Decay and interference processes in short-term retention of normal and brain-damaged patients. The present study examined decay and interference mechanisms in short-term verbal retention in brain damaged patients with and without clinical memory impairment. Petersons' distractor technique was modified for this purpose. Results indicated that decay and impaired consolidation of memory traces was greater in organic amnesics, accounting for a greater degree of forgetting than proactive interference. Absence of an interference function is discussed in terms of the modified technique utilizing repeated trials to recall and more rigorously eliminating covert rehearsal, in addition to employing a more representative sample of organic amnesics than in provious studies.


Symptoms in veterans considering compensation claims. The symptomatology of 56 male veterans considering compensation claims was compared to the symptomatology seen in 163 veterans not considering such claims. Antisocial behaviors, somatic/neurotic complaints, and persecutory ideas were all positively correlated with consideration of a claim. Although the symptom profile is consistent with diagnosis of antisocial personality, a correlation with this diagnosis was not established. Nonetheless, the clinical profile does suggest a link between noncompensable disorders and consideration of a claim. The implications of this finding for military and Veterans Administration psychiatry are discussed.


Tuberous sclerosis and autistic behavior. A patient with evidence of both infantile autism and tuberous sclerosis syndrome is presented. A possible relationship between the 2 syndromes is discussed. Examination of the skin of young, very disturbed children is recommended.


Neuroleptic malignant syndrome due to depot fluphenazine. The parenteral long-acting phenothiazines are drugs frequently used in the treatment of hospitalized and ambulatory psychiatric patients. The usual side effects of these drugs are familiar to psychiatrists. However, the "Neuroleptic malignant syndrome" reported in this paper can give rise to considerable diagnostic difficulty. The clinical characteristics and differential diagnosis of this syndrome are described with a care report. The need for prompt initiation of adequate therapy is stressed.


Hydrolysis and synthesis of wax esters by different systems of carp hepatopancreas preparation. It was found that the supernatant of hepatopancreas homogenate of the carp, Cyprinus carpio, contained enzymes involved in the hydrolysis and esterification of wax esters. The enzymes were salted out by 30% saturation with ammonium sulfate. This fraction also contained the highest activities of lipase and esterase. The enzyme for the hydrolysis of wax esters, together with lipase and esterase, was eluted in tube numbers 20 to 22 (void volume) from a Sephadex G-200 column. On the other hand, the enzyme for the synthesis of wax esters was eluted in tube numbers 33 to 35. It thus appears that the two activities belong to different enzyme proteins. The synthetic enzyme was activated by the addition of ATP, CoA, and NADH.


The action of trypsin on synthetic chromogenic arginine substrates. A new arginine derivative, N-benzyloxycarbonyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide hydrochloride (ZPVAPA.HCl) was synthesized by the condensation of N-benzyloxy-carbonyl-L-phenylalanyl-L-valine and L-arginine-p-nitroanilide dihydrochloride using dicyclohexylcarbodiimide as a coupling reagent and 1-hydroxy-benzotriazole as an additive. L-ZPVAPA.HCl was split by trypsin more readily than Na-benzyloxycarbonyl-L-arginine-p-nitroanilide hydrochloride (L-ZAPA, HCl), Na-benzoyl-L-arginine-p-nitroanilide hydrochloride (L-BAPA.HCl), Na-tosyl-L-arginine-p-nitroanilide hdyrochloride (L-TAPA.HCl) and Na-benzoyl-DL-arginine-p-nitroanilide hydrochloride (DL-BAPA.HCl) by factors of 100, 400, 600, and 1,200, respectively. Low concentrations of dimethyl formamide (DMF) enhanced the trypsin-catalyzed hydrolyses of L-ZAPA.HCl and L-TAPA.HCl, contrary to the findings of other authors that DMF has no effect on the tryptic hydrolysis.


Proteins exposed on the outer surface of the plasma membranes of AH-66 hepatoma ascites cells. The cell surface proteins of AH-66 hepatoma ascites cells were examined both by fluorescent labeling with cycloheptaamylose-fluorescamine complex and by radioiodination with lactoperoxidase. The labeled proteins were analyzed by measuring the distribution of fluorescence or radioactivity in polyacrylamide gels after electrophoresis. Although the degree of fluorescent labeling with cycloheptaamylose-fluorescamine complex was substantially different from that obtained by lactoperoxidase-catalyzed iodination, proteins with molecular weights of 130,000, 90,000, 86,000, 50,000--60,000, and 37,000 were strongly labeled in common by both reagents. It is suggested that at least these proteins are exposed on the surface of the plasma membranes of AH-66 cells. One of these proteins labeled strongly by both reagents is the major glycoprotein, with an apparent molecular weight of 130,000 in 10% polyacrylamide gels.


Studies on histone oligomers. I. Reconstitution and fractionation of homotypic histone oligomers. Histone oligomers were reconstituted from calf thymus whole histones by annealing from 2 M NaCl-5 M urea-0.05 M Tris buffer (pH 8.0)-1% beta-mercaptoethanol, and were fractionated in terms of solubility in ammonium sulfate solution. Fractionation occurred according to the histone compositions of oligomers. The oligomers thus obtained were (1) pentamer and trimer of H1, (2) tetramer of H2A and H2B, and (3) tetramer and higher aggregates of H3 and H4, under the following ionic conditions: 0.5 M NaCl-0.05 M phosphate buffer (pH 7.0). The aggregates of H3 and H4 could easily be converted to the tetramer by lowering of pH.


Tagging of sugars with a fluorescent compound, 2-aminopyridine. Potential aldehyde groups of several monosaccharides and oligosaccharides were coupled with 2-aminopyridine by reductive amination with sodium cyanoborohydride. The product was isolated by adsorption on a Dowex 50 (H+) column, followed by washing, elution with aqueous ammonia and evaporation. The specimen was analyzed by paper electrophoresis at pH 5.0. Each sugar derivative gave a single spot in addition to one corresponding to excess 2-amino-pyridine when the paper was scanned under a UV lamp. The migration rates of these fluorescent sugars were related to their molecular weights and were independent of their linkage points and anomeric configurations. The reducing end sugar units of oligosaccharide derivatives could be identified by gas-liquid chromatography after hydrolysis of the 2-aminopyridine derivatives of the oligosaccharides.


Amino acid sequences of the alpha and beta chains of adult hemoglobin of the European hedgehog, Erinaceus europaeus. Globin prepared from hemoglobin of the European hedgehog (Erinaceus europaeus) was separated into alpha and beta polypeptide chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and the resulting peptides were isolated. The sequences of all the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from their homology with the primary structures of alpha and beta chains of human adult hemoglobin. Comparing the primary structures of the alpha and beta chains of adult hemoglobin of the European hedgehog thus obtained with those of adult hemoglobin of the tupai (Tupaia glis), 35 amino acids substitutions in the alpha chains and 30 in the beta chains were recognized.


Studies on the protein components of 110S and total ribonucleoprotein particles of rat liver nucleoli. Proteins of rat liver nucleolar RNP, especially 110S RNP containing 45S RNA, were investigated and the following results were obtained. 1. Nucleolar extract was prepared from thioacetamide-treated rat liver nucleoli in the presence of 1 mg PVS per ml. Sucrose-density gradient centrifugation of the nucleolar extract showed RNP distributed between 60S and 110S. The 110S particles (110S RNP) contain 45S RNA as judged from the radioactivity profiles of nucleolar extract labelled in vivo with [3H]orotic acid for 10 min and 2 h. The protein components of 110S RNP and total RNP heavier than 60S (total RNP) were analyzed and compared. 2. Since the effects of PVS treatment on the extraction and the electrophoretic pattern of ribosomal proteins were removed by increasing the Mg2+ concentration during acetic acid extraction, as described in our preceding paper (1), proteins of 110S RNP as well as total RNP from PVS-pretreated nucleoli were extracted with 67% acetic acid containing 334 mM Mg2+ and analyzed by two-dimensional acrylamide gel electrophoresis. Ribosomal proteins masked by contaminating histone spots on the gel were identified by SDS-acrylamide gel electrophoresis. 3. 110S RNP contained a large portion of ribosomal proteins from large subunits; 24 protein spots were distinct, 6 protein spots were faint and 5 proteins (L3, L8, L30, L35, and L36) were missing completely. On the other hand, 110S RNP contained a small number of small subunit proteins; only 6 proteins showed distinct spots, 7 proteins showed faint spots and 12 protein spots were missing. 110S particles contained 11 non-ribosomal proteins, although it is difficult to rule out the possibility that some of them were contaminating chromatin proteins. 4. Total RNP showed electrophoretic patterns of 60S ribosomal proteins similar to those of 110S RNP, although L3 protein was present as a faint spot and 26 proteins showed distinct spots. Total RNP contained more small subunit proteins than 110S RNP; 7 proteins spots were distinct, 10 protein spots were faint and 8 protein spots were missing. The results suggest that some kinds of 40S proteins and L3 protein are attached to 110S RNP during the processing of 110S RNP.


Induction by parathyroid hormone of ornithine decarboxylase in rabbit costal chondrocytes in culture. The activity of ornithine decarboxylase (ODC) in rabbit costal chondrocytes in culture markedly increased after addition of parathyroid hormone (PTH), reaching a maximum 4 to 5 h after PTH addition. The effect of PTH was dose-dependent both in the presence and absence of fetal calf serum. Addition of cycloheximide or actinomycin D prevented the induction of ODC by PTH. Calcitonin, 1,25-dihydroxyvitamin D3, and vitamin A had no stimulatory effect on the activity of ODC, and did not inhibit the induction of ODC by PTH, when added with PTH.


Studies on the post-mortem fragmentation of myofibrils. 1. There was a close relationship between the fragmentation of myofibrils and the tension developed during post-mortem contraction of muscle. The extent of fragmentation was at its maximum when the sarcomeres attained a length of 2.0 to 2.2 micron. 2. The rate of fragmentation of myofibrils depended upon the calcium ion concentration within a range of 10(-5) to 2 x 10(-2) M, with a minimum at pH 6.5. The fragmentation of myofibrils free from muscle fibers was not affected by 10 mM iodoacetate, an irreversible inhibitor of calcium-activated factor (CAF). 3. Incubation of myofibrils with 10 mM CaCl2 caused the release of about 12% of the total myofibrillar proteins after homogenization. The protein solution contained little alpha-actinin, and considerable amounts of 54,000- and 76,000-dalton components which seem to originate from the Z-line. SDS-polyacrylamide gels of troponin prepared from the incubated myofibrils did not change with time of incubation. These findings are in contrast with the proteolytic degradation of Z-lines by CAF treatment, in which alpha-actinin and 87,000 dalton component are released. 4. These data directly demonstrate that the in vitro fragmentation of post-mortem muscle (i.e. duirng its conversion into myofibrils upon mechanical homogenization) is different from that induced by CAF. The possible role of calcium ions during in vitro fragmentation of myofibrils is discussed.


Ca2+-induced activation of succinate dehydrogenase and the regulation of mitochondrial oxidative reactions. 1. In rat liver mitochondria in vitro, an activation of succinate dehydrogenase [succinate: (2,6-dichloroindophenol)oxido-reductase], an inner membrane enzyme, was induced by Ca2+ at extramitochondrial concentrations (about 1.3 micron) close to those estimated in the cytosol. 2. The activation required both substrate (succinate) and ATP, and occurred whether mitochondria were coupled (Ca2+ could be accumulated) or uncoupled (Ca2+ could not be accumulated) by classical uncouplers. 3. The activation by Ca2+ of the uncoupled mitochondria was accompanied by a modest but significant change in the mitochondrial morphology as judged from light scattering measurements and electron microscopy. 4. In the uncoupled mitochondria, oxaloacetate added externally diminished the activation by Ca2+. In addition, the amount of oxaloacetate produced endogenously from succinate via malate fell after Ca2+ and ATP addition. However, the extent of the fall in mitochondrial oxaloacetate did not correlate with the degree of activation of succinate dehydrogenase. 5. The activation by Ca2+ of the uncoupled mitochondria was accompanied by a reductive shift of pyridine nucleotide and coenzyme Q, and an oxidative shift of flavoproteins and cytochromes b, c, and a-a3. 6. In the situation where the Ca2+-induced activation of succinate dehydrogenase (and consequently succinate oxidation) took place in the uncoupled mitochondria, oxidations of 3-hydroxybutyrate and pyruvate were markedly suppressed. 7. From the above findings, it is concluded that Ca2+ action on the mitochondrial inner membrane activates mitochondrial succinate dehydrogenase, and this action produces an inhibition of electron transport between NAD and flavoprotein. In view of the location of these reactions in the inner membrane, a conformation change of the membrane is suggested as a common cause.


Purification of Streptomyces chromofuscus phospholipase D by hydrophobic affinity chromatography on palmitoyl cellulose. Phospholipase D [phosphatidylcholine cholinehydrolase, EC 3.1.4.4] excreted from Streptomyces chromofuscus was purified from the culture supernatant by precipitation with acetone and column chromatographies on palmitoylated gauze (Pal-G), DEAE-cellulose, and Sephadex G-150 with an overall recovery of 46% and 1000-fold increase in specific activity. The purified enzyme preparation showed a single band on sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. The enzyme had a molecular weight of about 50,000 by gel filtration on Sephadex G-150 or about 57,000 by SDS-polyacrylamide disc gel electrophoresis and an isoelectric point (pI) of pH 5.1 on isoelectric focusing. The enzyme hydrolyses lecithin, lysolecithin, sphingomyelin, and cephalin; the relative reaction velocities and Km's for choline-phospholipids were 87% and 1.43 mM for lecithin, 100% and 1.67 mM for lysolecithin, and 22% and 0.56 mM for sphingomyelin. The enzymatic reaction was optimal at pH 8, and its velocity was appreciably increased by either detergent (Triton X-100, deoxycholate), Ca2+ or both detergent and Ca2+. Diethyl ether stimulated the enzymatic activity by 30%; SDS and EDTA inhibited the activity. Bovine serum albumin, Triton X-100, and lipids (lecithin, lysolecithin, phosphatidic acid, lysophosphatidic acid, palmitic acid, and oleic acid) inhibited adsorption of the purified enzyme onto palmitoyl cellulose (Pal-C) and affected both the enzyme activity and stability: albumin and Triton X-100 increased the activity and enhanced the heat-stability; lysophospholipids decreased the activity but other lipids increased the activity; all the lipids lowered the heat-stability. The enzyme adsorbed on Pal-C was active, although its activity was about one-ninth of that of free enzyme, and was protected from heat-inactivation. Thus this enzyme appears to possess a hydrophobic site distinct from its catalytic site and to be adsorbed onto Pal-C through the hydrophobic site. Albumin, Triton X-100, and lipids seem to bind to the hydrophobic site and to have an appreciable effect on the enzyme activity and stability.


Kinetics of the conformational change of troponin-C induced by magnesium-binding or removal. The kinetics of the conformation change of troponin-C (TN-C) induced by magnesium-binding or removal were studied in the absence of calcium ion by measuring the fluorescence intensity change of BIPM bound to TN-C by stopped-flow spectrofluorometry. The kinetic process of the conformational change was biphasic. The rate constants of the two phases were determined as a function of free magnesium ion concentration ([Mg]) of the solution. The [Mg]-dependence of the rate constants was explained by a simple molecular kinetic mechanism: (formula: see text) The dissociation constant of magnesium bound to TN-C was also determined to be 1 x 10(-3) M in the kinetic study.


Solubilization and reconstitution of dopamine-sensitive adenylate cyclase from bovine caudate nucleus. Dopamine-sensitive adenylate cyclase was reconstituted from the cholate-soluble components of caudate nucleus homogenate. Biological function was restored by precipitating the components from cholate and phospholipid with ammonium sulfate, dialyzing the resuspended precipitate, and activating the particulate complex with phospholipid in the assay. The reconstituted adenylate cyclase was stimulated 3- to 4-fold by dopamine and 8- to 12-fold by guanyl-5'-yl imidodiphosphate. The catecholamine stimulation was specific for dopamine and required the addition of GTP. The cholate-soluble component(s) of the basal adenylate cyclase were separated from the component(s) that conferred dopamine sensitivity by gel filtration chromatography. Dopamine-sensitive adenylate cyclase was also reconstituted from digitonin-soluble components. These were resolved into two fractions by DEAE-cellulose chromatography: one fraction contained adenylate cyclase, but both fractions were required for reconstitution of dopamine-sensitive adenylate cyclase.


Hydrogen bonding in solution between the uracil ring and the peptide backbone demonstrated by nuclear magnetic resonance spectroscopy. Proton and 13C nuclear magnetic resonance measurements indicate that uracil derivatives dissolved in chloroform bind to glycine and phenylalanine tripeptide derivatives through pairs of hydrogen bonds. The N(3)--H and C(4)=0 groups of the uracil ring appear to interact with the C=0 and N--H groups, respectively, of individual amino acid residues, suggesting a fundamental complementarity between uracil and the peptide backbone. The binding occurs in the same concentration range as the hydrogen bonding between derivatives of adenine and uracil under comparable conditions.


Ribulose-1,5-biphosphate carboxylase. Evidence in support of the existence of distinct CO2 activator and CO2 substrate sites. Ribulose-1,5-biphosphate carboxylase forms a complex with Mn2+ and CO2 that exhibits a considerably enhanced water proton relaxation rate. This effect is diminished upon interaction of the complex with the substrate, ribulose biphosphate, or with the competitive inhibitor, ribitol 1,5-biphosphate (Ki=0.55 mM). Included among the several mechanisms which can explain these observations is the possibility that a slow exchange of metal ligands occurs. Attempts at testing the feasibility of a slow exchange mechanism led to demonstration of a stable complex of enzyme with CO2 in the presence of metal and carboxyribitol biphosphate (CRBP), an analog of the hypothetical transition state intermediate. The complex formed upon mixing these components is stable to Sephadex G-75 chromatography and contains a nonexchangeable [14C]O2 bound stoichiometrically with respect to enzyme active sites. Mg2+ or Mn2+ can be used to form the E.M.[14C]O2.CRBP complex, which is stable with respect to CO2 exchange until the enzyme is denatured with sodium dodecyl sulfate. If the tight, functionally irreversible binding of the transition state analog is due to its occupancy of ribulose biphosphate and substrate CO2 sites, then simultaneous stoichiometric binding of [14C]O2 to enzyme indicates that 2 CO2 molecules participate in photosynthetic carbon fixation.


Participation of one isozyme of cytosolic glycerophosphate dehydrogenase in the adipose conversion of 3T3 cells. Growing surface cultures of 3T3 cells possess a low level of glycerophosphate dehydrogenase, an important enzyme in triglyceride synthesis. When 3T3-C2, a subline that does not undergo appreciable adipose conversion, reaches confluence, the level of the enzyme does not increase. In 3T3-F442A, a subline that undergoes the conversion with high frequency, the specific activity of the enzyme increases about 600-fold. The enzyme of the adipose 3T3 cells is different from that of non-adipose 3T3 cells in its thermal stability and its affinity for dihydroxyacetone phosphate. The enzyme of the adipose cells probably corresponds to the stable "adult" form of the enzyme, as described previously, and the enzyme of non-adipose 3T3 cells is probably the unstable "embryonic" form. For this reason, the change in the enzyme that takes place during the adipose conversion is greater than would be indicated simply by the total increase in specific activity. If, as seems likely, the two forms of glycerophosphate dehydrogenase are the products of independent genes, the adipose conversion may activate a hitherto silent gene for the stable enzyme.


Effect of hypophysectomy and growth hormone treatment on albumin mRNA levels in the rat liver. A complementary DNA copy of purified rat albumin mRNA was employed in RNA-excess hybridization reactions to examine albumin mRNA levels in the liver of the hypophysectomized rat. In normal rat liver, albumin mRNA sequences were found to be about 10% of the total poly(A)-containing RNA population. Hypophysectomy reduced albumin mRNA levels by approximately 50%. Growth hormone treatment of hypophysectomized animals returned albumin mRNA to nearly normal levels. These effects on albumin mRNA sequences agreed closely with the changes in the synthesis of albumin that are assoicated with these conditions. The results suggest that hypophysectomy and growth hormone influence albumin synthesis by affecting the level of albumin mRNA.


Multiple molecular forms of catechol-O-methyltransferase. Evidence for two distinct forms, and their purification and physical characterization. Catechol-O-methyltransferase (COMT: EC 2.1.1.6) has been shown to exist in the soluble fraction of rat liver as two distinct molecular forms, designated COMT I and COMT II, which are separable by gel filtration, ion exchange chromatography, and sedimentation. The predominant form, COMT I, has a smaller Mr of about 24,000, as determined by gel filtration and sedimentation, and less negative charge, whereas the minor form, COMT II, has a larger Mr of about 47,500 and more negative charge. The COMT I and COMT II have been purified 450- and 205-fold, respectively, from rat liver by a newly developed procedure which gives homogeneous enzyme preparations with respect to catechol-methylating activities. The molecular properties of the predominant form, COMT I, were: s20,w, 2.7; D20,W, 10.5; Stokes radius, 20.1 A; f/fo, 1.08; and pI, 4.9. For the minor form, COMT II, the values were s20,w, 3.8; D20,w, 7.3; Stokes radius, 28.7 A; f/fo, 1.23; and pI, 4.8. Catechol-O-methyltransferase was found to exhibit tissue-specific isozymic patterns in the distribution of its two variant forms. In the rat tissues, the liver and kidney exhibited the presence of the two physically separable forms. Catechol-O-methyltransferase was also found as two distinct molecular forms in human tissues, including liver, brain, and placenta. The two forms of human catechol-O-methyltransferase were not distinguishable by the criteria of gel filtration from their counterparts in rat liver, indicating that the two molecular forms of human and rat liver catechol-O-methyltransferase are homologous. No interconversion of one molecular form of catechol-O-methyltransferase into the other was observed under experimental conditions employed. Available evidence indicates that the two molecular forms of catechol-O-methyltransferase are genetically dissimilar proteins.


Human amidophosphoribosyltransferase. An oxygen-sensitive iron-sulfur protein. Glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase (EC 2.4.2.14), amidophosphoribosyltransferase, was partially purified from human placenta. Upon exposure to oxygen, both the glutamine and ammonia activities were lost in parallel. Inactivation by oxygen increased as the temperature of incubation rose and the partial pressure of oxygen increased. Molecular oxygen rather than a radical derivative was responsible for inactivation since scavengers of oxygen radicals did not protect against inactivation. AMP,GMP,PP-ribose-P, and inorganic phosphate partially protected both the glutamine and ammonia activities from inactivation by oxygen. Incubation with 1,10-orthophenanthroline, but not 1,7-metaphenanthroline or tiron, led to inactivation of amidophosphoribosyltransferase. Both the 1,10-orthophenanthroline- and oxygen-inactivated enzymes could be reconstituted by incubation with ferrous iron and inorganic sulfide in the presence of dithiothreitol under anaerobic conditions. The iron requirement could not be replaced by zinc, copper, cobalt, nickel, magnesium, or calcium. The sulfide requirement could not be replaced by higher concentrations of dithiothreitol. It is concluded from these studies that human amidophosphoribosyltransferase is an iron-sulfur protein and oxidation of this structure may be responsible for the marked lability of this enzyme in vitro.


Interaction of troponin subunits. The interaction between the inhibitory and tropomyosin-binding subunits. The interaction of troponin-I and troponin-T was demonstrated by circular dichroism and gel filtration. Troponin-I gives a negative circular dichroism band between 300 and 260 nm while troponin-T gives two weak positive bands, one at 290 nm and the other at 263 nm. When troponin-I and troponin-T were mixed, the complex produced a strong negative circular dichroism band with a maximum around 280 nm. This band was most intense with a molar ratio of troponin-T to troponin-I of 1:1. The intensity of the band was 2.4 times that expected from the separate components. The interaction was independent of salt concentration from 0.15 to 0.5 M KCl. Gel filtration on Sephacryl S-200 showed that a stable 1:1 complex was formed between troponin-T and troponin-I. When troponin-C was added to the complex of troponin-T.troponin-I; the reconstituted troponin had a circular dichroism spectrum identical to that of native troponin. The oxidation state of troponin-I was important in reconstituting troponin. Oxidized troponin-I produced less change in the near ultraviolet circular dichroism when added to troponin-T and the troponin-C than did reduced troponin-I. This showed the subunits were not assembled correctly with oxidized troponin-I. When the reconstituted complex was reduced, the circular dichroism was restored to that of native troponin. Troponin reconstituted with oxidized troponin-I did not confer calcium sensitivity on actomyosin ATPase; activity was restored by reducing the complex.


Determination of the apparent thermodynamic activities of saturated protein solutions. Although the solubility of a protein is a particularly informative solution parameter, little is known about the thermodynamics of protein solubilization. In these experiments, polyethylene glycol (PEG) is used to decrease the solubility of a number of proteins in a quantifiable manner. Simple thermodynamic considerations show that if the chemical potential of the PEG-induced solid phase is constant and plots of log protein solubility versus PEG concentration are linear, a valid extrapolation of the apparent solubility to zero PEG content can be made. Given the validity of these assumptions, extrapolated values should represent the activity of the protein in saturated solution. Evidence for the validity of this extrapolation includes (a) the experimentally observed linearity of log solubility versus PEG concentration plots, (b) the extrapolation of such plots to correct activities in the situation where protein activities can be experimentally determined, and (c) the independence of the extrapolated activities on protein concentration over a wide range. The utility of the PEG-determined activities, when applied in a comparative manner, is illustrated by application to various hemoglobin solutions. It is found that saturated solutions of the various hemoglobin forms, with the exception of deoxyhemoglobin S, manifest similar activities. In addition, all of the solutions demonstrate an apparent, surprising thermodynamic ideality.


Interaction of human apohemoglobin with inositol hexaphosphate. Experiments of sedimentation velocity and equilibrium indicate that in the presence of inositol hexaphosphate the degree of polymerization of apohemoglobin is shifted in favor of the formation of tetramers, with a maximum effect when the concentration of the polyphosphate is 1 mM. Above this concentration, a redissociation of the system into dimers is promoted. This phenomenon is probably due to the binding of inositol hexaphosphate to apohemoglobin with a stoichiometry higher than 1 mol of polyphosphate/4 subunits. The optical rotatory dispersion spectrum of apohemoglobin is also modified by its interaction with inositol hexaphosphate suggesting a small increase in the helical content of the protein. Measurements of circular dichroism in the near-UV region of the spectrum indicate that the environment of the aromatic chromophores of the protein such as tyrosine, phenyalanine, and tryptophan is not affected by the interaction. The presence of inositol hexaphosphate decreases the rate of reaction of the beta-93 cysteinyl residues of apohemoglobin with both p-mercuribenzoate and N-ethylmaleimide, suggesting a conformational change of the protein also at the level of its tertiary structure.


Purification and characterization of an inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. A heat-labile inhibitor protein of adenylate cyclase (EC 4.6.1.1) and phosphodiesterase (EC 3.1.4.17) has been purified to apparent homogeneity from bovine brain cerebrum by a simple two-column procedure. The inhibitor exerts its effect on adenylate cyclase or phosphodiesterase by forming a complex with the Ca2+-dependent activator protein, thereby competing with the apoenzyme for the activator. The protein was estimated to have a molecular weight of 80,000 and a Stokes radius of 39 A by gel filtration. The inhibitor was resolved in a sodium dodecyl sulfate-polyacrylamide gel into two equal molar subunits, with molecular weights of 60,000 and 18,500. In the presence of the activator and Ca2+, the thermal stability of the inhibitor was increased, indicative of a new conformation. The effectiveness of the inhibitor varied considerably, depending on its sequence of addition to the reaction mixture relative to phosphodiesterase and the activator protein, presumably because the activator appeared to have a greater affinity for the inhibitor than for phosphodiesterase.


Carboxylation and decarboxylation reactions. Anaplerotic flux and removal of citrate cycle intermediates in skeletal muscle. A system for in situ perfusion of rat hindquarters using a fluorocarbon for oxygen and CO2 exchange, and a polyol to provide oncotic pressure is described. Perfusion with glucose plus insulin resulted in no significant change in the tissue level of citrate cycle intermediates, phosphocreatine, ATP, ADP, AMP, and glycogen. Glucose was consumed at a linear rate, and lactate, pyruvate, alanine, glutamine, glutamate, and citrate were released into the perfusing medium. Inclusion of pyruvate resulted in elevation of citrate cycle intermediates and alanine, whereas acetate elevated the level of cycle intermediates without significant effect on tissue alanine or its release. Radioactivity from NaH[14C]O3 was incorporated into citrate cycle intermediates, glutamate, aspartate, and lactate by glucose-perfused hindquarters, the extent of which was markedly elevated as the tissue pyruvate was increased. When pyruvate was in the physiological range, acetate caused elevation in incorporation of CO2 into these metabolites, increased the concentration of citrate, and doubled the concentration of acetyl-CoA. Thirty-five to forty-four per cent of 14C incorporated into citrate was retained after enzymic degradation to 2-oxoglutarate. Perfusion with [2-14C-]propionate led to elevation in the level of citrate cycle intermediates, and radioactivity was incorporated into the latter, as well as glutamate, aspartate, lactate, pyruvate, alanine, and CO2. Two independent calculations estimated the rate of flux of 4-carbon cycle intermediates to 3-carbon metabolites of about 68 mumol/h (approximately 38 nmol/min/g of tissue), a rate in excess of those reported for alanine release from human or rat muscle during starvation. Arsenite blocked carbohydrate flux through the citrate cycle and effected accumulation of lactate, pyruvate, alanine, and 2-oxoglutarate. Flux from 4- to 3-carbon acids was diminished by arsenite, apparently as a result of lowered substrate concentration for decarboxylation. 3-Mercaptopicolinic acid, an inhibitor of phosphoenolpyruvate carboxykinase, was without effect on the parameters studied, suggesting that this enzyme is not involved in the decarboxylation reaction. It is concluded that (a) a constant level of citrate cycle intermediates is maintained in part by continuous flux of carbon into and out of the cycle by carboxylation and decarboxylation reactions; (b) the carbon skeleton of alanine released from skeletal muscle is derived in part from other amino acids which are catabolized to cycle intermediates; and (c) the subsequent removal of these intermediates is probably mediated by malic enzyme(s) (EC 1.1.1.40, or 1.1.1.36, or both.


Biological effects of cesalin, an anti-tumor protein, on cultured mammalian cells. The effects of the protein cesalin were studied to determine the site of its toxic action in KB cells for which the ID50 is 0.9 pg/ml. Several effects have been noted: (i) respiration in KB cells was reduced by greater than 50% within 1 h after cesalin addition, which could not be explained on the basis of mitochondrial damage or from elevated intracellular ATP levels, since these did not rise significantly until about 3 h after cesalin addition; (ii) both DNA and RNA synthesis were inhibited, starting about 3 h after addition of cesalin to KB cells, but the transport of nucleosides and amino acids does not appear to be affected; (iii) no decrease is observed in protein synthesis for at least 5 to 6 h; (iv) cesalin can inhibit DNA synthesis and mitotic division independently of one another. The interaction of cesalin with the cell surface is rapid and after 5 min at 37 degrees C less than 10% of treated cells can be rescued by addition of excess antiserum to cesalin. The cell surface receptors are exposed throughout the cell cycle and the toxicity of cesalin is not restricted to a particular phase of this cycle.


The amino acid sequence of dihydrofolate reductase from the mouse lymphoma L1210. The determination of the amino acid sequence of the dihydrofolate reductase (EC 1.5.1.3) from cells of the mouse lymphoma L1210 is described. The protein was cleaved by cyanogen bromide to produce the six fragments CB1 (residues 1 to 14), CB2 (residues 15 to 52), CB3 (residues 53 to 111), CB4 (residues 115 to 125), CB5 (residues 126 to 139), and CB6 (residues 140 to 186). One of the fragments, CB2, contained an internal homoserine derived from a methionine which was not cleaved by cyanogen bromide. The amino acid sequences and order of the cyanogen bromide fragments were determined by a combination of automatic and manual sequence analyses of the fragments and small peptides from tryptic, thermolytic, and Staphylococcus aureus protease digestions. The complete sequence comprises 186 residues in a single polypeptide chain of molecular weight 21,458. Comparison of the sequence of the L1210 dihydrofolate reductase with the sequences of the enzymes from Streptococcus faecium, escherichia coli RT500, and Lactobacillus casei indicates that all enzymes show some homology, which is strongest in the regions forming the substrate binding cleft.


Primary structure of human C-reactive protein. The complete amino acid sequence has been derived for human C-reactive protein (CRP). The protein yielded a unique sequence containing 187 amino acids in a single polypeptide chain. The NH2-terminal residue of CRP is pyrrolidonecarboxylic acid and the COOH terminus is proline. The 2 half-cystine residues at positions 36 and 78 are involved in a disulfide bond. Based on the amino acid composition derived from the sequence data, a minimal molecular weight of 20,946 has been calculated for human CRP. This value agrees well with the molecular weight of 21,500 established by gel filtration of CRP in 5.0 M guanidine Cl (Gotschlich, E.C., and Edelman, G.M. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 558--566). The primary structure of human CRP has been examined for internal homology and compared to all known proteins whose structures were published before April, 1978 by two computer programs; program SEARCH and program RELATE (Dayhoff, M. O., ed (1976) in Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 2, pp. 3--8, National Biomedical Research Foundation, Silver Spring, MD). The computer analyses showed no significant repeating sequences within the C-reactive protein molecule. This observation seems to rule out the possibility of gene duplication in the evolution of this protein. Distant homologies, which were statistically insignificant, have been noted to the CH2 domain of immunoglobulin G (IgG) and to C3a anaphylotoxin. The homologie noted are insufficient to support a common evolutionary origin of these proteins. No homology region in other heavy chains was observed. It is therefore preferable, at this point in time, to assign CRP and the protein known as 9.5 S alpha-glycoprotein, P component, and Clt to a new super family unrelated to any other proteins investigated. The homology between these proteins was demonstrated previously (Osmand, A.P., Friedenson, B., Gewurz, H., Painter, R.H., Hofmann, T., and Shelton, E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 739--743) on the basis of sequence data on approximately 20 NH2-terminal residues of rabbit C-reactive protein, of Clt, and a cyanogen bromide fragment of human CRP.


Isolation and characterization of single chain bovine factor V. A procedure for the isolation of bovine Factor V has been developed. The Factor V is isolated from bovine plasma by a series of steps including barium citrate adsorption, polyethylene glycol precipitation, QAE-cellulose adsorption, hydrophobic chromatography on octyl Sepharose, ammonium sulfate fractionation, preparative electrophoresis on acrylamide gels, and finally, phenyl Sepharose chromatography. During isolation, judicious use of inhibitors including benzamidine hydrochloride, soybean trypsin inhibitor, and diisopropylphosphorofluoridate has been applied to prevent activation of the Factor V TO Factor Va. The activity of the isolated protein increases by a factor of 80 when stimulated by catalytic amounts of thrombin. The specific activity of the material after thrombin activation is 1250 units/mg of protein when evaluated versus a bovine Factor V standard in human factor V-deficient plasma. The isolated protein is a single component when analyzed by a variety of electrophoretic techniques and has been characterized in terms of its gross physical and chemical properties. Bovine Factor V is a single chain glycoprotein which has a molecular weight of 330,000. The single chain nature of the molecule has been established by sedimentation equilibrium studies of the native molecule and on the molecule in 6 M guanidinium chloride with and without disulfide bond reduction. In addition to these mass measurements, the single chain nature of the molecule has been established by hydrodynamic estimation of the random coil volume by sedimentation velocity studies of the reduced carboxyamidomethylated protein in 6 M guanidinium chloride. Native Factor V has a sedimentation coefficient so20,w of 9.19 S, which indicates the molecule is highly asymmetric. The frictional ratio f/fmin for the molecule is estimated to be 2.01, and the axial ratio of the equivalent prolate ellipsoid is 25:1. Thus, present data suggest that Factor V is a rod-like molecule composed of a single chain.


Adrenocortical cytochrome P-450 side chain cleavage. Preparation of membrane-bound side chain cleavage system from purified components. Soluble cytochrome P-450 from bovine adrenocortical mitochrondria, capable of side chain cleavage, can be incorporated into membranes prepared by dispersion of phospholipids in aqueous buffer when cholate is added to the membrane suspension. In addition, the complete protein side chain cleavae system (i.e. including the ancillary proteins adrenodoxin and adrenodoxin reductase and the substrate cholesterol) can be incorporated into such membranes so that on addition of TPNH, pregnenolone is formed. These components remain in the membrane through gel filtration (which removes almost all the cholate) and sedimentation through sucrose density gradients which separate vesicles without protein and soluble enzyme from the membrane-bound P-450 remains associated with the membrane during and following lysis of vesicles. The vesicles which do not leak [14C]glucose were seen on electron microscopy to show a mean diameter of 350 to 450 A. A number of phospholipids are capable of accomodating P-450 in this manner: mitochondrial lipid extracts, synthetic dipalmitoyl phosphatidylcholine, synthetic dipalmitoyl phosphatidylserine, and egg lecithin, separately or in various combinations. Cholesterol is not necessary for incorporation of the side chain cleavage system. Membrane-bound P-450 shows a Vmax of 28.1 nmol of pregnenolone/min/mg of protein, more than 10 times that of soluble P-450. The spectral properties of the soluble P-450 are altered to become predominantly low spin in the membrane and the enzyme is more stable at 4 degrees C than is soluble p-450.


Self-association of synexin in the presence of calcium. Correlation with synexin-induced membrane fusion and examination of the structure of synexin aggregates. It has been proposed (Creutz, C. E., Pazoles, C. J., and Pollard, H. B. (1978) J. Biol. Chem. 253, 2858-2866) that synexin, an adrenal medullary protein that causes Ca2+-dependent aggregation of isolated chromaffin granules, might be the intracellular receptor for Ca2+ in the process of exocytosis. We now report that Ca2+ interacts directly with isolated synexin, inducing rapid self-association. Mg2+ or Sr2+ cannot substitute for Ca2+, and Ba2+ is only weakly effective at stimulating self-association. We have analyzed 90 degree light scattering data to determine a titration curve for the activation of synexin by Ca2+ in the self-association reaction. The curve has a Hill coefficient of 2.3 and is half-maximal at 200 micron Ca2+, which correlates exactly with the Ca2+ titration curve for the aggregation of chromaffin granules by synexin. Electron microscopy of negatively stained samples reveals that synexin monomers associate to form 50 A by 150 A rods which in turn associate side to side and end to end to form bundles of parallel rods. We suggest that synexin exists as a soluble monomer in the cytoplasm of the resting chromaffin cell and that when the cell is stimulated to secrete, the increase in the intracellular concentrations of Ca2+ causes synexin to form rods, similar to those formed in vitro, which bind to chromaffin granule and plasma membranes, joining them together to form the "pentalaminar" fusion complexes characteristic of the first step in exocytosis.


Immunological characterization of multiple weight forms of human cell plasminogen activators. Several molecular weight forms of plasminogen activator (PA) activity have been observed in serum-free conditioned media from human cells in culture. An antibody inhibition technique is described which combines inhibition of enzyme activity by anti-urokinase IgG with sodium dodecyl sulfate-gel electrophoresis to determine whether different molecular weight forms of human cell PA's are immunologically related to urokinase. Plasminogen activator forms with molecular weights of 85,000 to 95,000, 50,000 to 60,000, and 36,000 were inhibited by anti-urokinase IgG. In contrast, PA forms with molecular weights in the 73,000 range from three different types of human cells were not inhibited by comparable concentrations of the antibody. Human embryonic kidney cultures contain only anti-urokinase IgG-inhibitable PA forms, while melanoma-derived Malme-3M cultures contain only anti-urokinase IgG-resistant forms. Cultures of tumorigenic Detroit 562 cells and nontumorigenic IMR-90 cells contain a mixture of "antibody-sensitive" and "antibody-resistant" PA forms. The antibody-resistant 73,000-dalton PA form may be a precursor of the smaller antibody-sensitive, urokinase-related forms, or it may be the product of a second plasminogen activator gene which codes for a protein immunologically and structurally different from urokinase.


Nucleotide sequence at the 5' terminus of adenovirus 2 late messenger RNA. One major late promoter is transcriptionally active for the majority of adenovirus 2 late RNA synthesis. Nucleotide sequences proximal to this promoter are spliced onto the 5' termini of all messenger RNAs generated from it. We report here the complete nucleotide sequence of the capped 5-'terminal undecanucleotide common to these late mRNAs. The sequence m7G5'ppp5'm6AmCUCUCUUCCGp shows striking similarities to the 5'-terminal sequences of the eukaryotic alpha- and beta-globin mRNAs and contains the sequence 5'CUUCCG3' complementary to a six-base sequence near the 3' terminus of most eukaryotic 18 S ribosomal RNAs. On the basis of this sequence and the finding that this same sequence is present at the 5' end of adenovirus 2 late nuclear RNA precursor, it should now be possible to locate the major late promotor within the adenoviral genomic DNA sequence.


Nonenzymatically glucosylated albumin. In vitro preparation and isolation from normal human serum. Incubation of human serum with D-[6-3H]glucose resulted in the gradual accumulation of radioactivity in acid-precipitable material. Upon chromatography on Sephadex G-200, radioactivity was found associated with each of the major molecular weight classes of serum protein. Purified human serum albumin was also glucosylated in vitro upon exposure to D-[6-3H]glucose in phosphate-buffered saline. The glucosylated and unmodified albumins were separated by ion exchange chromatography. The physiological significance of these observations in vitro was confirmed by the isolation and quantitation of glucosylated albumin from normal human serum. Glucosylated albumin represents approximately 6 to 15% of total serum albumin in normal adults. The post-translational modification appears to occur by a nonenzymatic process analogous to that responsible for glucosylation of hemoglobin A to hemoglobin AIc, i.e. through Schiff base formation and Amadori rearrangement to a ketoamine derivative.


DNA binding, induction of unscheduled DNA synthesis, and excision of anthramycin from DNA in normal and repair-deficient human fibroblasts. The reaction of the antitumor antibiotic anthramycin with cellular DNA and the ability of normal human fibroblasts cells and xeroderma pigmentosum (XP) cells to respond to this injury has been evaluated. The binding of [15-3H]anthramycin to cellular DNA in human skin fibroblasts occurred in a linear manner up to 6 h. Treatment with unlabeled antibiotic resulted in unscheduled (repair) DNA synthesis in human skin fibroblasts maintained in hydroxyurea, whereas negligible unscheduled DNA synthesis was observed in cells of an excision-defective strain of XP. Confluent nondividing normal skin fibroblast cells were able to remove 86% of the bound anthramycin within 72 h, however XP cells were only able to remove 49% during the same incubation period. These results are discussed in terms of the types of DNA damage produced by anthramycin in vitro and the likely repair pathways involved in removing lesions produced on DNA by anthramycin.


A subset of trout testis nucleosomes enriched in transcribed DNA sequences contains high mobility group proteins as major structural components. Mononucleosomes greatly enriched in non-histone proteins were prepared by limited digestion of testis nuclei with micrococcal nuclease. Five to fifteen per cent of the chromatin was solubilized and could be separated by adjustment to 0.1 M NaCl, into a soluble fraction MN1, consisting of mononucleosomes containing the four inner histones and the small basic non-histone, H6, associated with a 140-base-pair DNA fragment. H1 was notably absent in MN1. The fraction insoluble in 0.1 M NaCl (MN2) comprised a mixture of mono-, di-, tri-, and oligosomes. MN2 monosome fraction contained the four inner histones plus H1 and lacked H6 and the length of its DNA was 170 base-pairs. Previous work had shown that limited micrococcal nuclease digestion of trout testis nuclei released a great proportion of the non-histone protein, high mobility group protein T (HMG-T). It seems likely that HMG-T is the major non-histone protein located in the linker regions of a subset of nucleosomes containing the non-histone protein H6 as a major structural component. Moreover, the presence of HMG-T renders this subset of nucleosomes very sensitive to micrococcal nuclease. Hybridization experiments were performed to demonstrate that the DNA from MN1 monosomes corresponds to a subset of the trout testis genome. This DNA subset is greatly enriched in sequences that are present in cytoplasmic RNA. Chromatin subunits enriched in their content of H6 and HMG-T could also be obtained by limited digestion of trout testis chromatin with DNase II followed by precipitation with MgCl2.


The biosynthetic route from ornithine to proline. It is shown by tracer experiments with DL-[2-3H,5-14C]- and DL-[(RS)-5-3H,5-14C]rnithine, that the metabolic conversion of ornithine into proline, in three plant species (Nicotiana tabacum, Datura stramonium, and Lupinus angustifolius), takes place with maintenance of the delta-hydrogen atoms but with loss of the alpha-hydrogen atom. This indicates a route via alpha-keto-delta-aminovaleric acid (5-amino-2-oxopentanoic acid) and disproves the accepted route via glutamic gamma-semialdehyde (2-amino-5-oxopentanoic acid).


Isolation and characterization of a double chain intermolecular cross-linked peptide from insoluble calf bone collagen. A double chain peptide containing the sodium borohydride-reduced intermolecular cross-link, hydroxylysinohydroxynorleucine, was isolated following sequential cyanogen bromide digestion and limited alkaline hydrolysis of insoluble calf bone collagen. Amino acid composition and NH2-terminal sequence analysis indicated that the peptide was highly acidic and consisted of 19 amino acid residues including the cross-link. Amino acid composition and automated sequence analysis of this peptide before and after cleavage of the cross-link, using periodic acid, provided the data from which the following structure was deduced. (formula: see text). The sequence of the larger peptide is identical with that of residues 8c to 19c in the COOH-terminal nonhelical region of the homologous skin collagen alpha1 chain. The hydroxylysine residue located at position 17c in the alpha chain of type I collagen appears to be a predominant site for intermolecular cross-link formation. Assignment of the smaller peptide component within the known primary structure of the collagen molecule currently cannot be made.


Sterol synthesis in variant Chinese hamster lung cells selected for resistance to 25-hydroxycholesterol. Cross-resistance to 7-ketocholesterol, 20alpha-hydroxycholesterol, and serum. Two lines of Chinese hamster lung (Dede) cells which are resistant to the killing effect of 25-hydroxycholesterol, and which grow to confluence in its presence, have been isolated. One of the resistant lines exhibited a high to normal growth rate and normal levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) activity and sterol synthesis in the presence of concentrations of 25-hydroxycholesterol that caused nearly complete suppression of these functions in wild type cells. The other variant line showed partial resistance to the inhibitory effects of the diol upon cell growth, sterol synthesis, and HMG-CoA reductase activity. The resistant phenotypes remained stable when the cells were maintained in the absence of the selecting agent. The Km (HMG-CoA), thermal stability, and susceptibility to inhibition by Mg2+ and ATP of HMG-CoA reductase were unaltered in both of the resistant lines. The two lines selected for growth in the presence of 25-hydroxycholesterol were also resistant to the inhibitory effects of 7-ketocholesterol, 20alpha-hydroxycholesterol, and serum upon HMG-CoA reductase and sterol synthesis, suggesting that suppression of cholesterol synthesis by these inhibitors involves a common step.


Structure of the high mannose oligosaccharides of a human IgM myeloma protein. II. The minor oligosaccharides of high mannose glycopeptide. The high mannose glycopeptide I from IgM (Patient Wa) contains, in addition to the two major oligosaccharides described earlier (Chapman, A., and Kornfeld, R. (1979) J. Biol Chem. 254, 816-823), four minor oligosaccharides. After release from glycopeptide I by endo-beta-N-acetylglucosaminidase Cu and reduction with NaBH4, all four oligosaccharides have been shown to have the basic structure: (see article of journal). Oligosaccharide IA-1 (Man9GlcitolNAc) has additional alpha1,2-linked mannose residues attached to positions a and b, and the sequence Man alpha1,2 yields to Man alpha1,2 yields to attached at position c. Oligosaccharide IA-2 (Man8GlcitolNAc) has additional alpha1,2-linked mannose residues attached to positions a, b, and c. Oligosaccharide IA-3 (Man7GlcitolNAc) has additional alpha1,2-linked mannose residues at positions b and c. In contrast, oligosaccharide IA-4 has a single N-acetyglucosamine residue beta1,2-linked to the mannose at position c. Oligosaccharides IA-1, -2 and -3 are thought to represent "processing" intermediates that are precursors of the major Man6 and Man5 oligosaccharides of glycopeptide I. IA-4 may arise from the Man5 oligosaccharide by the action of UDP-GlcNAc:glycoprotein N-acetylglucosaminyl-transferase, which catalyzes the first reaction leading to complex oligosaccharide synthesis.


The mechanism of indole-3-acetic acid oxidation by horseradish peroxidases. The oxygen-consuming oxidation of indole-3-acetic acid (IAA) occurred much faster in the presence of horseradish peroxidase C (neutral isoenzyme) than in the presence of horseradish peroxidase A (acidic isoenzyme). An intermediate oxidation product of IAA was found to be a hydroperoxide species that reacted with the ferric enzymes to form Compound I at second order rate constants of 6.8 X 10(3) M-1--S-1 for peroxidase A and 2.0 X 10(6) M-1--S-1 for peroxidase C at pH 4.4 The hydroperoxide concentration reached about one-half of the initial IAA concentration at the end of the oxygen-consuming reaction and then decreased slowly. The main intermediate of the enzyme observed during the oxygen-consuming reaction was Compound II, which oxidized IAA to its free radical at rate constants of 1.5 X 10(3) M-1--S1 for peroxidase A and 1.2 times 10(4) M-1--S-1 for peroxidase C at pH 4.4 The results supported the mechanism that the oxygen consumption occurs mainly through the reaction of oxygen with the IAA free radical formed from the peroxidatic oxidation of IAA. The ferric enzymes were not reduced by IAA under strict anaerobic conditions in the presence of carbon monoxide but were reduced upon addition to a small amount of oxygen or hydrogen peroxide to the systems. The results suggested that the ferric enzyme is reduced by the IAA free radical but not by IAA itself. From a comparison of reactivities of oxyperoxidase and Compound II we concluded that the catalytic cycle of ferrous and oxyperoxidases is not involved in the IAA oxidase reaction.


Isolation and characterization of proteoglycans from porcine ovarian follicular fluid. Monomer proteoglycan was isolated from porcine ovarian follicular fluid by isopycnic CsCl centrifugation in the presence of 4 M guanidine HCl and protease inhibitors. The elution profile of the D1 preparation on Sepharose 2B was similar to that of monomer proteoglycan from bovine nasal cartilage, indicating a similar molecular size. Follicular fluid proteoglycans consist of about 20% protein, 50% dermatan sulfate, and 20% oligosaccharides rich in sialic acid, galactose, mannose, glucosamine, and galactosamine. The amino acid composition of this proteoglycan is significantly different from that of cartilage proteoglycans, with a higher proportion of aspartic acid, threonine, and lysine, and lower amounts of proline and glycine. Alkali-released dermatan sulfate chains are larger on Sepharose 6B (average Mr = 56,000) than chondroitin sulfate chains from cartilage proteoglycans (average Mr = 25,000), and iduronic acid accounts for 9% of total hexuronic acid. Disaccharide units released by chondroitinase ABC consists of 67% 4-sulfated, 22% 6-sulfated, 5% non-sulfated, and 5% disulfated disaccharides. After treatment with 0.05 M NaOH, 1 M NaBH4 at 45 degrees C for 24 h, two major sialic acid-containing oligosaccharides were observed on Sephadex G-25, corresponding to penta- and hexasaccharides. The pentasaccharide contained sialic acid, galactose, glucosamine, and galactosamine in the proportions 1:2:1:1. The galactosamine is O-glycosidically linked to the protein core. This oligosaccharide accounts for approximately 77% of all the sialic acid in the follicular fluid proteoglycans. The hexasaccharide fraction contained sialic acid, galactose, mannose, and glucosamine in the proportions 1:2:1:2. It also contained a small amount of fucose and galactosamine. The linkage of these oligosaccharides to the protein core remains to be determined. The follicular fluid proteoglycans, unlike those from cartilage, do not interact with hyaluronic acid. Digestion with trypsin, chymotrypsin, or plasmin released dermatan sulfate-peptides nearly as small as those released by papain or alkali; in contrast, cartilage proteoglycans were resistant to plasmin and released peptides containing an average of more than four chondroitin sulfate chains after trypsin or chymotrypsin digestion.


Location of the single gene for the large subunit of ribulosebisphosphate carboxylase on the maize chloroplast chromosome. The structural gene for the large subunit of ribulosebisphosphate carboxylase (E.C. 4.1.1.39) in Zea mays is shown to be contained within a 2500 base pair sequence of chloroplast DNA. One copy of this DNA sequence is present in each circular maize chloroplast DNA molecule. It maps approximately 30,000 base pairs from the 5' end of the closest of two sets of rRNA genes and approximately 71,000 base pairs from the other set of rRNA genes.


Characterization of alkaline phosphatases from human first trimester placentas. Alkaline phosphatase from human first trimester placentas was purified, characterized, and compared with alkaline phosphatases from term placenta and liver. Three forms of first trimester placental alkaline phosphatase (I, IIa, and IIb) were isolated; their relative amounts were 35%, 39%, and 26%, respectively. Phosphatases I and IIa were found to be dimers, whereas phosphatase IIb appeared to be a tetramer consisting of two dimers of phosphatase I or IIa. Phosphatase I was indistinguishable from liver phosphatase by several criteria including apparent molecular weight (Mr = 165,000), size of the monomeric subunit (Mr = 77,000), heat liability, insensitivity to inactivation by antiserum against term placental alkaline phosphatase, and sensitivity to inactivation by antiserum against liver alkaline phosphatase. In addition, phosphatase I and liver phosphatase were equally sensitive to inhibition by amino acids, levamisole, l-p-bromotetramisole, and EDTA. Phosphatase IIa, in contrast, was indistinguishable from term placental alkaline phosphatase by the same criteria: apparent molecular weight (Mr = 115,000), size of the monomeric subunit (Mr = 63,000), heat stability, inactivation by antiserum against term placental alkaline phosphatase, and sensitivity to inhibition by various compounds. These findings clearly demonstrate the existence of two distinct placental alkaline phosphatases, one (phosphatase I) specific for the  first trimester placenta and the other (phosphatase IIa) occurring in both first trimester and term placentas.


Structural studies on human spectrin. Comparison of subunits and fragmentation of native spectrin. Native spectrin has trypsin-susceptible sites spaced at a constant molecular weight interval. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of spectrin treated with trypsin at low salt concentrations shows a ladder of fragments spaced at approximately 8,000-dalton intervals, from the intact Band 1 (240,000 daltons) and Band 2 (220,000 daltons) down to about 150,000 daltons. The five largest fragments were identified as products of Band 2 using tryptic 125I-peptide mapping of protein from gel slices. Endogenously incorporated [32P]phosphate is absent from the largest fragment, indicating that all phosphorylation sites on spectrin are within 8,000 daltons of a terminal of Band 2. Mapping of both [14C]carboxyamidomethylated cysteine-containing tryptic peptides and 125I-peptides reveals extensive sequence homology between the spectrin subunits. Further, only somewhat over half of the distinct spots expected from the cysteine content are found in both Band 1 and Band 2 peptides. These and the tryptic susceptibility results are interpretable as evidence for a repeating structure in spectrin.


The subunit structure of thrombin-activated factor V. Isolation of activated factor V, separation of subunits, and reconstitution of biological activity. Activated Factor V (Va) was prepared by treating a high molecular weight form of Factor V with thrombin. The activated Factor V was isolated by ion exchange chromatography and was composed of two polypeptide chains (Mr = 115,000 and 73,000). These chains were separated by ion exchange chromatography in the presence of EDTA. Biologically active Factor Va was restored from the inactive chains by incubation of the two chains in buffers containing MnCl2. Restoration of biological activity was correlated with formation of a complex between the chains as monitored by either disc gel electrophoresis or gel filtration chromatography. The apparent molecular weight of the activated Factor V was 290,000. Factor V was not dissociated in EDTA. However, this protein was split by thrombin to yield an activation intermediate composed of two chains (Mr = 210,000 and 115,000). Like activated Factor V, the two chains of the intermediate can be dissociated in EDTA and separated by gel filtration chromatography. The Factor V activity was restored by incubation of the two inactive chains in buffers containing MnCl2. Like Factor Va, restoration of the biological activity corresponds to formation of a complex between the chains with a higher molcular weight than either of the isolated chains. Incubation of the activation intermediate with thrombin increased the specific activity 3- to 4-fold. This increase in specific activity resulted from cleavage of the heavy chain of the Factor V intermediate by thrombin.


N-(Phosphonacetyl)-L-aspartate-resistant hamster cells overaccumulate a single mRNA coding for the multifunctional protein that catalyzes the first steps of UMP synthesis. We have investigated the mechanism of overproduction of the multifunctional protein catalyzing the first three steps of UMP biosynthesis in stable mutants of Syrian hamster cells in culture. The rate of degradation of this protein is unaltered in one mutant cell line which overproduces it by 118-fold. In all mutants tested, the increase in the rate of synthesis of this protein is equal to the increase in its steady state concentration. There is a similar correlation between steady state levels of this protein in vivo and the capacity of polysomal RNA isolated from these cells to direct the synthesis of the protein in vitro. The one mutant cell line studied contains large amounts of a polysomal poly(A)-containing RNA (Mr = 2.7 +/- 0.2 times 10(6)) that is not detected in wild type cells. This large RNA co-sediments in sucrose gradients with the capacity to direct the synthesis of the multifunctional protein in vitro.


The chemistry and biology of thymosin. II. Amino acid sequence analysis of thymosin alpha1 and polypeptide beta1. The amino acid sequences of two polypeptide components of thymosin Fraction 5 termed thymosin alpha1 and polypeptide beta1 have been established. The sequences were determined by automatic Edman degradation of the intact molecules as well as by manual sequence analysis of the enzymatic cleavage products. Thymosin alpha1, an immunologically active polypeptide, is highly acidic with an isoelectric point of 4.2. This molecule is composed of 28 amino acid residues with acetylserine as the NH2 terminus. A chemically synthesized molecule of thymosin alpha1 has been found to be as active as the natural molecule in our bioassay systems. Polypeptide beta1 is a molecule consisting of 74 amino acid residues and has an isoelectric point of 6.7. This peptide is not biologically active in our assay systems, suggesting that it is not involved in thymic hormone action. The sequence of beta1 was found to be identical with ubiquitin and a portion of protein A24, a nuclear chromosomal protein. The relationships among these proteins are discussed.


Purification and characterization of the sequence-specific endonuclease Bam HI. The specific endonuclease Bam HI from Bacillus amyloliquefaciens (RUB 500) has been purified to apparent homogeneity. Two active forms of the enzyme corresponding to the dimeric and tetrameric forms have been isolated. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme dissociated into Mr = 22,000 +/- 500 subunits. Bam HI has a broad pH optimum on the alkaline side and requires Mg2+ which can be partially replaced by Mn2+. The enzyme catalysis appears to be governed by a two-step mechanism.


A hybrid protein containing the toxic subunit of ricin and the cell-specific subunit of human chorionic gonadotropin. I. Synthesis and characterization. In order to study the mechanisms involved in the interaction of cell-surface components and effector proteins, we synthesized a double-labeled dimeric hybrid molecule consisting of the toxic A subunit of the lectin ricin and the cell-specific beta subunit of human chorionic gonadotropin (hCGbeta). Utilizing methyl-5-bromovalerimidate, a disulfide cross-linked conjugate of hCGbeta and toxin subunit A was prepared in 30% yield relative to hCGbeta input. The dimeric protein, purified by gel filtration and ion exchange chromatography, was characterized by: (a) sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol, (b) analysis by gel filtration chromatography in the presence and absence of 2-mercaptoethanol, and (c) precipitation with antibodies specific for hCG and subunit A.


A hybrid protein containing the toxic subunit of ricin and the cell-specific subunit of human chorionic gonadotropin. II. Biologic properties. The biologic properties of a purified hybrid protein containing the toxic subunit A of ricin and the cell-specific subunit of human chorionic gonadotropin (hCGbeta) in a disulfide conjugate is reported. The biologic activity of the hybrid has been studied by assaying inhibition of protein synthesis in rat R2C Leydig cells and mouse L-cells. The results indicate that for the A subunit to exhibit its toxic effect it must first bind to cells through receptors which are specific for the beta subunit of the hybrid. Thus, only cells with beta-type receptors are sensitive to the toxic effects, which hCG is capable of blocking, while cells which are not target cells for hCG are not subject to the toxic effects. Thus it is concluded that the disulfide conjugate may act as a functional analog of ricin toxin but with altered cell specificity. Or, alternately, the hybrid may be regarded as an analog of hCG but with drastically altered physiologic effects.


Subcellular membrane topology and turnover of a rat hepatic binding protein specific for asialoglycoproteins. Examination of the topological distribution of the rat liver binding protein specific for desialylated serum glycoproteins has revealed a subcellular distribution wherein this receptor is located largely, if not entirely, on the luminal membrane surface of the Golgi complex and smooth microsomes. In contrast, the receptor protein has been identified on the external, or cytosolic, surface of the lysosomal membranes. Recovery of the purified binding protein from whole rat livers established an average half-life survival time of approximately 88 h. Subsequently, injection in vivo of the readily catabolized ligand, asialo-orosomucoid, was found to be without effect upon the turnover rate of the receptor purified from isolated plasma membranes. These observations are interpreted as suggesting the existence of a recycling mechanism wherein the receptor is spared destruction in the lysosomes and remains available for subsequent reinsertion into the hepatocyte plasma membrane as part of a continuing process for the clearance of galactose-terminated ligands from the circulation.


Dissociation of complexes and their derivatives formed during inhibition of bovine thrombin and activated factor X by antithrombin III. The complexes formed by antithrombin III with activated bovine Factor X and thrombin have been studied by gel electrophoresis in dodecyl sulfate. When subjected to electrophoresis at pH 7, the complexes remain intact, whereas electrophoresis at pH 9 in the presence of Tris results in their dissociation. Dissociation of both the Factor Xa-antithrombin III complex and the thrombin-antithrombin III complex in dodecyl sulfate produces a modified form of antithrombin III which, unlike the native inhibitor, apparently consists of two chains. Gel electrophoresis of the dissociated complexes has also been used to study the sites where the complexes are cleaved by the respective enzymes. The cleavage of the Factor Xa-inhibitor complex by Factor Xa apparently results from hydrolysis of a single bond in the enzyme part of the complex and releases a 15,000-dalton NH2-terminal fragment of the heavy chain, with the light chain attached. Cleavage of the thrombin-inhibitor complex by thrombin involves several cleavages of the heavy (B) chain of the thrombin part of that complex. Neither enzyme-inhibitor complex is subject to cleavage by free enzyme in the inhibitor part of the complex under the conditions used.


Synthesis of ceramides and cerebrosides containing both alpha-hydroxy and nonhydroxy fatty acids from lignoceroyl-CoA by rat brain microsomes. The conversion of [1-14C]lignoceroyl-CoA to nonhydroxy- and alpha-hydroxyceramides and cerebrosides by brain microsomes of developing rat in the presence of NADPH was investigated. A new technique of thin layer chromatography for the separation of these lipids and unreacted substrate was developed for this assay. The synthesis of nonhydroxy- and hydroxyceramides was significantly stimulated by the addition of heat-stable factor, a factor which is essential in the alpha-hydroxylation of free lignoceric acid (I. Singh and Y. Kishimoto, manuscript in preparation). The addition of sphingosine also stimulated the ceramide synthesis to a great extent. When the microsomes or heat-stable factor were crude, the ceramides formed were further converted to cerebrosides, apparently by UDP-galactose contamination. The purification of these subcellular components resulted in the formation of only ceramides which, in turn, were converted to cerebrosides by the addition of UDP-galactose. These observations indicate that hydroxyceramide is the precursor of hydroxycerebroside and is formed by alpha-hydroxylation of lignoceroyl-CoA and N-acylation of sphingosine. However, lignoceroyl-CoA, like free lignoceric acid, does not appear to be the immediate substrate of the alpha-hydroxylation.


Active carrier-mediated transport of melphalan by two separate amino acid transport systems in LPC-1 plasmacytoma cells in vitro. Previous studies have shown that uptake of several alkylating agents occurs by independent transport mechanisms. Uptake of one of these agents, the phenylalanine derivative of nitrogen mustard (melphalan), has been investigated in LPC-1 plasmacytoma cells in vitro. Evidence suggesting that melphalan uptake is an active process is that uptake of free intact melphalan proceeds "uphill" against a concentration gradient, is temperature- and sodium-dependent, and is inhibited by several metabolic antagonists. Other findings supporting the concept that melphalan uptake is carrier-mediated is that uptake follows biphasic Michaelis-Menten kinetics, is chemically specific, and is mediated by at least two separate amino acid transport systems. Uptake by one system was sensitive to DL-beta-2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid, was sodium-independent, and appeared to be mediated by system L. The second unassigned system was characterized by sodium dependence, insensitivity to 2-aminoisobutyric acid and 2-(methylamino)isobutyric acid, and was inhibited by alanine, serine, cysteine, and other amino acids.


Histone molar ratios among different electrophoretic forms of mono- and dinucleosomes. The relative molar ratios of each of the histone classes and protein A24 have been determined in nuclei, chromatin, and different electrophoretic forms of mono- and dinucleosomes of cultured mouse cells. For this purpose, [3H]lysine- and [14C]arginine-labeled cells were used for sample preparations, and stoichiometries were estimated from protein radioactivity profiles and known amino acid compositions following sodium dodecyl sulfate (SDS)-gel electrophoresis. The results demonstrate that upper limits of one and two histone H1 molecules exist per mono- and dinucleosome, respectively. However, isolated nuclei contain less than one copy of histone H1 per nucleosome. In addition, among the chromatin subfractions studied, histones H3, H2B, and H4 are essentially equimolar, while histone H2A is less than equimolar by 19 +/- 9%. This latter finding offers direct support to the proposal of Goldknopf (Goldknopf, I. L., French, M. F., Musso, R., and Busch, H. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5492-5495) that protein A24 replaces histone H2A in the octamer protein core of the nucleosome, since about 10% of the total histone H2A of cultured mouse cells is in the form of protein A24 and is present in nucleosomes. From the results of the present study, it is concluded that electrophoretic fractionation of mono- and dinucleosomes is not due to variable molar ratios or amounts of the four smaller histone classes, but depends on part on DNA length, the number of associated histone H1 molecules, and non-histone chromosomal proteins.


Selective effects of membrane sterol depletion on surface function thymidine and 3-O-methyl-D-glucose transport in a sterol auxotroph. Using a sterol-specific auxotroph of LM cells (mouse fibroblasts), we obtained cells with progressively lower sterol contents. 3-O-Methylglucose and thymidine were shown to enter the cells by saturable as well as by nonsaturable processes and it was possible to measure these processes separately at various stages of sterol depletion. Sterol depletion inhibited the saturable as well as the nonsaturable components for both 3-O-methylglucose and thymidine uptake. The extent of inhibition of the saturable process was much more than that of the nonsaturable component at any stage of the sterol depletion. Furthermore, in the course of progressive sterol depletion the saturable component of 3-O-methylglucose or thymidine uptake was inhibited well before the nonsaturable process. Sterol depletion produced reductions in the Vmax values but no change in the Km values for the saturable uptake of these two substrates. The depletion of cellular sterol to 75% resulted in 70% and 90% reduction of the saturable uptake of 3-O-methylglucose and thymidine, respectively, and 30% and 50% reduction of the nonsaturable uptake of 3-O-methylglucose and thymidine, respectively. DNA and protein synthesis, however, were not affected at all when the saturable component of thymidine uptake was inhibited by 50% demonstrating that the observed decrease of transport activity was selective and not a reflection of a gross alteration in cellular physiology.


Effect of sterol depletion on LM cell sterol mutants. Changes in the lipid composition of the plasma membrane and their effects on 3-O-methlglucose transport. With the use of two sterol biosynthetic mutants, LM cells with various sterol levels were obtained after extended periods of growth with suboptimal sterol concentrations. As was found in the preceding paper, sterol depletion resulted in reduction of the apparent Vmax values for 3-O-methylglucose transport with no change in the apparent Km values. Analysis of the lipid compositions of isolated plasma membranes and crude endoplasmic reticulum membranes showed that depletion of the cellular sterol levels led to a reduction in the sterol content of both membranes. Furthermore, there were significant changes in the fatty acid composition of the phospholipids of both membranes. The amount of 18:1 of the plasma membrane phospholipids increased dramatically accompanied by a marked decrease in 16:0. The loss of transport activity could be reproduced in vitro by removing cholesterol from cells with the use of liposomes containing phosphatidylcholine. Furthermore, activity was restored to normal values when the cells were subsequently incubated with liposomes composed of phospholipids plus cholesterol. These findings indicate that 1) an inter-relation exists between the fatty acyl group structure of phospholipids and the sterol content of LM cell membranes which may serve to maintain membrane function and 2) that the reversible inactivation of a surface membrane transport system is directly related to reversible changes in the properties of membrane lipid.


N-Acetylglucosamine-6-sulfate sulfatase from human urine. N-Acetylglucosamine-6-sulfate sulfatase, which liberates sulfate from the N-acetylglucosamine 6-sulfate residue at the nonreducing terminus of a 3H-labeled trisaccharide prepared from heparan sulfate, was purified 136-fold from human urine. The final N-acetylglucosamine-6-sulfate sulfatase preparation was free of all lysosomal sulfatases known to act on sulfated polysaccharides and gave a single band in polyacrylamide gel electrophoresis. The enzyme appears to be a glycoprotein with a molecular weight of around 97,000 and displays considerable charge heterogeneity. Multiple forms with pI values between 5.4 and 8.3 with a maximum at pH 7.7 were detected. The enzyme acts on the 3H-trisaccharide with a pH optimum at 5.5 and is active towards the sulfated monosaccharides N-acetylglucosamine 6-sulfate and glucose 6-sulfate. Although predominantly in exosulfatase, the enzyme catalyzes hydrolysis of sulfate from internal N-acetylglucosamine 6-sulfate moieties at a low rate. The Km for the 3H-trisaccharide, N-acetylglucosamine 6-sulfate, and glucose 6-sulfate were 0.15, 1.5, and 7.7 mM, respectively. The enzyme is inhibited by albumin, Hg2+, PO43-, SO42-, and CN-. Enzyme activity was highest in kidney and cultured fibroblasts but could be demonstrated in all human tissues tested.


The rate of oxygen uptake by human red blood cells. Oxygen uptake into intact and reconstituted human red blood cells was measured using dual wavelength, stopped flow techniques. The rate of oxygen uptake by human erythrocytes is roughly 40 times slower (t 1/2 congruent to 80 ms at 0.125 mM O2, 25 degrees C) than the corresponding rate of oxygen combination with free hemoglobin. Oxygen transport through the red cell cytoplasm accounts for part of this difference and predicts a half-time of uptake of about 15 ms, which is still 5 times smaller than that observed experimentally. Further limitation of uptake appears to be due to the presence of unstirred layers of solvent adjacent to the red cell surface. Very rapidly after mixing, these layers form and become depleted of O2 due to uptake by the cells. This requires that the bulk of the oxygen molecules must diffuse over rather large distances, 1.0 to 5.0 micrometer, before they can penetrate the erythrocytes. A mathematical model was developed to take into account diffusion through an unstirred solvent layer which increases in thickness with time. This scheme can account quantitatively both for the dependence of the apparent rate of uptake on O2 concentration and for the shape of the observed time courses. In addition, the diffusion parameters which were developed for the O2 reaction can also be used to describe quantitatively the rates and time courses of CO and ethyl isocyanide uptake and the rates and time courses of O2 release from cells in the presence of sodium dithionite. Finally, the parameters used to describe the stopped flow results can also be used to simulate quantitatively O2 uptake time courses obtained from previous studies with thin films of red cells (Sinha, A. K. (1969) Ph.D. dissertation, University of California, San Francisco; Thews, G. (1959) Arch. Gesamte Physiol. Mens. Tiere (Pflufgers) 268, 308-317).


The mechanism of action of S-adenosylhomocysteinase. S-Adenosylhomocysteinase catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine (AdoHcy) to adenosine and L-homocysteine without added cofactors. A mechanism is proposed which involves oxidation of the 3'-hydroxyl group of AdoHcy by enzyme-bound NAD+. Following oxidation, L-homocysteine is eliminated, alpha-beta, to give 3'-keto-4'-5'-dehydroadenosine. This compound reacts with water in a Michael type addition to form 3'-ketoadenosine which is then reduced to adenosine. This mechanism is supported by these facts. 1) The enzyme contains 1 tightly bound NAD+ per subunit. Upon addition of substrate, this NAD is converted to NADH. 2) The enzyme catalyzes the exchange of the 4'-proton of substrate with solvent. This exchange is an integral part of the catalytic mechanism. 3) The hydrolysis of [4'-2H]S-adenosyl-L-homocysteine has a Vmax isotope effect of 1.44. This provides additional evidence that cleavage of the C-4' C-H bond is a step on the reaction pathway. 4) 4',5'-Dehydroadenosine is oxidized by the enzyme, then converted into adenosine or into AdoHcy in the presence of L-homocysteine. 5) An adenosine analog, 5'-deoxyadenosine, is oxidized by the enzyme to yield 3'-keto-5'-deoxyadenosine, and an analog of the proposed intermediate, 3'-ketoadenosine. 6) The enzyme catalyzes the exchange of the C-4' proton of 5'-deoxyadenosine. Since the enzyme catalyzes proton abstraction without OH elimination, it was concluded that the elimination of H2O from adenosine proceeds by a carbanion mechanism and not by a concerted elimination. Substrate analogs in which the 5'-OH group of adenosine is replaced by -F, -Cl, or -SMe are not substrates for the enzyme.


Characterization of iodoproteins secreted by phagocytosing human polymorphonuclear leukocytes. Several proteins, glycoproteins, and iodoproteins are secreted from human polymorphonuclear leukocytes during phagocytosis of inert latex particles. The amount of 125I-labeled proteins increases during 10 to 60 min of incubation. The 125I-iodoproteins secreted into the incubation medium during the phagocytosis were first separated on Sephadex G-150 column and then characterized by column chromatography on Sepharose 4B, Sephadex G-200, and G-100. Three 125I-iodoproteins were found with the molecular weights of 580,000, 100,000, and 22,000. The molecular mass of 15 protein subunits calculated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis ranged from 11,000 to 86,000 daltons. Four of the protein subunits were labeled with 125I. Their molecular weights were 63,000 to 69,000, 44,000 to 49,000, 22,000, and 11,000. In addition to iodoproteins, several 125I-labeled peptides and compounds were found by column chromatography on Bio-Gel P-2 and P-10. The 125I-iodoproteins are not secreted from resting or boiled granulocytes, and their production is 92 to 98% inhibited by 1 mM KCN or 1 mM sodium azide. The double-labeling technique with [125I]- and [131I]-iodine suggests that the iodoproteins formed in the phagocytosing granulocytes or secreted into the incubation medium are not identical.


Thrombin-catalyzed activation of single chain bovine factor V. The thrombin-catalyzed activation of single chain bovine Factor V was found to proceed by cleavage of the parent molecule (Mr = 330,000) to intermediates of apparent Mr = 205,000 and 150,000 followed by cleavage of the latter to end products of 94,000 and 74,000 apparent molecular weights. The generation of Factor Va activity coincides with the second cleavage. Further cleavages of the Mr = 205,000 component to apparent Mr = 74,000 and 32,000 fragments and possibly to others also occurred at a slower rate with little or no further increase in activity. The nature of the products and intermediates was unchanged regardless of whether the activation process was complete in either 1 or 10 min. Factor Xa did not catalyze the cleavage nor enhance the activity of Factor V under conditions in which the activation by comparable amounts of thrombin was complete. It was shown using either discrete clotting assays or continuous thrombin analysis with a chromogenic substrate that Factor V was subject to activation in situ by newly formed thrombin in a system initially containing prothrombin, Factor Xa, Ca2+, phospholipid, and unactivated Factor V. The rate of activation of prethrombin 1 was found to be saturable with added Factor Va when all other components were present at fixed levels. The amount of Factor Va required to achieve saturation was linearly dependent on the amount of Factor Xa present, indicating that the two factors interact stoichiometrically in the "prothrombinase" complex.


Isolation and characterization of two homoserine dehydrogenases from maize suspension cultures. Homoserine dehydrogenase in unpurified extracts of maize (Zea mays L.) cell suspensions is inhibited 73% by the feedback regulator threonine; the remaining 27% of the total activity is not affected even by high concentrations of threonine. The threonine-resistant and threonine-sensitive homoserine dehydrogenase activities were separated by affinity chromatography on Blue Sepharose columns, and the two distinct homoserine dehydrogenases were purified. The threonine-resistant enzyme is an Mr = 70,000 dimer of two Mr = 38,000 subunits and the threonine-sensitive enzyme is an Mr = 190,000 dimer containing two apparently different subunits with molecular weights of 89,000 and 93,000. The threonine-resistant enzyme exhibits normal Michaelis-Menten kinetics and its activity is not affected by any of the amino acid end products of the aspartate pathway. The threonine-sensitive enzyme exhibits positive cooperative kinetics with respect to NADPH and is inhibited by threonine and stimulated by isoleucine. All attempts to affect interconversion of the two purified enzymes have been unsuccessful. Because the purified enzymes correspond to activities present in crude extracts of various maize tissues, it is concluded that the two types of homoserine dehydrogenase are natural in vivo constituents of maize.


Environmental regulation of gene expression. In vitro translation of winter flounder antifreeze messenger RNA. The serum of winter flounder contains a group of antifreeze peptides which lower the freezing point of their body fluid in the winter. These antifreeze peptides disappear from their serum in the summer when the water temperature rises. Messenger RNA isolated from the liver of flounder collected in January directs the synthesis of predominantly one protein in an in vitro protein synthesis system. This protein can be precipitated by the antiserum made from the purified antifreeze peptides and was identified as the precursor(s) of these peptides. There is a seasonal variation in the concentrations of mRNA coding for the antifreeze peptides in the liver which can be correlated with the appearance and disappearance of these peptides in the serum. However, the changes in the levels of mRNA occur much sooner than the changes in the levels of antifreeze peptides in the serum, suggesting that an additional control mechanism might have taken place at the translational level.


Isolation of a collagen-binding fragment from fibronectin and cold-insoluble globulin. Limited proteolytic cleavage of fibronectin and plasma cold-insoluble globulin with cathepsin D produced two major fragments. The smaller, Mr = 72,000 fragment bound to collagen and contained most of the cysteine in the molecule. This region contains intrachain disulfide bonds which maintain a conformation that is necessary for interaction with collagen. Cleavage of the intact protein and the 72,000-dalton fragment with plasmin localized the collagen-binding region in cold-insoluble globulin to a sequence of about 42,000 daltons. This region is located approximately two-thirds of the linear distance from the NH2 terminus of each chain in the dimeric molecule.


NH2-terminal sequence of the chick proalpha1(I) chain synthesized in the reticulocyte lysate system. Evidence for a transient hydrophobic leader sequence. Translation of chick procollagen mRNA in a reticulocyte lysate system yields a larger proalpha1(I) chain than is observed in vivo. The NH2-terminal sequence of this putative precursor, determined by automated radiosequencing, is Met-Phe-Ser-Phe-Val-X-Ser-Arg-Leu-Leu-Leu-Leu-Ile-Ala-Ala-X-X-Leu-Leu. This sequence closely resembles the transient hydrophobic leader (signal) sequences observed on most secreted proteins. When synthesized in the presence of microsomal membranes from dog pancreas, which contain signal peptidase activity, proalpha chains with the electrophoretic mobility of underhydroxylated procollagen polypeptides synthesized in vivo are produced. Estimates of the molecular weight of the NH2-terminal extension of the precursor suggest that the leader sequence may be longer than those commonly found in precursors of secreted proteins.


Characterization of rabbit reticulocyte factor(s) that stimulates the translation of mRNAs lacking 5'-terminal 7-methylguanosine. Purified reticulocyte initiation factors were assayed for their ability to stimulate the translation of uncapped vesicular stomatitis virus (VSV) mRNA relative to capped VSV mRNA in reticulocyte lysates. Both eIF-3 and eIF-4B preparations contained such an activity. However, at least some of the activity was due to the presence of an as yet unidentified factor that contaminated the eIF-3 and eIF-4B preparations. A polypeptide of apparent molecular weight 24,000 that can be specifically cross-linked to the oxidized 5'-terminal cap structures of reovirus and other viral mRNAs co-purifies with this new factor, and may be a component of it. This stimulatory activity present in our eIF-4B preparations was found to be greater when lower concentrations of KCl were present in the reaction mixture, possibly explaining why uncapped VSV mRNA is translated relatively more efficiently at low K+ concentrations.


A kinetic analysis of D-glucose transport by adipocyte plasma membranes. The measurement of the initial rate of glucose uptake is of critical importance for the kinetic analysis of the effects of temperature, ions, or hormones on the uptake process. Presented here is a highly reproducible double isotope assay system which for the first time allowed the measurement of the initial rates of glucose uptake into isolated adipocyte plasma membrane vesicles by having time points as early as 1/2 s. Measurement of the initial rates of uptake at 1 s allowed the calculation of a Km of 9.0 mM and a Vmax of 3209 pmol/mg of protein/s for specific D-glucose transport (D-glucose minus L-glucose). The time course of specific D-glucose uptake is extremely rapid, reaching an equilibrium value in approximately 20 s with a half-time of 4 s. This uptake process is markedly inhibited by cytochalasin B. The similarity of these findings to those for intact adipocytes indicates that the plasma membrane preparation and the assay conditions used have retained the structural and functional integrity of the glucose transport system from the intact cell.


Enhanced transcription by RNA polymerases II and III after inhibition of protein synthesis. Intraperitoneal administration of cycloheximide (100 mg/kg) to rats produces a time-dependent rise in nuclear RNA polymerase II activity which is maximum at 30 min. This same concentration of cycloheximide also reduces RNA polymerase I activity to 64% of control within this time period. When 10 mg/kg of cycloheximide was administered, there was a 2-fold increase in both RNA polymerases II and III activities within 30 min as assayed in isolated nuclei. When these enzymes are solubilized from nuclei and resolved by DEAE-Sephadex, there is no significant change in the activity of RNA polymerase I or II when assayed on an exogenous template. It is suggested that the dual enhancement of nuclear RNA polymerase II and III activities is the result of a compensatory feedback relationship which exists between translation and transcription in vivo.


alpha-Clostripain. Chemical characterization, activity, and thiol content of the highly active form of clostripain. A highly active form of clostripain, composed of two polypeptide chains (Mr = 43,000 and 12,500), was isolated by hydrophobic chromatography from the culture medium of Clostridium histolyticum. It differs in amino acid composition, namely in the value for cyst(e)ine, from that previously reported. The analyses of the separated chains are given. Activity is related to the number of free cysteine residues and full activity is obtained only after complete reduction of the disulfide bonds. Specific modifications by sulfhydryl reagents and tosyl lysine chloromethyl ketone of one thiol group, the one implicated in the activity, are reported. High specificity of alpha-clostripain is restricted to arginyl peptide bonds as tested on parvalbumin.


Primary structure of a high potential, four-iron-sulfur ferredoxin from the photosynthetic bacterium Rhodospirillum tenue. The amino acid sequence of a high oxidation-reduction potential iron-sulfur protein (HiPIP) isolated from the purple photosynthetic bacterium Rhodospirillum tenue has been determined. This is the smallest of the HiPIP's, containing 63 residues, with only 3 residues apparently conserved in addition to the 4 cluster-binding cysteines. A minimum of four internal genetic gaps is postulated to align tenue high potential iron-sulfur protein with the previously known proteins. It is the most divergent of its class, with an average of only 25% identically matching residues in common with any of the other species.


The structure and stability of human plasma cold-insoluble globulin. The molecular properties of cold-insoluble globulin have been investigated by velocity centrifugation, circular dichroism, and fluorescence at neutral and alkaline pH. The stability of the protein to thermal and guanidine hydrochloride has been evaluated under both conditions. The close parallelism between the properties of cold-insoluble globulin and those of the cell surface protein (fibronectin) serve to establish the essential identity of the structures of the two proteins derived from different sources. It is suggested that the cold-insoluble globulin is composed of several domains connected by flexible polypeptide segments. The large increase in the frictional ratio observed between pH 7.0 and 11.0 can be explained by an expansion of the flexible segments without significant change in the domains. These domains are stable to about 55 degrees C at pH 7.0 but only to about 40 degrees C at pH 11.0.


Accumulation of two glycoasparagines in the liver in aspartylglycosaminuria. Two water-soluble glycoasparagine storage products were isolated from the liver of a patient with inherited deficiency of lysosomal N-aspartyl-beta-glucosaminidase (aspartylglycosaminuria). The compounds were characterized using gas chromatographic-mass spectrometric analyses of intact and specifically degraded derivatives. The storage compounds were identified as 2-acetamido-1-N-(4'-L-aspartyl)-2-deoxy-beta-D-glucopyranosylamine and alpha-D-mannopyranosyl-(1 leads to 6)-beta-D-mannopyranosyl-(1 leads to 4)-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-1-N-(4'-L-aspartyl)-2-deoxy-beta-D-glucopyranosylamine. Their amounts in the pathological liver were approximately 2.7 mg/g and 0.2 mg/g, wet weight, respectively. These glycoasparagines are structurally related to the inner core region of a number of different glycoproteins.