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Volume 81 (1977)
- Issue 6 Pages 1583-
- Issue 5 Pages 1181-
- Issue 4 Pages 813-
- Issue 3 Pages 533-
- Issue 2 Pages 277-
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Volume 81, Issue 6
Displaying 1-40 of 40 articles from this issue
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Keitaro HIROMI, Takumi KAWAGISHI, Masatake OHNISHI1977 Volume 81 Issue 6 Pages 1583-1586
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chemical modification of tryptophan residues of hen egg-white lysozyme by N-bromosuccinimide (NBS) was studied kinetically by the stopped-flow method, monitoring changes in absorbance and fluorescence. One most rapidly reacting tryptophan residue, probably Trp 62, was clearly distinguished from four other residues in terms of rate of modification. This residue was protected by ethylene glycol chitin, N-acetyl glucosamine (NAG), and tri-NAG, but not by gluconolactone. The dissociation constant Kd of the enzyme-ligand complex was obtained from the protection effects. These results are in good agreement with results previously obtained.View full abstractDownload PDF (1030K) -
Existence of a Glucoproteic Intermediate in the Initiation ProcessHidenari TAKAHARA, Kazuo MATSUDA1977 Volume 81 Issue 6 Pages 1587-1594
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA soluble enzyme preparation (20, 000 × g supernatant fraction), prepared from the mycelia of wild-type Neurospora crassa, was capable of transferring [14C]glucose from UDP-[14C]glucose into both trichloroacetic acid (TCA)-soluble and TCA-insoluble macromolecule products in the absence of added primer. These reactions did not require either high concentrations of salts or any other chemical reagents. Two labeled products were formed; one was a glycogen-like polysaccharide and the other was a glycoprotein with glucosyl chains bound to protein through an acid-labile bond. After mild treatment of the glucoprotein with acid, the product liberated from the protein behaved as a mixture of malto-oligosaccharides and α-1, 4-glucan with branches. The carbohydrate moiety of the glucoprotein seemed to be released upon prolonged incubation with the enzyme preparation. The glucan thus liberated from the glucoprotein may serve as a primer for the glycogen synthase. The results obtained are therefore suggestive of the existence of a glucoproteic intermediate in the initiation of glycogen biosynthesis.View full abstractDownload PDF (1256K) -
Density-gradient Ultracentrifugal and Gel Chromatographic Behavior of the Valvular Matrix Macromolecules under Dissociative and Associative ConditionsAtsushi HONDA, Yusuke KANKE, Yo MORI1977 Volume 81 Issue 6 Pages 1595-1603
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe density-gradient centrifugal and gel chromatographic behavior of the components (proteoglycans, glycoprotein, and hyaluronic acid) of a proteoglycan fraction (U-IIIa) isolated by DEAE-cellulose chromatography of a urea-containing solution of collagenase-indigestible material of bovine heart valves was examined to investigate the nature of the interactions of the components in U-IIIa in more detail.
A glycoprotein (buoyant density, about 1.32 g/ml) in U-Illa was separated from proteoglycans and hyaluronic acid by CsCl density-gradient (initial density 1.35g/ml) centrifugation in the presence of 4M guanidinium chloride. Observations under various density gradient conditions suggest that this glycoprotein in U-IIIa dose not interact with proteoglycans under associative conditions.
A glycoprotein (U-II) which was free from proteoglycans and contained about 2% hy-droxyproline was isolated by DEAE-cellulose chromatography. U-Il had a buoyant density of about 1.32g/ml and its amino acid composition was very similar to that reported in the literature for “structural glycoprotein.”
Chromatography of U-IIIa on Sepharose 4B under associative solvent conditions, together with the electrophoretic patterns of the resulting fractions, indicated that almost all the proteochondroitin sulfate and proteodermatan sulfate, and a small amount of hyaluronic acid were retained within the column, while a glycoprotein was eluted near the end of the total volume.
The present experiments indicate that heart valve proteoglycans from the collagenaseindigestible materials of bovine heart valves are not in the form of cartilage-type proteoglycan super-aggregates.View full abstractDownload PDF (2508K) -
Kunihiko IZUMI1977 Volume 81 Issue 6 Pages 1605-1611
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProton NMR spectra of the peracetylated derivatives of D-galactopyranoses and some of their aryl glycosides and glycosyl bromides were measured at 100 MHz in the presence of a shift reagent, tris (2, 2-dimethyl-6, 6, 7, 7, 8, 8, 8-heptafluoro-3, 5-octanedionato)-europium. Bound chemical shifts to lower fields were determined mainly by first-order analysis for all the methine, methylene, and acetyl protons in these compounds. Comparison of the shift data for these sugars of the galactopyranose series with those for the corresponding sugars of the glucopyranose series showed that the signals of H-4 in the former and H-2 (α-anomers) and H-6 (H-6') in the latter were most markedly shifted by the addition of the reagent. The highest values of the bound chemical shifts of the acetyl protons and the differences in values between H-6 and H-6' were further observed to be lower in the former sugars than in the latter sugars. These shift patterns, characteristic of the sugars of the galactopyranose series, were found to be more marked in their β-anomers. Based on these results, the preferred coordination structures for the complexes between these sugars and the reagent were inferred, and their stereochemical significance is discussed.View full abstractDownload PDF (579K) -
IV. Isolation and Characterization of Glycopeptides from Pig Serum Low Density LipoproteinJun-ichi AZUMA, Naoki KASHIMURA, Tohru KOMANO1977 Volume 81 Issue 6 Pages 1613-1619
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree glycopeptides were isolated from the pronase digest of the protein moiety of pig serum low density lipoprotein. The isolation procedure consisted of pronase digestion, gel filtration on Sephadex G-25 and G-50 columns, paper chromatography and DEAE-Sephadex A-50 column chromatography. Based on the carbohydrate analysis, the isolated glycopeptides were classified into two types. One type (GDI) consisted of mannose and N-acetylglucosamine residues in the molar ratio of 6:2 and had a molecular weight of about 2, 300. The other type (GDII and GDIII) consisted of sialic acid, mannose, galactose, fucose, and N-acetylglucosamine residues in the molar ratio of 1:4:2:1:3 and 2:4:3:1:3, respectively. The molecular weights of GDII and GDIII were about 2, 100 and 3, 100, respectively. The results on the strong alkaline treatment of these glycopeptides suggested that all carbohydrate chains were linked to the peptide chains through N-acetylglucosaminyl-asparagine linkages. Of these glycopeptides and pig serum lipoproteins, only glycopeptide GDI and native LDL strongly interacted with concanavalin A.View full abstractDownload PDF (564K) -
Setsuko ITO, Katsuko YAMASHITA, Robert G. SPIRO, Akira KOBATA1977 Volume 81 Issue 6 Pages 1621-1631
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structure of the carbohydrate moiety of a unit A glycopeptide of calf thyroglobulin was studied by glycosidase digestion, acetolysis, and methylation analysis. Based on the results, the whole structure of a unit A glycopeptide with the largest carbohydrate moiety was deter-mined as Manα1→2Manα1→6 (Manα1→2Manα1→3) Manα1→6 (Manα1→2Manα1→2Manα1→3) Manβ1→4GlcNAcβ1→4GlcNAc→Asn-peptide.View full abstractDownload PDF (795K) -
Hiromichi OKUDA, Etsuko TAKEDA, Yasushi SAITO, Nobuo MATSUOKA1977 Volume 81 Issue 6 Pages 1633-1637
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLipid micelles were prepared by incubating a mixture of glycerides (triolein, diolein, and monoolein), and lecithin in Krebs-Ringer phosphate buffer at 37°C for 30 min.
It was found that adrenaline stimulated the release of free fatty acids in a lipolytic system consisting of the lipid micelles and adipose tissue lipase. Adrenaline did not increase the cyclic AMP content of the reaction mixture.
Dibutyryl cyclic AMP, theophylline, and phospholipase C increased the rate of lipolysis in the system but cyclic AMP and phospholipase D did not.View full abstractDownload PDF (323K) -
Yosifumi HORIUTI, Sigeyuki IMAMURA1977 Volume 81 Issue 6 Pages 1639-1649
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPalmitoyl cellulose was used to adsorb the extracellular lipase [triacylglycerol acyl-hydrolase, EC 3.1.1.3] of Chromobacterium viscosum from crude enzyme solution, and the adsorbed enzyme was eluted with a suitable detergent, such as Adekatol 45-S-8 or Triton X-100. The enzyme was then purified by chromatography on a palmitoylated gauze column with an overall recovery of 71% and an increase in the specific activity of 11-fold from the supernatant fluid of bacterial cultures. Further purification procedures included fractionation with acetone, and chromatography on Sephadex G-150 and G-75 columns. Two isoenzymes were obtained, each in a homogeneous state on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: one had a molecular weight of 120, 000 and pI of 3.7 and the other a molecular weight of 30, 000 with a pI of 7.3.View full abstractDownload PDF (1361K) -
Akiko OSANAI, Toshio SAKAGAMI1977 Volume 81 Issue 6 Pages 1651-1659
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing [32P]-orthophosphate, the biosynthesis of phospholipids was studied with rat liver microsomes and cell supernatant in the presence and absence of rat liver mitochondria. Addition of mitochondria significantly stimulated the incorporation of [32P]-orthophosphate into phospholipids, especially into phosphatidylethanolamine. The incorporation pattern in the presence of mitochondria was similar to that found in vivo, that is, there was more incorporation of the isotope into phosphatidylethanolamine than into phosphatidylcholine, while the major radioactive phospholipid was phosphatidylcholine in the absence of mitochondria.
The stimulatory effect of mitochondria on the incorporation of the isotope was inhibited by cyanide or ultrasonic treatment of mitochondria.
Results obtained using [γ-32P]-ATP indicated that ATP concentration was not responsible for the stimulation by mitochondria of the incorporation of [32P]-orthophosphate into phosphatidylethanolamine relative to that into phosphatidylcholine.
An addition of 1mM EGTA (ethyleneglycol-bis-β-aminoethyl-ether-N, N'-tetraacetate), a specific chelator of Ca2+, stimulated the incorporation of the isotope into the phospholipids in the absence of mitochondria. The extent of the stimulation and the incorporation pattern were comparable to those found in the presence of mitochondria. On the other hand, an addition of 1mM Ca2+ inhibited the incorporation of the isotope into the phospholipids in the pres-ence of mitochondria.
The addition of 2-5mM Mn2+, in the presence of 10mM Mg2+, stimulated incorporation of the isotope into phosphatidylethanolamine but lowered that into phosphatidylcholine.
These results suggest that the regulation by mitochondria of the incorporation of the isotope into phospholipids might be due to changes in the concentrations of some divalent cations, particularly Ca2+, during incubation.View full abstractDownload PDF (653K) -
Hitoshi ARITA, Jun'ichi KAWANAMI1977 Volume 81 Issue 6 Pages 1661-1664
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNormal rat spleen contained a large amount of cytolipin R, N-acetylgalactosaminyl(β1→3)-galactosyl(α1→3)galactosyl(β1→4)glucosyl ceramide, together with a lesser amount of cytolipin K, N-acetylgalactosaminyl(β1→3)galactosyl(α1→4)galactosyl(β1→4)glucosyl ceramide, as in the case of rat kidney. Cytolipin R was confirmed to be not a rat tumor-dependent, but a species-specific glycolipid.View full abstractDownload PDF (265K) -
Jun NAGAI, Satoru KAWAMURA, Hirohiko KATSUKI1977 Volume 81 Issue 6 Pages 1665-1673
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFree sterols and their fatty acid esters in Saccharomyces cerevisiae were separated by TLC, and the sterol contents and compositions of both fractions were analyzed by GLC. During respiratory adaptation of anaerobically grown yeast cells, larger amounts of esterified sterols were accumulated than of free sterols. The sterol compositions of both fractions were quite different. Ergosterol was the predominant component in the free sterol fraction, whereas the esterified sterol fraction contained significant amounts of sterol intermediates in ergosterol synthesis, such as zymosterol, lanosterol, and two unidentified sterols, tentatively named x and y.
During respiratory adaptation, the radioactivity from L-[methyl-14C]methionine was incorporated into both free and esterified sterols. In the early period of incubation, incorporation of the radioactivity into free sterol y was marked but subsequently the radioactive sterol y was rapidly converted into ergosterol. In contrast, conversion of radioactive sterol intermediates to ergosterol in the esterified sterol fraction was much slower.
With the cell-free extracts of yeast, radioactivities of [14C]mevalonate and S-adenosyl-[14C]-methionine were incorporated mainly into the free sterol fraction. Active 14C interconversion between free and esterified sterols was not found in the cell-free system. Sterol ester did not serve as a substrate for partially purified S-adenosylmethionine: Δ24-sterol methyltransferase from yeast, while the free sterols obtained from the sample of sterol ester proved to be good methyl acceptors.View full abstractDownload PDF (692K) -
Masako OHASHI, Tamio YAMAKAWA1977 Volume 81 Issue 6 Pages 1675-1690
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe glycolipids of pig adipose tissue were isolated and analyzed. A method for their extraction, purification, and subsequent characterization is described. The glycolipids were extracted with hot ethanol from the acetone-defatted tissue and separated into one neutral and five acidic glycolipid groups on a DEAE-Sephadex column. Each group was further fractionated by column chromatography using silicic acid and ‘Iatrobeads’ silicate. Eleven species of ganglio-sides in addition to one neutral glycolipid were purified and partially characterized by TLC and GLC. Exact structures were established by direct mass spectrometry after permethylation of intact glycolipids and by GC-MS of the partially methylated carbohydrates after hydrolysis of the permethylated glycolipids. The pig adipose tissue was found to contain tetraglycosyl-ceramide (globoside) and disialoganglioside (GDla) as the predominant glycolipids, and furthermore, small amounts of hematosides (N-acetyl and N-glycolyl), three species of fucosecontaining and two species of glucosamine-containing monosialogangliosides, GM1 ganglioside, another type of disialoganglioside (containing both N-acetyl- and N-glycolyl-neuraminic acid) and two species of trisialogangliosides were also found. One of the trisialogangliosides possessed a carbohydrate composition Glc/Gal/Ga1NAc/NeuNAc=1:1:1:3 and appears to be a novel trisialoganglioside, GT2. The glycolipid content was about 12μmol/kg of the original tissue and more than a third of it corresponded to disialoganglioside (GDla). The adipose glycolipids had similar fatty acid compositions, in which docosanoic (22:0) and tetracosanoic (24:0) acids were predominant.View full abstractDownload PDF (1440K) -
VI. A Modified Method of Purification and Identification of IsozymesKazuo KAKII, Hiroshi YOSHIDA1977 Volume 81 Issue 6 Pages 1691-1697
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFusarium phosphodiesterase-phosphomonoesterase was purified 1, 630-fold with 19% yield from dried powder of the culture medium by a modified method consisting of seven steps. The purified preparation was shown to be devoid of inactive protein by disc electrophoresis. The preparation was homogeneous with respect to size as demonstrated by gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weights determined by gel filtration on Sephadex G-200 and by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis were 106, 000 and 100, 000, respectively. The sedimentation coefficient at infinite dilution was 5.71 S. Isoelectric focusing of the purified preparation showed the presence of at least four isozymes with isoelectric points of 6.6, 6.3, 6.2, and 5.9.View full abstractDownload PDF (1533K) -
Kazuko KIMURA, Mamoru NAKANISHI, Takeshi YAMAMOTO, Masamichi TSUBOI1977 Volume 81 Issue 6 Pages 1699-1703
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rate of reaction of chloroacetaldehyde (0.039 M) with the “free” adenine residue in deoxyadenosine-5'-phosphate (dAMP) at pH 6.5 has been found to be nearly equal to that at pH 4.5. Practically 100% of the adenine is converted to a fluorescent product (ε-adenine residue) on incubation for 60h at 37°C and pH 6.5. Of the adenine residues in “single-stranded” DNA, however, only 14% react with chloroacetaldehyde (0.039M) under the same incubation conditions. The reaction rate of this 14% is nearly equal to that of dAMP, but the fluorescence of the product is appreciably quenched; the quantum yield is only 0.45 times that of the “free” adenine residue. In “double-helical” DNA, on the other hand, no adenine residue has been found to react with chloroacetaldehyde. Possible application of these findings to structural studies of DNA is suggested.View full abstractDownload PDF (364K) -
Burghardt WITTIG, Stephanie REUTER, Hubert GOTTSCHLING1977 Volume 81 Issue 6 Pages 1705-1713
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour purified tRNALys species from 13-day-old chick embryo muscle have been characterized with respect to the following properties: qualitative oligoribonucleotide composition (polyacrylamide gel electrophoresis after RNase T1 digestion), anticodon response towards AAG and AAA (equilibrium dialysis and polylysine synthesis), strength of the aminoacyl bond (de-esterification kinetics), sedimentation coefficient, and temperature-dependent double helix-to-coil transition. The results confirm the existence of four molecularly independent lysine-specific tRNA's in this eukaryotic system.View full abstractDownload PDF (719K) -
Atsuo MIYAGAWA, Tsuneo OKUYAMA1977 Volume 81 Issue 6 Pages 1715-1720
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA direct spectrophotometric suspension method for the estimation of immobilized ligands on a gel matrix was developed. The method uses 0.05% agarose solution to prepare a practically stable suspension of various agarose beads. The quantity of agarose beads in the suspension was estimated turbidometrically from the attenuance at 650nm. The determination of immobilized ligands on agarose beads was performed colorimetrically at the wavelength of the absorption maximum against a control suspension of a similar amount of underivatized agarose beads. This method is more sensitive and simpler than previously known methods. The method is applicable to the estimation of absorption spectra and the measurement of the time course of reactions on agarose beads.View full abstractDownload PDF (466K) -
Yasuyuki SUZUKI, Eiji ITAGAKI, Hiromu MORI, Toichiro HOSOYA1977 Volume 81 Issue 6 Pages 1721-1731
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe testosterone-binding globulin (TeBG) from bovine serum was purified by affinity chromatography and hydroxylapatite chromatography. The affinity column used was prepared by coupling 17α-carboxyethynyl-l7-hydroxy-4-androsten-3-one to aminoethyl-Sepharose. The compound was replaceable by 17α-carboxyethynyl-l7-hydroxy-5α-androstan-3-one, but not by testosterone 17-hemisuccinate, estradiol 17-hemisuccinate, or testosterone 3-(O-carboxy-methyl)oxime. The TeBG isolated was homogeneous on analytical polyacrylamide gel electrophoresis and equilibrium centrifugation. The protein was a glycoprotein having a molecular weight of 89, 500 and a carbohydrate content of 17%. The association constant (M-1) at 4°C was 1.1 × 108 and the number of binding sites per molecule was 0.8. Treatment with guanidine-HCI dissociated the protein into subunits having a molecular weight of 28, 400 (about one-third of that of the original molecule). SDS-gel electrophoresis showed that two of the three subunits were slightly larger than the other. The dissociation into subunits could also be accomplished by GEDTA treatment with concomitant loss of testosterone-binding activity. The activity and molecular size were reversibly restored by incubation with excess Ca2+.View full abstractDownload PDF (1343K) -
Eiji ICHISHIMA, Michio TAKEUCHI, Ryuji YASUDA, Yutaka SUZUKI, Setsuko ...1977 Volume 81 Issue 6 Pages 1733-1737
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAspergillus saitoi acid carboxypeptidase hydrolyzed C-terminal peptidyl-L-proline bonds and released the C-terminal proline from Z-Gly-Pro-Leu-Gly-Pro and Z-Gly-Pro at pH 3.3. Proline liberated by the enzymic reaction was measured by a sensitive colorimetric ninhydrin method in glacial acetic acid at 513nm. A Km value of 1.0mM and a kcat value of 0.09s-1 for Z-GIy-Pro-Leu-Gly-Pro hydrolysis, and a Km value of 5.0mM and a kcat value of 0.0045s-1 for Z-Gly-Pro hydrolysis were calculated from Lineweaver-Burk plots.View full abstractDownload PDF (371K) -
I. Inactivation of Cladosporium Acid Protease by Diazoacetyl-DL-norleucine Methyl Ester as an Active-Site -Directed Irreversible InhibitorHaruo KANAZAWA1977 Volume 81 Issue 6 Pages 1739-1744
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acid protease from Cladosporium sp. No. 45-2 was rapidly inactivated with 1:1 stoichiometry by reaction with diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. Cupric ions were essential for this inactivation. The rate of inactivation was found to obey apparent first-order kinetics. Maximum inactivation was observed at pH 5.4. The completely inactivated enzyme contained about one residue of norleucine.
Saturation kinetics were observed with respect to the concentration of DAN. The enzyme-DAN complex (enzyme-substrate-like complex) in the presence of a 20-fold molar excess of cupric ions (200μM) has a dissociation constant, K1, of 0.55mM and is converted to inactive enzyme irreversibly with a rate constant of inactivation, k2, of 0.50min-1 at pH 5.4 and 30°C. Changes in the concentration of cupric ions had no effect on k2, though K1 decreased as the concentration of cupric ions increased. Saturation kinetics were not observed with respect to the concentration of cupric ions. In the presence of glycyl-DL-norleucine methyl ester (GN), a DAN analog, the development of irreversible inactivation was inhibited competitively. The enzyme-GN complex in the presence of a 20-fold molar excess of cupric ions (200μM) has a dissociation constant of 1.94mM.
These results suggested that DAN, as a copper-complexed carbene, reacts with the enzyme to form an intermediate complex (such as an enzyme-substrate-like complex), whereupon the formation of inactive enzyme occurs irreversibly.View full abstractDownload PDF (444K) -
Toshiharu HASE, Hiroshi MATSUBARA, Michael C. W. EVANS1977 Volume 81 Issue 6 Pages 1745-1749
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSReexamination of the amino acid sequence of Chromatium vinosum ferredoxin revealed that the original sequence proposed in 1970 (l) should be revised. Two segments in the sequence, residues 50-52 and residues 53-57, should be mutually displaced and therefore, the correct sequence in this region is concluded to be -Val-Glu-Val-Cys-Pro-Val-Asp-Cys-(residues 50-57). Another correction was the addition of one isoleucine residue between residues 57 and 58 and therefore, the total number of residues should be 82 instead of 81 originally counted. Sequence studies of other portions confirmed the previous results.View full abstractDownload PDF (380K) -
Hisataka KASAI, Kenji TAKAHASHI, Toshio ANDO1977 Volume 81 Issue 6 Pages 1751-1758
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reactions of ribonuclease T1 [EC 3.1.4.8] with N-acetylimidazole and p-diazobenzene-sulfonic acid have been investigated to obtain some information on the states and roles of tyrosine residues in the enzyme.
Three to four tyrosine residues in the enzyme were acetylated with N-acetylimidazole fairly readily at pH 7.5 without extensive loss of activity toward RNA. Of these, two residues appeared to be acetylated most easily. In the presence of phosphate anion, however, the reactivity of N-acetylimidazole was significantly lowered. Under the reaction conditions used, no acetylation took place in 0.2M phosphate buffer. On the other hand, one to two tyrosine residues were modified with p-diazobenzenesulfonic acid in 0.2M phosphate buffer, pH 7.0, without much loss of activity. In 8M urea all the tyrosine residues were acetylated with nearly complete loss of activity. These results indicated that at least two tyrosine residues are fully exposed while most of the remaining seven tyrosine residues are buried inside the molecule. Judged from the change in CD spectrum, some conformational change appeared to have already occurred when two tyrosine residues were acetylated. Upon removal of all the acetyl groups introduced by incubation with hydroxylamine, the fully acetylated, inactive enzyme regained full activity, although the original native structure did not appear to be completely regenerated from CD spectrum measurements.
These results indicated that several of the tyrosine residues in ribonuclease T1 are important in the formation and maintenance of the enzymatically active conformation although they are presumably not directly involved in the active site.View full abstractDownload PDF (558K) -
Tsunehiro KITAGAWA, Takako MIURA, Hyozo TANIYAMA1977 Volume 81 Issue 6 Pages 1759-1767
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConformational analyses of viomycin and related compounds were performed by means of PMR and CD spectroscopic investigations in relation to their antimicrobial activities. PMR signals for some α-methines and methylenes in the sixteen-membered ring of viomycin or dihydroviomycin showed different chemical shifts and splitting patterns from those of broxoviomycin. The differences can be explained by the different shielding effects on these protons by their neighbouring amide carbonyls due to the rigid or non-rigid conformations of the sixteenmembered ring. Measurements of temperature dependency in PMR signals and model investigations supported this view. The CD spectra of viomycin and related compounds, which were deduced to possess rigid conformations by PMR studies, showed a peak at 215nm, whereas compounds which were assumed not to have a rigid conformation did not show this peak. Modifications of the double bond of the 3-ureidodehydroalanine residue resulted in a conformational change of the sixteen-membered ring together with inactivation or reduction in antimicrobial activity. Thus, the dehydro residue in viomycin may have an important role in maintaining the rigid conformation. It is suggested that the rigid conformation is an important factor in the potency of the antibiotic, and the cyclic structure in the antibiotic is assumed to assist in maintaining the conformation. A possible role of D-form amino acids in peptide antibiotics is also discussed.View full abstractDownload PDF (1316K) -
I. Studies by Circular Dichroism and Viscosity MeasurementsTaro IZUMI, Hideo INOUE1977 Volume 81 Issue 6 Pages 1769-1779
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have measured the circular dichroism (CD) spectrum of lysozyme [EC 3.2.1.17] at 25 and 30°C and the viscosity at 25°C in water-dioxane mixtures within the range of apparent pH 2.0-2.4. Above 20% dioxane, three positive bands at 294, 289, and 283nm and a negative band at 262nm in the near-ultraviolet region gradually decreased in ellipticity until they were replaced by an overall negative broad spectrum above 30% dioxane. On the other hand, negative CD bands in the far-ultraviolet region with a shoulder at 222 nm and an extremum at 208nm first decreased slightly in ellipticity at 20-30% dioxane, and then gradually increased up to 40% dioxane, above which the ellipticity of these bands was still further enhanced. Such profiles were common to the two temperatures studied, but at 30°C a higher resolution of the far-ultraviolet CD bands was observed for lysozyme in 60 and 72% dioxane, suggesting the partial disruption of β-structure. These results indicate that the tertiary structure reflected by the near-ultraviolet CD bands was initially disrupted at 20-30% dioxane with possible slight disorganization of the ordered secondary structure, and then a marked increase in helical structure was induced at high dioxane concentrations. The helical content at 72% dioxane calculated from the ellipticity values at 222nm was about twice that of lysozyme in the absence of dioxane at both temperatures. The gross structural changes above 20% dioxane were confirmed by the intrinsic viscosities observed at 0, 20, 40, and 60% dioxane.
The ordered structure enhanced by dioxane in the lysozyme molecule was found to be more discernible when the difference CD spectrum was constructed from the observed spectra, taking the CD spectrum of lysozyme in the absence of dioxane as a reference. This procedure was also applied to investigate the effect of pH on the conformation of lysozyme at 60% dioxane. The appearance of β-structure at apparent pH 5.6 and of aggregated helices at apparent pH 7.5 was apparently induced by 60% dioxane.View full abstractDownload PDF (889K) -
Shoji SHIMOMURA, Toshio FUKUI1977 Volume 81 Issue 6 Pages 1781-1790
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLarge bands absorbing at 251 and 335 nm were observed in the difference circular dichroism and difference absorption spectra of the holo- versus the apo-enzyme of rabbit muscle phos-phorylase b. In addition to the previously known band at 335 nm, the band at 251 nm is here assigned to the bound pyridoxal 5'-phosphate (PLP). These observed difference spectra are simulated by the optical properties of the enol-imine form of the Schiff'base rather than those of the substituted aldimine. Pyridoxal and salicylaldehyde also bound to apophosphorylase in the same manner as PLP. Since analogs having no hydroxyl group at the ortho position to the aldehyde group did not bind to the apoenzyme, the hydroxyl group is thought to be important in stabilizing the interaction between apophosphorylase and PLP. The PLP bound at Site I of bovine serum albumin, which is known to be one of the PLP-binding proteins absorbing at 330-340nm, showed two prominent bands at 253 and 335nm. The circular dichroism and absorption spectra induced by the binding of PLP fit those of the enol-imine of the Schiff base, as in the case of phosphorylase.View full abstractDownload PDF (737K) -
Yoshitake MANO, Kazutaka KANO1977 Volume 81 Issue 6 Pages 1791-1801
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe relation between the KCl-soluble protein (KSP) which stimulates amino acid incorporation and proteins from the ribosomal wash fraction was studied in sea urchin systems.
KSP was found to differ from the elongation factor, EF-1. A similar factor was extracted from ribosomes at high salt concentration. KSP and the ribosomal wash fraction stimulated the binding of both phenylalanyl (Phe-) and acetylphenylalanyl (AcPhe-)-tRNA to 40 S ribosomes in poly U systems. Both bindings were dependent on poly U and eukaryotic initiation factor, EIF-1, but not on GTP. The ribosomal wash fraction was fractionated on DEAE-Sephadex, DEAE-cellulose, and CM-cellulose. One of the resulting fractions was similar to KSP, in that it stimulated the binding of both the aminoacyl-tRNA's to 40 S ribosomes in the presence of another fraction, which interacted with aminoacyl-tRNA to form a complex. The mechanism of stimulation of these bindings is considered to be as follows; AcPhe-tRNA interacts first with the interacting factor and GTP to form a ternary complex and then the complex is transferred to 40 S ribosomes with the aid of the KSP-like factor. The KSP-like factor showed ribosome-dependent GTPase activity. The interacting factor was found to be similar to EIF-1 and the KSP-like factor resembled another eukaryotic initiation factor, EIF-2. These were replaceable by the respective corresponding factors in AcPhe-tRNA binding, Met-tRNAMetF binding and polyphenylalanine synthesis. Overall activity of amino acid activation was observed in a differenct fraction of the ribosomal wash from the above fractions, suggesting that the binding activity and the activation activity found in KSP are derived from different proteins. The fraction activated phenylalanine and tryptophan among various amino acids tested. KSP, as detected by stimulation of the binding of AcPhe-tRNA, was found in both the postmicrosomal supernatant and the ribosomes. When isolated from ribosomes, KSP showed decreased PP1-exchange activity but its other activities in amino acid activation remained unchanged.View full abstractDownload PDF (819K) -
II. Extracellular Ovalbumin as an Inhibitor of Ovalbumin Synthesis in the OviductsTatsuo NAKAYAMA, Kinichi SUGAE, Kozo NARITA1977 Volume 81 Issue 6 Pages 1803-1812
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWashed, preincubated minccd hen's oviducts, which contained low levels of extracellular and intracellular proteins, synthesized egg-white proteins actively. The addition of ovalbumin to the incubation medium resulted in inhibition of the synthesis of egg-white proteins by the washed, preincubated oviduct cells, while the addition of bovine serum albumin seemed to stimulate protein synthesis and hen's egg-white lysozyme had no effect. The inhibitory or stimulatory effect on protein synthesis was proportional to the amount of protein added to the medium. The inhibitory effect of added ovalbumin was shown not to be due to the incorporation of ovalbumin into the oviduct cells from the incubation medium. Egg-white proteins added to the medium also inhibited protein synthesis inside the cells and the extent of the inhibition appeared to correspond to the amount of ovalbumin present in egg-white.View full abstractDownload PDF (757K) -
C. S. RAMADOSS, R. SELVAM, K. Radha SHANMUGASUNDARAM, E. R. B. SHANMUG ...1977 Volume 81 Issue 6 Pages 1813-1823
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlkaline phosphatase [EC 3.1.3.1] was isolated from rabbit kidney. Three distinct activities designated as Enz Ia (19%), Enz Ib (13%), and Enz II (68%) were separated on a DEAE-cellu-lose column. Enz Ia and Enz II were partially purified and their catalytic properties were studied. Both enzymes had the same optimal pH of 9.5. Enz Ia and Enz II had different Km values for the substrates phenyl phosphate and pyridoxal phosphate, but the same Km for α- and β-glycerophosphates and β-naphthyl acid phosphate. Among various cations studied, Mg2+ stimulated both enzymes to the greatest extent while Zn2+ ion completely inactivated them. Both phenylalanine and phosphate inhibited Enz II to a greater degree; phenylalanine had no effect on Enz Ia. The temperature of maximal activity was found to be 42°C for Enz Ia and 47°C for Enz II. Thermal inactivation studies showed that Enz Ia was heat-labile and Enz II heat-stable. The energy of activation using phenylphosphate as a substrate was calculated to be 11.9 kcal for both. Treatment with neuraminidase affected the electrophoretic mobility of Enz Ia but not that of Enz II, indicating that Enz Ia was a sialoprotein. Kinetic data obtained from the neuraminidase-treated enzyme were similar to those of the native enzyme. Immunological studies suggested that Enz Ia and Enz II had the same antigenic character. The above results suggest that Enz Ia is distinct from Enz II.View full abstractDownload PDF (1166K) -
C. S. RAMADOSS, E. R. B. SHANMUGASUNDARAM1977 Volume 81 Issue 6 Pages 1825-1831
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of citrinin poisoning on rabbit kidney alkaline phosphatase was investigated. After seven days administration of citrinin (2mg/kg body weight daily) the animals were sacrificed and the level of enzymes estimated in serum and kidney. Serum enzymes showed no variation in activity in the citrinin-treated animals, but in kidney, alkaline phosphatase activity decreased significantly. The decreased activity was mainly associated with the cytoplasmic fraction and in fractions Ib and II. The enzyme II obtained from citrinin-treated animal showed no kinetic difference in substrate specificity, inhibition by phenylalanine, phosphate, sodium-EDTA and Zn2+ ions, activation by Mg2+ ions, thermal inactivation and electrophoretic mobility to that of control Enzyme II. Immunological studies showed that the decrease in enzyme activity was due to existence of inactive enzyme protein. Hormones like cyclic AMP, prostaglandin E1 and parathyroid hormone reversed the decreased enzyme activity due to citrinin poisoning in mouse and rabbit. This study favours the possible existence of active and inactive forms of alkaline phosphatase in the system.View full abstractDownload PDF (490K) -
Taiichi SHIOTANI, Yukihiro SANADA, Nobuhiko KATUNUMA1977 Volume 81 Issue 6 Pages 1833-1838
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo distinct types of apoenzyme from native ornithine aminotransferase [EC 2.6.1.13], named forms I and II, have been found to be composed of a single type of subunit by means of Sephadex G-100 column chromatography in the presence of sodium dodecylsulfate, DE 52 cellulose column chromatography in the presence of 8M urea and urea-polyacrylamide gel disc electrophoresis. The amino-terminal residues of both apoenzymes were blocked, but several amino acids in sequence from the carboxylterminals were identical and the following sequence was found in both: Phe-Ser-Leu-IIe-Ala-(Val)-. In the native enzyme, 4SH groups out of a total 20SH groups/ tetramer could be titrated without loss of enzyme activity, while in the apo-form 1, 4SH groups out of a total 20SH groups/tetramer could be titrated with 80% loss of activity. In the apo-form II, in contrast, 6SH groups out of a total 10SH groups/dimer could be titrated and titration of the first 2SH groups caused 100% loss of activity.View full abstractDownload PDF (1148K) -
Shuichi FURUTA, Yasuko SHINDO, Takashi HASHIMOTO1977 Volume 81 Issue 6 Pages 1839-1847
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe purification procedures for α-keto acid dehydrogenase complexes from pigeon breast muscle have been improved. The sedimentation coefficients, SO20, W, were 77S for pyruvate dehydrogenase complex and 43S for 2-oxoglutarate dehydrogenase complex. The results of SDS-polyacrylamide gel electrophoresis and the ratios between the activities of the overall and component enzyme reactions of the purified preparation of pyruvate dehydrogenase complex indicate that the subunit structure of the pigeon breast muscle enzyme complex is nearly the same as that of the pig heart enzyme complex, except for a lower content of lipoamide dehy-drogenase component. The data on 2-oxoglutarate dehydrogenase complex indicate that the pigeon breast muscle enzyme complex contained more 2-oxoglutarate dehydrogenase and less lipoate succinyltransferase than the pig heart complex.
Michaelis constants for the reactants of the pyruvate dehydrogenase complex reaction were: pyruvate, 17μM; CoA, 27μM; NAD+, 200μm. Those of the 2-oxoglutarate dehydro-genase complex reaction were: 2-oxoglutarate, 150μM; CoA, 14μm; NAD+, 120μM. Products, acetyl-CoA, succinyl-CoA and NADH, were competitive inhibitors with respect to the corresponding substrates. Kynurenate and xanthurenate inhibited the activities of the two enzyme complexes by acting on the lipoamide dehydrogenase component.View full abstractDownload PDF (1366K) -
Yoshikatsu MOCHIZUKI, Samuel H. HORI1977 Volume 81 Issue 6 Pages 1849-1855
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHexokinase from pyloric caeca of the starfish, Asterias annirensis, was purified to a specific activity of 148 units/mg protein. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The molecular weight determined by SDS polyacrylamide gel electrophoresis and Ultrogel AcA 34 gel filtration was about 50, 000. The enzyme showed a broad pH optimum ranging from 7.4 to 9.5. The Km values for D-glucose, D-fructose, 2-deoxy-D-glucose, D-mannose, D-glucosamine and ATP were 0.045, 4, 0.21, 0.05, 0.35 and 0.3mM, respectively. N-Acetyl-D-glucosamine, D-xylose and D-galactose were not phosphorylated. The enzyme was strongly inhibited by the reaction products, glucose 6-phosphate and ADP, but not by high levels of D-glucose. The starfish hexokinase thus resembled mammalian isozyme A with respect to kinetic properties.View full abstractDownload PDF (470K) -
I. Phosphorylation of Histone FractionsMasahiro YAMAMOTO, Yoshimi TAKAI, Eikichi HASHIMOTO, Yasutomi NISHIZUK ...1977 Volume 81 Issue 6 Pages 1857-1862
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGuanosine 3', 5'-monophosphate (cyclic GMP)-dependent protein kinase partially purified from silkworm pupae reacts preferentially with Hl, H2A, and H2B histones but not with H3 and H4 histones. However, the latter can serve as substrates in the presence of a stimulatory modulator as described by Kuo and Kuo (J. Biol. Chem. 251, 4283-4286 (1976)). With H2B histone as substrate high Mg2+ concentrations (50-100mM) are necessary for the maximum rate of reaction. Although effects of the modulator and Mg2+ vary significantly with the histone fractions employed, analysis on the phosphorylation of histone fractions provides evidence that cyclic GMP-dependent protein kinase possesses an intrinsic activity that is similar to that of adenosine 3', 5'-monophosphate-dependent protein kinase.View full abstractDownload PDF (1044K) -
II. Phosphorylation of Ribosomal ProteinsMikeo CHIHARA-NAKASHIMA, Eikichi HASHIMOTO, Yasutomi NISHIZUKA1977 Volume 81 Issue 6 Pages 1863-1867
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGuanosine 3', 5'-monophosphate (cyclic GMP)-dependent protein kinase purified from silk-worm pupae reacts with rat liver ribosomal proteins when a stimulatory modulator (Kuo, W. N. & Kuo, J. F. (1976) J. Biol. Chein. 251, 4283-4286) is added to the reaction mixture. Judging from autoradiogram of the radioactive proteins separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide slab gel, the protein kinase utilizes the same proteins as those phosphorylated by adenosine 3', 5'-monophosphate (cyclic AMP)-dependent protein kinase. Fingerprint maps of the tryptic phosphopeptides of radioactive ribosomal proteins, which are phosphorylated by these two classes of protein kinases, are very similar. These results suggest that cyclic GMP-dependent protein kinase possesses an intrinsic activity that is similar to that of cyclic AMP-dependent protein kinase.View full abstractDownload PDF (955K) -
Akio ISHIDA1977 Volume 81 Issue 6 Pages 1869-1878
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA CO-binding hemochrome was accumulated in Escherichia coli cells, when intracellular heme concentration was increased by aerobic incubation of resting cell suspensions with ALA. Reduced minus oxidized difference spectrum of the hemochrome showed peaks at 560, 530, and 430nm and a shoulder at 575nm. The peaks of CO reduced minus reduced difference spectrum were located at 572, 540, and 422nm. The CO spectrum was similar to but not identical with the spectrum of cytochrome o, a known terminal oxidase in E. coli.
SDS-polyacrylamide gel electrophoresis of the CO-binding hemochrome showed its molecular weight to be about 33, 000. The hemochrome in crude cell-free extracts was oxidized by aeration and reduced by the addition of succinate or NADH. The reduction by succinate was inhibited by inhibitors of succinate dehydrogenase [EC 1. 3. 99. 1], and the reduction by NADH was inhibited by 2-heptyl-4-hydroxy-quinolin-N-oxide, which is an inhibitor of electron transport in E. coli cells.View full abstractDownload PDF (642K) -
Takashi SHIMIZU, Issei MABUCHI1977 Volume 81 Issue 6 Pages 1879-1888
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin was purified from bovine platelets without using chaotropic agents such as KI. Upon gel filtration on a Sepharose 4B column, ATPase activity appeared as a single peak corresponding to intact myosin. No activity was detected in fractions where myosin head would be eluted. Purified myosin exhibited high Ca- and K+EDTA-ATPase activity and low Mg-ATPase activity. Ca-ATPase activity increased with increasing Ca concentration up to 30mM, whereas K+EDTA-ATPase activity showed a maximum at 0.3mM EDTA. The effect of KCl concentration on Ca-ATPase activity was unique; a minimum was observed at physiologi-cal KCl concentration. Ca-ATPase activity showed two pH optima; 5.6-6.0 and 8.5, while K+EDTA-ATPase activity showed an optimum at pH 8.5. N-ethylmaleimide did not induce activation of Ca- or K+EDTA-ATPase activity, although 0.5-1.0M urea activated the Ca-ATPase activity to some extent. K+EDTA-stimulated enzyme activity was specific for ATP while Castimulated enzyme activity showed low substrate specificity.
Bovine platelet myosin consisted of one class of heavy chain and two classes of light chains. Upon electrophoresis in 4% acrylamide gel in the presence of SDS, the heavy chain migrated more slowly than that of rabbit skeletal muscle myosin. The larger light chain comigrated with LC2 of rabbit skeletal muscle myosin. The smaller light chain migrated more slowly than LC3. When platelet myosin was dialyzed against 0.1M KCl-0.5mM EDTA (pH 7), numerous bipolar filamentous aggregates about 250nm long and about 5nm wide were formed. On decreasing the KCl concentration to 0.02M, these bipolar filaments further associated at their ends to form huge aggregates.View full abstractDownload PDF (3112K) -
Hisami YAMADA, Shoji MIZUSHIMA1977 Volume 81 Issue 6 Pages 1889-1899
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn vitro assembly of membrane on the entire surface of peptidoglycan sacculus from dissociated components of the outer membrane of Escherichia coli has been achieved.
When the outer membrane dissociated in sodium dodecyl sulfate solution was mixed with the lipoprotein-bearing peptidoglycan sacculi and dialyzed against magnesium chloride solution, only large membranous sacculi were formed. The size and shape of the membrane formed were similar to those of the peptidoglycan sacculi used. When the peptidoglycan sacculi were replaced by sonicated fragments of the peptidoglycan, the size and shape of the membranes formed were similar to those of the fragmented peptidoglycan. Membranous vesicles resembling the original outer membrane preparation were recovered when the assembled membranepeptidoglycan complex was fragmented by brief sonication and treated with lysozyme.
When lipoprotein-free peptidoglycan sacculi were used, only small membranous vesicles resembling the original outer membrane preparation were formed, leaving the peptidoglycan sacculus as it was.
Lipopolysaccharide, phospholipid or a mixture of them can be assembled into membranous structures preferentially on the peptidoglycan sacculi; lipoprotein covalently, bound to the peptidoglycan sacculi was again essential for the assembly. However, the assembled membranes were often multilaminar and were not as clear and complete as those formed with all membrane components, including proteins.View full abstractDownload PDF (4686K) -
Reiko KAMEJI, Masuo OBINATA, Yasuo NATORI, Yoji IKAWA1977 Volume 81 Issue 6 Pages 1901-1910
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSButyric acid induces erythroid differentiation of cultured Friend leukemia cells when added to the culture medium. A high level of globin messenger RNA (mRNA) was detected in Friend leukemia cells treated with butyric acid by a liquid hybridization method using radioactive DNA complementary to reticulocyte globin mRNA.
The content of globin mRNA molecules induced in the cytoplasm of the butyric acidtreated Friend leukemia cells paralleled the hemoglobin content determined by the benzidine staining method. Therefore, this hemoglobin synthesis may be most reasonably explained in terms of transcriptional activation of globin genes, as previously proposed in the case of dimethylsulfoxide, another inducer of erythroid differentiation. Induced accumulation of globin mRNA in the nuclei also supports this interpretation.View full abstractDownload PDF (718K) -
Hiroyuki KONDO, Hisashi NIKI, Setsuro TAKAHASHI, Makoto ISHIMOTO1977 Volume 81 Issue 6 Pages 1911-1916
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIsethionate degradation in a bacterial extract was shown by the isolation of enzymes and by identification of an intermediate to take place in two steps; dehydrogenation to sulfoacetaldehyde and desulfonation leading to the formation of sulfite and acetate. The enzyme responsible for isethionate oxidation in the presence of FAD was particulate in nature and a solubilized preparation obtained by extraction with buffer of low ionic strength had oxidizing activities against only isethionate and n-butanol among compounds tested. The enzyme was inhibited by thiol and carbonyl reagents.View full abstractDownload PDF (436K) -
Shintaro SUZUKI, Takashi OSUMI, Hirohiko KATSUKI1977 Volume 81 Issue 6 Pages 1917-1925
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties and metabolic role of mesaconate hydratase [EC 4. 2. 1. 34] of an aerobic bacterium were studied. The enzyme was purified about 8-fold over the crude extract by (NH4)2SO4 fractionation, DEAE-Sephadex column chromatography, and Sepharose 6B gel filtration. The molecular weight of the enzyme was determined to be 160, 000 by Sephadex G-200 gel filtration, and the sedimentation coefficient was determined to be 8.0 S by sucrose density gradient ultracentrifugation. The enzyme was very unstable, but the inactivated enzyme could be reactivated by incubation with Fe2+ and 2-mercaptoethanol. The enzyme activity behaved similarly to S-malate-dehydrating activity on Sepharose 6B gel filtration, heat treatment, and other procedures, indicating that a single enzyme had S-malate-dehydrating activity in addition to S-citramalate-dehydrating activity. The pH optima for the two activities were about 9.0. Km values for S-citramalate, S-malate, mesaconate, and fumarate were 0.45, 0.55, 0.07, and 0.11mM, respectively, and the corresponding relative Vmax values were 100, 140, 185, and 300, respectively. The presence of phosphate increased these values. The enzyme was found to be a constitutive one, and no S-malate-dehydrating activity other than this enzyme could be detected in the crude extract. When Pseudornonas arvilla (ATCC 23974) or Alcaligenes faecalis (IRC 0800) was used in the above experiments instead of the test bacterium, similar results were obtained. It was concluded that the enzyme played a role in the interconversion between S-malate and fumarate in addition to that between S-citramalate and mesaconate in vivo.View full abstractDownload PDF (692K) -
Hitoshi UENO, Tatsuo OOI1977 Volume 81 Issue 6 Pages 1927-1929
Published: June 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour fragments of α-tropomyosin were prepared by specific cleavage at the Cys 190 by 2-nitro-5-thiocyanobenzoic acid and by tryptic digestion. These fragments were called the N-chain corresponding to residue 1 to 189 of the original chain, the C-chain from 190 to 284, the s-fragment from 13 to 149 and/or 128, and the p-fragment from 183 to 284, respectively. Fragments individually have little binding capacity to troponin as shown by gel electrophoresis. But a new band of the complex with troponin was detected using mixtures of the fragments, one from the N-terminal side and the other from the C-terminal side, i. e., the N- and C-chains, the s- and p-fragments, the N-chain and the p-fragment, and the s-fragment and the C-chain. Therefore, the troponin binding region of tropomyosin is thought to be located between residues 150 and 190.View full abstractDownload PDF (913K)
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