Mechanism of action of serine proteases: tetrahedral intermediate and concerted proton transfer. Stopped-flow spectrophotometry and proton inventory experiments have been used to define the reaction pathway for hydrolysis of a specific peptide substrate, Ac-L-Ala-L-Pro-L-Ala p-nitroanilide, by the serine proteases elastase and alpha-lytic protease. The stopped-flow studies reveal the existence and buildup of a tetrahedral adduct between the active site serine hydroxyl group and the sensitive carbonyl group of the substrate. The decomposition of this tetrahedral intermediate to the acyl enzyme and p-nitroaniline is the rate-limiting step for the hydrolytic reaction. The proton inventory data suggest the simultaneous transfer of two protons (presumably from the catalytic carboxyl of Asp-102 to N pi of the catalytic imidazole of His-57 and from N pi of the imidazole to the anilide NH) in the transition state leading to breakdown of the tetrahedral complex. That these proton transfers occur in a concerted, rather than stepwise, process attests to the ability of enzymes to lower the enthalpy of activation most effectively when the precise alignment of a highly specific substrate and catalytic groups minimizes the entropy of activation.


Deuterium and tritium exchange in enzyme kinetics. The theory of the isotopic exchange of deuterium and tritium between an enzyme-substrate complex and the solvent is derived for 16 different types of experiment involving measurements of initial velocities and of the isotopic content of the reactants and products as a function of the extent of reaction. It is shown how the data from these experiments can be analyzed to obtain the rate constants for the individual steps and thereby the Gibbs free energies of the intermediates and transition states in the reaction. The effects of isotopic substitution on each intermidiate and transition  state are also found and this allows conclusions to be drawn as to the extent to which a proton is in flight in a particular transition state. Neither substrate handling (that is, on-off steps), nor the isotopic exchange with the solvent, is assumed to be rapid.


Energetics of triosephosphate isomerase: the appearance of solvent tritium in substrate dihydroxyacetone phosphate and in product. When the isomerization of dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate is catalyzed by triosephosphate isomerase in tritiated water, both the substrate and product become labeled. The specific radioactivity of the product is about 80% that of the solvent, which shows that the protonation of the enediol intermediate at C-2 (to form the enzyme-bound product D-glceraldehyde 3-phosphate) is followed by a slower step not involving proton transfer. The specific radioactivity of the remaining substrate after partial reaction rises as the reaction proceeds and shows that the reaction intermediate that exchanges protons with the medium returns to dihydroxyacetone phosphate (picking up tritium) about one-third as often as it is converted to D-glceraldehyde 3-phosphate. These results allow a qualitative description of the relative heights of the energy barriers in the catalyzed reaction and contribute to the quantitative analysis of the energetics of the process.


Energetics of triosephosphate isomerase: the appearance of solvent tritium in substrate glyceraldehyde 3-phosphate and in product. When the isomerization of D-glyceraldehyde 3-phosphate to dihydroxyacetone phosphate is catalyzed by triosephosphate isomerase in tritiated water, both the substrate and the product become labeled. The specific radioactivity of the product is only about 13% that of the solvent, which shows that the protonation of the enediol intermediate at C-1 (to form the enzyme-bound product dihydroxyacetone phosphate) is a kinetically significant step, and that the rate of loss of dihydroxyacetone phosphate from the enzyme is relatively fast. The specific radioactivity of the remaining substrate after partial reaction rises as the reaction proceeds and shows that the reaction intermediate that exchanges protons with the medium returns to D-glyceraldehyde 3-phosphate about one-third as often as it is converted to dihydroxyacetone phosphate. These results confirm the qualitative description of the relative heights of the energy barriers in this reaction and further contribute to the quantitative analysis of the free-energy profile.


Energetics of triosephosphate isomerase: deuterium isotope effects in the enzyme-catalyzed reaction. The effect of isotopic substitution of the specifically labilized hydrogen in the substrates of triosephosphate isomerase on the steady-state rates of the enzyme-catalyzed reaction has been examined. The k cat value for the enzyme-catalyzed transformation of [1(R)-2H] dihydroxyacetone phosphate is 2.9 times smaller than that for the 1(R)-1H compound. Because of the rapid loss of 2H to solvent from the enzyme-enediol complex, this factor represents the full kinetic isotope effect of the proton abstraction step. The values of k cat and of Km for D-[2-2H]glyceraldehyde 3-phosphate are indistinguishable from those of the 2-1H material. This arises from the rapid loss of 2H from the enzyme-enediol intermediate, which results in 1H rather than 2H transfer in the rate-limiting step. The steady-state kinetic results reported in this paper qualitatively confirm and quantitatively extend the results from the previous papers on the variation of the free energy along the reaction path.


Energetics of triosephosphate isomerase: the nature of the proton transfer between the catalytic base and solvent water. The isomerization of specifically deuterium-labeled [1(R)-2H5dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate, catalyzed by the enzyme triosephosphate isomerase, has been studied. It is shown that the extent of transfer of the 2H label from the substrate to the product D-glyceraldehyde 3-phosphate is (after complete reaction) the same as that of the corresponding transfer of 3H. The absence of an isotope effect shows that the exchange process of the tstopically labeled enzyme carboxyl group, -COOL  H2O leads to -COOH + LOH, does not tnvolve a rate-limiting transition state in which L is the flight. Possible modes for the nature of the ionization of -COOL in 1H2O are discussed.


Free-energy profile of the reaction catalyzed by triosephosphate isomerase. The experimental results on the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate catalyzed by triosephosphate isomerase that are presented in the previous five papers are here collected and analyzed according to the theory presented in the first paper (Albery, W.J., Knowles, J.R. (1976), Biochemistry 15, the first of eight papers in a series in this issue). The rate constants and fractionation factors so derived allow the construction of theGibbs free-energy profile for this enzyme-catalyzed reaction.


On the identity of nuclear membrane and non-histone nuclear proteins. The fate of plasma and nuclear membrane polypeptides in preparations of acidic chromosomal protein from chicken erythrocytes has been investigated. It is shown that detergent extraction procedures (Nonidet P-40, Triton X-100, and saponin), commonly employed in the preparation of acidic chromosomal protein, cannot be relied upon to remove plasma and nuclear membrane polypeptides. These polypeptides persist in nuclear and chromatin preparations and subsequently fractionate as acidic chromosomal protein. In fact, the polypeptides in a preparation of erythrocyte acidic chromosomal protein are shown by gel electrophoresis in dodecyl sulfate to be almost identical to those in a preparation of erythrocyte nuclear membrane. The implication of these results for the preparation of acidic chromosomal protein is dicussed.


Nuclear magnetic resonance studies of D2O-substrate exchange reactions catalyzed by glutamic pyruvic and glutamic oxaloacetic transaminases. Nuclear magnetic resonance studies in D2O (greater than 90%) with glutamic pyruvate transaminase (GTP) (2.6.1.2) demonstrate that this enzyme catalyzes the rapid exchange of both the alpha and beta hydrogens of L-alanine, the exchange of only one alpha hydrogen of glycine, and the beta hydrogens of pyruvate and fluoropyruvate. When the beta hydrogens of L-alanine undergo the enzyme-catalyzed exchange, the product may have 1, 2 or 3 of beta hydrogens exchanged. The exchange is stimulated by the addition of catalytic  amounts of copartner of transaminations reaction. A mechanism is proposed for an extension of the conjugated system to include the alpha and beta carbons to explain the labilization of the beta hydrogens.


Intact photoreceptor membrane from bovine rod outer segment: size and shape in bleached state. Photoreceptor disk membranes isolated from bovine rod outer segments are suspended in dilute aqueous sucrose (4.36 X 10(-3%)) and bleached, and their size and shape are determined with quasielastic and elastic light scattering. They are found to be extremely homogeneous spherical vesicles with a radius of 0.49 +/- 0.07 mum which appear to have derived from the native disk shape by osmotic swelling.


Fluorescence lifetimes of Chlorella pyrenoidosa. The flucrescence decay of Chlorella pyrenoidosa has been investigated under a variety of conditions in the picosecond and nanosecond time regions. Most of the fluorescence is accounted for by an expression of the form I(t) = I0exp-(At+Bt1/2) though an additional exponential term is required to include a weak component of lifetime 32 ps observable only at the higher pulse intensities. This interpretation reconciles earlier and apparently conflicting data. The weak 32 ps component may be associated with Photosystem I, although the possibility that it is an artefact of the high intensity pulses used cannot be excluded at present. The main fluorescence, described by the equation above is attributed to the antenna chlorophyll and is of the form which would be expected from a single light harvesting array with trapping at randomly distributed sites.


Light-dependent development of thermoluminescence, delayed emission and fluorescence variation in dark-grown spruce leaves. Thermoluminescence profiles of spruce leaves grown under various light or dark conditions were measured after excitation at a low temperature (-70 to -20 degrees C) by 1-min illumination with red light, and the following results were obtained. Mature spruce leaves showed five thermoluminescence bands at -30, -5, +20, +40 (or +35) and +70 degrees C (denoted as Zv, A, B1, B2 and C bands, respectively), but dark-grown spruce leaves with a similar chlorophyll content showed only two bands, at -30 and +70 degrees C (the Zv and C bands) and were devoid of the three other bands (the A, B1, and B2 bands). On exposure of the dark-grown leaves to continuous red light, the A, B1and B2 bands were rapidly developed, and the development was accompanied by enhancement of delayed emission, fluorescence variation and the Hill activity (photoreduction of 2,6-dichlorophenolindophenol with water as electron donor). It was demonstrated that the dark-grown spruce leaves are devoid of the water-splitting system in Photosystem II, and that the latent water-splitting activity is rapidly photoactivated by exposure of the leaves to continuous red light. These results on the gymnosperm spruce leaves, in which greening proceeds in complete darkness, being independent of the development of the water-splitting system in light, were discussed in relation to previous observations on angiosperm leaves, in which both greening and the activity generation proceed in the light.


Relation between slow delayed light emission and acid base triggered luminescence in chloroplasts. Acid-base triggered luminescence in relation to slow delayed light emission (greater than 3 s) was studied in chloroplasts. After analyzing their time courses, the acid-base induced luminescence curve was found to return to the original curve of delayed light emission. Peaks of the acid-base triggered luminescence induced after various darkness periods following preillumination decreased parallel to the time course of delayed light emission without base treatment. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea enhanced both the delayed light emission and acid-base induced luminescence, while carbonyl cyanide m-chlorophenylhydrazone inhibited both. Several photophosphorylat ion uncouplers inhibited the acid-base induced luminescence wtihout any substantial effect on the delayed light emission. It is concluded that the acid-base triggered luminescence is not caused by the reversionof electrons from remote intermediates on the reducing side of Photosystem II. The possibility of the presence of an activation pathway for the acid-base triggered luminescence which differs from that of the delayed light emission is also discussed.


Energy transfer in the photochemical apparatus of flashed bean leaves. Fluorescence and energy transfer properties of bean leaves greened by brief, repetitive xenon flashes were studied at -196 degrees C. The bleaching of P-700 has no influence on the yield of fluorescence at any wavelength of emission. The light-induced fluorescence yield changes which are observed in both the 690 and 730 nm emission bands in the low temperature fluorescence spectra are due to changes in the state of the Photosystem II reaction centers. The fluorescence yield changes in the 730 nm band are attributed to energy transfer from Photosystem II to Photosystem I. Such energy transfer was also confirmed by measurements of the rate of photooxidation of P-700 AT -196 degrees C in leaves in which the Photosystem II reaction centers were either all open or all closed. It is concluded that energy transfer from Photosystem II to Photosystem I occurs in the flashed bean leaves which lack the light-harvesting chlorophyll a/b protein.


Studies on the pathway of cyclic electron flow in mesophyll chloroplasts of a C4 plant. 1. Cyclic photophosphorylation driven by white light, as followed by 14CO2 fixation by mesophyll chloroplast preparations of the C4 plant Digitaria sanguinalis, was specifically inhibited by disalicylidenepropanediamine (DSPD), antimycin A, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide (EDAC), and KCN suggesting that ferredoxin, cytochrome b563, plastoquinone, cytochrome f, and plastocyanin are obligatory intermediates of cyclic electron flow. It was found that 0.2 muM DCMU and 40 muM o-phenanthroline blocked noncyclic electron flow, stimulated cyclic photophosphorylation, and caused a partial reversal (40-100%) of the inhibition by DBMIB and antimycin A, but not DSPD. 2. Cyclic photophosphorylation could also be activated using only far-red illumination. Under this condition, however, cyclic photophosphorylation was much less sensitive to the inhibitors DBMIB, EDAC and antimycin A, but remained completely sensitive to DSPD and KCN. Inhibition in far-red light was not increased by preincubating the chloroplasts with the various inhibitors for several minutes in white light. 3. The striking correspondence between the effects of photosystem II inhibitors, DCMU and o-phenanthroline, on cyclic photophosphorylation under white light and cyclic photophosphorylation under far-red light (in the absence of photosystem II inhibitors) suggests that electrons flowing from photosystem II may regulate the pathway of cyclic electron flow.


The isolation of mitochondria from dipteran flight muscle. Procedures for the isolation of mitochondria from dipteran flight muscle have been investigated in an attempt to determine the extent and to identify the causes of deterioration associated with isolation. In the light of the results obtained isolation procedures have been improved by minimising mechanical damage, avoiding the development of anoxic conditions, and by the use of an isolation medium of a more physiological nature, containing the potassium salt of an organic anion as the principal osmoeffector, phosphate as the principal buffer, and low concentrations of free Mg2+. The oxidative capacity of mitochondria isolated by the improved method is adequate to support the in vivo requirements of the flight system.


Interaction of oxidized and reduced N-methylphenazonium methosulfate (PMS) with photosystem II. In 3-(3,4-dicholorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts, the restoration of the fluorescence induction is presumed to be due to a back reaction of the reduced primary acceptor (Q-) and the oxidized primary donor (Z+) of Photosystem II. Carbonylcyanide m-chlorophenylhydrazone (CCCP) is known to inhibit this back reaction. The influence of reduced N-methylphenazonium methosulfate  (PMS) in the absence of CCCP and of oxidized PMS in the presence of CCCP on the back reaction was investigated and the following results were obtained: (1) Reduced PMS at the concentration of 1 muM inhibits the back reaction as effectively as hydroxylamine, suggesting an electron donating function of reduced PMS for System II. (2) The inhibition of the back reactionby CCCP is regenerated to a high degree by oxidized PMS which led to assume a cyclic System II electron flow catalysed by PMS. (3) At concentrations of reduced PMS higher than 1 muM it is shown that both the fast initial emission and more significantly the variable emission are quenched.


Isolation of photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition. Partition in an aqueous Dextran-polyethylene glycol two-phase system has been used for the separation of chloroplast membrane vesicles obtained by press treatment of a grana-enriched fraction after unstacking in a low salt buffer. The fractions obtained were analysed with respect to chlorophyll, photochemical activities and ultrastructural charasteristics. The results reveal that the material partitioning to the Dextran-rich bottom phase consisted of large membrane vesicles possessing mainly Photosystem II properties with very low contribution from Photosystem I. Measurements of the H2O to phenyl-p-benzoquinone and ascorbate-Cl2Ind to NADP+ electron transport rates indicate a ratio of around six between Photosystem II and I. The total fractionation procedure could be completed within 2-3 h with high recovery of both the Photosystem II water-splitting activity and the Photosystem I reduction of NADP+. These data demonstrate that press treatment of low-salt destabilized grana membranes yields a population of highly Photosystem-II enriched membrane vesicles which can be discriminated by the phase system. We suggest that such membrane vesicles originate from large regions in the native grana membrane which contain virtually only Photosystem II.


Absorbance changes due to the charge-accumulating species in system 2 of photosynthesis. Absorbance changes are reported associated with Photosystem II and showing a periodicity of two and four as a function of flash number. The absorbance changes showing a periodicity of two were found to occur in the presence of artificial electron donors as well and are presumably caused by the secondary electron acceptor R of Photosystem II. The absorbance difference spectra suggest that R is a plastoquinone molecule, which is reduced to its semiquinone anion after an uneven number of flashes. After an even number of flashes, the semiquinone is reoxidized. The absorbance changes showing a periodicity of four are tentatively ascribed to the charge accumulating donor complex of Photosystem II.


Reactions of plastocyanin and cytochrome 553 with photosystem I of Scenedesmus. Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates. It is suggested that besides the reaction sequence cytochrome 553 leads to plastocyanin leads to Photosystem I reaction center, a second pathway cytochrome 553 leads to Photosystem I reaction center may operate additionally.


Protein-protein interactions of the light-harvesting chlorophyll a/b protein. II. Evidence for two stages of cation independent association. In a previous paper, we observed a two-stage cation-independent association of the light-harvesting chlorophyll a/b protein from spinach chloroplasts based on concentration-dependent changes in the sedimentation coefficient. The two stages of association occurred between (2-4) and (4-7) mug/ml chlorophyll. In this paper, we provide further evidence for this association. This includes: (1) A decrease in the number of divalent cation binding sites in the second stage of association. (2) A corresponding decrease in the extent of the cation-dependent association. (3) A positive deviation from Beer's law for chlorophyll b for both stages of the cation-independent association and a positive deviation for chlorophyll a for the second stage of association only. (4) A change in the fluorescence emission of both chlorophyll a and b. The change for chlorophyll b was observed for both steps of association whereas that for chlorophyll a was observed for the second step of association only. Therefore, the first stage of association affects only chlorophyll b whereas the second stage alters the environment of both chlorophyll a and b. (5) In addition, divalent cations quenched chlorophyll fluorescence. However, the quenching which required 200-300 muM divalent cation for half-maximal effects was related neither to divalent cation binding nor to the divalent cation-induced association of the protein.


An investigation of water splitting in the Kok scheme of photosynthetic oxygen evolution. The involvement of OH bond breaking in the 4 dark reactions of the Kok scheme of photosynthetic oxygen evolution was investigated using Chlorella and spinach chloroplasts. When the photosynthetic material was suspended in a 2H2O based medium, the reaction rates in all 4 cases were only slightly reduced as compared to the rates observed in an H2O based medium. This was evidence that these rate processes were probably not limited by the breaking of an OH bond. Observations were also made on the yields of O2 from dark adapted Chlorella subjected to a sequence of brief saturating light flashes. The oscillating flash yield sequence observed with algai suspended in 2H2O showed greater damping of the oscillations than when the algae were suspended in H2O. A computer fit of the Kok model to these results revealed a slightly higher proportion of misses, alpha (i.e. absorbed quanta that do not drive photochemistry) in the 2H2O case.


Photooxidation of P-960 and photoreduction of P-800 (bacteriopheophytin b-800) in reaction centers from Rhodopseudomonas viridis. The light excitation of P-960 results in the oxidation of P-960 and the reduction of P-800 (bacteriophytin b-800) in the reaction centers from Rhodopseudomonas viridis. A negative 847 nm band of the circular dichroism psectrum disappears under P-960 photooxidation, while a positive 827 nm band disappears under P-800 photoreduction. Exciton interaction of the pigment molecules in the reaction center is discussed.


Inhibition of the reoxidation of the secondary electron acceptor of photosystem II by bicarbonate depletion. In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R2- by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q-). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-),)-dimethylurea)- induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR-, but also if added in the state QT2-.


Synthesis and loss of activity of glucose-6-phosphate dehydrogenase in dividing plant cells. 1. Using the technique of density-labelling with deuterium oxide, evidence has been obtained for the de novo synthesis of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADPH+ 1-oxidoreductase, EC 1.1.1.49), during the culture of synchronously growing plant cells. 2. The entire increase in enzyme activity during the early cell cycles in this material can be accounted for by the appearance of an enzyme species with increased buoyand density. 3. A method is described for resolving overlapping distribution profiles after density centrifugation, which allows estimation of the amount of each species present at different times, and calculation of the loss of activity of the light species present from the start of culture. 4. Loss of activity of glucose-6-phosphate dehydrogenase in normal growing conditions in the presence of 2,4-dichlorophenoxyacetic acid is very much faster than in conditions which do not lead to cell division: in the absence of 2,4-dichlorophenoxyacetic acid, or in the presence of the inhibitor of RNA synthesis, 6-methylpurine.


The last two pathway-specific enzyme activities of hexosamine biosynthesis are present in Blastocladiella emersonii zoospores prior to germination. Forward direction assays have been developed for the last two pathway-specific enzymes of hexosamine biosynthesis using crude extracts from Blastocladiella emersonii zoospores. The specific enzyme activities measured are substantially higher than those reported with enzyme preparations from other organisms. During the development of one of the assays, another enzyme activity was observed which converts one of the intermediates of the pathway, N-acetylglucosamine-6-phosphate, to N-acetylglucosamine. The finding of these three enzyme activities in zoospore extracts completes the demonstration that all the enzyme activities necessary to synthesize some 2% by weight as chitin early during zoospore germination (de novo cell wall formation) pre-exist in the zoospore. This demonstration is consistent with the conclusion that the hexosamine pathway is regulated at the post-translational level during zoospore germination.


Purification and properties of blood group A-active glycoprotein from oyster viscera. A blood group A active substance was isolated from an acetone-dried powder of oyster viscera by extraction with 0.1 M NaCl after heating a homogenate with extraction medium, in boiling water. After the removal of the acidic fraction with cetylpyridinium chloride, the separated neutral fraction was digested successively with alpha-amylase and amyloglucosidase to remove glycogen. The blood group A-active portion was eluted from a Sepharose 4B column and purified by DEAE-Sephadex column chromatography. The purified active substance was homogeneous by polyacrylamide gel electrophoresis, and its molecular weight was estimated as 100 000 by sedimentation equilibrium. The sugar content of the purified active substance, expressed in percentage of dry weight, was galactosamine, 16.6; galactose, 12.5; fucose, 9.9; glucosamine, 4.6; and glucose, 3.3. Sialic acid was not detected. Total amino acid content was 23.0% and the main constituents were threonine, proline and serine. The ORD spectrum indicated that the hexosamines were N-acetylated. Absence of glycolipid was confirmed by the analysis of fatty acid and sphingosine base. This active substance had a strong blood group A activity (0.04 mug/ml) but neither B nor H activity; it interacted with lima bean lectin but not with concanavalin A.


Structure of chondroitin sulfates. Analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum. The structures of chondroitin sulfate A from whale cartilage and chondroitin sulfate C from shark cartilage have been examined with the aid of the chondroitinases AC and C from Flavobacterium heparinum. The analyses of the products formed from the chondroitin sulfates by the action of the chondroitinases have shown that three types of oligosaccharides compose the structure of chondroitin sulfate A, namely, a dodeca-, hexa- and a tetra-saccharide, containing five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue, respectively. The polymer contains an average of 3 mol of each oligosaccharide per mol of chondroitin sulfate A. Each mol of chondroitin sulfate C contains an average of 5 mol of 4-sulfated disaccharide units. A tetra-saccharide containing one 4-sulfated disaccharide and one 6-sulfated disaccharide C indicating that the 4-sulfated disaccharides are not linked together in one specific region but spaced in the molecule.


Biosynthesis of chondroitin sulfate side chains and hyaluronate. Time-course studies with cartilage slices of calf ribs under different conditions. The time course of double labeling with 35SO24- and [3H]glucosamine was followed in a semi-in vitro system of cartilage slices from calf ribs whose chondroitin sulfate peptide pool consists of (A) less than 1% of very short under sulfated side chains of less than 10 disaccharide units length, (B) 3--5% of short under sulfated longer side chains (16 to 25 disaccharide units), (C) 3--5% of short, slightly oversulfated side chains (16--23 dissacharide units, very probably containing some dermatan sulfate), (D) the bulk material (74--82% of total uronate) of longest, slightly undersulfated or equally sulfated side chains (22--42 disaccharide units). After 10 min incubation rapid chain elongation with [3H]glucosamine and prelabeling with 35SO24- of endogenous acceptors are apparent. Chains of type A exhibit highest specific radioactivities. During 30--60 min incubation it is mainly chains of type B that show highest specific radioactivities, after 90 min chains of type C. On the after hand, chains of type D always incorporated the highest total amount of both precursors. Preincubation of slices for 40 min at 37 degres C strongly enhances labeling rates of all types A and B. After 10 min preincubation followed by 35SO24- labeling for 60 min, a decrease of radioactivity of type A and a distinct increase with type B are observed during the post incubation period. After pulse chase experiments type B exhibits highest specific radioactivities. The data make it evident that undersulfated short chondroitin sulfate side chains form very rapidly in a well organised manner and grow, by elongation and proceeding sulfation processes, to longer higher sulfated chains. The labeling of the hyaluronate pool is about half of that of the chondroitin sulfate pool after a lag phase of 10 min. The latter increases linearly after 35--45 min incubation time. However, after preincubation and chase experiments the hyaluronate pool is more highly labeled. The data indicate different precursor pools of both biosynthesis mechanisms, probably located in different cell compartments and/or different cartilage cells.


The effect of hormones on synthesis of the region linking chondroitin sulfate to protein. 1. Particulate fractions of costal cartilage from young rats are capable of catalyzing the formation of the first two monosaccharide units of the chondroitin sulfate-protein linkage region. 2. Hormonal imbalance has been shown to influence the activity of the glycosyltransferases responsible for the sequential transfer of xylose and galactose from UDPxylose and UDPgalactose, respectively, in the formation of the linkage region. 3. The activity of xylosyltransferase was found to be decreased in costal cartilage of diabetic, thyroidectomized and hypophysectomized rats, but not in rats injected with either testosterone or hydrocortisone. In the latter two treatment groups, galactosyltransferase activity was decreased only in the group receiving hydrocortisone. 4. The combined results of this and previous studies suggest that decreased levels of chondroitin sulfate in diabetic, thyroidectomized and hypophysectomized animals are due to interference in the synthesis of the linkage region of the proteoglycan at the xylosyltransferase level whereas hydrocortisone acts primarily at the level of the galactosyltransferase.


Glycosaminoglycans and glycoproteins associated with rat brain nuclei. The concentration, composition and sulfate labeling of glycosaminoglycans and glycoproteins have been studied in purified nuclei isolated in bulk from rat brain. The concentration of total glycosaminoglycans is 0.142 mumol hexosamine/100 mg protein, comprising 57% chondroitin 4-sulfate, 7% chondroitin 6-sulfate, 29% hyaluronic acid and 7% heparan sulfate. Control experiments demonstrated that less than 5% of the sulfated glycosaminoglycans associated with nuclei could be accounted for by the nonspecific adsorption of soluble acidic proteoglycans to basic nuclear proteins. Glycoprotein carbohydrate is present at a level of 206 mug/100 mg protein, and has an average composition of 30% N-acetylglucosamine, 29% mannose, 19% N-acetylneuraminic acid, 15% galactose, 4% N-acetylgalactosamine, and 3% fucose. Labeling studies also indicated the presence of ester sulfate residues on the glycoprotein oligosaccharides.


Cell protein degradation in cultured rat embryo fibroblasts. Suppression by vinblastine of the enhanced proteolysis by serum-deficient media. Rat embryo fibroblasts grown in Eagle's minimal essential medium with 10% serum were labeled with L-[14C]leucine. After a 24 h cold chase, rates of proteolysis were evaluated by measuring the appearance of trichloroacetic acid-soluble 14C in the media. Cells remaining in minimal essential medium with 10% serum (basal) showed a proteolysis rate of 1% per h, whereas cells placed in minimal essential medium alone (serum-deficient) showed a stimulation of proteolysis to 3--4% per h. This enhanced proteolysis was transitory, occurring only for the first 4--8 h after cells were placed in the serum-deficient media. Vinblastine 10-5 M inhibited the enhanced proteolysis 40% but had no effect on basal proteolysis. Control experiments showed no detectable hydrolysis of extracellular proteins, nor did vinblastine affect the rate of protein synthesis. These data suggest that basal and enhanced proteolysis have at least partially distinct mechanisms in the cell and that only enhanced proteolysis involves microtubules.


The effect of poly(ethyleneglycol) esters on the partition of proteins and fragmented membranes in aqueous biphasic systems. The hydroxyl groups of poly(ethyleneglycol) have been esterified (partly) with a number of carboxylic acids. When these esters are included in dextranpoly(ethyleneglycol)-water biphasic systems the partitions of proteins and membranes between the two phases (and the interface) are in some cases strongly affected. The affinity of serum albumin for the poly(ethyleneglycol)-rich phase is strongly increased when the fatty acid group consists of more than 10 carbon atoms. The partition also depends on the number of double bonds in the fatty acid. A corresponding relationship is found for membranes from spinach chloroplasts. The partitions of ovalbumin, lysozyme (EC 3.2.1.17) and ribonuclease (EC 3.1.4.22) are not influenced by the fatty acid esters. Esters of dibasic carboxylic acids show a minute but marked effect on the partition of proteins in general while malate and tartrate esters affect strongly the partition of chloroplast membranes. The partitions of both proteins and membranes are influenced by poly(ethyleneglycol) deoxycholate. Experiments with malate dehydrogenase (EC 1.1.1.37), lactate dehydrogenase (EC 1.1.1.27), fumarase (EC 4.2.1.2), enolase (EC 4.2.1.11) and glutamate-ocaloacetate transaminase (EC 2.6.1.1) show that their partitions, measured on enzymic activity basis, is changed when esters of benzoic, linolenic, tartaric or deoxycholic acid are included in the biphasic system. The mechanism behind the effect of the esterified poly (ethyleneglycol) on the partition of biomaterial, in this type of aqueous biphasic systems, is discussed in terms of a direct binding of the esters to the partitioned material.


Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity. A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity. The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes. By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [3H]thymidine injection it was found that the label moved largely into the tetraploid compartment. Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation. The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.


Heterogeneity of chondrocyte mitochondria. A study of the Ca2+ concentration and density banding characteristics of normal and rachitic cartilage. Previous morphological studies of the mineralizing epiphysis suggested that some mitochondria were concerned with Ca2+ accumulation while others were associated with cellular energetics and metabolism. To determine if there was mitochondrial heterogeneity in chondrocytes of the epiphyseal growth plate, mitochondria were isolated from four different regions of the plate and subjected to continuous sucrose gradient centrifugation. Centrifugation of the organelles in a narrow density sucrose gradient (1.5--2.0 M) in the presence of inhibitors of Ca2+ transport (ruthenium red and 5,5'-dithiobis-(2-nitrobenzoic acid)) revealed that considerable heterogeneity existed. In the least calcified zone 20% of the mitochondria formed a low density band of low Ca2+ concentration (309 nmol/mg protein). Organelles isolated from more calcified tissue zones showed a concomitant increase in Ca2+ concentration (up to 5700 nmol/mg protein) as well as an increase in the total percentage of mitochondria sedimenting in 2.0 M sucrose. The banding patterns of mitochondria isolated from rachitic and hypertrophic cartilage were similar. In addition, similarities were also noted in the Ca2+ concentration and the cytochrome oxidase activities of mitochondria of these tissues. During recovery from the rachitic condition, there was a change in the density centrifugation characteristics of this tissue and a substantial increase was noted in the proportion of mitochondria sedimenting in 2.0 M sucrose. The Ca2+ concentration of mitochondria of this rapidly calcifying tissue suggested that the critical Ca2+ concentration necessary for initiation of the calcification mechanism was 4 mumol/mg protein.


Fluorimetric determination of the resting potential changes associated with the chemotactic response in Paramecium. (1) Evidence is presented which indicates that the carbocyanine dye (3,3'-dipropyl thiadicarbocyanine) can be used as a spectroscopic probe for monitoring the resting potential across tha plasma membrane of the ciliated protozoan Paramecium. (2) The dye at low concentrations (less than or equal to 1 muM) does not affect either the viability or the motility of the cells, nor does it induce a chemotactic response. (3) The fluorescence of the dye bound to the cells alters as the potential across the membrane is changed by increasing the external cation concentration. (4) The absorbance of the bound dye also changes in response to an alteration of the membrane potential. (5) The membrane potential changes as measured by the fluorescence method have been correlated with the measurements of the potential estimated by microelectrode methods. (6) Both cations which induce a negative chemotactic response in Paramecium (K+, Na+, Ba2+) and several non-toxic cations bring about a rapid depolarization of the plasma membrane. The significance of these rapid changes in relation to the swimming behaviour of the ciliate is discussed.


Nuclear magnetic resonance of tissue 23Na. Correlation time. Shporer and Civan (Biochim. Biophys. Acta (1974) 354, 291--304) reported the effect of magnetic-field strength on the NMR relaxation times of 23Na in frog skeletal muscle. From these data, they estimated the correlation time c for bound 23Na whose tumbling is severely restricted, and they suggested that the fraction of bound 23Na does not exceed some few percent of the total 23Na population. However, a step in their theoretical approach seems oversimplified. With an improved approach, we obtained an effectivec of 4--9 ns for bound 23Na. This value is some 10 times shorter than the corresponding value estimated by them from the same data. On the other hand, their conclusion concerning the amount of bound 23Na seems to remain valid. The origin of the observed difference between the two transverse relaxation times of tissue 23Na is also discussed.


A comparison of negatively and positively charged liposomes containing entrapped polyinosinic-polycytidylic acid for interferon induction in mice. Intravenous injection of negatively and positively charged liposomes containing entrapped poly(I)-poly(C) induced a vigorous interferon response in mice with serum titers of interferon reaching twenty times those observed with comparable dosages of free poly(I)-poly(C). The response did not persist over an extended time period as observed earlier for enhanced interferon production stimulated by positively charged liposomes containing the inducer. Both negatively and positively charged liposomes containing [14C]poly(I)-poly(C) were taken up chiefly by the liver when given intravenously. Negatively charged particles were concentrated somewhat preferentially by the spleen (7--9% of the dose compared to 4--6%). Less radioactivity was found in liver and spleen when negatively charged particles were given intraperitoneally than was the case when positively charged particles were injected by this route. Free [14C]poly(I)-poly(C) was extensively metabolized to low molecular weight materials within four hours of injection, while encapsulation of the polymer provided protection against in vivo degradation. When both preferential localization and protection were considered, from three to five times as much high molecular weight E114C]poly(I)-poly(C) was recovered from liver at four hours after intravenous injection when the compound was given in encapsulated form compared to free polymer. Similarly, for spleen, seven times and three times as much polymeric [14C]poly(I)-poly(C) was recovered following injection of negatively charged liposomes and positively charged liposomes respectively compared to free [14C]poly(I)-poly(C). At 48 h after an intravenous injection of positively charged liposomes, as much as four percent of the dose remained in high molecular weight form in the liver and one percent in the spleen. Following intraperitoneal injections, polymeric [14C]poly(I)-poly(C) recovered from the liver never exceeded 4.3% of the dose, showing that most of the radioactivity in the liver consisted of metabolites. These results suggest that elevated and prolonged production of interferon in animals treated with encapsulated inducer results from a combination of factors including preferential tissue location and protection of the inducer from hydrolytic cleavage.


Fractionation and characterization of low molecular weight solubilized proteins of newborn rat keratohyalin granules. Newborn rat epidermis was extracted using methods reported to extract keratohyalin granules. All extraction techniques yielded preparations of solubilized proteins with similar sodium dodecyl sulfate-polyacrylamide electrophoretograms. The solubilized proteins were fractionated on a Sephadex G-200 column and six low molecular weight protein fractions (apparent molecular weights between 10000 and 18000) have been identified. Four of these have been isolated and partially characterized. Two of the fractions are characterized by high histidine, arginine, serine and glutamic acid concentrations and have an amino acid composition similar to that of the histidine-rich protein characteristic of keratohyalin granules. One of these histidine-rich fractions (molecular weight 13700) has ribonuclease activity. The other two isolated fractions are basic proteins, one of which (molecular weight 12800) is a basic lysine-rich protein. This protein is not found in any other tissues of the new born or adult rat.


The quaternary structure of a molluscan (Helisoma trivolvis) extracellular hemoglobin. The hemoglobin (erythrocruorin) of the planorbid mollusc Helisoma trivolvis has a molecular weight of 1.7-10(6) and a sedimentation coefficient (s0 20, w) of 33.8 S at pH 7.0. At pH 2.0, the pigment consists of 32 S and 13 S material. The hemoglobin exists as a 350 000 molecular weight submultiple in 6 M guanidine and can be further dissociated into a 175-200 000 dalton polypeptide in 6M guanidine, 0.1 M 2-mercaptoethanol or by sodium dodecyl sulfate gel electrophoresis of globin, performic acid oxidized globin or carboxymethylated globin. Electron microscope observations show a ten-membered ring structure measuring 200 A in diameter. It is proposed that Helisoma hemoglobin consists of a 1.7-10(6) dalton circular assembly of ten 175-200 000 dalton polypeptide chains. The amino acid composition of the pigment is reported. The hemoglobin contains one heme per 18-19 000 g protein. Limited proteolysis of the intact pigment shows 60 000,  40 000 and 17 000-18 500 dalton components when analyzed by sodium dodecylsulfate gel electrophoresis. It is likely that the 175-200 000 dalton polypeptide consists of a linear arrangement of 8-12 heme-containing domains, each domain having a molecular weight of 18-19 000.


Conformational changes in two neurotoxic proteins from snake venoms. alpha-Neurotoxin from Naja nigricollis and erabutoxin b from Laticauda semifasciata, two homologous neurotoxic proteins, are studied by circular dichroism, ultraviolet spectroscopy and fluorescence in various water/trifluoroethanol mixtures. The data obtained show that the beta structure of alpha-neurotoxin is conserved in water as well as in the organic solvent. By contrast, erabutoxin b changes from the beta-structure in water to the helix type in trifluoroethanol. The latter induces similarly for both toxins a structural modification around tryptophan 29, a residue common to all neurotoxins known to date. The vicinity of tyrosine 25, another common amino acid, is also altered by the presence of the organic solvent as demonstrated by the sudden increase of reactivity of the phenolic ring towards iodine. The present work affords some evidence for the presence of a particular structure located around the two aromatic residues, which is common to all neurotoxins and able to rearrange independently from the rest of the molecule. Biological importance of this peculiar region is highly probable.


Demonstration and isolation of the hybrid hemoglobins alpha 2 A beta A beta C and alpha 2 A beta S beta C. The corss-linking reagent p,p'-difluoro-m,m'-dinitrodiphenylsulfone has been used to fix in the tetramer form the various species of hemoglobin present in mixtures of hemoglobin A and hemoglobin C and of hemoglobin S and hemoglobin C. Following reaction, the presence of the hybrid hemoglobins alpha 2 A beta A beta C and alpha 2 A beta S beta C in these hemoglobin mixtures was demonstrated electrophoretically and the hybrids were isolated by ion-exchange chromatography. The identity of the alpha 2 A beta A beta C hybrid was further verified by peptide analysis. The success in cross-linking alpha 2 A beta 2 C, alpha 2 A beta A beta C, and alpha 2 A beta S beta C with p,p'-difluoro-m,m'-dinitrodiphenylsulfone shows that the distance between the alpha chain amino terminals in solution for these hemoglobin species is the same as in normal hemoglobin.


Formiminotransferase cyclodeaminase from porcine liver. An octomeric enzyme containing bifunctional polypeptides. The forminiminotransferase-cyclodeaminase complex contains 2.8% carbohydrate and is composed of identical subunits based on molecular weight, isoelectric focusing and the number of CNBr-generated peptides. Electron microscopy demonstrated that the complex is a ring-shaped molecule when negatively stained both in solution and in crystalline structures. Rotational reinforcement of electron micrographs reveals that the molecule is composed of eight subunits arranged in a planar ring. These results indicate that each polypeptide of the complex must contain both enzymic activities.


Studies on metrizamide-protein interactions. 1. The apparent density of catalase after isopycnic centrifugation in metrizamide gradients is dependent on the metrizamide concentration into which the enzyme is dissolved at the beginning of the centrifugation. 2. This different behaviour of the enzyme in metrizamide gradients is due to the formation of a metrizamide-protein complex which is more dense than the uncomplexed catalase. 3. A bimodal distribution of the catalase, with additional heavy bands, was only observed in metrizamide gradients in light water, where rather high metrizamide concentrations are needed even for a banding of the uncomplexed enzyme. 4. The half-life of the metrizamide-protein complex is less than 5 min. This was shown by spectroscopical measurements and band sedimentation analysis in an analytical ultracentrifuge.


[Quantitative analysis of ovalbumin by thin-layer isoelectric focusing (author's transl)]. A new method for quantitation of ovalbumin by thin layer isoelectric focusing in polyacrylamide gel is described. This technique separates ovalbumin from avian-magnum homogenates or egg white and allows the quantitative determination of each ovalbumin fraction. Considering the very small amount (7 mul) of sample needed for an analysis, the sensitivity and the reproducibility of the proposed method are good. The application of this technique to quail egg white ovalbumin analysis leads to the characterization of four fractions A3, A2, A1 and A0 containing, respectively, 0, 1, 2 and 3 atoms of phosphorus per mole and with isoelectric points that range from 4.95 (A3) to 4.65 (A0).


NMR relaxation investigation of water mobility in aqueous bovine serum albumin solutions. T1 and T2 dispersion measurements of proton spin relaxation on aqueous solutions of bovine serum albumin as a function of both protein concentration and temperature, covering Larmor frequencies from 3 kHz to 75 MHz, will be presented as a most conclusive test for a three-phase fast-exchange relaxation model. By means of a computer curve fitting of this model to the experimental data, we were able to separate 3 distinct water environments and, in addition, the rotational tumbling of the bovine serum albumin molecules.


Characterization of components of heat-treated serum albumin with bromcresol green. The interactions of bromcresol green with various components isolated from heat treated bovine serum albumin at 65 degrees C and pH 9.0 were studied spectrophotometrically. The difference spectra of the dye induced by native albumin and component 1, which has the same electrophoretic mobility as native albumin, had a trough at 616 nm and peak at about 668 nm, whereas those induced by the denatured monomer (component 1'), denatured dimer (component 2) and denatured trimer (component 3) had a trough at 610 nm and peak at 655 nm. From these difference spectra, it was concluded that component 1 is a mixture of at least two components (native and modified native forms) and that components 1', 2 and 3, formed during the process of denaturation, have essentially the same conformation in the environment of the binding sites for bromcresol green. A possible mechanism for the heat denaturation of serum albumin is proposed.


Acid phosphomonoesterase of human prostate. Carbohydrate content and optical properties. Human prostatic acid phosphatase I, a glycoprotein, has been analyzed with respect to its quantiative carbohydrate composition and its fluorescence, optical rotatory and circular dichroic spectra. The protein of 89 000 molecular weight has 38 to 41 carbohydrate residues attached, of which 3 residues are fucose, 4 are galactose, 11 mannose, 15 glucosamine and there are 7 to 8 residues of sialic acid. The native glycoprotein contains about 30% alpha-helix as estimated from the rotational and dichroic spectra. Upon removal of sialic acid by neuraminidase treatment, there is a small increase in this value, while the fluorescence intensity at he emission maximum (357 nm) is distinctly increased. These effects suggest that an interaction exists of the sialic acid group with parts of the native protein. Allowance is made for the contribution of the carbohydrate components in interpreting the spectra in structural terms.


Preparation and properties of carp muscle parvalbumin fragments A (residues 1 leads to 75) and B (residues 76 leads to 108). The calcium-binding protein (parvalbumin), isolated from carp (Cyprinus carpio) muscle, has been specifically fragmented into two polypeptides by tryptic hydrolysis at the single arginine residue at position 75. Fragment A contains residues 1 leads to 75 and fragment B is composed of residues 76 leads to 108. The fragments have been characterized according to size, amino acid composition, carboxyl- and aminoterminal analysis. Both fragments appear to be homogeneous by these criteria. The intact protein is known to bind 2 mol of calcium per mol of parvalbumin, and although each fragment alone contains all of the essential ligands for the coordination of one Ca2+, neither fragment displays calcium binding activity. Attempts to reconstitute the two fragments, under a variety of conditions, into a functional complex which can bind calcium have been unsuccessful. The side chain of Arg-75 is known to occupy an internal position in the crystalline structure of parvalbumin (Kretsinger, R.H. and Nockolds, C.E. (1973) J. Biol. Chem. 248, 3313), where it is stabilized by an intricate network of hydrogen bonding involving the side chain of Glu-81. Although this internal salt bridge is approx. 20 A from either calcium binding site, it has been suggested that this structural feature of the molecule plays an essential role in the reversible binding of Ca2+. That the side chain of Arg-75 likewise occupies an internal position in the solution structure is indicated by its unavailability for reaction with 1,2-cyclohexanedione under conditions of physiological pH and temperature. However, in the presence of EDTA and at pH 8, it is readily modified by cyclohexanedione. This modification is accompanied by a concomitant loss in calcium binding activity. Reversal of the modification by treatment with hydroxylamine is accompanied by restoration of calcium binding activity. The serum of these data support the hypothesis that Arg-75 plays a critical role in the structural organization and calcium binding activity of the molecule, and in addition, suggests that the integrity of the peptide bond between Arg-75 and Ala-76 may be necessary for establishing the proper micro-environment required for formation of the internal salt bridge between Arg-75 and Glu-81.


Denaturation of oxyhemoglobulin S by mechanical shaking. The oxy form of sickle hemoglobin precipitates rapidly during mechanical shaking. The rate of precipitation depends on shaking conditions such as stroke amplitude, frequency, angle between the vial axis and the shaking motion, volume and viscosity of hemoglobin solution and temperature. The rate increases linearly with either stroke (S) or frequency (n) above a certain value (S = 5 mm or n = 10Hz). The rate constant was maximum when the angle 0 between the vial axis and the shaking direction was 30 degrees. The rate of precipitation of sickle oxyhemoglobin was faster at high temperatures and always about 10 times greater than that of normal hemoglobin. The activation energies of precipitation of hemoglobin S and A are 20.3 and 21.3 kcal/mol, respectively. The thermogram of hemoglobin was measured by a differential scanning calorimeter. The denaturation temperatures determined from the peak of denaturation curves were 83.5 degrees C for oxyhemoglobin S and 85 degrees C for oxyhemoglobin A. The precipitated hemoglobin after mechanical shaking did not show any denaturation peak indicating that the protein molecules are irreversibly denatured by shaking.


Studies on phytohemagglutinins. XXVIII. Chemical modifications of the lectin from seeds of the lentil (Lens esculenta Moench.). The reaction of lentil lectin with maleic anhydride yields maleyl derivatives with a degree of modification depending on the amount of used maleic anhydride. Hemagglutinating activity of maleyl derivatives decreases with the increasing number of modified lysine residues, but even at a maleylation of 90% of free -NH2 groups, the maleyl derivative of the lectin still retains a hemagglutinating activity. Maleylation of the lentil lectin is not accompanied by dissociation; the chemically modified lectin also retains its bound Mn. Maleyl derivatives of the lectin retain the ability to interact specifically with polysaccharides (Sephadex, mannan). After removal of maleyl groups in acidic medium, the hemagglutinating activity does not differ from that of the native lectin. Acetylation of the lentil lectin with N-acetylimidazole results in formation of a modified protein containing 3-5 acetylated -OH groups of the tyrosyl residues out of the total of 15 groups and 7-17 acetylated -NH2 groups out of the total of 26 free -NH2 groups, depending upon the reaction conditions. The presence of D-mannose in the reaction mixture does not affect the extent of modification of tyrosyl groups, but it decreases the modification of -NH2 groups. Acetyl derivatives of the lentil lectin do not possess hemagglutinating activity, but they retain the ability to interact with polysaccharides and to bind to erythrocytes. Acetylation does not affect the content of protein bound Mn. After de-O-acetylation, the hemagglutinating activity of the modified protein increases to the value of the native protein.


Effect of iron on sedimentation-velocity and gel filtration behaviour of transferrins from several vertebrates. 1. The effect of iron binding on the conformation of transferrins has been studied using analytical gel filtration and differential velocity sedimentation. 2. In all cases where bicarbonate is the anion the transferrin molecules undergo changes in Stokes' radius and sedimentation coefficient of the same order of magnitude, but of opposite sign. 3. Replacement of the bicarbonate ion with oxalate results in smaller changes in these two parameters. 4. In all cases except ovotranferrin, sheep serum transferrin, and human transferrin with oxalate as the anion, changes in Stokes' radius for the addition of the first and second iron atoms are similar. 5. For ovotransferrin and sheep transferrin it is necessary to postulate interaction between the iron-binding sites to account for the change in Stokes' radius caused by the second iron atom binding being greater than for the first. 6. Human transferrin with oxalate as the anion shows a greater change for the first atom bound than for the second which is consistent with the increased size of the oxalate ion compared with bicarbonate.


Conformational and biological properties of a covalently linked dimer of glucagon. Reaction of mono- and bifunctional sulfenyl halides. The tryptophan residue of glucagon was modified by reaction with a mono-functional sulfenyl chloride (2-nitrophenylsulfenyl chloride) and with a bifunctional sulfenyl chloride (2,4-dinitro-1,5-phenyldisulfenyl chloride) to produce a monomeric form of glucagon with a modified tryptophan, glucagon-nitrophenylsulfenyl and a dimeric form (glucagon)2-dinitrophenyldisulfenyl respectively. The dimeric form was isolated by chromatography on Sephadex G-50. The circular dichroism spectra of pH and low temperature. The derivatives activated adenylate cyclase from rat liver to an extent comparable to that of the native hormone, indicating that a glucagon dimer is capable of biological activity and that an intact tryptophan residue is not essential for biological response.


Biochemical aspects of the visual process. XXXIV. Relation between sulfhydryl groups and properties of rhodopsin studied by means of methylmercuric iodide. 1. Treatment of isolated bovine rod outer segment membranes with the very reactive, small and uncharged sulfhydryl reagent methylmercuric iodide shows all six SH groups of rhodopsin can be modified without loss of its typical absorbance spectrum. 2. The partial loss of regeneration capacity, which occurs under these circumstances, can be attributed to secondary conformational changes following the actual modification reaction. 3. These observations plead against a direct involvement of SH groups in these two important parameters of rhodopsin. 4. Upon modification of SH groups, the normal photolytic sequence of rhodopsin is increasingly disturbed. When four or more SH groups are modified, the appearance of a photointermediate resembling metarhodopsin I dominates under conditions where native rhodopsin yields the transition of metarhodopsin II to metarhodopsin III.


Isolation, properties and primary structure of coypu and chinchilla pancreatic ribonuclease. Pancreatic ribonucleases from the hystricomorph rodent species: coypu and chinchilla were isolated using chromatography on carboxymethyl-cellulose. The amino acid sequences were determined from tryptic digests of the aminoethylated proteins. The tryptic peptides were positioned in the sequence by homology with other pancreatic ribonucleases. Coypu pancreas contains one carbohydrate-containing ribonuclease component. From chinchilla pancreas two carbohydrate-containing ribonuclease components were obtained; one homogeneous and the other heterogeneous. The latter differs from the first in being more acidic; it exhibits heterogeneity both in its carbohydrate moiety (glycopeptides both with and without sialic acid were isolated) and in amino acid sequence (probably glycine at position 32 has been partially substituted by aspartic acid). In both ribonucleases the carbohydrate is attached to asparagine 34. Earlier results on the titration behaviour of histidine residues in both proteins obtained by nuclear magnetic resonance spectroscopy are discussed. An ion bridge between the invariant glutamic acid 49 and histidine 80 may explain the high pK value of the latter.


Sheep prothrombin: purification and partial characterization. A procedure for the preparation of highly purified sheep prothrombin is described. The purified zymogen, when subjected to disc gel electrophoresis in polyacrylamide, gave rise to one single band. Only alanine was found as N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. The isoelectric point, as determined by isoelectric focusing in polyacrylamide gel slab, was shown to be 4.9-5.0. Non-chromatographed, but not the purified zymogen, could be converted into active thrombin in half-saturated trisodium citrate seeded with thrombin. Pure sheep prothrombin showed 5.6% of neutral sugars and the following amino acid composition: Ala35, Arg44, Asx54-55, -Cys24, Glx72, Gly53-54, His8, Ile19, Leu45, Lys31, Met7, Phe23, Pro36, Ser34, Thr29-29, Trp16, Tyr19 and Val33, which accounts for a molecular weight of about 66 000 (amino acids only). The molecular weight as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis after reduction by 2-mercaptoethanol, was shown to be 77 000 +/- 3000 (carbohydrates included).


Amino acid sequences of lamprey fibrinopeptides A and B and characterizations of the junctions split by lamprey and mammalian thrombins. The amino acid sequences of the fibrinopeptides A and B from lamprey fibrinogen have been determined. The fibrinopeptide A is the shortest fibrinopeptide ever isolated, being comprised of only six amino acids. The fibrinopeptide B, on the other hand, is the largest fibrinopeptide characterized to date, having 36 amino acid residues and a cluster of covalently bound carbohydrate. As reported previously, lamprey fibrinogen is readily clotted by mammalian thrombins, but only the fibrinopeptide B is released during the process. Lamprey fibrinopeptide A is not released by mammalian thrombins and could only be removed with the use of lamprey thrombin. Firm proof that the lamprey fibrinopeptides A and B are the amino segments of the alpha and beta-chains respectively was obtained by a series of stepwise degradations on lamprey fibrinogen and lamprey fibrins produced in turn by the action of mammalian thrombin (fibrin B) and lamprey thrombin (fibrin A). These studies were supplemented by stepwise degradations on the individual Aalpha and Bbeta-chains. It the case of the lamprey Aalpha-chain it was also possible to release the 6-residue fibrinopeptide A from the isolated chain with lamprey thrombin and demonstrate that the newly exposed amino-terminal sequence begins with the Gly-Pro-Arg sequence characteristic of mammalian fibrin alpha-chains. In fact, the sequences on the fibrin side of both of the junctions split by thrombin(s) are highly conserved and virtually identical with those found in mammalian alpha and beta-chains.


Hemoglobin Dakar = Hb Grady: demonstration by a new approach to the analysis of the tryptic core region of the alpha chain and oxygen equilibrium properties. Preliminary studies have suggested that in Hb Dakar, histidine alpha112 was substituted by a glutamine. A re-investigation on this hemoglobin is presented in this report. A structural study has been performed using a new approach to analyse the tryptic core region of the human hemoglobin alpha chain. After tryptic digestion of the aminoethylated alpha chain, a secondary digestion of the tryptic core was carried out with chymotrypsin and with another protease, thermolysin. Analyses of the chymotryptic and thermolytic peptides indicated that the structure of Hb Dakar was identical to that of Hb Grady previously described by Huisman et al. who showed the insertion of three amino acid residues in position alpha115 or alpha118. The insertion, which was localized near two residues involved in the alpha1beta1 contact, did not produce a dissociation into dimers. Functional studies demonstrated a a slightly increased oxygen affinity, a lowered cooperativity and a normal Bohr effect. The low amount of the abnormal hemoglobin (8%) may in part be explained by a slight instability of the molecule.


Ultraviolet difference spectral studies of human serotransferrin and lactotransferrin. In order to determine the conformational relationship of iron binding of human serotransferrin and lactotransferrin, ultraviolet difference spectral studies were performed in the presence of guanidine chloride and perturbants as deuterium oxide, ethylene glycol, glycerol and polyethylene glycol. In the presence of guanidine chloride solution the molar absorption differences at 292 nm of iron-saturated forms versus iron-free forms of human serotransferrin and lactotransferrin are respectively -16000 +/- 1000 and -14000 +/- 775. These modifications may be attributed to the involvement of tryptophan residues in the iron-binding sites of the two proteins. However, the results do not demonstrate that these tryptophan residues are bound directly to iron. Difference spectral studies in the presence of perturbants show that the apparent exposed tryptophan and tyrosine residues are higher with shorter range perturbants in iron-free forms of both transferrin molecules. The most important modification of exposed tyrosine residues has been noticed upon removing iron from human lactotransferrin than from serotransferrin.


Low temperature magnetic circular dichroism spectra of met- and myoglobin derivatives. 1. Low temperature magnetic circular dichroism spectra of high and low spin derivatives of metmyoglobin and myoglobin have been measured in the Soret and high wavelength regions. 2. The large difference in intensity of the Soret magnetic circular dichroism bands suggest that a correlation exists between the signal intensity and spin state of the heme-iron. 3. From a comparison of the high and low spin sepctra of the myoglobin derivatives it is concluded that oxymyoglobin contains between 10 and 20% of a ferrous high spin component below 100 degrees K.


Laser Raman investigation of the conformation of human immunoglobulin G. Laser Raman spectra of human immunoglobulin G in neutral solution, as well as in the lyophilized and alkaline-denatured states are presented. In the spectrum of the native protein, the amide III band appears at 1240 cm-1 and is assigned to the presence of beta-sheet structure. From its intensity, using a procedure described in this paper, we evaluate the beta-structure content to 37 +/- 4%. This result is supported by the strong amide I' band at 1667 cm-1 and by the presence in the spectra of two bands at 991 and 1078 cm-1, respectively assigned to the C-C and C-N skeletal stretching modes. The differences between the spectrum of the lyophilized powder and that of the solution show that the lyophilization process induces conformational changes that perturb the local environment of some of the tryptophan residues and alter the secondary structure of immunoglobulin G. The beta-structure appears to be more uniform and more abundant in solution. When the protein is denatured at pH 11, the amide III and amide I'bands, which become weaker and broader, shift in frequency from 1240 to 1248 cm-1 and from 1667 to 1656 cm-1 respectively. These changes indicate a decrease in the amount of beta-structure and a transition toward a much more disordered conformation. During the denaturation, the intensities of many bands of the aromatic chromophores change, notably the tryptophan peaks at 879, 1359 and 1573 cm-1.


An experimental and theoretical investigation of the core level spectra of a series of amino acids, dipeptides and polypeptides. A series of simple amino acids, dipeptides and polypeptides have been studied experimentally by electron spectroscopy for chemical analysis and the results theoretically quantified using the charge potential model and semi-empirically computed charge distributions, and non-empirical calculations of absolute and relative binding energies for model systems. Core binding energies are shown to be characteristic of given structural features and the experimental data taken in conjunction with the model theoretical calculations indicate a zwitterionic structure with extensive intermolecular hydrogen bonding for simple amino acids in the solid state.


The structure of Miracil D, a DNA-binding drug. The crystal structure of the drug Miracil D has been determined. Although the accuracy of the analysis is limited by disorder, it is apparent that the thioxanthone ring system is planar. The proximal nitrogen atom of the side-chain probably forms an intramolecular hydrogen bond with the carbonyl oxygen. The rest of the side-chain has a large degree of conformational mobility.


Interaction of a fluorescent reagent, fluorescein mercuric acetate, with nucleic acids. Fluorescein mercuric acetate (fluorescein Hg Ac), which is a fluorescent thiol reagent, was shown to bind to various nucleic acids by measuring the changes in its absorption and fluorescence properties. Up to a critical concentration of free fluorescein Hg Ac (1-10(-7) M for calf thymus DNA, with 42% GC, and 2-10(-7) M for Micrococcus lysodeikticus DNA, with 72% GC) this reagent appears to bind selectively to single-stranded sections in DNA. Above this critical concentration, cooperative binding to double helical DNA occurs, and denatured DNA is obtained after removal of bound fluorescein Hg Ac by dialysis against 1 M KCl. These facts indicate that fluorescein Hg Ac causes the denaturation of double helical DNA prior to binding as has been shown in the case of methylmercuric hydroxide. The binding of fluorescein Hg Ac to DNA is much stronger than that of methylmercuric hydroxide. The number of total binding sites for fluorescein Hg Ac is close to the number of base pairs for both calf thymus DNA and M. lysodeikticus DNA. Furthermore, it was shown that fluorescein Hg Ac binds to thymidine, deoxyguanosine, poly(U) and poly(G). Since fluorescence quenching of fluorescein Hg Ac accompanies its complex formation with DNA and the affinity is markedly high as indicated by the association constant of 6.8-10(7) M(-1) for single-stranded calf thymus DNA, fluorescein Hg Ac can be used for the structural studies of small amounts of nucleic acids.


Mechanism of action of the 12,13-epoxytrichothecene, anguidine, an inhibitor of protein synthesis. Anguidine, muconomycin A, T-2 toxin, crotocin and trichodermin, a group of 12,13-epoxytrichothecenes, inhibit protein synthesis in HeLa cells and in rabbit reticulocyte lysates. These five mycotoxins can be divided into two groups on the basis of the reversibility of their effects in HeLa cells, and kinetics of inhibition and effects on polyribosome structure in rabbit reticulocyte lysates. Anguidine, muconomycin A and T-2 toxin are irreversible inhibitors of protein synthesis; crotocin and trichodermin are reversible inhibitors of protein synthesis. After addition of low concentrations (1 muM) of anguidine, muconomycin A or T-2 toxin to rabbit reticulocyte lysates, polyribosomes are broken down to monosomes. At higher concentrations, 1 mM, these drugs begin to freeze the polyribosomes. Crotocin and trichodermin freeze the polyribosomes at a concentration of 10 muM. We conclude that anguidine, muconomycin A and T-2 toxin act primarily as inhibitors of initiation of protein synthesis, whereas crotocin and trichodermin inhibit the process of chain elongation.


Fractionation on the microscale of brain polyribosomes by electrophoresis on acrylamide gels. A method is described for brain polyribosome fractionation by acrylamide gel electrophoresis. Brain polyribosomes were run in 2.0% gels in quartz capillaries of 800 mum inner diameter where the gels were supported by capillary force. The gels could then be ultraviolet-scanned in situ. Amounts of brain polyribosomes as small as 10-10(-3) A260nm unit could be analysed by this method. The method was checked by running a macroscale-prepared brain polyribosome sample. The various electrophoretic bands obtained showed a favourable A260nm: A280 ratio. A short RNase treatment caused the disappearance of the slowly migrating bands and the emergence of a predominant band migrating faster than the dimer. The various polyribosomal bands were then identified by comparison with the mobility of polyribosome fractions taken from a sucrose gradient fractionation. Finally, the electrophoretic pattern of brain polyribosomes compared favourably with the pattern obtained by the classic method of sucrose gradient sedimentation. The electrophoretic fractionation of polyribosomes prepared from one rat hippocampus (80 mg) is presented.


The isolation and characterization of intact polyribosomes from a cell wall mutant of Chlamydomonas reinhardi. A technique is described for obtaining intact polyribosomes from a cell-wall mutant of Chlamydomonas reinhardi. When cells were lysed by nonionic detergent in buffers containing high salt and Mg2+-EDTA, polyribosomes of up to 25 ribosomes per polyribosome were obtained on sucrose density gradients. Under these conditions, nascent polypeptide radioactivity was associated with the polyribosomes and not with monoribosomes, and inactive monoribosomes were dissociated to ribosomal subunits. Whole cell lysates contain a mixture of cytoplasmic and chloroplastic polyribosomes. The relative contribution of the two types of polyribosomes was evaluated by polyacrylamide gel electrophoretic analysis of ribosomal RNA extracted from polyribosomes. This analysis showed that less than 15% of the polyribosomes from detergent-lysed cells were from chloroplasts. The contribution of chloroplast polyribosomes to total polyribosomes was increased to about 30% by incubation of the cells in chloramphenicol. When cells were disrupted mechanically (in a French pressure cell) only about 10% of the resulting free polyribosomes were chloroplastic.


The distribution of nascent polypeptide chains among intact polyribosomes from Chlamydomonas reinhardi. A model of polyribosome function based on tape theory has been applied to the analysis of intact polyribosomes from Chlamydomonas reinhardi. The distribution of nascent polypeptide chains found on polyribosomes does not conform to the expected pattern in which small polypeptides are synthesized on small polyribosomes and large polypeptides on correspondingly large polyribosomes. This discrepancy was revealed in the analysis of specific activity of polyribosomes (radioactivity in nascent chains per ribosome) versus polyribosome size at labeling saturation. It was found that the specific activity of small polyribosomes was higher than predicted and that of large polyribosomes was lower. This finding was validated by measuring the sizes of nascent chains from various polyribosome size classes by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The presence of large polypeptides on small polyribosomes could be partially accounted for by the synthesis of polypeptides on chloroplast (chloramphenicol-sensitive) polyribosomes. A maximum peptide interval time of 10 s was estimated from the labeling kinetics of the nascent chains of mid-sized (cytoplasmic) polyribosomes. This rate of translation is comparable to that reported in other eucaryotic cells.


Studies on liver chromatin RNA from rats treated with alkylating agents. We have examined firstly some properties of rat liver chromatin RNA and nuclear sap RNA and secondly the incorporation of [3H]orotic acid into the RNA in vivo in control rats and in rats treated with the alkylating agents, N,N-dimethylnitrosamine or methyl methane sulphonate. Half or more of the nuclear RNA is associated with the chromatin and consists mainly of two species: one is labelled and probably comprises "nascent" RNA, and the other is unlabelled and of lower molecular weight. Neither species is attributable to cytoplasmic contamination. Studies with added polylysine with RNAase A and with DNAase I suggest that both species are ironically bound to protein and that the labelled species is not associated with the part of the chromatin DNA most readily degraded by DNAase I. After dimethylnitrosamine treatment, the amount of unlabelled RNA remains constant but the amount of labelled RNA increases after a low dose, and decreases after a high dose. After methyl methane sulphonate treatment, no change occurs in either species. These results can be explained by changes in extent of association of the DNA and protein within the chromatin complex.


Polypyrimidine sequences found in eukaryotic DNA have been conserved during evolution. L-cell DNA contains an unexpectedly large amount of long pyrimidine tracts. Hydroxyapatite chromatography has been employed to show that these polypyrimidines hydridize extensively to the reiterated DNA of a large number of eukaryotes but fail to hybridize to prokaryotic DNA. This reaction is sequence specific and not the result of a special property of polypyrimidines since random 3H-labelled poly(dC-dT) shows poor hybridization to eukaryotic DNA. The hybrids formed by L-cell polypyrimidines and heterologous repeated sequences have a higher thermal stability than the corresponding hybrids of total repeated DNA indicating that sequences related to these polypyrimidines have been conserved during evolution. Furthermore, at least some of these tracts are transcribed because they are capable of reacting extensively with total cellular RNA. Although the function of these sequences is not yet known, the fact that they are widely conserved in evolution and also transcribed leads us to speculate that they play an an important role in eukaryotic cells.


Hybridization of bean chloroplast transfer RNAs to chloroplast DNA. Bean (Phaseolus vulgaris) chloroplast tRNAsLeu and tRNAsPhe hybridize to chloroplast DNA, whereas the corresponding cytoplasmic tRNA species do not, suggesting that chloroplast transfer RNAs are coded for by chloroplast DNA. The hybridization of the three chloroplast tRNAsLeu or of the two tRNAsPhe isoacceptors is not additive, and the isoacceptors compete with each other in the hybridization to chloroplast DNA, suggesting that these isoacceptors are coded for by the same gene(s) and differ only in the extent of post-transcriptional modification. Although hererologous aminoacylation reactions and comparisons of base composition suggest a resemblance between chloroplast and procaryotic tRNAs, only a slight cross hybridization reaction was observed between chloroplast and Escherichia coli leucyl- or phenylalanyl-tRNAs and DNAs.


Isolation and translation of non-crystallin messenger RNA from calf lens. Affinith chromatography of lens polyribosomal RNA on oligo(dT)-cellulose yields three fractions. As arule the second fraction has been neglected in other studies reported in the literature. According to our investigations this fraction in particular contains the messengers for the non-crystallin lens proteins.


Studies on the formation and stability of aminoacyl-tRNA synthetase complexes from Ehrlich ascites cells. Nine aminoacyl-tRNA synthetases from Ehrlich ascites cells were examined with respect to their ability to be isolated as high molecular weight complexes, soluble enzymes, and ribosome-bound enzymes. Several different methods were employed for cell homogenization and enzyme isolation, with particular attention paid to the effects of hypotonic, isotonic, and hypertonic buffers on enzyme isolation. The binding of all synthetases to ribosomes was eliminated if the low ionic strength of the isolation buffer was raised to isotonic levels. In contrast, neither the ionic strength or composition of the buffers, nor the procedures used for cell homogenization or enzyme isolation had any significant effect on the isolation of the high molecular weight synthetase complex. Certain enzymes (lysyl-, methionyl- and isoleucyl-tRNA synthetases) formed very stable complexes and high molecular weight species were the predominant forms of these enzymes under all conditions of cell homogenization and enzyme isolation. Other enzymes (glycyl-, tyrosinyl- and threonyl-tRNA synthetases) formed complexes very weakly, if at all, and always appeared predominately in the soluble enzyme fraction. Isolated soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases did not associate to form significant amounts of complex upon re-isolATION, SUGGESTING THAT A COMPONENT NECESSARY FOR COMPLEX FORMATION WAS MISSING FROM THE SOLUBLE ENZYME FRACTION. However, the soluble forms of these enzymes, but not the glycyl-, tyrosinyl- and threonyl-tRNA synthetases, did for complexes when mixed with ribosomal RNA or polyuridylic acid. Preliminary experiments showed no significant differences between the complexed and soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases with respect to Km values or ability to charge different isoaccepting tRNAs.


Near-ultraviolet difference absorption and circular dichroism studies on partially synthetic ribonucleases S'. Near-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-,1epsilon,7epsilon-diguanidono-[Asn14]-, [Phe(F)8, Orn10]- and 1epsilon, 7epsilon-diguanidino-S-peptide, with S-protein. Environmental alterations of Phe-8 in the S-peptide and Tyr-25 in the S-protein, derived from the association process, lead to strong optical signals whose location and magnitude were clearly defined by means of a comparative analysis of the above spectra. Additionally, the spectroscopic effects resulting from insertion of a tyrosyl residue into an hydrophobic environment in the presence or absence of hydrogen-bonding partners were identified and compared with similar findings obtained from the model compound p-cresol.


Far-ultraviolet difference absorption and circular dichroism studies on partially synthetic ribonucleases S'. Far-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-, 1epsilon, 7epsilon-diguanidino-[Asn14]-, [Phe(F)8, Orn10]-, [Cha8, Orn10]- and 1epsilon, 7epsilon-diguanidono-S-peptide, with S-protein. The aromatic chromophores contributions to the absorption spectra in the 220-250 nm wavelengths interval strongly exceed the hyperchromism due to the random coil to right handled alpha-helix transition of the S-peptide, accompanying the association process. Contributions resulting from peptide transitions constitute a large portion of the total dichroism, nevertheless substitution of the non aromatic cycloexylalanine residue in position 8 of the S-peptide with phenylalanine, p-fluorophenylalanine and tyrosine leads to CD negative maxima located at 215, 215 and 212,5 nm, respectively. The strongest ellipticity increment (28%), relative to that of the Cha-derivative, was observed at 212,5 nm for the [Tyr8]-S-peptide analog, while in the narrow 222 nm range minimal differences were found upon insertion into the position 8 of the S-peptide of the above aromatic residues. Information derived from above data and from a comparison of RNAase A, S and S-protein CD spectra enabled us to assign the positive CD band at 240 nm in RNAase A spectrum to transitions of phenylalanines and inaccessible tyrosines.


Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA). poly (dT) method. The hybrid plasmid PBETAG, consisting of plasmid PMB9 DNA with an insert of rabbit globin DNA (about 600 base pairs) flanked by poly(dA) poly(dT) regions (Maniatis, T., Kee, S.G., Efstratiadis, A. and Kafatos, F.C. (1976) Cell 8, 163-182), was cleaved into two fragments by endonuclease S1 under conditions of partial denaturation. Only the smaller fragment (575 base pairs) contained globin-specific sequences, showing that excision had occurred in the A-T-rich regions. This method of cleavage provides a useful procedure for assessing the length of inserts in hybrid plasmids prepared by the poly(dA)-POLY(DT) tail method, and allows the preparative recovery of the insert.


Interaction of glucose oxidase with phospholipid vesicles. The interactions between glucose oxidase and phospholipid vesicles were investigated. The investigations were carried on molecules adsorbed on the outer surfaces as well as entrapped in the interior of the vesicles . The adsorption of glucose oxidase on the surfaces of egg egg licithin vesicles, containing varying amounts of cholesterol and stearoylamine was measured by determining the free fraction of glucose oxidase detected in the filtrates. In general an enhancement of enzymic activity was observed upon interaction with the vesicles. The enhancement depends on the lipid composition of the vesicles and the surface concentration of the adsorbed glucose oxidase. It reached a maximal value at a surface concentration of 1.4-10(11) molecules/cm2 (approximately 7.1 - 10(4) A2/molecule) on pure phosphatidylcholine vesicles and about 6.5 - 10(10) molecules/cm2 (approximately 16 - 10(4) A2/molecule) when the vesicles contained cholesterol or cholesterol and stearoylamine. CD measurements indicated that the change in enzymic activity of the adsorbed glucose oxidase was accompanied by conformational modification of the enzyme. In order to entrap glucose oxidase into the vesicles, the lipid was sonicated in the presence of the enzyme. After removal of the free and adsorbed enzyme the amount of the entrapped enzyme was determined by measuring its activity after disintegration of the vesicles with Triton. The enzymic activity of the entrapped glucose oxidase served as a measure for the permeability of the bilayer membrane of the lipid vesicles to glucose. Addition of insulin to the suspension of vesicles containing the entrapped glucose oxidase increased the permeability of glucose by up to 9 - 10(-8) cm/s. This value is the lowest estimate based on the assumption that one glucose oxidase molecule was entrapped in every vesicle.


Lipid-protein interactions with native and modified myelin basic protein. The basic protein of central nervous system myelin has been shown to form complexes with acidic lipids in vitro. We measured the interaction of myelin basic protein with several charged and neutral lipids in a biphasic chloroform/methanol/water system and investigated the effect of decreasing the electrical charge of the basic amino groups of the myelin basic protein by acetylation. The modified myelin basic protein, which has an average of eight acetyl residues incorporated, was characterised by gel electrophoresis and circular dichroism. Complexes formed between the acetylated myelin basic protein and acidic lipids exhibited a reduction in the amount of lipids bound, a value that could be correlated with the number of modified amino groups. The significance of these experiments with reference to protein-lipid interaction in the myelin membrane is discussed.


Effects of incorporated trypsin on anion exchange and membrane proteins in human red blood cell ghosts. Varying concentrations of trypsin were sealed into human red cell ghosts and the effects on membrane proteins and sulfate equilibrium exchange were studied. After incubation for 45 min at 37 degrees C, pH 7.2, the following observations were made: above 10 ng/ml the ghosts undergo fragmentation without lysis. Dodecyl sulfate gel electrophoresis shows that the digestion of spectrin and of the protein in band 2.1 (nomenclature of Steck (1974) J. Cell. Biol. 62, 1-19) is nearly complete at 50 ng/ml, that of the protein in band 3 at 25 mug/ml. After digestion at 25 mug/ml, about 60% of the total protein of the membrane is released and the original bands of conventional dodecyl sulfate gel electropherograms of the remaining protein are nearly completely abolished. In their place three new bands appear representing peptides with molecular weights of 58 000, 48 000 and 34 000, respectively. Sometimes a fourth peptide with a molecular weight of approx. 13 000 is also observed. Using a radioactive labeling technique it is shown that the two peptides with the highest molecular weights are derived from the protein in band 3. Labeling with diazo[35S]sulfanilic acid indicates that in addition to the peptides in the described four Coomassie blue-stainable bands, other peptides with molecular weights up to 100 000 are still present in the exhaustively trypsinized ghosts. External trypsin has no effect on the sulfate equilibrium exchange in ghosts while internal trypsin causes inhibition. Inhibition becomes apparent at trypsin concentration exceeding those required to produce a complete digestion of spectrin. It remains incomplete, even at the highest intracellular concentrations which cause maximal effects on all membrane proteins, including the protein in band 3. Under these conditions strong further inhibition can be produced by agents which are known to inhibit anion transport in untreated red cells and ghosts. These agents include the penetrating 1-fluoro-2,4-dinitrobenzene and the nonpenetrating phlorizin, 4-acetamido-4'-isothiocyanato stilbene-2,2'-disulfonic acid, 4,4'-diacetamido stilbene-2,2'-disulfonic acid, and 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonic acid (APMB). Unlike the other nonpenetrating inhibitors APMB is not only capable of inhibiting at the outer but also at the inner membrane surface. Treatment with internal trypsin does not significantly reduce the inhibition by incorporated APMB. The described observations suggest that after exhaustive tryptic digestion of the major membrane proteins, the receptor sites for typical inhibitors of anion transport continue to exert their function.


Biogenesis of microsomal membrane glycoproteins in rat liver. IV. Characteristics of a cytoplasmic lipoprotein having properties of a membrane precursor. The supernatant fraction from a rat liver homogenate contains a lipoprotein complex which can be incorporated into microsomal membranes in vitro. The lipoprotein can be purified by gel filtration and flotation in a NaBr solution. The isolated lipoprotein has an equilibrium density in the range of 1.07-1.14 g/ml, a sedimentation coefficient of 4.9 S and a calculated molecular weight of 210 000. The isolated complex contains 44% lipid by weight, half of which is phospholipid. The protein also has covalently bound sugar moieties, namely mannose, galactose, glucosamine, and sialic acid. The complex is unstable and may dissociate during the isolation procedure. The complex and its fragments contain two peptides with molecular weights of 11-13000 and 67-69000. These two components have practically identical amino acid compositions.


Molecular arrangements in sphingolipids. Conformation and hydrogen bonding of ceramide and their implication on membrane stability and permeability. The preferred conformation of the ceramide part of sphingolipids has been deduced from single crystal structures of a series of sphingolipid constituents: N-tetracosanoylphytosphingosine, glycosylphytosphingosine hydrochloride, sphingosine hydrochloride, triacetylsphingosine, DL-2-hydroxytetradecanoic acid and N-stearoylethanolamine. The amide group of the ceramide, which serves as a link between the hydrocarbon chains, has a basic significance for the contormation of the entire molecule. This rigid group, which comprises six atoms in a planar conformation, adopts a perpendicular orientation towards the axes of the two hydrocarbon chains. The carbonyl oxygen thereby turns into an eclipsed position with the hydrogen atoma at carbon atom 2 of the sphingosine. A parallel chain stacking is achieved by a sharp perpendicular bend of the fatty acid. This bend is produced by a sequence of two --60 degrees rotations about the C-C bonds at both sides of the alpha-carbon atom. The orientation of the hydrogen bond donors and acceptors of the amide group and the hydroxyl groups allow lateral interaction with other lipid molecules. The proposed models are supported by infrared spectra, thin-layer chromatographic behaviour and monolayer studies of synthetic model ceramides. The functional role of the hydrogen bonding groups in the ceramide part of sphingolipids is emphasized and their significance for the formation of lateral hydrogen bonds within the membrane layer and thereof arising effects on membrane stability and permeability are discussed.


Calorimetric and freeze fracture analysis of lipid phase transitions and lateral translational motion of intramembrane particles in mitochondrial membranes. Differential scanning calorimetry combined with freeze fracture electron microscopy reveals that thermotropic lipid phase transitions and lateral translational motion of intramembrane particles occur in both membranes of whole, intact rat liver mitochondria and in isolated inner and outer membranes. The onset temperature of the liquid crystalline to gel state lipid phase transition in whole mitochondria and in the isolated outer membrane fraction is biphasic with an initial transition exotherm occurring at 9 degrees C. The onset temperature of the transition exotherm of the isolated inner membrane occurs at -4 degrees C. The onset temperature of the lipid transition endotherm is -15 degrees C for whole mitochondria, the inner membrane, ane the outer membrane fractions. These calorimetric analyses reveal that the bilayer lipid in the inner, energy transducing membrane is more fluid than in the outer membrane. Mitochondrial membranes cooled to temperatures in the region of their transition exotherms and then frozen reveal striking lateral separations between smooth, intramembrane particle-free regions (rich in gel state lipid) and particle-dense regions (rich in integral proteins) in their hydrophobic fracture faces. Such thermotropic lipid-protein lateral separations are completely reversible. These freeze fracture observations suggest that both mitochondrial membranes are naturally fluid to the extent that the integrat membrane proteins can diffuse laterally in the bilayer lipid.