sulfated chondroitin sulfate in human blood and urine. Blood and urinary low-sulfated chondroitin sulfate from healthy young and aged volunteers have been characterized by gel chromatography, two-dimensional electrophoresis on cellulose acetate strips and by chemical and enzymatic analysis. No difference in content of the material (24 nmol hexosamine per ml plasma) was observed regardless of age. Chemical composition (approximately 40% sulfation at 4-position of galactosamine) and molecular weight (about 8000) of blood and urinary low-sulfated chondroitin sulfates were found to be the same, though urinary excretion of the material was much higher in the aged than in the young adults (Ohkawa et al. (1972) J. Biochem. 72, 1495--1501). Low-sulfated chondroitin sulfate in serum was in a bound form with a molecular weight of more than 100000, irrespective of age. These results suggest that increase in urinary excretion of low-sulfated chondroitin sulfate in the aged is mainly due to renal dysfunction. Low-sulfated chondroitin sulfate was also the main component of acidic glycosaminoglycans in blood from patients with Hurler's syndrome who excreted excessive amounts of dermatan sulfate and heparan sulfate in urine. This suggests that low sulfated chondroitin sulfate in blood is not merely a precursor of urinary glycosaminoglycans in the case of healthy young adults.


Binding of thrombin to human platelet plasma membranes. Binding of 125I-labeled thrombin to isolated human platelet plasma membranes was studied. Two classes of sites, one with high and one with low affinity for thrombin, were demonstrated. The apparent dissociation constants for the high and low affinity sites were 3.2 and 600 nM, respectively, similar to values obtained with intact platelets. Maximum binding was within 10 s, the shortest time measured, and then decreased with time to a constant level of binding within 45 s. When the equilibrium was perturbed by dilution, the  system re-equilibrated with less thrombin bound than in a control that was dilute before mixing thrombin and membranes. Neither the time-dependent decrease nor the dilution effect were observed with phenylmethylsulfonyl-125I-labelled thrombin, an irreversibly inhibited thrombin, suggesting that these phenomena may involve a thrombin-catalyzed modification of the membranes leading decreased binding.


Iron uptake by Chang cells from transferrin, nitriloacetate and citrate complexes: the effects of iron-loading and chelation with desferrioxamine. Iron uptake by Chang liver cells in culture is about thirty times as great when ferric nitriloacetate is used as a donor as when iron-transferrin is used. Iron uptake from ferric citrate is no greater than from iron-transferrin. Most of the intracellular iron derived from transferrin is found in the supernatant after 20 000 x g centrifugation of the cell homogenate for 40 min: about half of this is in the form of ferritin. Iron derived from ferric nitriloacetate is found largely in the membranous pellet after centrifugation and very little of this is in the form of ferritin. Iron incorporated in cytosol ferritin is easily available for chelation by desferrioxamine and this process is facilitated by ascorbic acid. Membrane-bound iron is less available for chelation. This tissue culture model forms a convenient basis for the study of iron overlead and iron chelation.


Effect of cytosol fractions from lutropin-stimulated rat testes on pregnenolone production by mitochondria from normal rat testes. The rate limiting step in the production of steroids in the testis is the mitochondrial conversion of cholesterol to pregnenolone. This conversion can be stimulated by lutropin, but the precise interaction between lutropin-induced cytoplasmic factors and the mitochondrial activity in steroid production is as yet unknown. The results described in the present paper concern the steroid production of isolated mitochondrial fractions in recombination experiments with isolated supernatant fractions from total testes homogenates. Cyanoketone as well as SU-10603, an inhibitor of steroid 17 alpha-hydroxylase activity are required to block pregnenolone metabolism. The results show that the cytoplasm contains lutropin-induced factor(s) which can exert its effect in vitro on the cholesterol side-chain cleavage activity in intact mitochondria isolated from control testes.


Precocious induction of pepsinogen in the stomach of suckling mice by hormones. Effects of hormones on pepsinogen activity in mouse stomach were investigated by enzyme assay and electron microscopy. Administration of hydrocortisone alone to mice on days 5--10 increased the enzyme activity in the stomach to as much as 4.5-fold that of untreated mice and the increase was dose dependent. Thyroxine also evoked precocious differentiation of the stomach. The effects of thyroxine and hydrocortisone were additive. Injections of insulin had little effect when given alone, or in combination with other hormones. Injection of hydrocortisone alone or plus thyroxine also caused morphological differentiation of the chief cells in the stomach mucosa. Administration of thyroxine to mice on days 15--20 induced as much enzyme activity as that induced by hydrocortisone, but neither of these hormones had any effect when injected after day 23. These results suggest that besides hydrocortisone, thyroxine is also involved in differentiation of the stomach in mice for the first 20 days after birth and that the normal increase of pepsinogen activity in the stomach of mice during the late suckling period is brought about by serum glucocorticoids, possibly with thyroxine.


Water binding by glycogen molecules.

Water binding by glycogen molecules. Low temperature NMR spectra have been obtained of the water bound to glycogen. These data have allowed the evaluation of the amount of water bound and the energy and entropy associated with this bonding. High molecular weight glycogen (approx. 1 - 10(9)) exhibits water binding properties analogous to those previously found for other glycoproteins. Low molecular weight glycogen (approx. 1 - 10(7)), however, shows anomalous binding characteristics, with large amounts of associated "non-freezing" water. These findings are discussed in terms of previously proposed molecular architecture.


An ultramicro method for analysis of lithium and other biologically important cations. A new method for Li+ analysis by flameless atomic absorption spectroscopy is several orders of magnitude more sensitive than previous methods. Li+ quantities as small as 1 - 10(-12) mol, or Li+ concentrattions as low as 1 - 10(-8) M, can determined with a coeffient of variation of 2--4%. The same technique can determine approx. 1 - 10(-14) mol of Ca2+ and Mg2+, and approx. 1 - 10(-13) mol of Na+ and K+.


Alterations in the pyruvate dehydrogenase complex during adaptation to glucose by Neurospora. A 20-fold induction of the pyruvate dehydrogenase complex, pyruvate dehydrogenase (EC 1.2.4.1) plus dihydrolipoate S-acetyltransferase, (lipoyltransacetylase) (EC 2.3.1.12) plus dihydrolipoyl dehydrogenase, NADH : lipoamide oxidoreductase, (EC 1.6.4.3), from a specific activity of 3.5-65.0 was observed in mitochondrial extracts during adaptation of Neurospora to glucose from acetate media. The extent of ATP-dependent, time-dependent inactivation of the pyruvate dehydrogenase complex was approximately the same in both acetate- and glucose-grown cells, thereby indicating that the low pyruvate dehydrogenase complex activities in acetate-grown cells did not represent phosphorylated pyruvate dehydrogenase complex molecules. High levels of dihydrolipoyl transacetylase (EC 2.3.1.12) were observed in mitochondrial extracts from acetate-grown cells; this lipoyltransacetylase was analyzed on sucrose density gradients and found to be associated with the pyruvate dehydrogenase complex. Digitonin fractionation of mitochondria revealed that both the pyruvate dehydrogenase complex and lipoyltransacetylase were primarily associated with the mitochondrial outer membrane.


Characterization of a vasoactive intestinal peptide-sensitive adenylate cyclase in rat intestinal epithelial cell membranes. A vasoactive intestinal peptide-sensitive adenylate cyclase in intestinal epithelial cell membranes was characterized. Stimulation of adenylate cyclase activity was a function of vasoactive intestinal peptide concentration over a range of 1 . 10(-10)-1 . 10(-7) M and was increased six-times by a maximally stimulating concentration of vasoactive intestinal peptide. Half-maximal stimulation was observed with 4.1 +/- 0.7 nM vasoactive intestinal peptide. Fluoride ion stimulated adenylate cyclase activity to a higher extent than did vasoactive intestinal peptide. Under standard assay conditions, basal, vasoactive intestinal peptide- and fluoride-stimulated adenylate cyclase activities were proportional to time of incubation up to 15 min and to membrane concentration up to 60 microgram protein per assay. The vasoactive intestinal peptide-sensitive enzyme required 5-10 mM Mg2+ and was inhibited by 1 . 10(-5) M Ca2+. At sufficiently high concentrations, both ATP (3 mM) and Mg2+ (40 mM) inhibited the enzyme. Secretin also stimulated the adenylate cyclase activity from intestinal epithelial cell membranes but its effectiveness was 1/1000 that of vasoactive intestinal peptide. Prostaglandins E1 and E2 at 1 . 10(-5) M induced a two-fold increase of cyclic AMP production. Vasoactive intestinal peptide was the most potent stimulator of adenylate cyclase activity, suggesting an important physiological role of this peptide in the cyclic AMP-dependent regulation of the intestinal epithelial cell function.


Maturation dependent decline of adenylate cyclase in rabbit red blood cell membranes. Adenylate cyclase (EC 4.6.1.1) was studied in membrane preparations of reticulocyte-rich blood obtained from phenylhydrazine-treated rabbits and compared to that of untreated animals. Basal and fluoride-stimulated activities were decreased 2- and 4-fold, respectively, during the process of maturation. Catalytic parameters such as time course, protein, ATP, Mg2+ concentration curves and Km have been determined and were found to be similar in the reticulocyte and the erythrocyte. Adenylate cyclase was sensitive to GTP, 5'-guanylyl imidodiphosphate, prostaglandin E1 and prostaglandin E2. Activation by prostaglandin E1 was higher than that produced by prostaglandin E2. Only additive effect was found when 5'-guanylyl imidodiphosphate or GTP was added to hormone-stimulated activity. The sensitivity of the enzyme to these effectors was decreased over the transition reticulocyte-erythrocyte. In either cell the enzyme was not activated by catecholamines (epinephrine, norepinephrine, isoproterenol).


Catabolism of threonine in mammals by coupling of L-threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase. There is doubt about the L-threonine 3-dehydrogenase (EC 1.1.1.103) and threonine aldolase (EC 2.1.2.1) catabolic pathways of L-threonine in mammals which are believed to produce aminoacetone and glycine plus acetaldehyde, respectively. L-Threonine 3-dehydrogenase in disrupted guinea-pig liver mitochondria was investigated in a reaction mixture containing L-threonine without and with CoA and oxaloacetate; L-[U-14C]threonine was included in four similar experiments for autoradiograms. Threonine aldolase was examined in similar mitochondria from liver and kidney. CoA reduced the aminoacetone formed from L-threonine to 10-14% and CoA plus oxaloacetate produced citrate (from CoASAc) in approximately equal amounts to the decrease in aminoacetone. Autoradiograms confirmed the decrease in aminoacetone with the simultaneous appearance of citrate and glycine. No evidence was obtained that threonine aldolase catabolised L-threonine at the concentration used to assay the dehydrogenase. It is concluded that 2-amino-3-oxobutyrate (precursor of aminoacetone), which is produced from L-threonine by L-threonine 3-dehydrogenase, undergoes CoA-dependent cleavage to glycine and CoASAc by 2-amino-3-oxobutyrate-CoA ligase. The results suggest that the coupling of these enzymes provides a new pathway for the catabolism of threonine in mammals.


Alphatic 3,4-epoxyalcohols. Metabolism by epoxide hydrase and mutagenic activity. Rabbit hepatic microsomal epoxide hydrase catalyzes the rapid hydrolysis of 1,2-epoxy-4-heptanol to 1,2,4-heptanetriol. Both diastereomers of the substrate are hydrolyzed, and both product diastereomers are formed. Similarly both cis- and trans-3,4-epoxy-1-hexanol are hydrolyzed, albeit more slowly, to give 1,3,4-hexanetriol. The trans isomer gives exclusively one diastereomer (erythro) of the triol, while the cis isomer gives the other diastereomer (threo). The product expected if a primary cationic intermediate were to be formed and trapped intramolecularly during the hydrolysis of 1,2-epoxy-4-heptanol, 2-propyl-4-tetrahydrofuranol, was not observed. A comparison of the mutagenic activity in the Ames test of 1-heptane, 1-hepten-4-ol, 1,2-epoxyheptane, and 1,2-epoxy-4-heptanol revealed that only the latter is a detectable mutagen. A vicinal hydroxyl therefore does not interfere significantly with enzymatic epoxide hydrolysis, but it does enhance the bioalkylating potential of even an aliphatic epoxide.


Metabolism of sulfated glycosaminoglycans in cultured endothelial cells and smooth muscle cells from bovine aorta. The glycosaminoglycan metabolism of cultured endothelial cells and of cells grown from the intima and from the media layer of bovine aorta thoracia was investigated in a comparative study. The following results were obtained: 1. Endothelial cells have in common with intima and media cells the distribution of newly formed sulfated glycosaminoglycans into extracellular, pericellular and intracellular compartments. Endothelial cells, however, synthesize lower amounts of glycosaminoglycans and distribute them in a different ratio into the three pools. 2. Though all the various cell lines synthesize chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparan sulfate and small amounts of keratan sulfate, endothelial cells exhibit a unique distribution pattern of sulfated glycosaminoglycans in each of the three compartments. Generally, a high proportion of heparan sulfate and chondroitin 6-sulfate and a very low dermatan sulfate content was detected. 3. Heparan sulfate produced by endothelial cells has a higher N-sulfonate content when compared with that from other sources. The cell membrane-associated heparan sulfate, especially, exhibits some heparin-like features as judged by nitrous acid degradation and susceptibility towards heparitinase.


Uterine and lung uteroglobins in the rabbit. Two similar proteins with differential hormonal regulation. Previous studies have shown that several rabbit tissues contain proteins which cross-react in the radioimmunoassay for uteroglobin, a progestin-regulated protein in rabbit uterus (Torkkeli et al. (1977) Mol. Cell. Endocrinol. 9, 101-118). In the present study, a uteroglobin-like protein was purified to an apparent homogeneity from an extra-uterine tissue, rabbit lung, by successive chromatographies on hydroxyapatite, Sephadex G-75, SP-Sephadex, DEAE-cellulose and CM-cellulose. The final preparation behaved homogeneously in various polyacrylamide gel electrophoretic systems and in isoelectric focusing. The uteroglobin-like protein isolated from the lung had very similar physicochemical and immunological properties to those of uteroglobin present in the rabbit uterine fluid. The two proteins had: (i) the same molecular weight, of approx. 13 000, with a two subunit structure (each approx. Mr 7000); (ii) identical behavior in polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions; (iii) the same isoelectric point at pH 5.4; (iv) absence of carbohydrate in the molecule; (v) very similar amino acid compositions; (vi) lack of tryptophan among the amino acids; (vii) the same N-terminal amino acid (glycine), and (viii) indistinguishable immunological characteristics. Collectively, these data strongly suggest that uterine and lung uteroglobins are identical proteins. In contrast to the induction of the uterine uteroglobin by steroids with progestational activity, the synthesis of extra-uterine uteroglobins was no affected by these steroid hormones to any major extent. In keeping with the concept that lung is a target tissue for glucocorticoid action, cortisol and dexamethasone were capable of increasing the concentration of lung uteroglobin 3-fold (from 3 to 9 microgram/mg soluble protein). These compounds did not, however, alter the secretion of the uterine protein. Administration of high doses of testosterone and 5alpha-dihydrotestosterone elevated significantly the content of both uterine and lung uteroglobin. Only approx. one-fifth of the adult pulmonary uteroglobin levels were present in lungs of newborn rabbits indicating that developmental changes occur in the lung uteroglobin content.


Interaction of anthelmintic benzimidazoles and benzimidazole derivatives with bovine brain tubulin. The binding and inhibitory properties of 11 benzimidazoles for bovine brain tubulin were investigated. The effects of the benzimidazoles on the initial rates of microtubule polymerization were determined by a turbidimetric assay. The median inhibitory concentrations (I50) for nocodazole, oxibendazole, parbendazole, mebendazole and fenbendazole ranged from 1.97 . 10(-6) to 6.32 . 10(-6) M. Benomyl, cambendazole and carbendazim had I50 values from 5.83 . 10(-5) to 9.01 .10(-5) M. Thiabendazole had an I50 value of 5.49 . 10(-4) M. Inhibitor constants (Ki) were determined by the colchicine binding assay. Oxibendazole, fenbendazole, and cambendazole had Ki values of 3.20 . 10(-5), 1.73 . 10(-5) and 1.10 . 10(-4) M, respectively. Oxibendazole and fenbendazole were competitive inhibitors of colchicine. In contrast, cambendazole was a noncompetitive inhibitor of colchicine. The ability of these benzimidazoles to inhibit microtubule polymerization and the mode of action for the anthelmintic benzimidazoles is discussed.


Photodynamic inactivation and its repair in mycoplasmas. Photodynamic inactivation is the loss in viability observed when organic dye-treated cells are exposed to visible light and molecular oxygen. The photodynamic inactivation of mycoplasmas, the smallest free living cells, has been studied. Depending on the extent of inactivation in Acholeplasma laidlawii, photodynamic induced damage can be repaired if the irradiated cells are incubated in the dark in buffer. Analysis of the DNA of these cells shows that photodynamic inactivation induces single strand breaks which can be repaired during liquid holding. To examine possible damage to the cell membrane, glucose uptake was studied as a permeability measure. Neither acriflavine nor photodynamic inactivation had any measurable effect on membrane permeability.


The effect of warfarin and vitamin K-1 on the carboxylation and glycosylation of prothrombin in vivo. The effect of warfarin and vitamin K-1 on the carboxylation and glycosylation of prothrombin in the rat in vivo was investigated. Neither warfarin nor vitamin K-1 had an effect on carboxylation. However, warfarin inhibited glycosylation 80-90% and this inhibition was readily reversed by the administration of vitamin K-1.


Degradation of rat liver metallothioneins in vitro. The degradation of zinc and cadmium-induced hepatic metallothionineins was investigated in vitro. Both zinc-thionein and cadmium-thionein were labeled in vivo with [35S]cystine. The labeled proteins were isolated and purified by gel filtration and DEAE-ion exchange chromatography. Purified zinc[35S]thionein and cadmium-[35S]thionein were incubated with trypsin, chymotrypsin and pronase for varying times up to 24 h. The rate of degradation of zinc-thionein was twice that of cadmium-thionein when the proteins were incubated with trypsin. Virtually no digestion occurred when the proteins were incubated with chymotrypsin, whereas the rates of degradation were about equal when they were incubated with pronase. In contrast, degradation of zinc-thionein was twice that observed with cadmium-thionein when the proteins were incubated at pH 5.0 with a purified lysosomal extract. Degradation of these proteins by the lysosomal proteases was 77 and 46% within 3 h for zinc-thionein and cadmium-thionein, respectively. Thionein, the metal-free form of metallothionein, was degraded extremely rapidly by both neutral and lysosomal proteases. Chromatography of the digestion products on Sephadex G-25 demonstrated that all three forms of metallothionein were degraded to species of approximately 100-300 daltons. These data indicate that metals stabilize thionein polypeptides and suggest that the degradation of metallothionein in vivo is regulated in part by the species of metal bound.


Isolation and identification of beta-citryl-L-glutamic acid from newborn rat brain. An unknown compound containing glutamic acid residue was found in newborn rat brain. The compound occurred predominantly in brain. Its concentration was approx. 1 mumol/g tissue at birth and decreased to one-tenth 24 days after birth. The compound was isolated from newborn rat brains, and subjected to elementary analysis and to infrared and mass spectrometric analysis. Glutamic acid and citric acid were formed from the compound on acid hydrolysis. The compound was presumed to be a citrylglutamic acid. Two isomers, alpha- and beta-citrylglutamic acid, were synthesized. The unknown compound was identified as beta-citryl-L-glutamic acid. The occurence of this compound has not been reported in nature.


Isolation and characterization of surface glycopeptides from adult rat hepatocytes in an established line. The fractionation of glycopeptides isolated from the surface of an established line of adult rat hepatocytes, after 48 h metabolic labeling with [14C] glucose leads to the demonstration of the presence of two types of glycopeptides: i) O-glycopeptides containing N-acetylneuraminic acid, galactose and N-acetylgalactosamine, ii) N-glycopeptides with mannose, galactose, N-acetylgluosamine, fucose and N-acetylneuraminic acid. The former yield, by alkaline reductive degradation, a trisaccharide: sialyl-galactosyl-N-acetyl-galactosaminitol, a tetrasaccharide : di-sialyl-galactosyl-N-acetyl-galactosaminitol and a small amount of the disaccharide: galactosyl-N-acetyl-galactosaminitol. The latter are alkali-stable and can be divided by ion-exchange chromatography on DEAE-cellulose into i) acidic glycopeptides, not retained on a Concanavalin A-Sepharose column and belonging to the N-acetyllactosaminic type glycopedtides and ii) neutral glycopeptides which can be further fractionated on the Con-A-Sepharose column into non-retained glycopeptides belonging to the N-acetyllactosaminic type, and glycopeptides retained on the Sepharose-Con A column, containing only N-acetylglucosamine and mannose residues and related to the oligomannosidic type glycopeptides.


Structure of the three major fucosyl-glycoasparagines accumulating in the urine of a patient with fucosidosis. Fifteen fucosyl-oligosaccharides and fucosyl-glycoasparagines have been isolated from the urine of a patient with fucosidosis. The structure of the three most abundant glycoasparagines are as follows: alpha-Fuc-(1 lead to 6)-beta-GlcNAc-Asn; alpha-Man-(1 leads to 6)-beta-Man-(1 leads to 4)-beta-GlcNAc-(1 lead to 4) [alpha-Fuc-(1 leads to 6)]-belta-GlcNAc-Asn; beta-Gal-(1 leads to 4) [alpha-Fuc-(1 lead to 3)] beta-GlcNAc-(1 leads to 2)-alpha-Man-(1 lead to 6)-beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4) [alpha-Fuc-(1 leads to 6)] beta-GlcNAc-Asn. The structures are related to the class of fucosyl-glycoproteins (e.g.: IgG immunoglobulin, lactotransferrin and alpha 1-acid glycoprotein). The terminal sequence: beta-Gal-(1 leads to 4) [alph-Fuc-(1 leads to 3)] beta-GlcNAc-(1 leads to 2)-a-Man leads to R is novel for carbohydrate moieties in glycoproteins.


Role of spermidine in casein gene expression in the rabbit. Spermidine concentration in rabbit mammary gland was estimated during pregnancy, lactation and after the induction of milk synthesis by prolactin and glucocorticoids in vivo and in vitro. It was observed that mammogenesis and lactogenesis during preganancy and the initiation of milk secretion at parturition are accompanied by an enhancement of spermidine concentration in the mammary gland. By contrast, the initiation of these phenomena by hormone injections does not require such variations of spermidine concentration. In organ culture, a slight increase in spermidine concentration was obtained under the influence of an hormonal combination including insulin, prolactin and cortisol. Spermidine added to the culture medium was unable to mimic cortisol action. An amplification of casein synthesis and a parallel increase of casein mRNA concentration was provoked by cortisol even when spermidine synthesis was blocked. Thus, one of the major actions of glucocorticoids during lactogenesis in the rabbit is not mediated through an increase in spermidine concentration in the mammary gland.


[Role of lipids in the function of microsomal electron transport chains of potato]. Microsomal membranes from potato tubers were extracted by acetone solutions of increasing concentrations (5, 10, 15, 20, 30, 40, 50, 70 and 90 p. cent). Microsomal lipids were progressively extracted: acetone concentrations exceeding 30 p. cent extracted large amounts of membraneous phospholipids (figure). Lipid extraction reduced NADH-cytochrome c reductase activity but did not affect NADH-ferricyanide reductase and NADPH-cytochrome c reductase activities. This was confirmed by experiments using increasing concentrations of sodium deoxycholate. After lipid extraction with acetone (or solubilization by triton X100), NADH-cytochrome c reductase activity of microsomal membranes could not be recovered by adding back lipids under various experimental conditions. These results strongly suggest that, in potato microsomes, lipids are undispensable components of the electron transport chain starting from NADH especially in the portion involving cytochrome b5. On the contrary, the second microsomal electron transport chain, starting from NADPH, is not regulated by lipids. However, plant microsomal membranes would be much more disturbed by liped extraction than animal microsomes and suitable relipidation conditions remain to be found to prove definitely the lipid dependence of plant microsomal electron transport.


Phenylalanine analogues as inhibitors of phenylalanine-hydroxylase from rat liver. New conclusions concerning kinetic behaviors of the enzyme. The conversion of phenylalanine to tyrosine is catalysed by phenylalanine-hydroxylase. The substrate phenylalanine shows two effects: (1) allosteric transition at low phenylalanine concentrations, (2) excess substration inhibition. The molecular structure of phenylalanine-hydroxylase has not yet been elucidated. However, a tetrameric structure has been proposed. The Kinetic analysis with respect to substrate analogues suggest the existence of three types of sites on each protomer: (1) a catalytic site, (2) a non-competitive inhibitory site, (3) a positive cooperative site. Use of the enzyme's natural cofactor, tetrahydrobiopterin, has been emphasized to ensure good interpretation of the kinetic results of the phenylalanine-hydroxylase effectors.


Self-observation and self-reinforcement as sources of self-control in children. Eight fifth- and sixth-grade black males engaged in a set of single-subject, multiple-baseline studies to determine the relative effects of self-observation and self-reinforcement. A black male college student employed the children as research assistants who would study their own behavior. The employer negotiated a series of individualized contracts with each boy. The contracts specified what self-regulation procedures the subject would perform. Overall, the children were more consistent in carrying out their contracts when they were on self-reinforcement than when they were on self-observation. Second, the children were more effective in increasing behavioral deficits than they were in decreasing excesses. Third, self-reinforcement was clearly a superior means of improving their own behavior than was self-observation.


Learning processes in multilayer threshold nets. An algorithm of learning in multilayer threshold nets without feedbacks is proposed. The net is built of threshold elements with binary inputs. During a learning process each input vector chi is accompanied by a teacher's decision omega (omega epsilon(1,...,M)). The pairs (chi[n], omega[n]) appear in successive steps independently according to some unknown stationary distribution p(chi, omega). The problem of learning of a threshold net has been decomposed to a series of problems of learning of the threshold elements. The proposed learning algorithm of the threshold elements has a perceptron-like form. It was proven that a decision rule of the threshold net stabilizes after a finite number of steps. For definite classes (p(chi,omega))K of distributions p(chi, omega), an optimal decision rule stabilizes after a finite number of steps. These classes (p(chi, omega))K also contain distributions describing learning processes with perturbations.


Performance of a model for a local neuron population. A model of a local neuron population is considered that contains three subsets of neurons, one main excitatory subset, an auxiliary excitatory subset and an inhibitory subset. They are connected in one positive and one negative feedback loop, each containing linear dynamic and nonlinear static elements. The network also allows for a positive linear feedback loop. The behaviour of this network is studied for sinusoidal and white noise inputs. First steady state conditions are investigated and with this as starting point the linearized network is defined and conditions for stability is discovered. With white noise as input the stable network produces rhythmic activity whose spectral properties are investigated for various input levels. With a mean input of a certain level the network becomes unstable and the characteristics of these limit cycles are investigated in terms of occurence and amplitude. An electronic model has been built to study more closely the waveforms under both stable and unstable conditions. It is shown to produce signals that resemble EEG background activity and certain types of paroxysmal activity, in particular spikes.


Pattern separability and the effect of the number of connections in a random neural net with inhibitory connections. It has been claimed that pattern separation in cerebellar cortex plays an important role in controlling movements and balance for vertebrates. A number of the neural models for cerebellar cortex have been proposed and their pattern separability has been analyzed. These results, however, only explain a part of pattern separability in random neural nets. The present paper is intended to study an extended theory of pattern separability in a new model with inhibitory connections. In addition to this, the effect of the number of connections on pattern separability is cleared up. It is also shown that the signal from the inhibitory connections has crucial importance for pattern separability.


Properties of length-selective and non-length-selective adaptation mechanisms in human vision. The contrast threshold elevation effect has been measured for one dimensional (grating) and for two dimensional (spot) stimulus patterns. It has been shown previously (Burton and Ruddock, 1978) that such stimuli elicit, respectively, non-length-selective and length-selective adaptation effects. It is established that, unlike the frequency shift effect, the contrast threshold elevation effect is sensitive to the width of the light but not to that of the dark elements of the stimulus patterns. Adaptation to spot patterns elicits a significant threshold elevation for detection of both spot and grating test stimuli, but only under monoptic viewing conditions. The experimental findings are summarized in a block-diagram and it is shown that adaptation to grating patterns is successfully described by the spatial frequency response data given by Maudarbocus and Ruddock (1973).


Silent endocrine tumors. A steady-state analysis of the effects of changes in cell number for biological feedback systems. Some tumors of hormonal organs are clinically active, while others are not. The "silent" tumors may be discovered by accident or because of effects due to their increase in size. From a simple steady state analysis of hormonal feedback systems follows that hormonal cell multiplication does not significantly influence the systems steady state behaviour (hence the clinical silence).--Exceptions to this rule occur in three situations: when the gain of the system is low; when the growth concerns cells with isolated sensor or reference functions; or because of the growth of autonomous cells. In many biological systems the dangerous situation of clamping to low levels upon sensor cell multiplication has been prevented by lumping, such as the combination of sensor and comparator functions into sensor-comparator cells.


Computer simulated discharges of thalamic ventrobasal neurones during sleep and wakefulness. A theoretical model is described which in response to combinations of poissonian pulse trains with different mean frequencies on three independent incoming lines, generated output signals simulating spontaneous discharges of thalamic ventrobasal (VB) neurones during sleep and wakefulness. Some dynamic neuronal properties as refractoriness, facilitation, short term memory were simulated and characteristics of response to single pulses on different lines properly selected to reproduce those exhibited by VB neurones upon artificial stimulation of thalamic afferent systems. The data obtained from the model are briefly discussed in relation to possible contributions of specific and nonspecific afferent systems in producing spontaneous VB discharges characteristic of different levels of vigilance.


Visual discrimination of textures with identical third-order statistics. We found a new class of two-dimensional random textures with identical third-order statistics that can be effortlessly discriminated. Discrimination is based on local "granularity" differences between these iso-trigon texture pairs. This is the more surprising since it is commonly assumed that texture granularity (grain) is determined by the power spectrum which, in turn, can be obtained from the second-order statistics. Because textures with identical third-order statistics must have identical second-order statistics (i.e., identical power spectra), visible texture granularity is not controlled by power spectra, and not even by third-order statistics.