Showing papers in "Journal of Biochemistry in 1975"
TL;DR: It is found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column.
Abstract: Using [3H]-labeled oligosaccharides, we found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column. This finding is also applicable to N-[14C]acetylated glycopeptides. Thus, the concanavalin A-Sepharose column might become a useful tool for structural studies of glycopeptides and oligosaccharides and for their fractionation. Glycopeptides prepared from the trypsinate of rat fibroblasts, which has been purified by paper electrophoresis, were further separated into two fractions by chromatography on a concanavalin A-Sepharose column.
403 citations
TL;DR: FHD (flavin-hypoxanthine dinucleotide) has coenzymatic activity equal to that of FAD (FAD) and could be a new treatment for central nervous system disorders according to EMT results reported in EMT4.4.
Abstract: Glucose oxidase [&-D-glucose: oxygen 1-oxidoreductase, EC 1. 1. 3. 4] from Aspergillus niger was purified by DEAE-cellulose chromatography and Sephadex G-200 gel filtration using phosphate buffer, pH 7.0.1. The preparation has a specific activity of 172 μmoles oxygen consumed/min/mg of enzyme at 30° and pH 5.6, and is essentially catalase-free. The constituents of the enzyme are protein (74.0±2.8%), neutral sugar (16. 4±0.3%), amino sugar (2.4±0.5%), and 2 moles of iron per 160, 000 daltons, in addition to FAD.2. Optical data for the newly purified holoenzyme show that the ratio A280/A450 is 11.1, and e for enzyme-bound FAD at 450nm is 1.52×l04.3. The holoenzyme (molecular weight, 160, 000) consists of two identical subunits with molecular weights of 79, 000±4, 000, and the molecular weight of the apoenzyme seems to be identical with that of the subunit, as shown by SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-200.4. FHD (flavin-hypoxanthine dinucleotide) has coenzymatic activity equal to that of FAD.
148 citations
TL;DR: The mode of interaction between sodium dodecyl sulfate (SDS) and protein polypeptides with special reference to SDS-polyacrylamide gel electrophoresis is clarified and the isotherms obtained were different for each kind of protein.
Abstract: To clarify the mode of interaction between sodium dodecyl sulfate (SDS) and protein polypeptides with special reference to SDS-polyacrylamide gel electrophoresis, the binding of SDS to several protein polypeptides was investigated by the equilibrium dialysis technique. Each of the binding isotherms was characterized by the presence of two phases: an initial gradual increase in the amount of binding to 0.3-0.6 g/g (first phase) and a subsequent steep increase to 1.2-1.5 g/g (second phase). The binding was completed at a concentration of SDS below the critical micelle concentration. Throughout the first and second phases, the isotherms obtained were different for each kind of protein. On the basis of experiments with bovine serum albumin and ribonuclease (EC 3.1.4.22], the isotherms were profoundly affected by the method used for modification of the sulfhydryl groups. The claim of Reynolds and Tanford (Proc. Natl, Acad. Sci. U.S., 66, 1002 (1970)) that the isotherms are virtually identical for many kinds of proteins was not supported by the present data. Changes in the gross and local conformations were examined with reference to the isotherms by measurements of CD spectrum, free boundary electrophoresis, and gel filtration. The results obtained were collectively interpreted based on the model of SDS-protein polypeptide complexes proposed by the present authors (J. Biochem., 75, 309 (1974)).
136 citations
TL;DR: It was observed that protein and RNA contents in dystrophic muscle decreased at the early stage of the disease, in disagreement with the reported results on a wet weight basis, in which RNA content in dyStrophic muscle had been found to increase.
Abstract: Protein and RNA contents in muscle of normal and hereditary dystrophic mice C57BL/6J-dy/dy were reexamined on the basis of DNA. It was observed that protein and RNA contents in dystrophic muscle decreased at the early stage of the disease, in disagreement with the reported results on a wet weight basis, in which RNA content in dystrophic muscle had been found to increase. Rates of protein and RNA systhesis in the early stage of the disease were also determined with a concomitant check of the specific activities of free amino acids and free nucleotides. The rates of both protein and RNA synthesis (i.e., specific activities of protein and RNA) were higher in the dystrophic muscle, but when they were expressed on a DNA basis, the total protein synthesis per cell was the same as that of normal muscle and the total RNA synthesis per cell showed a smaller increase in dystrophic muscle. These apparent increases of protein and RNA synthesis were discussed in connection with the decreased protein and RNA contents in the cells of dystrophic muscle. The synthesized RNAs seemed to contain mRNA on the basis of sedimentation character and Millipore filter binding ability. However, no particular RNA was mainly synthesized in dystrophic muscle.
130 citations
TL;DR: The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy and the advantages of this method for large scale preparation are discussed.
Abstract: A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E. coli. The characteristics of both membrane fractions were studied chemically, biologically, and morphologically. Spheroplasts of E. coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme [EC 3.2.1.17], were disrupted in a French press. The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation. The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide. The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction. Cytochrome b1, NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.5.1.3], and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction. In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction. The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy. The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.
122 citations
TL;DR: Beta-Galactosidase has been purified from a culture of Aspergillus oryzae by 2-propanol fractionation, column chromatography on DEAE-Sephadex A-50 and SephadeX G-200 and showed pH optima of 4.5 with ONPG-1 as a substrate and 4.8 with lactose.
Abstract: Beta-Galactosidase [EC 3.2.1.23] has been purified from a culture of Aspergillus oryzae by 2-propanol fractionation, column chromatography on DEAE-Sephadex A-50 and Sephadex G-200. The preparation was homogeneous on ultracentrifugation and disc electrophoresis. The enzyme showed pH optima of 4.5 with ONPG-1 as a substrate and 4.8 with lactose as a substrate. The stable pH range was from 4.0 to 9.0 and the optimum temperature was 46 degrees. The Michaelis constants were 7.2 X 10-minus 4 M with ONPG and 1.8 X 10-minus 2 M with lactose. Hg-2+, Cu-2+, N-bromosuccinimide, and sodium laurylsulfate caused marked inhibition. The apparent molecular weight was calculated to be about 105,000 by Sephadex gel filtration and sucrose density gradient centrifugation.
122 citations
TL;DR: A proteinase from the sarcocarp of melon was purified by a three-step procedure involving batch-wise treatment with CM-cellulose fibers, column chromatography on CM- cellulose powder and gel filtration on Sephadex G-75 and was homogeneous on acrylamide gel electrophoresis.
Abstract: A proteinase from the sarcocarp of melon (Cucumis Melo L. var. Prince) was purified by a three-step procedure involving batch-wise treatment with CM-cellulose fibers, column chromatography on CM-cellulose powder and gel filtration on Sephadex G-75. The final enzyme preparation was homogeneous on acrylamide gel electrophoresis. Its molecular weight was estimated by two different methods to be about 50,000. Anlayses indicated tha presence of 475 amino acid residues and at least 7 moles of hexose. The maximum activity was found in the alkaline pH region against casein as a substrate. The optimum temperature against casein was 70 degrees at pH 7.1. The enzyme was strongly inhibited by diisopropyl fluorophosphate, partly inhibited by HgCl2 and not inhibited by EDTA, p-chloromercuribenzoic acid, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor. The reduced and carboxymethylated insulin B-chain was cleaved at the peptide bonds of Asn3-Gln4, Cm-Cys7-Gly8, Glu13-Ala14, Leu15-Tyr16, Cm-Cys19-Gly20, Phe25-Tyr26, Pro28-Lys29, and Lys29-Ala30 by the enzyme.
95 citations
TL;DR: A series of partially O-methylated N-methylglucosamines was synthesized by limited-time methylation of methyl-2-N-methylacetamido- 2-deoxyglucopyranoside by Kuhn's procedure, followed by acid hydrolysis, and a mass fragmentographic method was established for specific analysis of the methylated aminosugar derivatives.
Abstract: A series of partially O-methylated N-methylglucosamines was synthesized by limited-time methylation of methyl-2-N-methylacetamido-2-deoxyglucopyranoside by Kuhn's procedure, followed by acid hydrolysis. These partially O-methylated N-methylglucosamines were separated satisfactorily by gas chromatography on a column of OV-17 on Gas-chrom Q as amino alditol acetates and identified from their mass spectra. For specific analysis of the methylated aminosugar derivatives, a mass fragmentographic method was established. Methylated aminosugars can be successfully determined in amounts as low as about 1 ng by this method.
94 citations
TL;DR: The intrinsic viscosity ([eta]) and the molecular weight (M) by sedimentation equilibrium were determined for hyaluronic acids of low and high molecular weights by double logarithmic plot of [eta] against M.
Abstract: The intrinsic viscosity ([eta]) and the molecular weight (M) by sedimentation equilibrium were determined for hyaluronic acids of low (M=104--7.2X10(4)) and high (M=3.1X10(5)--1.5X10(6)) molecular weights. Double logarithmic plot of [eta] against M gave different lines for the two groups. The relationship between [eta] and M was [eta]=3.0X10(6)XM1,20 for the former and [eta]=5.7X10(-4)XM0.46 for the latter group. The molecular weight at the point of intersection of the two lines was about 1.5X10(5). The rheological behavior of the hyaluronic acids below M=2.1X10(4), for which the value of reduced viscosity was independent of concentration, was different from that of the hyaluronic acids above M=5.1X10(4), for which the value of reduced viscosity increased with concentration.
85 citations
TL;DR: It is suggested that Mg2+ serves as a regulator for the enzyme activity that is more sensitive to inhibition at pH 9.2 or 9.1, and the effects of pH on the Km value for M g2+ activation and on the Mg1+ inhibition contributed to an apparent shift of the pH optimum for activity induced by EDTA.
Abstract: EDTA at a concentration of 1 muM produced a threshold effect in the activation of purified rabbit liver fructose-1, 6-diphosphatase [EC 3.1.3.11] in the presence of 5 mM Mg2+ at pH 7.2. Without EDTA, biphasic activation curves were produced by Mg2+. A double-reciprocal plot of the data gave the Km values corresponding to the two linear regions. They were 0.19 and 0.83 mM at pH 7.5, and 0.055 and 0.83 mM at pH 9.1. In the presence of 5muM EDTA a sigmoidal curve was obtained for Mg2+ activation in the range of noninhibitory Mg2+ concentrations at pH 7.2. The apparent Km value for Mg2+ was 0.15 mM, and the Hill coefficient was 2.0. At pH 9.1 cooperativity among the Mg2+ sites disappeared, and the apparent Km value for Mg2+ was 0.055 mM. These Km values at pH 7.2 or 9.1 corresponded to the smaller of the biphasic Km values obtained without EDTA. In the absence of EDTA, no inhibition by Mg2+ was observed in the Mg2+ concentration range below 10 mM. In the presence of EDTA, the enzyme was inhibited markedly by Mg2+ at concentrations above 0.5 mM at pH 7.2, and was more sensitive to inhibition at pH 9.1. The effects of pH on the Km value for Mg2+ activation and on the Mg2+ inhibition contributed to an apparent shift of the pH optimum for activity induced by EDTA. Cooperative interaction among fructose-1, 6-diphosphate sites was observed for the enzyme in the presence of EDTA. The Hill coefficient was approximatley 1.8, and the apparent Km value for the substrate was 0.74 muM. EDTA appears to make liver fructose-1, 6-diphosphatase very sensitive to various effectors. It is suggested that Mg2+ serves as a regulator for the enzyme activity.
83 citations
TL;DR: D-Fructans synthesized from sucrose by cell-free systems of strains of Streptococcus mutans and Strep have been shown by methylation and enzymatic studies to have different glycosidic linkages, and the solubility properties of these fructants are discussed.
Abstract: D-Fructans synthesized from sucrose by cell-free systems of strains of Streptococcus mutans and Strep salivarius have been shown by methylation and enzymatic studies to have different glycosidic linkages The cold water-insoluble D-fructans from Strep mutans strain BHT and JC-1 have inulin-type structures consisting beta-(2 leads to 1)-D-fructofuranosidic linkages, with average repeating units of 8 and 27 sugar residues, respectively, whereas the water-soluble fructan from Strep salivarius strain HHT has a levan-type branched structure consisting beta-(2 leads to 6)-D-fructofuranosidic linkages with an average repeating unit of 9 sugar residues The solubility properties of these fructants are discussed on the basis of the structural differences
TL;DR: Kinetic parameters for carboxypeptidase Y confirmed that the enzyme released COOH-terminal proline and beta-alanine at an appreciable rate, as well as neutral amino acids with aromatic and aliphatic side chains at a very high speed.
Abstract: Kinetic parameters for carboxypeptidase Y [EC 3.4.12.1], characterized as a nonspecific enzyme, are given for the hydrolysis of a series of acylated peptides, acylated amino acid esters, and amides. We confirmed that the enzyme released COOH-terminal proline and beta-alanine at an appreciable rate, as well as neutral amino acids with aromatic and aliphatic side chains at a very high speed. The rates of hydrolysis of ester and amide substrates were compatible with those produced by chymotrypsin [EC 3.4.21.1]. Stereospecificity was also demonstrated by the failure to hydrolyze peptide, ester, amide, and anilide substrates containing a D-amino acid. The effects of pH, solvents, and salt concentrations on the kinetic parameters of hydrolysis of peptide and ester substrates are also described.
TL;DR: It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety is responsible for the tight binding of the reduct enzyme to microsomes and for its normal functioning in the membrane.
Abstract: NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.
TL;DR: An enzyme that releases acylamino acid from amino terminal acylated peptides and proteins has been isolated from rat liver in a highly purified form by six-step procedure comprising extraction from liver homogenate, ammonium-sulfate fractionation, heat treatment, chromatography on columns of DEAE-cellulose and hydroxylapatite and gel filtration on a Sepharose 6B column.
Abstract: An enzyme that releases acylamino acid from amino terminal acylated peptides and proteins has been isolated from rat liver in a highly purified form bya six-step procedure comprising extraction from liver homogenate, ammonium-sulfate fractionation, heat treatment, chromatography on columns of DEAE-cellulose and hydroxylapatite and gel filtration on a Sepharose 6B column. About 1,500-fold purification was achieved from the liver homogenate. The purified enzyme preparation showed a single band on polyacrylamide gel disc electrophoresis. The enzyme specifically released acylamino acids from several amino terminal acylated peptides and proteins with different rates of hydrolysis depending on the acyl groups, terminal amino acid sequences and tertiary structure of the acyl protein substrates. The present enzyme may be useful for the removal of the N-terminal acylamino acid from some N-terminal blocked peptides and proteins in amino acid sequence analysis. The molecular weight of the purified enzyme was estimated to be 360,000-420,000 by gel filtration and sucrose density gradient ultracentifugation. Disc electrophoresis of the acylamino acid-releasing enzyme on SDS-polyacrylamide gel suggested that the enzyme consisted of five or six identical subunits having a subunit weight of about 75,000. The N-terminal residue of the subunit, which consisted of a single polypeptide chain, was glycine. Other properties of the enzyme, including isoelectric point, the effects of metal ions and several chemical reagents on the enzyme activity, pH optimum, and amino acid composition were also examined.
TL;DR: ESR spectra of the frozen solution and lyophilized powder of the nitrite reductase predominantly showed the presence of two kinds of copper; the bond between the oxidized enzyme and nitrite seems to be ionic.
Abstract: 1. Dialysis against cyanide at pH 7 of Achromobacter cycloclastes nitrite reductase [EC 1.7.99.3] of a dissimilatory type led to the removal of about 50% of the copper from the enzyme molecule, with a concomitant decrease of the enzymatic activities. It was inferred that enzyme-bound copper atoms play an essential role in the catalytic activities of the enzyme. 2. The amino acid composition of the enzyme was determined after acid hydrolysis. 3. ESR spectra of the frozen solution and lyophilized powder of the nitrite reductase predominantly showed the presence of two kinds of copper: Type 1 Cu2+, which had narrow and sharp hyperfine splitting, and Type 2 Cu2+, which had broader hyperfine splitting. The bond between the oxidized enzyme and nitrite seems to be ionic.
TL;DR: Pork insulin was subjected to mercaptosuccinylation and then coupled to beta-D-galactosidase [EC 3.2.1.23] from Escherichia coli using N,N'-o-phenylenedimaleimide as mentioned in this paper.
Abstract: Pork insulin was subjected to mercaptosuccinylation and then coupled to beta-D-galactosidase [EC 3.2.1.23] from Escherichia coli using N,N'-o-phenylenedimaleimide. The competitive binding of the conjugate and insulin to anti-insulin antibody was tested. Results showed that formation of an insulin-beta-D-galactosidase conjugate could be used for immunoassay of insulin.
TL;DR: It is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine and being a competitor of L-ornithine induced by IPR in mouse parotid glands.
Abstract: The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.
TL;DR: Preincubation of bleomycin with these reducing or oxidizing agents reduced the DNA-degrading activity of the antibiotic, however, this reduction in activity was observed even in the absence of oxygen, or in preincubsation mixture supplemented with radical scavenger.
Abstract: The effects of reducing agents, such as 2-mercaptoethanol, dithiothreitol, L-ascorbic acid, or sodium borohydride, and oxidizing agents, such as hydrogen peroxide or dehydroascorbic acid, on the in vitro action of bleomycin were investigated. After the incubation of DNA with a low concentration of bleomycin and a reducing or oxidizing agent, single strand breaks were mainly caused in the DNA molecules. The degradation of DNA was largely prevented by the removal of oxygen, or by the addition of divalent cations or of S-(2-aminoethyl)isothiuronium bromide hydrobromide, a radical scavenger, to the incubation mixture. Preincubation of bleomycin with these reducing or oxidizing agents reduced the DNA-degrading activity of the antibiotic. However, this reduction in activity was observed even in the absence of oxygen, or in preincubation mixture supplemented with radical scavenger.
TL;DR: The spectral changes of cytochrome P-450 caused by the addition of small molecules to liver microsomes were investigated precisely and the following conclusions were reached.
Abstract: The spectral changes of cytochrome P-450 caused by the addition of small molecules to liver microsomes were investigated precisely and the following conclusions were reached. 1. The Type I spectral change was entirely due to the interaction of the cytochrome with a hydrocarbon residue in a ligand. To induce the modified Type II spectral change, the presence of a hydroxyl group in a ligand was required. Compounds which contain a basic amino group induced the Type II spectral change. 2. The Type I spectral change was caused by the interaction of a ligand with the 419-nm form of cytochrome P-450, with its concomitant conversion to the 394-nm form. Whereas, compounds inducing modified Type II spectral change interacted with the 394nm form of the cytochrome. In this case, however, the 394-nm form was not converted back to the 419-nm form but was converted to a new state showing an absorption peak at 416 nm. The Type II spectral change-inducing interaction of a ligand with the cytochrome could occur with all forms of the cytochrome. 3. Both Type II and modified Type II compounds bound to the cytochrome at heme iron, and converted the cytochrome into modified ferrihemochromes. On the other hand, the Type I interaction occurred ina protein moiety of the cytochrome, and probably caused a conformational change of the cytochrome accompanied either by weakening of the internal ligand interaction or by displacement of the ligand with another one having a weaker field at the heme iron. 4. Type I and each of other two types of binding of compounds with cytochrome P-450 could occur simultaneously.
TL;DR: It was conclusively shown that the active component stimulating serum protein biosynthesis is saponin and was directly proportional to the dose in the case of ginsenoside-Rd, which had the highest activity.
Abstract: Systematic isolation and purification of the biologically active component of ginseng extract were followed by observing the incorporation of labeled leucine into serum protein at 6 hr after a single intraperitoneal injection in a mouse. Ginseng saponin mixture (fraction 5) exhibited high activity for such incorporation. Seven saponins were isolated from fraction 5 by means of preparative TLC, and assayed. Administration of all these saponins (ginsenoside-Rb2, Rc, Rc2, Rd, Re, and Rg1)except for ginsenoside-Rb1, caused an increase of leucine incorporation over that in control animals. The incorporation rate was directly proportional to the dose in the case of ginsenoside-Rd, which had the highest activity. The increase specific radio-activity of serum protein was not due to a decrease in the pool size of free amino acids in the liver. It was conclusively shown that the active component stimulating serum protein biosynthesis is saponin.
TL;DR: The localization of specific antiboidies against troponin components was studied by the use of an electrom microscope and stained with anti-TN-I or anti- TN-C formed narrow striations.
Abstract: 1. The localization of specific antiboidies against troponin components, i.e., troponin T(TN-T), troponin I(TN-I, and troponin C(TN-C), was studied by the use of an electrom microscope. 2. Every antibody was distributed along the thin filament with a period of 38 nm. 3. Staining with anti-TN-I or anti-TN-C formed narrow striations. The location of the first striation was 26 nm from the free end of the thin filament. 4. The width of individual striations formed by anti-TN-T was 14--20nm The H-band-side end of each striation coincided with the location of anti-TN-I or anti-TN-C.
TL;DR: Urea synthesis was studied using the isolated liver perfusion with ammonium cholride and glutamine as nitrogen sources and a marked additive effect on the rate of urea formation was observed with the combined addition of ornithine and N-acetylglutamate.
Abstract: Urea synthesis was studied using the isolated liver perfusion with ammonium cholride and glutamine as nitrogen sources. The rate of urea formation increases with ammonium cholorde concentration up to 5mM, and the rate remained constant in the range between 5 and 20mM of ammonium chloride as the substrate. The concentration of ammonia in the medium to support the half-maximum velocity of urea formation was 0.7mM. The rate of urea formation was stimulated by the addition of 2.5mM ornithine, and the greater part of the ornithine which was taken up into the liver was accumulated as citrulline in the presence of ammonia. A considerable accelerating effect of N-acetylglutamate on the synthetic rate was observed, but a rather high concentration of N-acetylglutamate was required in order to obtain the maximum effect possibly, because its permeability into liver cells may be limited. A marked additive effect on the rate of urea formation was observed with the combined addition of ornithine and N-acetylglutamate. The metabolic conversion of glutamine nitrogen to urea in the perfused rat liver and the effect of several compounds which stimulated urea synthesis with ammonia were further examined. The process of conversion of glutamine nitrogen to urea might be composed of the following three steps. In the first lag phase, a small amount of glutamine was removed from the medium. In the second stage, the glutamine level decreased rapidly and ammonia was accumulated in the perfusate. The third stage was a period in which glutamine concentration remained at a constant low level, and the accumulated ammonia was rapidly conversed to urea. The rate of urea formation in this third stage was found to be much higher than that with ammonia as the substrate. The maximum rate of glutamine removal was obtained at pH 7.7 of the perfusate and at a concentration of 10mM glutamine. Urea formation with glutamine was also stimulated by the addition of ornithine, malate, or N-acetylglutamate, which had accelerating effects on the urea synthesis with ammonia. This stimulation was due to an effective conversion of ammonia to urea, but no change in the rate of removal glutamine was obtained.
TL;DR: The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.
Abstract: Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.
TL;DR: Physicochemical studies of this modified enzyme (OL-I) revealed that the ozonization of lysozyme in aqueous media resulted in little change of the gross molecular conformation, and it was deduced that the modified tryptophan residue in OL-I was possibly located in position 62 (or 63) of the protein.
Abstract: A tryptophan residue in hen's egg-white lysozyme [EC 3.2.1.17] was modified by ozone in an aqueous solution. One of the six tryptophan residues in the enzyme was oxidized to N'-formylkynurenine with concomitant loss of the enzymatic activity. Physicochemical studies of this modified enzyme (OL-I) revealed that the ozonization of lysozyme in aqueous media resulted in little change of the gross molecular conformation. It was deduced that the modified tryptophan residue in OL-I was possibly located in position 62 (or 63) of the protein.
TL;DR: In the jejunum, the activities of all the enzymes tested exhibited clear circadian variations with the highest activity at 0000-0400 h and the lowest at 1200 h when the rats were fed ad libitum, but the amplitude of the rhythm was smaller than that in the jejenum.
Abstract: The activities of the digestive enzymes, maltase [EC 3.2.1.20], sucrase [EC 3.2.1.26], trehalase [EC 3.2.1.28], Leucine aminopeptidase [EC 3.4.11.1], and alkaline phosphatase [EC 3.1.3.1] were measured in various regions of the small intestine of rats. The activities of all these enzymes were much higher in the jejunum than in the ileum, and in the distal regions of the ileum no sucrase, trehalase or alkaline phosphatase activity was detected. In the jejunum, the activities of all the enzymes tested exhibited clear circadian variations with the highest activity at 0000-0400 h and the lowest at 1200 h when the rats were fed ad libitum. In the ileum, maltase and sucrase also exhibited circadian variations, but the amplitude of the rhythm was smaller than that in the jejenum. Trehalase and alkaline phosphatase did not show any circadian variation in the ileum. Leucine aminopeptidase showed a circadian variation in the ileum with the same amplitude as in the jejunum. The phase of the circadian variations shifted about half a day when the rats were fed in the daytime, but the amplitude of the rhythm did not change.
TL;DR: A ganglioside containing fucose (fucogangliosides) was obtained from boar testis and purified by silicic acid and DEAE-cellulose column chromatographies and preparative thin layer chromatography.
Abstract: A ganglioside containing fucose (fucoganglioside) was obtained from boar testis and purified by silicic acid and DEAE-cellulose column chromatographies and preparative thin layer chromatography. The structure of this ganglioside, determined by chemical and enzymatic methods was: (see article). Its fatty acids were mainly long chain saturated ones (20 : 0, 22 : 0, 24 : 0). Its long chain bases consisted of 27% C(16:1) sphingosine and 68% C(18:1) sphingosine.
TL;DR: A rapid and sensitive assay for [3H]GTP binding activity of tubulin has been developed and it was found that bovine brain tubulin is markedly stablized by glycerol and GTP against denaturation.
Abstract: A rapid and sensitive assay for [3H]GTP binding activity of tubulin has been developed. This assay method is based on the quantitative retention of [3H]GTP. Tubulin complex on a nitrocellulose membrane filter. It was also found that bovine brain tubulin is markedly stablized by glycerol and GTP against denaturation. A large-scale purification of bovine brain tubulin was achieved using the new assay procedure and by the inclusion of glycerol and GTP in a buffer solution used for column chromatograph. The purified tubulin could be stored at -80degrees in the presence of glycerol and GTP for at least a year without any apprecialbe loss of [3H]GTP- and [3H]colchicine binding activities. The interaction of tubulin with guanine nucleotides was also studied using the nitorcellulose membrane filter procedure. It was found that the binding of [3H]GTP to tubulin with an empty exchangeable site proceeded promptly within k sec while the exchange of [3H]GTP- with a GTP-tubulin complex in which the exchangeable site had been occupied with unlabeled GTP occured more slowly. The dissociation constants for GTP and GDP at the exchangeable site of tubulin were determined as 0.5 times 10-6M and 1.9 times 10-6M, respectively. 5'-Guanylylimidodiphosphate could interact, although less strongly, with tubulin at this site, whereas the interaction of other nucleoside triphosphates includint ATP, CTP, UTP, and 5'-guanylyl methylenediphosphonate was very weak, if it occured at all. The presence of Mg2+ and a free sulfhydryl group was found to be essential for binding of [3H]GTP to tubulin. Ca2+ was found to replace Mg2+ in this binding reaction.
TL;DR: The subcellular distribution of phosphatidate phosphatase in rat liver was studied by assaying the activity of the enzyme in the presence of Mg2+ and phosphatidylcholine, and in contrast ot the results of previous studies, most of the enzymes activity was found in the soluble fraction.
Abstract: Phosphatidate phosphatase (EC 3.1.3.4Y was purified 15- to 20-fold from the soluble fraction of rat liver. The purification procedure involved calcium phosphate gel adsorption and elution, ammonium sulfact precipitation, and molecular-sieve chromatography. For the enzyme assay, and aqueous dispersion of phosphatidate, rather than "membrane-bound" phosphatidate, was used as substrate. The partially purified enzyme depends almost entirely on the presence of Mg2+ for its activity. Morover, the activity of the enzyme is stimulated by phosphatidylcholine. The enzyme exhibits a high substrate specificity for phosphatidate. The apparent Km for phosphatidate is approximately 0.05 mM. The optimum pH is between 7.4 and 7.6. The enzyme is inhibited by fluoride and by p-chloromercuribenzoate. The subcellular distribution of phosphatidate phosphatase in rat liver was studied by assaying the activity of the enzyme in the presence of Mg2+ and phosphatidylcholine. In contrast ot the results of previous studies, most of the enzyme activity was found in the soluble fraction.
TL;DR: It is suggested that the heme iron of the metmyoglobin peroxide compound is in an oxidation state other than the ferric state and that the porphyrin structure of met myoglobin may be modified by the reaction with hydrogen peroxide.
Abstract: The circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of horse heart metmyoglobin and the following derivatives were measured in the Soret and near ultraviolet regions: metmyoglobin and its peroxide compound, and hydroxide, cyanide, azide, and fluoride derivatives. The heme-related CD bands in the Soret and near ultraviolet wavelength regions were altered by ligand substitution, though their relationships to the magnetic moment were quite different. In the Soret region, the CD peak had no definite relation to the magnetic moment, while in the near ultraviolet region the magnitude of the CD peak decreased with the magnetic moment. The MCD peak in the Soret and near Ultraviolet regions also varied with ligand substitution. The magnetic ellipticity decreased with the magnetic moment in both wavelength regions. There was a more quantitative correlation between the magnetic ellipticity and the magnetic moment in the near ultraviolet region than in the Soret region. Metmyoglobin peroxide compound exhibited slightly different behavior in the MCD spectrum from other derivatives. It is suggested that the heme iron of the metmyoglobin peroxide compound is in an oxidation state other than the ferric state and that the porphyrin structure of metmyoglobin may be modified by the reaction with hydrogen peroxide.