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Volume 84 (1978)
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Volume 84, Issue 4
Displaying 1-37 of 37 articles from this issue
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I. Isolation and General PropertiesIchio II, Kazuo DEGUCHI, Shizue KAWASHIMA, Sachiko ENDO, Nobuo UETA1978 Volume 84 Issue 4 Pages 737-749
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMost of the water-soluble lipoproteins in sea urchin eggs (Hemicentrotus puicherrimus) were localized within yolk granules. Under hypotonic conditions, yolk granules released lipoproteins and a 24S protein species as high molecular weight components; the lipoproteins constituted about 40% of the total materials released. Three yolk lipoproteins (YLP-1, 2, and 3, in order of quantity) were isolated by ultracentrifugation and gel filtration. The hydrated densities of YLP-1, 2, and 3 were 1.027, 1.062, and 1.009 g/cm3, respectively. YLP-1, 2, and 3 contained glyceride as a major lipid in quantities of 3.1, 1.8, and 4.3 times the amount of each protein, respectively. These lipoproteins contained large amounts of carbohydrate. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed four major periodic acid-Schiff (PAS) positive polypeptide bands common to the three lipoproteins. All the constituent polypeptides of the 24S protein were also PAS positive. Electron microscopy of negatively stained YLP-1, 2, and 3 revealed the average diameters to be 36, 29, and 48nm, respectively. The 24S protein appeared to be cylindrical in shape with average exterior dimensions of 10-20nm. Thin-section micrographs showed that yolk granules are packed with particles around 30 nm in diameter, suggesting that these particles are not the 24S proteins but the lipoprotein particles.View full abstractDownload PDF (2323K) -
Toshiaki ARATA, Yasuo MUKOHATA, Yuji TONOMURA1978 Volume 84 Issue 4 Pages 751-761
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycerol-treated muscle fiber bundles were fixed at their rest length in 50 mM KCl, 2 mM MgCl2, and 10μM CaCl2 at pH 7.8 and 0°C in the presence of sufficient amounts of ATP, creatine kinase, and creatine phosphate. The fiber bundles were stretched linearly with time for 0.3 sat a constant amplitude, suddenly released, then fixed at the rest length for a constant time interval (α seconds). The stretch-release cycle was repeated, and the ATPase activity (the rate of ADP liberation) [EC 3.6.1.3] was measured. It was found that:
1. ATPase was activated by repeated stretch-release. As repetitive stretch-release of 1-2% of the rest length caused maximum activation, we usually selected a value of 2.5% of the rest length. The activation of ATPase was found to be a function of the duration, α, of the isometric phase after sudden release from stretching. The ATPase activity of fiber bundles was almost unaffected when they were oscillated by a simple stretch-release without an isometric phase after the sudden release (α=0).
2. The ATPase activity of oscillated muscle fibers increased with increase in the value of a, reached a maximal level, then decreased gradually with further increase of α to a value slightly larger than that of static fibers. At 0°C, the value of α for the maximum activation was observed at about 2 s, and the maximum activity was about 2.5 times that of static fibers. At 20°C, the α value for maximum activation was about 0.5 s, and the maximum activity was about 1.8 times that of static fibers.
3. The time course of ADP liberation after one stretch-release cycle could be easily calculated from the ATPase activity of the summed durations of the isometric phase, α, assuming that the ATPase activation was turned off and on by the stretching and release, respectively, and that the state of cross-bridges immediately after the stretch-release was independent of α of the cycle. The rate of ADP liberation after stretch-release thus obtained showed a short lag phase, a sigmoidal increase, a decrease to almost zero, then a return to nearly the original level (the rate of static fibers). About 1.3 mol of ATP per mol of myosin was hydrolyzed at both 0°C and 20°C during one cycle of the changes in the rate of ADP liberation.View full abstractDownload PDF (828K) -
Kazuhiro SOGAWA, Kenji TAKAHASHI1978 Volume 84 Issue 4 Pages 763-770
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNeutral proteinase activity in subcellular fractions of rat liver was measured with heat-denatured casein as a substrate before and after repeated freezing and thawing, followed by extraction of each fraction with 1 M KCl. By this treatment, most of the proteinase activity in each fraction was solubilized except for that in the microsomal fraction. More than 50% of the neutral proteinase activity in the microsomal fraction remained insoluble, which indicated that the 1 M KCl-insoluble neutral proteinase activity was predominantly localized in the microsomal fraction. The activity was present in both the rough and smooth microsomes.
The neutral proteinase(s) was largely solubilized from the 1 M KCl-washed microsomal fraction by 0.5% at NaCl containing 0.5% deoxycholate and 0.25% cholate. The solubilized proteinase(s) was eluted from a Sepharose CL-6B column as a single but rather broad peak, giving a molecular weight of about 84, 000. It showed maximal activity at pH 8.0 toward heat-denatured casein as a substrate. It was markedly inhibited by diisopropyl phosphorofluoridate, but was not significantly inhibited by chymostatin, soybean trypsin inhibitor, p-chloromercuribenzoate or EDTA. o-Phenanthroline was somewhat inhibitory. Among urea-denatured proteins tested as substrates, calf thymus histone was hydrolyzed most rapidly, followed by casein and hemoglobin, whereas ovalbumin, bovine serum albumin, and γ-globulin were not hydrolyzed to any significant extent.
These results demonstrate that rat liver microsomes contain a unique serine proteinase(s) firmly bound to their membranes.View full abstractDownload PDF (635K) -
III. Carbohydrate Moiety of Japanese Monkey Pepsinogens and the Amino Acid Sequence around the Site of Its Attachment to ProteinTakashi KAGEYAMA, Kenji TAKAHASHI1978 Volume 84 Issue 4 Pages 771-778
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPurified Japanese monkey pepsinogens I and II contain carbohydrate as a part of the enzyme molecule. By gel filtration on Sephadex G-100, chromatography on DE-32 cellulose, and polyacrylamide disc gel electrophoresis, the carbohydrate moiety could not be separated from the enzyme protein, and the content did not decrease on repeated chromatography. Glycopeptides were obtained by successive digestion of pepsinogens with thermolysin and aminopeptidases and isolated by chromatography on Sephadex G-25 and G-50. Identification and determination of carbohydrate components was performed by paper and gas-liquid chromatographies. The presence of 4 glucosamines, 6 galactoses, 6-8 mannoses, and 8-11 fucoses per molecule of the glycopeptide of both pepsinogens was observed, of which the high content of fucose is especially unique. The molecular weight of the carbohydrate chains should be around 4, 000-5, 000. The amino acid sequence of a major glycopeptide was deduced to be Ile-Gly-Ile-Gly-Thr-Pro-Gin-Ala-Asn, in which the asparagine residue is the site of attachment of the carbohydrate chain.View full abstractDownload PDF (1598K) -
Masashi KOSAKAI, Zensaku YOSIZAWA1978 Volume 84 Issue 4 Pages 779-785
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe color yields with hexuronic acids and hexuronic acid-containing substances were studied by means of the carbazole method of Bitter and Muir with or without 0.025 M borate. The carbazole-borate to carbazole (CB/C) ratios thus obtained indicated not only the degree of the borate effects on the color yields with these materials but also the anomalous nature of some of these substances in the carbazole reaction. The present data indicate that the high color yield with heparin in the carbazole reaction may be due to the production of free amino groups from acid-labile sulfamino groups in the early stage of the reaction, resulting in suppression of protonation on the α-D-glucosaminidic oxygen atoms attached to the hexuronic acid residues in the molecule. Moreover, it is suggested that the degree of the unusual color yields in the carbazole reaction with glycosaminoglycans is greatly influenced by the anomeric configuration of the hexosaminyl linkages attached to the hexuronic acid residues in these polymers.View full abstractDownload PDF (524K) -
Takashi UENO, Takamitsu SEKINE1978 Volume 84 Issue 4 Pages 787-794
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFluorescence changes of 1-anilino-8-naphthalenesulfonic acid and 3, 3'-dipropyl-2, 2'-thiadicarbocyanine during Ca transport by sarcoplasmic reticulum vesicles were studied.
The fluorescence of both probes is enhanced corresponding to the rapid initial Ca uptake. The enhancement could be interpreted in terms of increased binding of the former (anionic) dye and decreased binding of the latter (cationic) dye to the vesicles, suggesting a change in the surface charge of the membranes associated with Ca transport.
Under limited conditions where Na, K, and Mg were not added exogenously, Ca could be transported without concomitant counter-transport of these cations.View full abstractDownload PDF (591K) -
Essential Arginyl Residues for Catalytic and Regulatory FunctionsIsamu KAMESHITA, Masanobu TOKUSHIGE, Hirohiko KATSUKI1978 Volume 84 Issue 4 Pages 795-803
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChemical modification of phosphoenolpyruvate carboxylase [EC 4.1.1.31] of Escherichia coli W with 2, 3-butanedione in borate buffer resulted in inactivation of the enzyme. The reaction proceeded following pseudo-first-order kinetics, showing a maximal rate at pH 8.5. This inactivation was reversed when excess butanedione and borate were removed. DL-Phospholactate, a structural analog of the substrate, effectively protected the enzyme against inactivation. Concomitant with the butanedione modification, the enzyme was completely desensitized to fructose 1, 6-bisphosphate and GTP, allosteric activators of the enzyme. Desensitiza-tion also occurred to acetyl coenzyme A, another allosteric activator and to L-aspartate, an allosteric inhibitor, to a lesser extent. Desensitization to these effectors occurred upon modi-fication both in the presence and absence of DL-phospholactate. After the butanedione modification, the pH optimum for the activity and the Km value for the substrate remained unchanged, indicating that the desensitization to the effectors was not due to alteration of these kinetic properties. Amino acid analysis showed that 7 out of 49 arginyl residues per subunit were modified concomitant with a complete loss of activity. Modification of other amino acids was not detected under the experimental conditions used. Examination of the protective effect of DL-phospholactate against the butanedione inactivation and the order of reaction suggested that one arginyl residue is essential for the catalytic activity of the enzyme. Similarly, modification of the enzyme in the presence of fructose 1, 6-bisphosphate and in its absence suggested that at least one, possibly two, arginyl residues are essential for the regulatory interaction with fructose 1, 6-bisphosphate.View full abstractDownload PDF (661K) -
Gilbu SOE, Nozomu NISHI, Tomisaburo KAKUNO, Jinpei YAMASHITA1978 Volume 84 Issue 4 Pages 805-814
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt is known that the coupling factor purified from the acetone powder of chromatophores from Rhodospirillum rubrum shows ATPase activity in the presence of Ca2+, but not in the presence of Mg2+ or Mn2+- The present study deals with conditions, under which the Ca2+-ATPase activity is reversibly converted into Mg2+- and Mn2+-ATPase activities with the purified coupling factor.
1. Of the pH indicators tested, 6 kinds converted the Ca2+-ATPase activity into Mg2+- and Mn2+-ATPase activities in the order, ethyl orange>tropaeolin 000≥metanil yellow>tropaeolin 00>ethyl red≥bromthymol blue.
2. Of the detergents tested, those other than Triton X-100 and Brij 58 caused the conversion described above; dodecylsulfonate was most effective, whereas dodecylpyridinium chloride was moderately effective.
3. 2, 4-Dinitrophenol stimulated approximately two-fold the Ca2+-ATPase activity, but not the Mg2+- or Mn2+-ATPase activity at all. However, in the presence of dodecylpyridinium chloride, the pH indicator remarkably stimulated the Mg2+- and Mn2+-ATPase activities, accompanied with a partial inhibition of the Ca2+-ATPase activity. Methyl red and ethyl red showed similar effects.
4. All the nucleoside triphosphates tested can serve as the substrate. ATP was most effective for the Ca2+-ATPase activity, whereas dATP was most effective for the Mg2+- and Mn2+-ATPase activities induced by ethyl orange.
5. In the presence of ethyl orange, the ATPase activity was induced by various divalent cations in the following order of effectiveness, Mg2+>Zn2+>Co2+>Mn2+>Ni2+.
6. The mechanism of the reversible conversion from the Ca2+-ATPase activity to the Mg2+-and Mn2+-ATPase activities by pH indicators and detergents is discussed.View full abstractDownload PDF (760K) -
Kazunori ANZAI, Yutaka KIRINO, Hiroshi SHIMIZU1978 Volume 84 Issue 4 Pages 815-821
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe temperature dependence of the Ca2+-dependent ATPase activity and of the conformational fluctuation of the ATPase molecule has been measured for four kinds of preparations: fragmented sarcoplasmic reticulum, MacLennan's enzyme (purified ATPase preparation), and DOL and egg PC-ATPase (purified ATPase preparations in which lipids are replaced with dioleoyllecithin and egg yolk lecithin, respectively). It has been found that Arrhenius plots of the Ca2+-dependent ATPase activity show a break at about 18°C for all the preparations. Hydrogen-deuterium exchange kinetics of the peptide NH protons were used to measure the conformational fluctuation of the protein molecules. Van't Hoff plots of the conformational fluctuation amplitude of a region near the surface of the ATPase molecule also show a break at about 18°C for all the preparations. It is concluded that the break at around 18°C is not related to a gel-liquid crystalline transition of lipids but to a change in the conformation of the ATPase molecule existing in fluid lipids.View full abstractDownload PDF (528K) -
Evidence of the Occurrence of X-Antigenic Determinant in Serum-type Sugar Chains of GlycoproteinsMasanori NISHIGAKI, Katsuko YAMASHITA, Ichiro MATSUDA, Shinichiro ARAS ...1978 Volume 84 Issue 4 Pages 823-834
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUrine of a fucosidosis patient contained a large amount of fucosyl oligosaccharides and fucoserich glycopeptides.
Six major oligosaccharides were purified by a combination of Bio-Gel P-2 and P-4 column chromatographies and paper chromatography. Structural studies by sequential exoglycosidase digestion and by methylation analysis revealed that their structures were as follows: Fucα1-→6GlcNAc, Fucα1→2Galβ1→4Glc, Gαlβ1→4(Fuca1→3)GlcNAcβ1→2Manα1→3Manβ1→4GIcNAc, Gαlβ1→4(Fucα1→3)GlcNAcβ1→4Manα1→3Manβ1→4G1cNAc, Gαlβ1→4(Fucα1→3)GlcNAcβ1→2Mana1→6Manβ1→4GlcNAc, and Galβ1→4(Fucαl→3)GlcNAc-βl→4Manal→6Manβ1→4GlcNAc.
In addition, the structure of a minor decasaccharide was found to be Galβ1→(Fuca1→)-G1cNAcβ1→Manα1→Galβ1→(Fucα1→)GlcNAcβ1→Manα1→Manβ1→4GlcNAc.View full abstractDownload PDF (860K) -
XIV. Preparation of 6-Deoxy-6-Halogenomaltotrioses and Their Hydrolysis by Taka-Amylase AKaoru OMICHI, Yoshio MATSUSHIMA1978 Volume 84 Issue 4 Pages 835-841
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. O-6-Deoxy-α-D-glucopyranosyl-(1→4)- O-α-D-glucopyranosyl-(1→4)-D-glucopyranose, O-6-chloro-6-deoxy-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(l→4)-D-glucopyranose, O-6-bromo-6-deoxy-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyrano syl-(1→4)-D-glucopyranose, and O-6-deoxy-6-iodo-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose were prepared, taking advantage of the substrate specificities of Taka-amylase A and glucoamylase, and the action of Taka-amylase A on these substrates was investigated.
2. The Michaelis constant Km and the molecular activity k0 were determined at 37°C and pH 5.2 using the modified maltotrioses. The values of Km and k0 decreased upon modification of maltotriose and those of k0/Km were in agreement with the comparative initial rates for the corresponding derivatives of phenyl α-maltoside at low substrate concentrations. This result suggested that a subsite of the enzyme may have a specific interaction with halogen atoms in the substrate.
3. All halogenomaltotrioses examined showed substrate inhibition at high substrate con-centrations.View full abstractDownload PDF (1518K) -
Kuniyo INOUYE, Ben'ichiro TONOMURA, Keitaro HIROMI, Tadao KOTAKA, Hiro ...1978 Volume 84 Issue 4 Pages 843-853
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular weight of Streptomyces subtilisin inhibitor (SSI), a protein proteinase inhibitor, and that of the complex of SSI and subtilisin BPN' [EC 3.4.21.14] were determined by a sedimentation equilibrium method in 25 mm phosphate buffer, at pH 7.0, ionic strength 0.1 M (NaCl), 25.0°C. The molecular weight of SSI was found to be 23, 000 over a wide concentra-tion range, 0.01-10 mg/ml, the range used for inhibitory, spectrophotometric, and kinetic measurements. Based on the amino acid sequence, the molecular weight of SSI has been cal-culated to be 11, 500 (Ikenaka, T., et al. (1974) J. Biochem. 76, 1191-1209); therefore, the molecular weight of 23, 000 obtained above suggests that SSI is in a dimeric form under usual conditions in the concentration range of 5×10-7-5×10-4 M.
The molecular weight of the subtilisin BPN'-SSI complex was determined to be 78, 000 in the concentration range of 0.03-5.0 mg/ml by sedimentation equilibrium of the crystallized preparation and by that of a mixture of subtilisin BPN' and SSI treated as a multicomponentpolydisperse system. The molecular weight obtained here, combined with the results of binding stoichiometry (Inouye, K., et al. (1977) J. Biochem. 82, 961-967) that showed that one mol of SSI (molecular weight, 11, 500) and one mol of the enzyme (molecular weight, 27, 500) are tightly bound (Kd<1 nM), demonstrate that one mol of dimeric SSI binds two mol of the enzyme to form a stable complex, E2I2.View full abstractDownload PDF (1502K) -
Keiko YAMADA, Terukatsu SASAKI, Toshio SAKAGAMI1978 Volume 84 Issue 4 Pages 855-863
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cytosol fraction of rat small intestinal mucosa stimulated the transfer of [32P]phosphatidylcholine and [32P]phosphatidylinositol from donor liposomes to acceptor liposomes. The proteins which catalyzed the exchanges were separated into three fractions by gel filtration on a Sephadex G-75 column and chromatography on DEAE-cellulose and CM-cellulose. One of the fractions was purified 340-fold and stimulated phosphatidylcholine exchange but not phosphatidylinositol exchange. The other two fractions were active in the stimulation of phosphatidylcholine exchange as well as phosphatidylinositol exchange. These two fractions were purified 35-fold and 44-fold over the cytosol fraction with respect to the phosphatidylinositol exchange activity.View full abstractDownload PDF (1324K) -
I. Four Classes of Patterns of Expression in Bacteriophage T4 Early GenesMasaki ITO, Katsuji HORI, Mutsuo SEKIGUCHI1978 Volume 84 Issue 4 Pages 865-871
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe studied the process of transcription of T4 early genes by analyzing the synthesis of T4-induced proteins by gel electrophoresis. The simultaneous addition of the antibiotic rifampicin and a 3H-labeled amino acids mixture to infected cells at various times allowed us to estimate the amounts of mRNAs for T4 early genes formed during the course of infection, assuming that mRNAs are translated at a constant rate. According to the patterns of transcriptional activity, we classified T4 early genes as follows. Class I (genes 39, rIIA, 42, y, and A6-1); the transcription takes place immediately and ceases within 1 min after phage infection. Class II (genes 43, 46, and MB); there are significant levels of transcription throughout the process (two peaks of activity are usually observed). Class III (gene 52); an initial high level of transcription is followed by a slow decline and a second transcriptional peak at the later stage (10-13 min after infection). Class IV (gene 32); the transcriptional level increases gradually and ceases at the later stage (12 min after infection). On infection with ultraviolet-irradiated phage, significantly altered patterns of transcription were observed. The levels of transcriptional activity for most genes increased continuously. In addition, it was found that the initiation of transcription of some of the genes was delayed (no transcription of genes 43 and 46 took place within 5 min after infection). Based on these results, we proposed that T4 early genes are controlled by at least four classes of promotors.View full abstractDownload PDF (1004K) -
II. Roles of the β' Subunit of RNA Polymerase and Termination Factor ρ of the Host in the Expression of T4 Early GenesMasaki ITO, Mutsuo SEKIGUCHI1978 Volume 84 Issue 4 Pages 873-879
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to clarify the role of host factors in the regulation of T4 early transcription, we studied T4 early protein synthesis in T4D-infected Escherichia coli K12 strains having mutations in the genes for the β' subunit of RNA polymerase and for the termination factor ρ. Gel electrophoretic analysis revealed that in E. coli T16 (rpoC1), a temperature-sensitive β' subunit mutant, the synthesis of various T4 early proteins was equally inhibited at both 30°C and 42°C. Since RNA synthesis was almost completely inhibited under these conditions, it is likely that the reduced levels of protein synthesis are due to decreased amounts of T4 mRNA. Thus, the normal function of β' subunit of E. coli RNA polymerase is required for T4 early transcription. When E. coli HD152 (nitA702) cells, which produce thermolabile ρ factor, were exposed to 45°C and then infected with T4D, the level of T4 early protein synthesis was reduced to less than 5 % of the level at 30°C. The same heat treatment of the wild-type cells resulted in only a slight decrease in protein synthesis (70% of the level at 30°C). In T413-infected HD152 cells, normal amounts of RNA were produced at either 30 or 45°C. It is possible that RNA produced by the ρ factor mutant under restrictive conditions is non-functional. It was found that some of the T4 early proteins were overproduced in the ρ factor mutant when it was exposed to 30°C. Larger amounts of gene 32 and 46 proteins were formed in the mutant than in the wild-type strain, while other proteins, such as gene rIIA, rIIB, 39, 52, and 42 proteins, were produced at the normal levels. This result may be explained by the assumption that there are two classes of ρ-dependent termination sites, weak and strong ones, in early regions of the T4 genome and that read-through occurs at the weak termination sites when ρ factor is partially suppressed.View full abstractDownload PDF (1601K) -
Use of the Wide Substrate Specificity of AcetylornithinaseMasahiko KISUMI, Tsutomu TAKAGI, Ichiro CHIBATA1978 Volume 84 Issue 4 Pages 881-890
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Arginine biosynthesis in Serratia marcescens Sr41 was found to be controlled by (a) feedback inhibition of N-acetylglutamate synthetase and (b) repression of some L-arginine biosynthetic enzymes, and an L-arginine-degrading system was found to exist. Accordingly, an L-arginineproducing mutant (aru argR argA) of S. marcescens+ Sr41 was constructed as follows. A mutant incapable of L-arginine utilization (aru) was obtained from the wild strain. Subsequently, from the lysine auxotroph (lysA) of aru mutant, a mutant having derepressed L-arginine biosynthetic enzymes (argR) was isolated by screening for colonies that could utilize Na-acetyl-L-lysine in the presence of L-arginine. This selection was based on the finding that acetylornithinase of S. marcescens hydrolyzed Na-acetyl-L-lysine. On the other hand, to obtain a mutant with feedback-resistant N-acetylglutamate synthetase (argA), the proAB argD argR triple mutant was isolated from the indirectly suppressed revertant (proAB argD) of the proline auxotroph (proAB). Next, the argA mutant was isolated from the triple mutant by selection for resistance to 3, 4-dehydro-DL-proline in the presence of L-arginine. The argA mutation was introduced into the aru lysA argR strain by PS20-mediated cotransduction with lysA+. The aru argR argA IysA+ transductant produced 25 mg/ml of L-arginine in the medium.View full abstractDownload PDF (724K) -
Itsuo OKUMURA, Takehiko YAMAMOTO1978 Volume 84 Issue 4 Pages 891-895
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAllantoin racemase was isolated from cells of Candida utilis, and purified by chromatography on columns of DEAE-cellulose and Sephadex G-100. Using this purified enzyme, the racemization of allantoin in deuterium oxide was investigated. Polarimetric and PMR spectroscopic analyses showed that racemization of allantoin by the enzyme proceeded in parallel with release of the hydrogen atom (5-H) attached to the asymmetric carbon (C-5) of allantoin. Non-enzymic racemization of allantoin, which was examined for comparison, however, was accompanied by much less or almost no release of allantoin 5-H. This indicates that the mechanism of racemization by the enzyme differs from that of non-enzymic racemization.View full abstractDownload PDF (318K) -
Yoshinori SATOW, Yukio MITSUI, Yoichi IITAKA1978 Volume 84 Issue 4 Pages 897-906
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of a protein proteinase inhibitor, SSI (Streptomyces subtilisin inhibitor), which strongly inhibits bacterial alkaline proteinases specifically, was determined at 4 Å resolution using four heavy-atom derivatives. The SSI molecule can be described as an ellipsoid of about 30×40×65 Å composed of two identical subunits each having dimensions of about 35×25×40 Å and a molecular weight of 11, 483. The subunit has an extensive β-sheet structure, but no long a-helices are present. Based on the binding sites of platinum reagents known to form coordination complexes with methionine, it is speculated that the P1 residue, Met 73, of the reactive site is at the protruding edge of the subunit. At the subunit-subunit interface, a β-sheet of one subunit is stacked on top of the corresponding β-sheet of the other suhnnit.View full abstractDownload PDF (1617K) -
Failure of the Usual Anti-Ig Antibody Method for Quantitative Measurement of Low Affinity AntibodiesSachiko TANIUCHI, Koichi TAMOTO, Jiro KOYAMA1978 Volume 84 Issue 4 Pages 907-915
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe usual anti-Ig antibody method, consisting of the precipitation of soluble antigen-antibody complexes by heterologous anti-Ig antibody, was applied for quantitative estimation of guinea pig IgG2 anti-ovalbumin and anti-2, 4-dinitrophenyl (DNP) antibodies by measuring the maximum amounts of antibody-bound antigens. However, the amounts of antibodies estimated were less than those obtained by other methods: the precipitin reaction, the precipitation of antigen-antibody complexes with 50% saturated ammonium sulfate, and equilibrium dialysis. In particular, the anti-Ig antibody method greatly underestimated the amount of anti-DNP antibody with low affinity for ε-DNP-L-lysine. Thus, it was concluded that partial dissociation of the antigen-antibody complexes occurring upon precipitation with anti-Ig antibody made the anti-Ig antibody method unsuitable for quantitative determination of antibodies.View full abstractDownload PDF (677K) -
Sizue UENO, Hideki TACHIBANA, Yuzuru HUSIMI, Akiyoshi WADA1978 Volume 84 Issue 4 Pages 917-924
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe origin of cooperatively melting regions in DNA, which appear as fine structures in the optical melting profile, has been examined for DNA fragments of known base sequences from bacteriophages φX174 and fd.
Thermal stability maps, which indicate the states of base pairs along these DNA strands, were constructed within the established theoretical framework using the parameters which best reproduce the melting profiles obtained by high temperature resolution experiments.
By comparing these stability maps with genetic maps, it was found that several coopera-tively melting regions which span several hundred bases have some correlation with the gene locations.View full abstractDownload PDF (549K) -
Yasuzo NISHINA, Teizo KITAGAWA, Kiyoshi SHIGA, Kihachiro HORIIKE, Yuic ...1978 Volume 84 Issue 4 Pages 925-932
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH3 RF), 3-carboxymethyl (3-CH2COOH RF), and 7, 8-dichioro riboflavins (7, 8-Cl RF), in H2O and D2O were observed in the 700-1700cm-1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253cm-1 line of RF was shifted to ca. 1300cm-1 for 3-D RF, 3-CH3 RF, and 3-CH2COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632cm-1 line of RF was shifted for 7, 8-C1 RF and was assigned to a Ring I mode. No Raman line mainly due to C=O stretching mode was observed in the present resonance Raman spectra.View full abstractDownload PDF (591K) -
Takahiko MORIGUCHI, Daisaburo FUJIMOTO1978 Volume 84 Issue 4 Pages 933-935
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPyridinoline is a crosslinking amino acid of collagen fibers. The age-related changes in the content of pyridinoline were followed for collagens from human and rat costal cartilage and Achilles tendon. The pyridinoline content of the collagens in fetal or newborn animals was very low and increased markedly with growth of the animals. In rat tissues, the pyridinoline content continued to increase after the animal had reached maturity. On the other hand, in human tissues, it began to decrease after about 30 years of age. Pyridinoline may serve as an interesting index for the aging of connective tissues.View full abstractDownload PDF (170K) -
I. Hormonal Effects on Cell Viability and Protein SynthesisKeiji TANAKA, Mitsuru SATO, Yumiko TOMITA, Akira ICHIHARA1978 Volume 84 Issue 4 Pages 937-946
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLiver parenchymal cells were isolated from adult rats by digesting liver slices or perfusing liver with collagenase. The cell yields were 1.5×107 and 1.0×108 cells/g liver from slices and perfused liver, respectively, and in both cases the cell viabilities and attachment efficiencies were over 90%. and 60%, respectively. The cells were viable for more than one week when cultured in Williams medium E with 10%. fetal bovine serum, and addition of insulin and dexamethasone enhanced the maintenance of cell viability. Various biochemical functions of freshly isolated cells and cultured cells were compared in this medium.
In freshly isolated cells, induction of tyrosine transaminase [EC 2.6.1.5] by dexamethasone was low and none of the hormones examined stimulated protein synthesis; but when the cells had been cultured for a few days, induction of tyrosine transaminase became prominent, and insulin and dexamethasone stimulated protein synthesis and glucagon inhibited their effect. About half the synthesized proteins were secreted into the medium, and among these proteins, albumin, transferrin, fibrinogen, and lipoproteins were identified immunochemically and electrophoretically. It was also shown that the polysomes in freshly isolated cells were almost completely disaggregated, but that in cells after a few days culture they were reaggregated.
These results showed that freshly isolated cells have impaired functions, but that after culture for a few days the cells recover various liver functions and thus become more suitable for use in biochemical studies on liver functions.View full abstractDownload PDF (1220K) -
Asao MAKIOKA, Tamio HIRABAYASHI1978 Volume 84 Issue 4 Pages 947-955
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. An inhibitory protein for Mg2+-activated actomyosin ATPase from rabbit skeletal muscle was prepared from frozen chicken gizzard and purified by DEAE-Sephadex chromatography and gel filtration.
2. The inhibition by this protein was released by the addition of skeletal muscle troponin C and was independent of gizzard tropomyosin.
3. Localization of the inhibitory protein in gizzard muscle tissue and gizzard thin filaments was demonstrated by immunohistological techniques and immunodiffusion tests.View full abstractDownload PDF (2455K) -
Hiroshi KAGAWA, Hiroshi ASAI1978 Volume 84 Issue 4 Pages 957-963
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe diffusion phenomena of myosin (myosin A, H-meromyosin or subfragment-1) in F-actin plus ATP solutions were investigated. The upper part of the diffusion cell was filled with F-actin plus ATP, and the lower part was filled with F-actin, ATP, and myosin, then both parts were brought into contact so that a boundary of the two solutions was formed and the diffusion of myosin in F-actin plus ATP solutions started. The diffusion pattern was observed with a schlieren lens system. When almost all the ATP in the lower part of the cell had been consumed by actomyosin, a hyper-sharp schlieren pattern appeared near the boundary. On analyzing this pattern, it was found that a local fast migration of proteins was occurring. Simple Brownian motion of myosin molecules could not explain the hyper-sharp phenomenon. This phenomenon occurred in ther pesence of Mg2+ or Ca2+, but very little in the presence of EDTA. Although it is well known that the superprecipitation of myosin B suspension occurs only at physiological ionic strength, this phenomenon occurred over a relatively wide range of ionic strengths.
The molecular mechanism of this phenomenon is discussed in relation to the basic mechanism of the interaction between myosin and F-actin.View full abstractDownload PDF (526K) -
Masanobu JANADO, Reiko NAKAYAMA, Yuki YANO, Hiroko NAKAMORI1978 Volume 84 Issue 4 Pages 965-969
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe osmotic effect operative in frontal gel chromatography was quantitatively studied. When mixtures of a non-penetrating solute (Kav=0) and a partially penetrating solute (0<Kav<1) were subjected to frontal gel chromatography, the latter formed a coextensive concentration gradient across the trailing boundary of the former, leading to the formation of a second plateau where the concentration exceeded that of the original solution plateau. It was shown that this anomaly, which we have previously predicted, was a direct consequence of osmotic perturbation of the bead size of the Sephadex gel and could be satisfactorily described by an equation based solely on the osmotic distention of the gel beads. Finally, the implications of the osmotic effect in the frontal chromatographic analysis of acceptor-ligand interactions is discussed and a method for correcting this effect is presented.View full abstractDownload PDF (405K) -
Nobuhito MASUDA, Shizuko KOBAYASHI, Kensaku MIZUNO, Fumio SAKIYAMA1978 Volume 84 Issue 4 Pages 971-975
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFor the isolation of human lysozyme from the urine of leukemia patients, a simple method has been established which involves precipitation of urinary proteins by 60% saturation with ammonium sulfate, fractionation of crude lysozyme on Sephadex G-50 and purification by CM-Sepharose chromatography. By this method approximately 60% of the lysozyme in the urine was isolated in a pure state in ten days.View full abstractDownload PDF (928K) -
Yukio SUZUKI, Takashi MURACHI1978 Volume 84 Issue 4 Pages 977-984
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat peritoneal macrophages are known to contain a chymotrypsin-like neutral protease associated with a specific inhibitor. By homogenizing the cells in 0.25M sucrose (pH 8.0) containing 0.5% Triton X-100, both the protease and the inhibitor were found to be localized in the nuclei, particularly in chromatin. The inhibitory factor in chromatin was then separated from the protease by hydroxylapatite gel chromatography in the presence of 2M NaCl and 5 M urea. The inhibitor fraction obtained was deproteinized by digestion with Pronase and subsequent extraction with phenol; these treatments did not alter the inhibitory potency. The deproteinized inhibitor fraction had a UV absorption ratio, A280/A260, of 0.61, but it was resistant to digestion with various nucleases, including DNase 1, nuclease P1, and snake venom phosphodiesterase. However, when it was incubated with poly(ADP-ribose) glycohydrolase from calf thymus, the inhibitory potency was markedly decreased. An authentic poly(ADP-ribose), with a mean chain length of approximately 30 ADP-ribose units, produced significant inhibition of the neutral protease isolated from macrophage chromatin. No such inhibition was produced by DNA, single-stranded DNA, RNA, polyadenylate, polyuridylate, polycytidylate, or monomeric ADP-ribose.View full abstractDownload PDF (608K) -
Tatsuo SENSHU, Mitsugu FUKUDA, Mochihiko OHASHI1978 Volume 84 Issue 4 Pages 985-988
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe assembly of newly synthesized histones into chromatin during replication of MH-134SC cells was studied. Cells pulse-labeled with iododeoxyuridine and [3H]lysine were mixed with an equivalent number of normal cells labeled with [14C]lysine. Nuclease chromatin obtained from pooled cells was fractionated by buoyant-density centrifugation in a gradient containing Metrizamide and 3-iodo-1, 2-propanediol. Histones extracted from heavy and normal chromatin regions of the gradient were fractionated by acid-urea gel electrophoresis, and 3H/14C ratios of individual histones were compared. The results showed highly preferential association of newly synthesized H3 and H4 with newly replicated DNA.View full abstractDownload PDF (262K) -
Atsuhiko KUNITA, Minoru KOSHIBE, Yasuo NISHIKAWA, Keiichi FUKUYAMA, To ...1978 Volume 84 Issue 4 Pages 989-992
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA chloroplast-type ferredoxin containing two non-heme iron and two labile sulfur atoms per molecule was prepared from Aphanothece sacrum. Crystals were obtained by dialysis against 75% saturated ammonium sulfate solution, and belong to the tetragonal system with cell dimensions a=b=92.2Å and c=47.6Å, containing four molecules in an asymmetric unit.
The electron density map at 5Å resolution was calculated by using the best phase angles determined by the single isomorphous replacement method coupled with the anomalous dispersion effect. An anomalous dispersion difference Fourier map for the native crystal clearly showed four humps corresponding to the iron atoms in an asymmetric unit. The electron density map indicates that the active site of the molecule is close to its surface.View full abstractDownload PDF (634K) -
Jun OKUDA, Ichitomo MIWA, Kazuhiro INAGAKI, Masatoshi UEDA, Kazuhisa T ...1978 Volume 84 Issue 4 Pages 993-995
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe D-glucose anomeric preference of hexokinases isolated from rat liver, brain, and skeletal muscle, and bovine retina was studied using the glucose-6-phosphate dehydrogenase-NADP system. The ratios of maximum phosphorylation rates of β-D-glucose to those of α-D-glucose were 1.33, 1.46, and 1.54 for hexokinase types I, II, and In from rat liver, 1.45 and 1.63 for type I from rat brain and bovine retina, 1.53 for type II from rat skeletal muscle, and 0.55 (when determined at 5mM) for type IV (glucokinase) from rat liver, respectively.View full abstractDownload PDF (192K) -
Junko IGARASHI, Norio HAYASHI, Goro KIKUCHI1978 Volume 84 Issue 4 Pages 997-1000
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCobalt protoporphyrin inhibited the drug-induced increase of δ-aminolevulinate synthase as well as its transfer from the cytosol fraction to the mitochondria in rat liver in a similar way to protoheme. Cobalt chloride given to animals in a large dose exhibited similar effects. Cobalt protoporphyrin was isolated from the liver of rats treated with cobalt chloride. The observed regulatory effects of cobalt chloride with respect to the induction and the intracellular translocation of δ-aminolevulinate synthase may be mediated by cobalt protoporphyrin synthesized in vivo.View full abstractDownload PDF (273K) -
Yukio KATO, Norimitsu NASU, Toshiyuki TAKASE, Fujio SUZUKI1978 Volume 84 Issue 4 Pages 1001-1004
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMultiplication-stimulating activity (MSA), a substance obtained from conditioned medium of Buffalo rat liver cells, stimulated replication of rabbit costal chondrocytes in culture and their DNA synthesis, sulfation of glycosaminoglycans, protein synthesis, and collagen synthesis. These stimulatory effects of MSA were dose-dependent in serum-free medium, indicating that MSA has intrinsic somatomedin activity. Even after several successive passages, cultured chondrocytes were more responsive to MSA than other organ- and cell-culture systems reported. Therefore, cultured rabbit costal chondrocytes proved a good in vitro system for analysis of somatomedin actions.View full abstractDownload PDF (268K) -
Noboru OTOTANI, Zensaku YOSIZAWA1978 Volume 84 Issue 4 Pages 1005-1008
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChondroitinase C, chondroitinase AC, heparinase, and heparitinase separated from an extract of Flavobacterium heparinum were subjected to affinity chromatography with glycosaminoglycan-bound AH-Sepharose 4B, previously coated non-covalently with glycosaminoglycan, as the matrix. The results suggested the importance of coating the matrix with glycosaminoglycan in the binding of the enzyme protein to the matrix.View full abstractDownload PDF (259K) -
Hitoshi UENO1978 Volume 84 Issue 4 Pages 1009-1012
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBinding abilities of troponin components to two fragments of rabbit skeletal α-tropomyosin, one the N-chain (residues 1-189) obtained by specific cleavage at Cys 190 and the other the p-fragment (residues 183-284) of the tryptic product, were investigated by gel electrophoresis. The mixture of the tropomyosin fragments showed a new band of complex with either troponin-(T+C) or whole troponin, troponin-(T+I+C), irrespective of the presence of Ca2+ in solution. On the other hand, troponin-T and troponin-(T+I) as well as troponin-C, -I, and -(I+C) had little binding capacity to the tropomyosin fragments. Thus, troponin-C enhances the binding capacity of troponin-T to tropomyosin fragments.
A two-site binding of troponin-T to tropomyosin is proposed.View full abstractDownload PDF (661K) -
Kazuyuki SHIMADA, Yoshio YAZAKI1978 Volume 84 Issue 4 Pages 1013-1015
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAngiotensin H binding sites were demonstrated in human mononuclear leucocytes by use of [125I]angiotensin II. The binding of [125I]angiotensin II to mononuclear leucocytes was rapid and reversible. The abilities of unlabeled compounds to displace [125I]angiotensin II were proportional to their abilities to displace labeled hormone in adrenal and smooth muscle membrane preparations. The Scatchard plot revealed two apparent orders of binding sites. The affinity constants were comparable with those for binding sites in other main target tissues of angiotensin II.View full abstractDownload PDF (210K) -
Koscak MARUYAMA, Kazuyo OHASHI1978 Volume 84 Issue 4 Pages 1017-1019
Published: October 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe value of flow birefringence of F-actin was greatly decreased by filamin due to precipitate formation. This precipitate could be dispersed into birefringent filaments by sonication. Tropomyosin inhibited precipitation of F-actin induced by filamin, and no decrease in birefringence occurred when filamin was added to tropomyosin-bound F-actin. Hence it appears that filamin acts on F-actin in non-muscle cells similarly to α-actinin.View full abstractDownload PDF (641K)
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