The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 79 , Issue 1
Showing 1-30 articles out of 30 articles from the selected issue
  • Masahiko INDO, Zensaku YOSIZAWA
    1976 Volume 79 Issue 1 Pages 1-4
    Published: January 25, 1976
    Released: November 18, 2008
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    Uterine slices obtained from estrogen-treated rabbits were incubated in vitro with N-acetyl-D-[l-3H] glucosamine together with D-[U-14C] glucose. The isotope-labelled acidic complex saccharides were then isolated by pronase digestion, Dowex 1 column chromatography and preparative electrophoresis on cellulose acetate membrane, in succession. In this way, individual acidic complex saccharides (hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, sulfated glycopeptide, and sialoglycopeptide) were separated into 2-5 subfractions.
    The specific radioactivity of hexosamine in the subfractions indicated that the metabolic rate of the uterine complex saccharides was as follows: hyaluronic acids sulfated glycopeptide>heparan sulfate>chondroitin sulfate A>chondroitin sulfate C> dermatan sulfate. In addition, metabolic heterogeneity of heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, and dermatan sulfate was suggested.
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  • Masahiko ENDO, Osamu NAMIKI, Zensaku YOSIZAWA
    1976 Volume 79 Issue 1 Pages 5-10
    Published: January 25, 1976
    Released: November 18, 2008
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    Euglobulin fraction of human plasma was extracted with 0.01M phosphate buffer (pH 7.3) (PB) containing 1M NaCl. The substances in the extract were separated by DEAE-cellulose column chromatography into two uronic acid-containing fractions (0.5 Fr and 0.75 Fr). The major fraction (0.75 Fr) was eluted at the void volume in gel filtration on Sephadex G-200, yielding 0.75-G Fr. 0.5 Fr and 0.75 Fr and their pronase digestion products (0.5-P Fr and 0.75-P Fr) together with 0.75-G Fr were examined by electrophoresis on cellulose acetate membrane. The results of electro-phoresis and the digestibility with mucopolysaccharidases of these fractions indicated that 0.5 Fr and 0.75 Fr contained proteochondroitin sulfate A. It is suggested that chondroitin sulfate A in 0.5 Fr might be less sulfated than that in 0.75 Fr. Further-more, analytical data and the infrared spectrum of 0.75-G Fr supported the above view.
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  • Masatake OHNISHI, Keitaro HIROMI
    1976 Volume 79 Issue 1 Pages 11-16
    Published: January 25, 1976
    Released: November 18, 2008
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    Chemical modification of glucoamylase [EC 3.2.1.3] from Rhizopus niveus by N-bromosuccinimide was carried out to investigate the role of tryptophan residues in the enzyme-catalyzed reaction and their location in the enzyme subsites. Of the ten tryptophan residues of the enzyme four could be modified. The two more reactive residues were confirmed not to be essential for the catalytic activity for the hydrolysis of maltodextrin and phenyl α-maltoside,
    Complete loss of the catalytic activity, however, was brought about by modifying the two less reactive residues, and the modification of these residues was prevented by the substrates. The characteristic difference spectrum produced by maltose (7) disappeared in parallel with the loss of the catalytic activity.
    These results suggest that the tryptophan residue(s) responsible for the maltose-induced difference spectrum may be located at one of the subsite near the catalytic site and plays an important role in the catalytic activity of the enzyme.
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  • Atsushi HONDA, Yusuke KANKE, Yo MORI
    1976 Volume 79 Issue 1 Pages 17-25
    Published: January 25, 1976
    Released: November 18, 2008
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    Proteoglycan complexes from collagenase [EC 3.4.24.3]-indigestible materials of bovine heart valves were extracted with 4M guanidinium chloride, purified by ion-exchange column chromatography in a urea-containing solution, then fractionated by density-gradient centrifugation under dissociative conditions.
    Electrophoretic characteristics and enzymic susceptibility of the density-gradient fractions revealed that the glycosaminoglycans constituting the proteoglycan complexes in this indigestible materials were mainly dermatan sulfate in the top three fractions, and dermatan sulfate and chondroitin sulfates in the bottom fraction; a minor con-stituent which was common to all the fractions was hyaluronic acid.
    A gel-like substance (Fr. Ig) at the top of the gradient, amounting to about 25% of the loaded dry sample, contained only a trace of hydroxyproline (<1%) and was composed of proteodermatan sulfate, glycoprotein, and a small amount of hyaluronic acid.
    Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of Fr. Ig with 2-mercaptoethanol showed that the major part of the proteins in this gel-like sub-stance was cross-linked by disulfide bridges.
    Chromatography of Fr. Ig on Sepharose 4B in buffered 4M guanidinium chloride containing 2-mercaptoethanol, together with the electrophoretic patterns of the resulting fractions, suggested that proteodermatan sulfate was not associated with hyaluronic acid through covalent bonds.
    The amino acid composition of Fr. Ig was very similar to that reported in the literature for “dermatan sulfate-protein complex, ” and “structural glycoprotein” or “acidic structural protein.”
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  • Hiroshi HATANAKA, Yoko OGAWA, Fujio EGAMI
    1976 Volume 79 Issue 1 Pages 27-34
    Published: January 25, 1976
    Released: November 18, 2008
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    Two glycosulfatases [EC 3.1.6.3], I and II, were purified 31.3- and 33.9-fold respectively, from a crude extract of the liver of Charonia lampas. The purification was carried out by the following chromatographic procedures; phosphocellulose, Sephadex G-150, Concanavalin A-Sepharose and isoelectric focussing. The enzyme preparations obtained were practically free from arylsulfatase [EC 3.1.6.1] contamination. Both glycosulfatases are probably glycoproteins differing in their carbohydrate moieties. The molecular weights of glycosulfatase I and II were estimated to be about 112, 000 and 79, 000, respectively. They had the same optimum pH of 5.5, and the same Km value of 25.0mM for glucose 6-sulfate.
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  • Akio HASEGAWA, Naoto MIWA, Tairo OSHIMA, Kazutomo IMAHORI
    1976 Volume 79 Issue 1 Pages 35-42
    Published: January 25, 1976
    Released: November 18, 2008
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    An amylase-producing thermophilic bacterium was isolated from a Japanese hot spring. The thermophilic cells were gram-negative, nonsporulating, nonpigmented rods and were motile with flagella. α-Amylase [EC 3. 2. 1. 1] purified from the thermophile was studied. The enzyme was more heat-stable than the corresponding enzymes from mesophilic sources, and 50% loss of activity was observed after incubation for 1 hr at 90°. The thermophilic enzyme resembled the corresponding mesophilic enzymes in many respects, such as kinetic parameters, physicochemical properties, and amino acid composition. The molecular weight of the enzyme was 50, 000, and the enzyme contained 2 moles of half-cystine residues per mole of protein, but there were no disulfide crosslinkages. The α-helix content of the enzyme was estimated to be about 20% from CD spectra, a value similar to those of other α-amylases. The isoelectric point of the enzyme was at pH 9.2.
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  • Yoshinori KIDANI, Junzo HIROSE, Hisashi KOIKE
    1976 Volume 79 Issue 1 Pages 43-51
    Published: January 25, 1976
    Released: November 18, 2008
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    The mechanism of removal of the zinc ion from bovine carbonic anhydrase [EC 4.2.1.1] (BCA) by a chelating agent was studied. It was shown that the removal of the zinc ion from BCA took place through the formation of a ternary complex involving the enzyme, chelating agent, and metal ions. The formation constant of the ternary complex (KEML) was 102 M-1. This value was lower than the formation constant assumed by Wilkins. The reaction of zinc-2, 6-pyridinedicarboxylate complex with the apoenzyme also took place through the formation of the ternary complex and the species which reacted with apo-BCA was a 1:1 complex of zinc and 2, 6-pyridine-dicarboxylate. The theoretical equilibrium equation derived from the reaction mechanism showed a good fit with observed equilibrium dialysis data.
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  • Tsuguo MIZUOCHI, Hirosuke FUKASAWA
    1976 Volume 79 Issue 1 Pages 53-60
    Published: January 25, 1976
    Released: November 18, 2008
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    The properties of poly(G) polymerase and poly(A) polymerase activities in the DNA-dependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase EC 2.7.7.6] I fraction from cauliflower (Brassica oleracea var. botrytis) were comparatively investigated. The pH optimum, the effect of ionic strength, the effect of substrate concentration on the rate of synthesis, the effect of divalent metal ion concentration, and the time course of synthesis at different temperatures were all different for the three polymerase activities. The enzyme fraction preferentially utilized denatured DNA. Synthetic poly(C) and poly(U) were more effectively utillized for the synthesis of polyguanylate and polyadenylate, respectively. Further, it was found that poly(G) and poly(A) formed in vitro by the enzyme fraction had chain length of 25-28 and 84-89 nucleotides, respectively, and that poly (adenylate-gluanylate) chain was hardly formed when ATP and GTP were added together as substrates in the same reaction medium.
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  • Yoichiro IWAKURA
    1976 Volume 79 Issue 1 Pages 61-68
    Published: January 25, 1976
    Released: November 18, 2008
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    Poly(A)•poly(U) synthesis in the absence of template DNA is a unique reaction catalyzed by the RNA polymerase [EC 2.7.7.6] holoenzyme II of Escherichia coli. As one approach to investigating the physiological role of the enzyme, the molecular mechanism of poly(A)•poly(U) synthesis was studied.
    Streptolydigin, an inhibitor of the elongation of RNA chains, was shown to inhibit poly(A)•poly(U) synthesis, and the inhibition was released by a streptolydigin-resistant mutation on the β subunit. These observations indicate that the active site for the reaction might be located on the β subunit. Another antibiotic, rifampicin, which is known to be a specific inhibitor of the initiation of DNA-dependent RNA synthesis, effectively inhibited both initiation and elongation steps in poly(A)•poly(U) synthesis. This suggests that the enzyme conformation during the chain elongation reaction might be different in this case from that in DNA-dependent RNA synthesis. Analysis of the products formed during the initiation reaction indicated that the rate-determining reaction in poly(A)•poly(U) synthesis was the formation of primers of short chain length, and that holoenzyme I was unable to form the first phosphodiester bond in this reaction. Functional properties of holoenzyme II are discussed in connection with these observations.
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  • Ken-ichi ARAI, Shun NAKAMURA, Takao ARAI, Masao KAWAKITA, Yoshito KAZI ...
    1976 Volume 79 Issue 1 Pages 69-83
    Published: January 25, 1976
    Released: November 18, 2008
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    The digestion of EF-Tu•DP (or EF-Tu•TP) by trypsin [EC 3.4.21.4] under native conditions has been shown to proceed through two different and characteristic stages.
    1. In the first phase, the protein is transformed into a fragment (Fragment A) with a molecular weight of 39, 000 by exposure to trypsin for a relatively short period of time. Fragment A is unable to catalyze the binding of aminoacyl-tRNA to ribosomes. The ability to promote two partial steps of the binding reaction, i.e., formation of the aminoacyl-tRNA•F-Tu•TP ternary complex as well as the methanol-stimulated, ribosome-dependent GTPase reaction, was rapidly destroyed. On the other hand, the ability to interact with guanine nucleotides as well as EF-Ts survived well during prolonged digestion.
    2. In the second phase of digestion, a nick is introduced in Fragment A to yield two subfragments (Fragments B and C). These two fragments exist as a hybrid molecule which migrates as a single peak on a Sephadex G-75 column, and which dissociates into Fragments B and C only in the presence of 6 M guanidine hydro-chloride or 5% sodium dodecyl sulfate. The molecular weights of Fragments B and C, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, were 22, 000 and 12, 000 respectively. The hybrid molecule still retained one mole of bound guanine nucleotide and was resistant to further tryptic digestion.
    3. Three sulfhydryl groups of EF-Tu were found to be present in Fragment B, both by amino acid analysis of the purified fragments and also by electrophoresis of Cryptic digests labeled with N-ethyl[14C]maleimide.
    4. The tryptic digestion of EF-Tu•DP (or EF-Tu•TP) labeled with N-(1-anilino-naphthyl-4)maleimide (ANM) at SH2 (the second SH), caused a 30% decrease in the fluorescence emission during the first rapid phase of digestion. This indicates that destruction of the hydrophobic environment near SH2 of EF-Tu occurred in the early phase of tryptic digestion.
    5. The kinetic studies on the reaction of ANM with EF-Tu before and after tryptic digestion indicated that both Fragment A and the hybrid molecule reacted with ANM in the presence of GTP three to four times more rapidly than in the presence of GDP. Thus, it appears that the ability to induce conformational transition near SH2 by a change of the nucleotide ligands is still retained in the hybrid molecule con-sisting of Fragments B and C.
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  • Kazushi TANABE, Taijo TAKAHASHI
    1976 Volume 79 Issue 1 Pages 85-90
    Published: January 25, 1976
    Released: November 18, 2008
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    DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment.
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  • Fumio KISHIDA, Takachika AZUMA, Kozo HAMAGUCHI
    1976 Volume 79 Issue 1 Pages 91-105
    Published: January 25, 1976
    Released: November 18, 2008
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  • Kazumitsu HANAI
    1976 Volume 79 Issue 1 Pages 107-116
    Published: January 25, 1976
    Released: November 18, 2008
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    When the competitive inhibitor benzeneboronic acid (BBA) forms a complex with α-chymotrypsin [EC 3.4.21.1] protons are released in the acidic pH region. The proton release can be measured by a difference potentiometric technique. The proton release is also observed in chymotrypsinogen A but not in TRCK-, DIP-, and anhydro-chymotrypsins. Based on these observations, a simple procedure to estimate the equilibrium constants of the trigonal-tetrahedral interconversion of BBA is proposed. Thermodynamic parameters of the ionization of His 57 and of each step involved in BBA binding can be estimated from the temperature dependence of the proton release. Those of His 57 are essentially the same as those of imidazole in water. Regarding the interconversion of BBA on the enzyme, the value of ΔS is similar to ΔS_??_of the deacylation step of nonspecific substrates, and ΔH is remarkably reduced from that for the ionization of BBA in water. The enthalpic gain of the enzymic process is suggested to be due to the change of the proton acceptor, which is water in the case of the ionization of BBA in water, to imidazole on the enzyme.
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  • Susumu KURIOKA
    1976 Volume 79 Issue 1 Pages 117-123
    Published: January 25, 1976
    Released: November 18, 2008
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    By treating fibrin with cationic detergent in a medium containing 5 M urea, a fibrin-cationic detergent complex was formed. The complex was soluble in distilled water, and was observed to be in a dispersed state by electron microscopy. The complex was precipitated and resolubilized in the presence of salt. This behavior of the complex was due to adsorption of the salt anions onto the complex. When an aqueous solution of the complex was incubated with fresh serum, the complex soon aggregated and then was converted into a firm clot which was not soluble in distilled water.
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  • Koei HAMANA, Koichi IWAI
    1976 Volume 79 Issue 1 Pages 125-129
    Published: January 25, 1976
    Released: November 18, 2008
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    Polyacrylamide gel electrophoresis of whole histones of calf thymus, chicken erythro-cytes, and Tetrahymena was carried out in the absence or presence of a nonionic surfactant, Triton X-100 (up to 6mM), in 0.9M acetic acid and 6.25M urea-0.9M acetic acid. Calf thymus whole histone was also chromatographed on Bio-Gel P-200, in the absence or presence of 6mM Triton in 0.01M HCl and 8M urea-0.9M acetic acid. Triton reduced the electrophoretic mobility and distribution coefficient of various histone species in the following order of decreasing effect; H2A>H3, H4>H2B>Hl in the absence of urea. Hl and specific histones for chicken erythrocytes and Tetra-hymena were almost unaffected. Urea antagonized the surfactant effect more for H4 and H2B, and less for H2A and H3. Such surfactant effects can be correlated with the helical contents of histone species under the experimental conditions used, rather than their total hydrophobicites, suggesting that Triton binds to helical regions.
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  • Hiroaki YAMANISHI, Yasushi SATO
    1976 Volume 79 Issue 1 Pages 131-144
    Published: January 25, 1976
    Released: November 18, 2008
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    1. Non-collagenous substances in newborn calf dermis were extracted with solutions of various concentrations of MgCl2. The total protein and hydroxyproline contents in MgCl2 extracts increased with increase in the concentration of MgCl2 in the solutions. In particular, steep increases of their contents were observed at concen-trations of MgCl2 from 0.5 to 1.0M. Total amounts of hydroxyproline in 1.0, 2.0, and 3.0M MgCl2 extracts were equivalent to 40-50% of the hydroxyproline content in the whole connective tissue. Hexose and hexosamine contents of MgCl2 extracts increased with increase of the MgCl2 concentration. Hexuronic acid was hardly present in the residues after extractions with 0.5, 1.0, 2.0, and 3.0 M MgCl2.
    2. Plasma proteins, hyaluronic acid, and dermatan sulfate were extracted at low concentrations of MgCl2. A non-collagenous protein and MgCl2-soluble collagen were extracted with 1.0, 2.0, and 3.0M MgCl2 solutions. The disperson of collagen fibrils was observed in the residue extracted with 1.0M MgCl2 solution by electron micro-scopy; the fibril structure of collagen was disordered by extraction with 2.0 and 3.0M MgCl2. These results suggest that the dispersion and disorder of collagen fibrils lead to the release of a non-collagenous protein. Furthermore, it is suggested that the removal of hyaluronic acid and dermatan sulfate was not very effective for the solubilization of a large amount of collagen, but was suitable as a pretreatment to the extraction of a non-collagenous protein accompanied by the solubilization of a large amount of collagen.
    3. The non-collagenous protein was purified by DEAE-cellulose column chromatog-raphy. Polyacrylamide gel electrophoresis of this protein at pH 8.5 showed a single band moving to the cathode. The non-collagenous protein contained 3.7% hexose, 1.8% hexosamine, and no hexuronic acid. This protein is rich in glycine, glutamic acid, and alanine, and contains neither hydroxyproline nor hydroxylysine. Sedimen-tation analysis showed a single peak with 1.8S and the molecular weight was approx. 43, 000 as determined by SDS polyacrylamide gel electrophoresis.
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  • Kiyoshi KAWAI, Takeshi AKITA, Sansei NISHIBE, Yoshinori NOZAWA, Yukio ...
    1976 Volume 79 Issue 1 Pages 145-152
    Published: January 25, 1976
    Released: November 18, 2008
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    The effects of xanthomegnin and O-methylxanthomegnin on the oxidative phospho-rylation of rat liver mitochondria were compared. The n-octanol/water partition coefficient of xanthomegnin was markedly enhanced by O-methylation, but O-methyl-ation of xanthomegnin reduced the uncoupling effect on the respiratory system of mitochondria. Analogous results were obtained in the uncoupling action of 5-hydroxy-1, 4-naphthoquinone (juglone) and 5-methoxy-1, 4-napthoquinone (O-methyljuglone) on the oxidative phosphorylation of rat liver mitochondria. These data indicate that the phenolic hydroxyl groups of xanthomegnin might contribute to its uncoupling action on the oxidative phosphorylation of mitochondria.
    Bovine serum albumin (BSA) improved the efficiency of oxidative phosphorylation of mitochondria which were uncoupled by xanthomegnin. Spectroscopic observations revealed that xanthomegnin interacted with BSA by means of hydrophobic and ionic forces but O-methylxanthomegnin showed only hydrophobic interaction. Analogous interactions between mitochondria and xanthomegnin or O-methylxanthomegnin were observed. These results indicate that the uncoupling action of xanthomegnin on the respiratory system in mitochondria might involve ionic interaction of xanthomegnin with cationic residues in the hydrophobic region of mitochondrial membrane proteins.
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  • Tohru OHYAMA
    1976 Volume 79 Issue 1 Pages 153-158
    Published: January 25, 1976
    Released: November 18, 2008
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    The effects of phthalate esters on the oxidation of succinate, glutamate, β-hydroxy-butyrate and NADH by rat liver mitochondria were examined and it was found that di-n-butyl phthalate (DBP) strongly inhibited the succinate oxidation by intact and sonicated rat mitochondria, but did not inhibit the State 4 respiration with NAD-linked substrates such as glutamate and β-hydroxybutyrate. However oxygen uptake accelerated by the presence of ADP and substrate (State 3) was inhibited and the rate of oxygen uptake decreased to that without ADP (State 4). It was concluded that phthalate esters were electron and energy transport inhibitors but not uncouplers. Phthalate esters also inhibited NADH oxidation by sonicated mitochondria.
    The degree of inhibition depended on the carbon number of the alkyl groups of phthalate esters, and DBP was the most potent inhibitor of respiration. The activity of purified beef liver glutamate dehydrogenase [EC 1.4.1.3] was slightly inhibited by phthalate esters.
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  • Hiroshi KONDO, Shin'ichi ISHIWATA
    1976 Volume 79 Issue 1 Pages 159-171
    Published: January 25, 1976
    Released: November 18, 2008
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    It was shown that the growth of F-actin occurs uni-directionally in vitro; that is, actin monomers (G•ADP-actin) polymerize onto only one of the two ends of a pre-existing seed in the absence of ATP. Fragments of the F-actin-heavy meromyosin complex prepared by sonication were used as seeds. By studying a large number of electron micrographs, we concluded that growth occurs on the tail end of the arrow-head structure of the seed. This result suggests that the thin filament grows in vivo from the center of the sarcomere towards the Z-line. It was further shown that the fragments of F-actin combine with both ends of the seed with nearly equal probability. These results are well interpreted according to the theory deveoloped by Oosawa (1, 2).
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  • Isamu SHIIO, Shin-ichi SUGIMOTO
    1976 Volume 79 Issue 1 Pages 173-183
    Published: January 25, 1976
    Released: November 18, 2008
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    The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetasel) [EC 4.1. 2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [pre-phenate hydro-lyase (decarboxylating), EC 4.2.1.15] were compared in three phenyl-alanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously(1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mm tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2×105, a half of that of the wild-type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity.
    On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.
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  • Tomohiro MEGA, Yoshio MATSUSHIMA
    1976 Volume 79 Issue 1 Pages 185-194
    Published: January 25, 1976
    Released: November 18, 2008
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    A number of adsorbents useful for purifying glycosidases were synthesized and their adsorption characteristics were examined using partially purified glycosidase mixtures from Takadiastase or soybean. These adsorbents were prepared by coupling di-ε-aminocaproyl-p-aminophenyl N-acetyl-l-thio-β-D-glucosaminide, β-D-glucoside, β-D-galactoside or α-D-mannoside with CNBr-activated Sepharose 4B.
    Many glycosidases were adsorbed on the four adsorbents at low ionic strength, and increase of the ionic strength caused the enzymes to be eluted. However, the specificity of the adsorbents, contrary to our expection, was very low. All of these adsorbents adsorbed Taka N-acetyl-β-D-glucosaminidase [EC 3.2.1.30], Taka β-D-glucosidase [EC 3.2.1.21], and Taka β-D-galactosidase [EC 3.2.1.23] at low ionic strength. The order of elution of these three enzymes by a linear gradient of ionic strength was the same in the four adsorbents, the order being β-D-galactosidase, β-D-glucosidase, and N-acetyl-β-D-glucosaminidase.
    Soybean glycosidases also showed nearly the same elution pattern, though the ionic strength of the eluate was slightly lower than with Taka glycosidases. Soybean α-D-mannosidase [EC 3.2.1.24] was also adsorbed on these adsorbents, and was eluted between β-D-glucosidase and N-acetyl-β-D-glucosaminidase.
    These adsorption phenomena were not specific as regards the structure of the glycoside moiety, but they were useful for purifying glycosidases, possessing good reproducibility with easy activation and mild operating conditions.
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  • Masahiko ONODERA, Hiroyuki SHIOKAWA, Toshio TAKAGI
    1976 Volume 79 Issue 1 Pages 195-202
    Published: January 25, 1976
    Released: November 18, 2008
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    1. Rabbits were immunized with N-methyl-2-anilinonaphthalene-6-sulfonyl (MANS) bovine serum albumin (MANS-BSA) prepared by the reaction of bovine serum albumin with N-methyl-2-anilinonaphthalene-6-sulfonyl chloride to obtain IgG fraction contain-ing anti-MANS antibodies.
    2. Both N-methyl-2-anilinonaphthalene-6-sulfonate (MANSate) and N-methyl-2-anilino-naphthalene-6-sulfonamide (MANSamide), which are virtually non-fluorescent in aqueous solution, became strongly fluorescent, with a blue shift of about 100nm, when they were specifically bound to the antibodies against the hapten group contained in the IgG fractions.
    3. IgG fractions were obtained from five immunized rabbits at intervals after the primary immunization. The fluorescence characteristics of the hepten were carefully examined with preparations of IgG fraction differing in source and time of bleeding. One of the rabbits showed a very interesting immune response. The rabbit produced two groups of anti-MANS antibodies which were significantly different in their effect on the fluorescence properties of the hapten group. One of the groups was exclusively produced at the early stage in the immune response. The other group appeared at the late stage. It is suggested that the two groups differ as regards the structure of the hepten combining sites.
    4. The above findings indicate that the MANS group can be successfully used as a hapten with useful properties as a fluorescent probe.
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  • Noriyuki MUROYA, Yosinobu NAGAO, Kaoru MIYAZAKI, Katsuzo NISHIKAWA, Ta ...
    1976 Volume 79 Issue 1 Pages 203-215
    Published: January 25, 1976
    Released: November 18, 2008
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    Pyruvate kinase [EC 2.7.1.40] in various tissues of rats was separable into seven kinds of pI-isozymes by isoelectric separation with Ampholine carrier ampholytes; pI 5.4-isozyme, pI5.6-isozyme, pI6.2-isozyme (2 kinds), pI6.6-isozyme, pI7.4-isozyme, and pI 7.8-isozyme. Some of these pI-isozymes contained bound fructose 1, 6-diphos-phate (FDP).
    The bound FDP was completely dissociated when the pI-isozymes were salted out with ammonium sulfate. In the FDP-free form, pyruvate kinase was classified into three types, liver-type (type L) of pI 6.2, muscle-type (type M) of pI 7.4, and spleen-type (type M2) of pI 7.8.
    The liver-type isoenzyme had two kinds of FDP-binding sites; the pI 5.6-iso-zyme and pI5.4-isozyme were obtained when one and two kinds of sites were bound with FDP, respectively. The association and dissociation of FDP at both sites were reversible in the presence and absence of 0.15M KCl (high ionic strength).
    The muscle-type isoenzyme had no FDP-binding site.
    The spleen-type isoenzyme had two kinds of FDP-binding sites, like the liver-type isoenzyme. When the ionic strength of solutions containing the enzyme and FDP was sufficiently low, one and two kinds of the sites could bind with FDP, converting the enzyme into pI 6.6-isozyme and pI 6.2-isozyme, respectively. FDP bound with one kind of site (the 2nd site) was easily dissociable, but FDP bound with the other kind of site (the 1st site) was not. Provided that the 1st site carried bound FDP, the 2nd site was associable at high ionic strength.
    The liver-type isoenzyme free of FDP and the spleen-type isoenzyme bound with FDP at both sites had similar pI values of 6.2 and were not separable by isoelectric separation.
    Some properties of these pI-isozymes were compared.
    When Rhodamine sarcoma was transplanted in rats, the content of spleen-type isoenzyme in the livers increased. When rats were injected with chromatin prepared from either Rhodamine sarcoma or spleen, the content of spleen-type isoenzyme in the livers again increased. This was not observed on the injection of chromatin prepared from liver, indicating that the factor capable of controlling the gene expression was present in chromatins of sarcoma and spleen but barely or not at all in chromatin of liver.
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  • Yoshiyuki TANAKA, Ikuzo URITANI
    1976 Volume 79 Issue 1 Pages 217-219
    Published: January 25, 1976
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Antibody toward phenylalanine ammonia-lyase (PAL) [EC 4.3.1.5] was obtained by immunization of a rabbit with highly purified PAL. The antibody reacted specifically with RAL, as demonstrated by the Ouchterlony double diffusion test, immuno-electrophoresis, and SDS polyacrylamide gel electrophoresis of the immunoprecipitate. Experiments using anti-PAL showed that PAL was not present in fresh sweet potato tissue, but appeared in response to cut injury, reaching a maximum, and then decreasing, in parallel with PAL activity. The results suggest that the development of PAL activity was due to de novo synthesis of PAL and that the decrease of PAL activity after reaching a maximum was due to proteolytic degradation of PAL.
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  • Masako HAYASHI, Yasuo NATORI
    1976 Volume 79 Issue 1 Pages 221-224
    Published: January 25, 1976
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Degradation of rat liver cytosolic proteins at a neutral pH in the presence of 0.1%0 SDS was demonstrated by SDS-acrylamide gel electrophoresis. This proteolysis in vitro mimics molecular-size-dependent proteolysis in vivo; larger proteins were degraded more rapidly than smaller ones. Evidence is presented that the proteolysis is not due to contaminating lysosomal cathepsins in the cytosol.
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  • Fumio SAKIYAMA, Reiko NATSUKI
    1976 Volume 79 Issue 1 Pages 225-228
    Published: January 25, 1976
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The position of a single N'-formylkynurenine residue in ozone-inactivated hen egg-white lysozyme[EC 3.2.1.17] was determined by two methods. One involved identification of an amino-terminus of the C-peptide obtained by selective cleavage with hydrazine of the kynurenyl peptide linkage in oxidized lysozyme. The other was to analyze a tryptic peptide containing kynurenine in the modified enzyme. Both methods showed that tryptophan 62 in lysozyme was so sensitive to ozone as to be selectively oxidized to N'-formylkynurenine with concomitant loss of the lytic activity.
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  • Setsuro EBASHI
    1976 Volume 79 Issue 1 Pages 229-231
    Published: January 25, 1976
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    A simple method was developed for preparing actin-free myosin in good yield from gizzard. This method also provided a fairly pure F-actin preparation.
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  • Tsunehiro KITAGAWA, Tadaomi AIKAWA
    1976 Volume 79 Issue 1 Pages 233-236
    Published: January 25, 1976
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    A novel coupling reagent, meta-maleimidobenzoyl N-hydroxysuccinimide ester was synthesized. Using this reagent, insulin was conjugated very easily with β-D-galactosidase [EC 3.2.1.23] in neutral, aqueous solution. No reduction of the enzyme activity was observed during the coupling procedure. The competitive bindings of the conjugate and insulin to anti-insulin serum were tested. The results indicated that the conjugate has enough immune reactivity for use in enzyme coupled immunoassay. Using this assay 20-800 pg of insulin were detectable.
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  • 1976 Volume 79 Issue 1 Pages e1a
    Published: 1976
    Released: November 18, 2008
    JOURNALS FREE ACCESS
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  • 1976 Volume 79 Issue 1 Pages e1b
    Published: 1976
    Released: November 18, 2008
    JOURNALS FREE ACCESS
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