Showing papers in "Journal of Biochemistry in 1980"
TL;DR: The aliphatic index of proteins of thermophilic bacteria is significantly higher than that of ordinary proteins and may be regarded as a positive factor for the increase of thermostability of globular proteins.
Abstract: A statistical analysis shows that the aliphatic index, which is defined as the relative volume of a protein occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine), of proteins of thermophilic bacteria is significantly higher than that of ordinary proteins. The index may be regarded as a positive factor for the increase of thermostability of globular proteins.
897 citations
388 citations
TL;DR: Invertebrate calmodulins of the sea anemone and scallop muscle were isolated and their properties were compared with those of vertebrate cal modulins from rabbit muscle and pig brain.
Abstract: Invertebrate calmodulins of the sea anemone and scallop muscle were isolated and their properties were compared with those of vertebrate calmodulins from rabbit muscle and pig brain. The molecular weights estimated by SDS-polyacrylamide gel electrophoresis were similar to the molecular weight (16,500) of the vertebrate calmodulins. Every calmodulin contained 1 mol each of trimethyllysine and histidine, and high contents of acidic amino acids. The marine invertebrate calmodulins contained only one tyrosine in contrast to two tyrosines in the vertebrate ones. As a result, the UV absorption spectra were clearly different. The Ca2+-induced difference UV absorption spectra of the invertebrate calmodulins were indistinguishable from those of the vertebrate ones in spite of the difference in tyrosine contents. In tryptic peptide maps of invertebrate calmodulins, a few spots different from those of vertebrate calmodulins were observed in the basic and acidic peptide regions. The calmodulins of invertebrate muscles and that of rabbit skeletal muscle were almost indistinguishable in terms of the activation profile of rabbit skeletal myosin light chain kinase.
265 citations
TL;DR: Acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was purified from rat liver and it was concluded that components B and C were formed from component A, probably by proteolytic cleavage, based on the result of amino acid analysis of each component.
Abstract: Acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was purified from rat liver. The final preparation was judged to be nearly homogeneous from the results of sedimentation analysis. Ultrogel AcA-34 column chromatography, and phosphocellulose column chromatography. The molecular weight of the enzyme was determined to be 139,000 by the sedimentation equilibrium method and Ultrogel AcA-34 column chromatography. The S020,W of the enzyme was 7.85S. Three protein components, A, B, and C, were found in the enzyme preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weights, 75,500, 50,100, and 19,000) and high-pressure liquid chromatrography in the presence of 6 M guanidine . HCl (molecular weights, 71,900, 51,700, and 20,500). It was concluded that components B and C were formed from component A, probably by proteolytic cleavage, based on the result of amino acid analysis of each component. The pI of the enzyme was 9.2. The enzyme contained FAD as a prosthetic group, and exhibited absorption maxima at 278, 378, and 450 nm. The FAD content was 1.22 mol/mol of enzyme. When palmitoyl-CoA was added to the enzyme solution under anaerobic conditions, the bound FAD was reduced. The Km values were lower for C14 to C18 acyl-CoA's than for others tested, whereas Vmax values were roughly the same for C8 to C18 acyl-CoA's. The Km value for O2 was 5 microM. The optimal pH was 8. 3-Ketohexadecanoyl-CoA inhibited the enzyme (Ki=0.47 microM), forming a charge-transfer complex with the enzyme.
225 citations
TL;DR: Studies on the titration of the rat liver enzyme with acetoacetyl-CoA, the effects of salts, SH titration and proteolytic inactivation suggest that the active centers for these two reactions are located at different sites.
Abstract: Mitochondrial and peroxisomal enoyl-CoA hydratases were purified from rat liver. The mitochondrial enzyme, with a molecular weight of 161,000, was composed of 6 identical subunits. The molecular structure of the rat liver enzyme was very similar to that of the bovine liver enzyme. Acetoacetyl-CoA was a competitive inhibitor of the mitochondrial enzymes. The results of titration of the rat liver enzyme with acetoacetyl-CoA suggest that 3 subunits of the enzyme exhibit catalytic activity. The catalytic properties of the enzyme were studied. The peroxisomal enzyme was composed of one polypeptide with a molecular weight of 70,000-81,000. Some of the enzyme molecules were shown to be cleaved to two polypeptides in the cell by the following methods: amino acid analysis, peptide mapping and immunoprecipitin reaction. The catalytic properties of the peroxisomal enzyme were different from those of the mitochondrial enzyme. The peroxisomal enzyme is a bifunctional enzyme exhibiting 3-hydroxyacyl-CoA dehydrogenase activity. Studies on the titration with acetoacetyl-CoA, the effects of salts, SH titration and proteolytic inactivation suggest that the active centers for these two reactions are located at different sites.
146 citations
TL;DR: In this paper, a method for the separation and purification of different forms of cytochrome P-450 from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-pretreated rabbits was described.
Abstract: A method is described for the separation and purification of different forms of cytochrome P-450 from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-pretreated rabbits. It consists of solubilization of microsomes with cholate, followed by successive chromatography on omega-aminooctyl Sepharose 4B, hydroxylapatite and CM-Sephadex C-50 columns. Separation of different forms of cytochrome P-450 is achieved in the aminooctyl Sepharose and hydroxylapatite chromatograhy steps. This method permits the separation of three forms of cytochrome P-450, i.e. "P-450(1)," "P-450(2)," and "P-488(1)," from PB-induced microsomes; P-450(1), the main cytochrome P-450 component in these microsomes, and P-448(1) can each be obtained in a gel-electrophoretically homogeneous state, whereas P-450(2) can be obtained in a partially purified state. Application of the same method to MC-induced microsomes led to the purification of P-448(1), the main component in these microsomes, to homogeneity and to partial purification of a fourth form, i.e. "P-450(3)." P-448(1) from MC-induced microsomes seems to be identical with P-448(1) from PB-induced microsomes in monomeric molecular weight (54,000), amino acid composition and chromatographic behavior. However, P-448(1) from MC-induced microsomes, but not P-448(1) from PB-induced microsomes, contains 0.18 to 0.88 mol of tightly bound MC per mol of protein. P-450(1) has a molecular weight of 49,000 and its amino acid composition is clearly different from that of P-448(1). Although P-450(2) is similar in molecular weight, they differ from each other in chromatographic behavior. P-450(3) seems to be different from P-450(1) in molecular weight, though they are similar to each other in chromatographic behavior. All the cytochrome P-450 preparations can be freed from the detergents used in the purification procedure by CM-Sephadex C-50 chromatography. Detergent-free P-450(1), P-450(2), and P-448(1) exist in aqueous solution as oligomeric aggregates.
118 citations
TL;DR: A completely sodium dodecyl sulfate-insoluble protein was obtained from plasmodia of Physarum polycephalum and the amino acid composition was very similar to that of connectin, an elastic protein of muscle, which may act as a cytoskeleton at the cytoplasmic surface of the plas modium cell membrane.
Abstract: A completely sodium dodecyl sulfate-insoluble protein was obtained from plasmodia of Physarum polycephalum. The amino acid composition of this protein was very similar to that of connectin, an elastic protein of muscle. When the protein suspension was treated with 6 M guanidine-HCl in the presence of 0.1 M 2-mercaptoethanol, entanglements of very thin filaments, less than 5 nm in width, were observed under an electron microscope. Since the network structure is almost the same as that of isolated muscle connectin, this plasmodium protein can be regarded as forming an elastic net structure. Using an antiserum against the insoluble protein, it appeared to be located at the peripheral region of the plasmodium. This elastic net structure may act as a cytoskeleton at the cytoplasmic surface of the plasmodium cell membrane.
110 citations
TL;DR: Kinetic studies indicated that E-64 was an irreversible inhibitor of these enzymes, and E- 64 binds to an equimolar amount of active SH residues of cathepsin B in parallel with inactivation of the enzyme.
Abstract: The mechanism of inhibition of cathepsin B [EC 3.4.22.1] and cathepsin L [EC 3.4.22.-] by E-64 was investigated. Kinetic studies indicated that E-64 was an irreversible inhibitor of these enzymes. [3H]E-64 is incorporated into cathepsin B in a one/one molar ratio in parallel with inactivation of the enzyme. Titration of one of the 10 SH groups of native cathepsin B with 2,2'-dithiodipyridine resulted in complete loss of enzyme activity. Decrease of titratable SH groups and activity of cathepsin B was proportional to the concentration of E-64 added, indicating that E-64 binds to an equimolar amount of active SH residues of cathepsin B. The effects of E-64 and its derivatives on lysosomal cathepsin B and cathepsin L in rat liver were studied in vitro and in vivo. The D form of E-64 inhibited the cathepsin both in vitro and in vivo, although its inhibitory effects were less than those of E-64-(L). E-64-b(RR), in which the terminal agmatine of E-64 is replaced by leucine, was as active as E-64-(L) in vitro, but was completely inactive in vivo. Among the E-64 derivatives tested, E-64-c(SS), in which the terminal agmatine of E-64 is replaced by isoarylamide, showed strong inhibitory activity in vivo, like E-64-(L).
103 citations
TL;DR: A newly-identified muscle protease which degraded myosin, alpha-actinin, and actin and removed the Ca-sensitivity of the ATPase activity of myofibrils was purified from rabbit skeletal muscle and showed that the enzyme thus obtained was almost homogeneous.
Abstract: A newly-identified muscle protease which degraded myosin, alpha-actinin, and actin, and removed the Ca-sensitivity of the ATPase activity of myofibrils was purified from rabbit skeletal muscle by ammonium sulfate fractionation, Sephadex G-75 chromatography, P-cellulose chromatography, Sephadex G-75 rechromatography, and Ultrogel AcA 54 chromatography. Polyacrylamide gel electrophoresis showed that the enzyme thus obtained was almost homogeneous. The molecular weight of the enzyme was found to be 24,000 by gel filtration on Sephadex G-75. The optimum pH for the Ca-sensitivity-removing activity was pH 7.0, and that for the activity to degrade myosin was pH 4.1. The enzyme was stable at pH 4.5--6.5. It was strongly inhibited by iodoacetate, leupeptin, and antipain, but not by pepstatin or phenylmethane sulfonyl fluoride. EDTA was essential for the activity of the enzyme. The enzyme did not split alpha-N-benzoyl-DL-arginine-beta-naphthylamide. Since these properties resemble those of cathepsin L isolated from rat liver lysosomes, the enzyme was regarded as muscle cathepsin L.
100 citations
TL;DR: The titration curves obtained by the above three groups were consistently explained in terms of the same set of pKint values and showed that no buried and untitratable groups are present in the native lysozyme molecule.
Abstract: The acid-base titration curves of hen egg-white lysozyme [EC 3.2.1.17] obtained by three groups (Sakakibara & Hamaguchi (1968) J. Biochem. 64, 613--619; Tanford & Roxby (1972) Biochemistry 11, 2192--2198; Pfeil & Privalov (1976) Biophys. Chem. 4, 23--32) were analyzed using the empirical formula of Linderstrom-Lang. Hen lysozyme has 32 ionizable groups including the alpha-amino and alpha-carboxyl groups. Of the 21 groups other than the 11 arginyl residues, the pK values of 17 ionizable groups have been determined by various methods. Using these pK values, the pK values of the other four ionizable groups were estimated. The apparent pK values obtained were 2.0 (pKmit = 3.4), 2.1 (pKint = 3.5), 2.5 (pKint = 3.8), and 7.9 (pKint = 8.5) at 0.1 ionic strength and 25 degrees C. The titration curves obtained by the above three groups were consistently explained in terms of the same set of pKint values. The results obtained also showed that no buried and untitratable groups are present in the native lysozyme molecule.
98 citations
TL;DR: Calcium-activated neutral protease (CANP) was demonstrated in myofibrils, especially at the Z-band in chicken glycerinated myofibils, by an immunofluorescent method and a possible role of myofilbril-bound CANP in the physiological turnover of the my ofibrillar proteins is discussed.
Abstract: Calcium-activated neutral protease (CANP) was demonstrated in myofibrils, especially at the Z-band in chicken glycerinated myofibrils, by an immunofluorescent method. The amount of CANP bound to myofibrils was approximately 4 percent of that contained in the whole muscle homogenate. No immunological difference was recognized between the myofibril-bound CANP and the soluble one when examined by Ouchterlony's immunodiffusion analysis. A possible role of myofilbril-bound CANP in the physiological turnover of the myofibrillar proteins is discussed.
TL;DR: The results show that bovine pancreatic ribonuclease B has one of the following sugar chains as asparagine-linked carbohydrate.
Abstract: The sugar chain of bovine pancreatic ribonuclease B was released from the polypeptide moiety by hydrazinolysis and by endo-beta-N-acetylglucosaminidase H digestion, and reduced with NaB[3H]4 after N-acetylation. The radioactive oligosaccharide mixtures thus obtained were fractionated by Bio-Gel P-4 column chromatography, and their structures were studied by sequential exoglycosidase digestion, methylation study, periodate oxidation and acetolysis. The results show that bovine pancreatic ribonuclease B has one of the following sugar chains as asparagine-linked carbohydrate. Formula (See Text).
TL;DR: In the livers of fasted rats, the activity of peroxisomal palmitocyl-CoA oxidation (NADH production) was increased more rapidly and markedly than that of mitochondrial carnitine palmitoyltransferase, which is the rate limiting enzyme of mitochondrial beta-oxidation.
Abstract: In the livers of fasted rats, the activity of peroxisomal palmitocyl-CoA oxidation (NADH production) was increased more rapidly and markedly than that of mitochondrial carnitine palmitoyltransferase, which is the rate limiting enzyme of mitochondrial beta-oxidation. The peroxisomal oxidizing activity was about twice that of the control throughout the period of fasting (1-7 days). carnitine acetyltransferase activity was increased to a similar extent in both peroxisomes and mitochondria. A possible physiological role of liver peroxisomes may thus be as an effective supply of NADH2, acetyl residues and short and medium-length fatty acyl-CoA in the cells on the enhancement of peroxisomal beta-oxidation of the animals under starvation; these substances thus produced may be transported into the mitochondria as energy sources.
TL;DR: Nucleotide specificities supported the idea that the catalytic sites are located in the beta subunits and the allosteric sites are Located in the alpha subunits, and suggested that the tight binding sites areLocated in thealpha subunits.
Abstract: The catalytic and allosteric sites of proton translocating adenosine triphosphatase (ATPase) were studied by measuring the binding of nucleotides to the ATPase, and its alpha and beta subunits purified from thermophilic bacterium PS3, with a circular dichroic spectrometer. In contrast to mesophilic ATPases, this thermophilic enzmye contained no tightly bound nucleotides, and its subunits were stable after their purification. These properties were advantageous for analyzing both catalytic and allosteric sites. The former site showed rapid and loose binding, but the latter slow (t 1/2 = 1 h, for ADP) and tight binding. When a nucleotide was bound, the beta subunits showed a negative ellipticity at 275 nm corresponding to a tyrosyl residue, while the alpha subunits showed an ellipticity change corresponding to the absorption curve of the bound nucleotide. This difference enabled us to distinguish the binding sites in ATPase. At a low concentration, ADP selectively bound to alpha subunits in the ATPase, while at a high concentration, it bound to both subunits. This finding suggests that the tight binding sites are located in the alpha subunits. Although ADP and ATP bound to both the purified alpha and beta subunits, CTP did not bind to beta but only to alpha subunits, and ITP bound to beta but hardly to alpha. These nucleotide specificities also supported the idea that the catalytic sites are located in the beta subunits and the allosteric sites are located in the alpha subunits.
TL;DR: The results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys- lysine residues, which were found to be useful for the specific and sensitive assay of plasmin.
Abstract: Fluorogenic peptides, peptidyl-4-methylcoumaryl-7-amides (MCA), containing COOH-terminal lysine residues, were newly synthesized and tested as substrates for plasmin. Among six peptidyl-MCA's, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA were found to be useful for the specific and sensitive assay of plasmin. The Km values estimated from Line-weaver-Burk plots for these substrates using human and bovine plasmins were in the region of 10(-4) M. Boc-Glu-Lys-Lys-MCA was slightly hydrolyzed by bovine plasma kallikrein, and Boc-Val-Leu-Lys-MCA was slightly hydrolyzed by human and hog urinary kallikreins and hog pancreatic kallikrein. However, both of the fluorogenic peptides were essentially unaffected by urokinase, alpha-thrombin, Factor Xa, Factor IXa, Factor XIa, and Factor XIIa. It was confirmed that plasmin hydrolyzed Boc-Glu-Lys-Lys-MCA, cleaving the lysyl-MCA bond, but not the lysyl-lysyl bond. These fluorogenic peptides were resistant to human plasmin activated by streptokinase. Boc-Glu-Lys-Lys-MCA was not hydrolyzed by human plasmin or plasminogen in the presence of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys- of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys-MCA to human plasmin was also reduced, but plasmin retained 35% of the maximum activity even in the presence of a 20-fold molar excess of streptokinase. These results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys-Lys-MCA.
TL;DR: A cathepsin D-like acid proteinase from human gastric mucosa was purified for the first time to homogeneity as examined by polyacrylamide disc gel electrophoresis as discussed by the authors.
Abstract: A cathepsin D-like acid proteinase from human gastric mucosa was purified for the first time to homogeneity as examined by polyacrylamide disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 85,000 by gel filtration on Sephadex G-150 and, after treatment with 2-mercaptoethanol, about 38,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. These results indicate that the native enzyme is composed of two apparently identical monomeric units. The protein band could also be stained with the Schiff reagent after periodate oxidation, indicating that the proteinase is a glycoprotein. Analyses showed that the enzyme contains approximately 4 glucosamine and 16 mannose residues per molecule (M.W. 85,000). The amino acid composition generally resembled those of cathepsins D except for the lower contents of basic amino acid residues, especially lysine. It also showed some similarity to pepsinogens. The optimal pH was 2.3--3.5 with hemoglobin as a substrate. The enzyme was strongly inhibited, like pepsin, by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as well as by pepstatin and diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. The proteinase was stable in alkaline solution up to pH 9.0, and was rather stable to sequential acidification and neutralization. The acidification resulted in an increase of electrophoretic mobility towards the anode.
TL;DR: It is suggested that the reduction of nitrobenzene to an intermediate, possibly nitrosobenzenes or phenylhydroxylamine, limits the rate of aniline formation, and such an initial step of nitromine reduction can be catalyzed by NADPH-cytochrome c reductase alone.
Abstract: 1. The subcellular distribution of nitrobenzene reduction activity in rat liver cells indicated the existence of two different enzyme systems, one localized in microsomes and the other localized in cytosol. The activity in the cytosol was mainly attributable to xanthine oxidase, judging from its substrate specificity and the inhibition by allopurinol. 2. The participation of the microsomal electron transport system in nitrobenzene reduction was examined by using antibodies against four components of the system, NADPH-cytochrome c reductase (fpT), NADH-cytochrome b5 reductase (fpD), cytochrome b5, and cytochrome P-450. Both NADH- and NADPH-dependent nitrobenzene reduction activities were strongly inhibited by anti-fpT IG and also by anti-P450 IG, but not inhibited by anti-fpD IG or anti-b5 IG. The reduction of nitrosobenzene and phenylhydroxylamine, which are supposed to be the intermediates of nitrobenzene reduction, was also examined, and it was found that NADH- and NADPH-dependent reduction of both compounds were strongly inhibited by anti-fpT IG and anti-P450 IG, but not by anti-fpD IG or anti-b5 IG. 3. Reconstruction experiments using purified NADPH-cytochrome P-450 reductase and cytochrome P-450 were also carried out and it was confirmed that the reduction of nitrobenzene, nitrosobenzene, and phenylhydroxylamine to aniline could be effected by these two components. 4. Nitrobenzene reduction by microsomes exhibited a short initial time lag and was activated by the addition of purified NADPH-cytochrome c reductase, whereas nitrosobenzene and phenylhydroxylamine reductions did not show any initial time lag and were not activated by the reductase. These observations suggest that the reduction of nitrobenzene to an intermediate, possibly nitrosobenzene or phenylhydroxylamine, limits the rate of aniline formation, and such an initial step of nitrobenzene reduction can be catalyzed by NADPH-cytochrome c reductase alone. Cytochrome P-450 is essential at least in the final step of nitrobenzene reduction to aniline. This conclusion was further confirmed by determination of these intermediates in nitrobenzene reduction.
TL;DR: A tubulin-tyrosine ligase was purified from porcine brains using DEAE-cellulose chromatography, Sepharose-sebacic acid hydrazide-ATP affinity chromatography and SepharOSE-tubulin affinity Chromatography to catalyze the tyrosination of the alpha subunit of tubulin in vitro.
Abstract: A tubulin-tyrosine ligase was purified from porcine brains using DEAE-cellulose chromatography, Sepharose-sebacic acid hydrazide-ATP affinity chromatography and Sepharose-tubulin affinity chromatography. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 46,000. The apparent molecular weight was 37,000 on a high speed liquid chromatograph equipped with gel filtration columns. The pH optimum for the activity was around 8 and a second peak was observed at around 6.5 8.5 microM ATP or 30 microM tyrosine gave half-maximal activity. The purified enzyme catalyzed the tyrosination of the alpha subunit of tubulin in vitro.
TL;DR: The results suggest that O-demethylation of p-nitroanisole by this particular form of cytochrome P-450 absolutely requires the intact form of Cytochrome b5 and that the second electron needed for the demethylation may be donated to the cyto chrome P- 450 only by way of cy to chrome b5.
Abstract: A reconstituted system containing a form of cytochrome P-450, cytochrome b5, NADPH-cytochrome P-450 reductase, and NADH-cytochrome b5 reductase, all purified from rabbit liver microsomes, could catalyze O-demethylation of p-nitroanisole in the presence of both NADPH and NADH. Omission of either cytochrome P-450 or cytochrome b5 from the system led to complete loss of the activity. The reconstituted activity was sensitive to carbon monoxide, metyrapone, phenyl isocyanide, and cyanide, indicating that the cytochrome P-450 used is cyanide-sensitive and is involved in the catalytic process. The maximal demethylase activity was attained when the system contained cytochrome P-450 and cytochrome b5 at a 1 : 1 molar ratio. Trypsin digestion of cytochrome b5 abolished the capacity of this cytochrome to reconstitute the demethylase activity. These results suggest that O-demethylation of p-nitroanisole by this particular form of cytochrome P-450 absolutely requires the intact form of cytochrome b5 and that the second electron needed for the demethylation may be donated to the cytochrome P-450 only by way of cytochrome b5.
Journal Article•
TL;DR: The covalently bound FAD was reduced on addition of either choline or betaine aldehyde under anaerobic conditions and was reoxidized by aeration and the enzyme was found to contain 1 mol of FAD per mol enzyme.
Abstract: Choline oxidase from Alcaligenes sp. catalyzed the oxidation of choline and betaine aldehyde to betaine with concomitant consumption of oxygen and production of hydrogen peroxide. The values of Km for choline and betaine aldehyde were 0.87 and 6.2 mM, respectively. The molecular weight of the enzyme was estimated to be 66,000 by SDS-gel electrophoresis and 72,000 by gel-filtration using a high performance liquid chromatograph. The prosthetic group of the enzyme was identified as 8 alpha-[N(3)-histidyl]-FAD from the electrophoretic mobility at pH 6.25 of the hydrolysate of the methylated histidylflavin. The visible absorption spectrum of the enzyme showed peaks at 358 and 453 nm and a shoulder at about 480 nm. The covalently bound FAD was reduced on addition of either choline or betaine aldehyde under anaerobic conditions and was reoxidized by aeration. The enzyme was found to contain 1 mol of FAD per mol enzyme. Amino acid analysis of a purified flavin peptide gave the following molar ratios of amino acids to flavin: pro(1), Asp + Asn(3), Ser(1), His(1), and Arg(1). Aspartic acid was the N-terminal amino acid. The partial sequence of amino acids in the flavin peptide was as follows: Formula (See Text).
TL;DR: A purothionin homolog was isolated from barley flour and purified by CM-52 column chromatography and showed potent lethal activity towards brewer's yeast and its complete amino acid sequence was determined to be as follows.
Abstract: A purothionin homolog was isolated from barley flour and purified by CM-52 column chromatography. It showed potent lethal activity towards brewer's yeast and its complete amino acid sequence was determined to be as follows. Lys-Ser-Cys-Cys-Arg-Ser-Thr-Leu-Gly-Arg-Asn-Cys-Tyr-Asn-Leu-Cys-Arg-Val-Arg-Gly-Ala-Gln-Lys-Leu-Cys-Ala-Gly-Val-Cys-Arg-Cys-Lys-Leu-Thr-Ser-Ser-Gly-Lys-Cys-Pro-Thr-Gly-Phe-Pro-Lys. It thus consists of 45 amino acid residues with 8 cysteines. The number of amino acid residues and the positions of the 8 cysteines are identical with those of wheat purothionins. There is a high degree of homology in the primary structures of these proteins.
TL;DR: The defective coupling factor F1 ATPase from a mutant strain (KF11) of Escherichia coli was purified to a practically homogeneous form and results indicate that the mutation is in the beta subunit.
Abstract: The defective coupling factor F1 ATPase from a mutant strain (KF11) of Escherichia coli was purified to a practically homogeneous form. The final specific activity of Mg2+-ATPase was 6-9 units/mg protein, which is about 10-15 times lower than that of F1 ATPase from the wild-type strain. The mutant F1 had a ratio of Ca2+-ATPase to Mg2+-ATPase of about 3.5, whereas the wild-type F1 had ratio of about 0.8. The mutant F1 was more unstable than wild-type F1: on storage at -80 degrees C for 2 weeks, about 80% of its activity (dependent on Ca2+ or Mg2+) was lost, whereas none of the activity of the wild-type F1 was lost. The following results indicate that the mutation is in the beta subunit. (i) High Mg2+-ATPase activity (about 20 units/mg protein) was reconstituted when the beta subunit from wild type F1 was added to dissociated mutant F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. (ii) Low ATPase activity having the same ratio of Ca2+-ATPase to Mg2+-ATPase as the mutant F1 was reconstituted when a mixture of the beta subunit from the mutant F1 and the alpha and gamma subunits from wild-type F1 was dialyzed against the same buffer. (iii) Tryptic peptide analysis of the beta subunit of the mutant showed a difference in a single peptide compared with the wild-type strain.
TL;DR: Results of immunochemical neutralization studies and immunolectrophoresis indicated that anti-enolase III was specific to the subunit of enolases III and reacted with enolase II and enol enzyme III.
Abstract: Three forms of rat brain enolase (Fletcher, L., Rider, C.C., & Taylor, C.B. (1976) Biochim. Biophys. Acta 452, 245-252), which were separable by DEAE-cellulose chromatography (referred as enolase I, enolase II, and enolase III in order of the elution), were purified with a high yield by use of column chromatographies of Sephadex G-150, Blue Sepharose CL-6B, and hydroxylapatite. About ten mg of each isozyme was obtained from 220 g of the brain with a yield of 30-50%. Sodium dodecyl sulfate gel electrophoresis of purified enolase I and enolase III showed single bands with relative mobilities corresponding to molecular weights of 49,000 (enolase I) or 46,000 (enolase III), and that of purified enolase II showed two bands corresponding to the above molecular sizes. Amino acid analysis of three forms of enolase revealed that the amount of each amino acid enolase II was midway between those of enolase I and enolase III. Antiserum to enolase I or enolase III was raised in New Zealand white rabbits. Results of immunochemical neutralization studies and immunolectrophoresis indicated that anti-enolase III was specific to the subunit of enolase III and reacted with enolase II and enolase III. However, anti-enolase I reacted not only with enolase I and enolase II, but also with enolase in various other tissues. These results support previous reports on crude preparations that enolase II was a hybrid molecule consisting of one enolase I subunit and one enolase III subunit.
TL;DR: It is shown that the control of the enzyme reaction in vivo is considerably different from that expected from the in vitro experiments, and that deficiencies of "coarse control" are covered by a "fine control."
Abstract: Intracellular concentrations of phosphoenolpyruvate (PEP) and five kinds of allosteric effectors (acetyl-CoA, fructose 1,6-bisphosphate, GTP, L-aspartate, and L-malate) of PEP carboxylase were measured in E. coli cells grown on various compounds as a carbon source. Based on the data obtained, reaction systems which contained a definite concentration of the enzyme and the ligands at the concentrations found in vivo were constructed and the enzyme activities were measured. The ratio of each activity thus obtained to the maximal activity attainable with the same concentration of enzyme and saturating concentrations of the activators was estimated. For the cells grown on glucose, glycerol, or lactate, the extent of exhibition of the enzyme activity was 2-15% of the maximal activity. For the cells grown on acetate or oleate, the extent was 1-3%. For the cells grown on succinate, L-aspartate, L-malate, or glucose plus L-aspartate, the extent was less than 0.4%. Consideration of the data obtained in the present studies, together with those obtained in our previous studies on the enzyme level (Teraoka, H. et al. (1970) J. Biochem. 67, 567-575), showed that the control of the enzyme reaction in vivo is considerably different from that expected from the in vitro experiments, and that deficiencies of "coarse control" are covered by a "fine control."
TL;DR: Results indicate that the enzyme is synthesized as a larger precursor which may be imported into mitochondria in association with post-translational proteolytic processing to the mature form of the enzyme.
Abstract: A putative precursor of rat liver ornithine transcarbamylase [EC 2.1.3.3] which was about 3,400 daltons larger than the subunit of the mature enzyme (36,000 daltons) was synthesized in a rabbit reticulocyte cell-free system and immunoprecipitated using an antibody against the bovine enzyme and fixed Staphylococcus aureus cells. The mature enzyme of rat liver competed effectively with the putative precursor for interaction with the antibody. Digestion of the putative precursor by S. aureus protease gave a pattern of peptide fragments similar to that of the mature enzyme. A rat liver mitochondrial preparation converted the putative precursor to a polypeptide which comigrated with the mature subunit on sodium dodecyl sulfate/polyacrylamide gels. The "processed" product was recovered in sedimented mitochondria and was no longer susceptible to externally added proteases. These results indicate that the enzyme is synthesized as a larger precursor which may be imported into mitochondria in association with post-translational proteolytic processing to the mature form of the enzyme.
TL;DR: The pH difference absorption spectra of human lysozyme were measured and a time-dependent spectral change observed above pH 11 was not due to the exposure of buried tyrosyl residues on alkali denaturation but was due mainly to disulfide cleavage and Exposure of buried tryptophyl residues.
Abstract: The pH difference absorption spectra of human lysozyme [EC 3.2.1.17] were measured. The difference spectra in the acidic region had a peak at 300 nm, as observed for hen and turkey lysozymes. The pH dependence curve of the extinction difference at 300 nm was well interpreted in terms of the pK values of the catalytic groups (3.4 for Asp 52 and 6.8 for Glu 35 at 0.1 ionic strength and 25 degrees C) determined from the pH dependence of the circular dichroism at 303.5 nm (Kuramitsu et al. (1974) J. Biochem. 76, 671--683) and the fluorescence excited at 305 nm (Kuramitsu et al. (1978) J. Biochem. 83, 159--170). The difference spectra of human lysozyme in the alkaline pH region were characteristic of tyrosyl ionization. The perturbation of tryptophyl residues, which had been observed for hen and turkey lysozymes (Kuramitsu & Hamaguchi (1979) J. Biochem. 85, 443--456), was not observed for human lysozyme. On the basis of the pH dependence curves of the extinction difference at 245 and and 295 nm, we roughly estimated the apparent pK values of the six tyrosyl residues as 9.2 9.2, 10.5, 10.9, 12.4, and 12.5. A time-dependent spectral change observed above pH 11 was not due to the exposure of buried tyrosyl residues on alkali denaturation but was due mainly to disulfide cleavage and exposure of buried tryptophyl residues.
TL;DR: Comparison of the melting temperatures of T. thermophilus tRNAf2Met and tRNAmMet at different magnesium concentrations showed that magnesium was also a factor in the thermostability of the thermophile tRNA.
Abstract: Three species of methionine tRNAs and phenylalanine, tyrosine, and isoleucine tRNAs were purified from an extreme thermophile, Thermus thermophilus HB8. Formylation studies of the three methionine tRNAs and their codon-specific binding activities to ribosomes showed that two of them (named tRNAf1Met and tRNAf2Met) were initiator tRNAs and the other (named tRNAmMet) was a non-initiator. The tRNAs from T. thermophilus all had melting temperatures of up to ten degrees higher than the corresponding species from E. coli. Most of the species also had slightly higher G+C contents than the corresponding species of E. coli, and each of them contained one mol each of the modified nucleosides, O2'-methylguanosine (Gm), 2-thioribothymidine (s2T), and 1-methyladenosine (m1A). Their high melting temperatures could be explained by their high G+C contents and the presence of the modified nucleosides, espically s2T. Comparison of the melting temperatures of T. thermophilus tRNAf2Met with those of E. coli tRNAfMet and tRNAmMet at different magnesium concentrations showed that magnesium was also a factor in the thermostability of the thermophile tRNA.
TL;DR: The coupling between Cbz-Phe-OH and Leu-NH2 catalyzed by thermolysin was examined under various experimental conditions and strict stereospecificity was observed at this position.
Abstract: The coupling between Cbz-Phe-OH and Leu-NH2 catalyzed by thermolysin was examined under various experimental conditions. The highest yield (ca. 80%) was obtained in the reaction mixture containing 0.05 M each of the carboxyl and amine components and 10 muM enzyme at pH 7 and 37 degrees C for 5 h. The reactivity was ca. 100 times higher than that of alpha-chymotrypsin. Amino acid derivatives or peptides were useful as amine components, though a hydrophobic or bulky amino acid residue was required at the N-terminal position. Strict stereospecificity was observed at this position. A hydrophobic or bulky amino acid residue occupying the C-terminal position of carboxyl components was also favorable for synthesis. The specificity requirements for synthesis were the same as those for hydrolysis.
TL;DR: It is concluded that OM cytochrome b is distinct from microsomal cyto Chrome b5, and a clear difference was detected in amino acid composition, particularly in the contents of basic amino acids and methionine.
Abstract: A cytochrome b5-like hemoprotein associated with the outer membrane of rat liver mitochondria, OM cytochrome b, was purified to homogeneity after solubilization with trypsin. OM cytochrome b was separated from microsomal cytochrome b5 by hydroxylapatite chromatography, though they were indistinguishable in DEAE-cellulose chromatography and in Sephadex G-75 gel filtration. The absorption spectra of reduced OM cytochrome b and cytochrome b5 in the visible region at liquid nitrogen temperature showed small but significant differences between them. A clear difference was also detected in amino acid composition, particularly in the contents of basic amino acids and methionine. Using rabbit antibodies prepared against purified OM cytochrome b and cytochrome b5, immunochemical comparison was also carried out. No immunological cross reaction was observed by Ouchterlony double diffusion in agar gel or in a quantitative immunoprecipitation test. It is thus concluded that OM cytochrome b is distinct from microsomal cytochrome b5.