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Volume 93, Issue 1
Displaying 1-41 of 41 articles from this issue
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Tamao ENDO, Keizo INOUE, Shoshichi NOJIMA, Takashi SEKIYA, Yoshinori N ...1983 Volume 93 Issue 1 Pages 1-6
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStructures of aqueous dispersions prepared from monoglycosyldiglycerides and from mixtures of monoglycosyldiglyceride and phosphatidylcholine were studied by freeze-fracture electron microscopy. Aqueous dispersions of glucosyl or galactosyl dipalmitylglycerol formed lamellar structures when quenched both from below and above the gel-liquid crystalline phase transition temperature. With mixtures of monoglycosyl dipalmitylglycerol and dipalmitoylphosphatidylcholine, tubular structures could be observed in the extended bilayer structures. Diglucosyl dipalmitylglycerols showed only a smooth fracture surface, however, when mixed with dipalmitoylphos-phatidylcholine. In the fractured face of dispersions composed of egg yolk phosphatidylcholine and glucosyl dipalmitylglycerol, particles having a diameter of 3-6 nm, instead of tubular structures, were observed, suggesting that the occurrence of tubular structure may depend on the fatty acid compositions of the mixtures. A mixture of dipalmitoylphosphatidylcholine and galactosylceramide did not show any distinct tubular or globular structures on the fractured face. These findings indicate that structure in the polar group of molecules as well as structure in the nonpolar group may be important for the formation of a non-bilayer, tubular structure.View full abstractDownload PDF (1971K) -
Hayao TAGUCHI, Masaru HAMAOKI, Hiroshi MATSUZAWA, Takahisa OHTA1983 Volume 93 Issue 1 Pages 7-13
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus Thermus, and tentatively named T. caldophilus sp. n. strain GK24. The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth. Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about I IS. The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for N-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH2). The enzyme was stable in the pH range of 5 to 11 for 1 day at 4°C or for 1 h at 70°C. The enzyme sample showed a maximal activity at 90°C and had an extreme stability toward treatment by heat and denaturing reagents. The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis-(β-aminoethyl ether)N, N'-tetraacetic acid (EGTA). From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin. The enzyme also was hydrolytic active toward an ester compound, N-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward N-benzoyl-L-arginine ethyl ester (BAEE).View full abstractDownload PDF (486K) -
Keiichi TSUKAHARA, Yasuo YAMAMOTO1983 Volume 93 Issue 1 Pages 15-22
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetics of the reduction of metmyoglobins by ascorbic acid (H2A) were studied under a nitrogen atmosphere at 25°C, at an ionic strength of 0.30M (NaCl), and between pH 7.18 and 8.09. Neither Tris-HC1 nor phosphate buffers had any effect on the reduction of metmyoglobin. Imidazol and 1-methylimidazol accelerated this reaction, but N3- and CN- ions inhibited it. It is concluded that the reduction of imidazolmetmyoglobin or 1-methylimidazolmetmyoglobin is faster than that of aquametmyoglobin and that neither azidometmyoglobin nor cyanometmyoglobin can be reduced by ascorbate under the present experimental conditions. The second-order rate constants were determined for the reductions of aqua-, imidazol-, and 1-methylimidazolmetmyoglobin by ascorbate (HA- and A2-). The higher reactivity of imidazolmetmyoglobin with ascorbate may be due to the easy transfer of an electron of ascorbate to partially exposed imidazol or porphyrin ring because of expansion of the heme pocket induced by the coordination of imidazol.View full abstractDownload PDF (1024K) -
II. The Effect of Dissociation and Reassociation of Phospholipids on the Activity of Fcγ Receptors of MacrophagesYoshitomi AIDA, Kaoru ONOUE1983 Volume 93 Issue 1 Pages 23-32
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe receptor for the Fc portion of immunoglobulin G (Fc receptor) of guinea pig macrophages was solubilized with a detergent and partially delipidated to the point where the ligand binding activity was essentially lost. Delipidation of the Fc receptor was done by fractionating the macrophage lysate by gel filtration in the presence of detergent. The elution behavior of Fc receptor-detergent complex and phospho-lipid-detergent mixed micelles varied depending on the kind of detergents used for membrane solubilization and for gel filtration. Separation of phospholipids from Fc receptor was best achieved when octylglucoside-solubilized fraction was chromatographed on Sepharose CL-6B in the presence of deoxycholate; the phospholipid peak emerged at Kav=0.55 and the Fc receptor at Kav=0.45. The fraction of Kav=0.45 showed only a marginal activity when the activity was measured after removal of detergents, but activity was clearly shown when phospholipid fraction was added to this fraction prior to removal of the detergents. Reappearance of the Fc receptor activity was shown to be due to association of phospholipids with the Fc receptor. Three kinds of phospholipids with different polar head groups examined, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine, were all able to reconstitute active Fc receptor, although phosphatidylethanolamine was somewhat less effective than the others. Thus, our study demonstrated the amphipathic nature of the Fc receptor, the binding of which is dependent on the interaction with phospholipids.View full abstractDownload PDF (705K) -
Takashi IGARASHI, Tetsuo SATOH, Koichi UENO, Haruo KITAGAWA1983 Volume 93 Issue 1 Pages 33-36
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAge and sex differences in the hepatic glutathione level and related enzyme activities in rats of both sexes were investigated. At 7 weeks of age there was no significant difference in GSH levels between males and females, although a higher level of intracellular GSH was observed in male rats at 12 weeks of age. Hepatic γ-glutamyl transpeptidase activities were significantly higher in females than in males at only 7 weeks of age. Glutathione peroxidase showed higher activities in females than in males. Conversely, glutathione S-transferase, glutathione reductase and glutathione synthesis rates were markedly higher in males than in females.View full abstractDownload PDF (269K) -
Toshiaki NOCE, Koji OKAMOTO, Ikuo TAKEUCHI1983 Volume 93 Issue 1 Pages 37-45
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExtracellular phosphodiesterase for adenosine 3':5'-monophosphate [EC 3.1.4.17] was purified from the supernatant of aggregation phase culture of Dictyostelium discoideum, and two types (type I and type II) of the enzyme were found. The type I enzyme was not absorbed on DEAE-Sephacel at pH 8.5 and had an apparent molecular weight of about 67, 000 daltons. In contrast, the type II enzyme was adsorbed on DEAE-Sephacel and had an apparent molecular weight of about 120, 000 daltons. The Km values of the two types were similar (2-4 μM).
Upon SDS polyacrylamide gel electrophoresis analyses, however, both types produced the same bands with molecular weights of 55, 000 and 57, 000, indicating that they are two different forms composed of common constituents.
During the growth phase, the two types of the enzyme were present in culture supernatant in roughly equal amounts, but type II accumulated predominantly in the aggregation phase, suggesting that the ratio of activity of the two forms is under developmental control.
Rabbit antiserum prepared against purified type II enzyme cross-reacted with type I as well as membrane-bound enzyme, indicating that the three classes of the enzyme possess some common sequence.View full abstractDownload PDF (1000K) -
Keiko KITAGISHI, Keitaro HIROMI, Kohei ODA, Sawao MURAO1983 Volume 93 Issue 1 Pages 47-53
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding between thermolysin and its specific inhibitor, talopeptin (MKI), was found to show a fluorescence increase when excited at 280 nm and 295 nm, and a difference spectrum characterized by two peaks at 294 nm and 285 nm with a shoulder around 278 nm, indicating a microenvironmental change in tryptophan residue(s) of thermolysin and/or talopeptin.
The inhibitor constant of talopeptin against thermolysin, K1, was determined over the pH range 5-9 from the inhibition of the enzyme activity towards 3-(2-furylacryloyl)-glycyl-L-leucine amide (FAGLA) as a substrate. The dissociation constant of thermolysin-talopeptin complex, Kd, determined directly from fluorometric titration was in good agreement with the inhibitor constant, Kt, between pH 6 and 8.5. The pH dependence of K1 and Kd suggested that at least two ionizable groups of thermolysin in their protonated forms are essential for the binding between thermolysin and talopeptin.
The temperature dependence of K1 at pH 5.5 indicated that the binding is largely exothermic (ΔH°=-12 kcal/mol) and essentially enthalpy-driven.View full abstractDownload PDF (427K) -
Kinetics of Binding between Thermolysin and Streptomyces Metalloprotease Inhibitor, Talopeptin (MKI)Keiko KITAGISHI, Keitaro HIROMI1983 Volume 93 Issue 1 Pages 55-59
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of binding between thermolysin with its specific inhibitor, talopeptin (MKI), was studied kinetically with the stopped-flow method by monitoring the enhancement of tryptophan fluorescence caused by the complex formation. Only one relaxation obeying first-order kinetics was observed. The dependence of the apparent first-order constant, kapp, on the inhibitor concentration is consistent with a minimum two-step mechanism, including a fast bimolecular binding step followed by a slow unimolecular step. It was found that the increase in tryptophan fluores-cence occurs solely in the slow unimolecular step. The apparent second-order rate constant, (kon)app, in the low inhibitor concentration range, was determined over the pH range between 5 and 8.5 and decreases with increasing pH. The activation parameters for the overall binding process were obtained from the temperature dependence of (kon)app.View full abstractDownload PDF (287K) -
Kazufumi KURODA, Ritsuko KAGIYAMA, Makoto KAGEYAMA1983 Volume 93 Issue 1 Pages 61-71
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPseudomonas aeruginosa strain NIH S produced a bacteriophage, KF1, immunologically cross-reactive with F-type pyocins. Phage KF1 was neutralized by both anti-pyocin F1 and anti-pyocin F3 sera, although the efficiency was very low. About eleven polypeptides were detected by SDS-polyacrylamide gel electrophoresis of the phage. Most of the subunit proteins were different from those of F-type pyocins, but the molecular weights of minor subunit proteins P3 and P6 seemed to be the same as those of band 1 and band 5 of F-type pyocins, respectively.
The head of the phage appeared to have an icosahedral structure, approximately 63 nm in diameter, with a long (190 nm, 11 nm wide and about 45 striations) flexuous tail connected to a fiber structure (about 53 nm in length). The density in CsCI and the sedimentation coefficient of the phage were 1.54g/ml and 392S, respectively. Some other biochemical properties were described.
The nucleic acid of the phage was linear, double stranded DNA of molecular weight 4×107. The density of the DNA in CsCI was 1.719g/ml, the melting temperature was 95.4°C. The guanine plus cytosine content was calculated to be 60 to 64%.View full abstractDownload PDF (2624K) -
Makoto NISHIKATA1983 Volume 93 Issue 1 Pages 73-79
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA Sepharose derivative coupled with a chymostatin analogue, Gly-Gly-L-Leu-L-phenylalaninal (Pheal), was prepared. A number of native and chemically modified proteases were applied on a column of the adsorbent. Bovine chymotrypsins [EC 3. 4. 21. 1] and Streptomyces griseus protease B were adsorbed strongly at pH 8.2. The affinities of these enzymes under various conditions were measured quantitatively by frontal chromatography in terms of the dissociation constant (Kd) of the enzyme-immobilized ligand complex. The pH dependence of the Kd value of α-chymotrypsin was consistent with that of the inhibition constant (K1) of the enzyme for a corresponding soluble peptide aldehyde. Anhydro-chymotrypsin, in which the active site Ser-195 is converted to dehydroalanine, was not adsorbed. Ser-195 proved to be essential for the binding. The frontal chromatography method also gave the amount of the immobilized ligand that can interact with the enzyme. It was extremely small compared with the amount of the immobilized ligand determined by amino acid analysis. This was explained on the basis of the structural features of the agarose gel.View full abstractDownload PDF (428K) -
Toshihiro AIUCHI, Makoto ARAI, Kazuyasu NAKAYA, Yasuharu NAKAMURA1983 Volume 93 Issue 1 Pages 81-86
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe sulfate transport in AH-66 hepatoma ascites cells was examined under various controlled conditions using 35SO42- as a tracer. The sulfate efflux rate was dependent on temperature, pH and anion species of the cell suspending medium. The efflux rate became saturated as the concentration of extracellular anions was increased. The efflux of anion was inhibited by some chemical reagents specifically reactive with amino or sulfhydryl groups. The results obtained in this study suggest that sulfate anions were transported by a facilitated transport system(s), and that some membrane protein(s) is involved in the anion transport system(s) of AH-66 cells. Both amino and sulfhydryl groups are thought to play a determinant role at the sulfate transport site in AH-66 cells.View full abstractDownload PDF (366K) -
II. Purification, Characterization and Interactions with Microtubules and Ca-CalmodulinShin-ichi HISANAGA, Hikoichi SAKAI1983 Volume 93 Issue 1 Pages 87-98
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPurification of cytoplasmic dynein from unfertilized sea urchin eggs was performed in the presence of protease inhibitors to avoid proteolysis throughout the purification procedure, which comprised several chromatographies, including a calmodulin-Sepharose 4 B affinity column chromatography. This is the first report of the purification of cytoplasmic dynein to near homogeneity. The purified fraction was composed of a single high molecular weight polypeptide and some minor low molecular weight polypeptides. The high molecular weight polypeptide comigrated with flagellar dynein Aβ chain from sperm on SDS-polyacrylamide gels. There was no polypeptide stainable with periodic acid-Schiff reagent (PAS) in the purified cytoplasmic dynein fraction. Cytoplasmic dynein showed characteristics quite similar to those of axonemal dynein, i.e. high substrate specificity for ATP and inhibition by low concentrations of vanadate, though its Ca-ATPase activity showed almost the same dependence on the concentration of either divalent cations or KCl as the Mg-ATPase activity. The purified enzyme seemed to possess functional form as judged from its properties: 1) pH dependence of the ATPase activity, 2) dependence of the ATPase activity on MgCI2 and KCl concentration, 3) Km for Mg-ATP, and 4) binding to flagellar doublet microtubules. Cytoplasmic dynein bound to calmodulin-Sepharose 4B only in the presence of Ca2+ and was eluted with EGTA. Furthermore, the ATPase activity was enhanced 6-fold by calmodulin in a Ca2+-dependent manner. The activation by calmodulin was prevented by a stoichiometric amount of trifluoperazine.View full abstractDownload PDF (2348K) -
Kihachiro HORIIKE, Hiromasa TOJO, Toshio YAMANO, Mitsuhiro NOZAKI1983 Volume 93 Issue 1 Pages 99-106
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrom a comparison of the gel chromatographic properties of large randomly-coiled polypeptides in 6M guanidine hydrochloride and of large globular proteins, we found that the distribution coefficient was more closely correlated with the intrinsic viscosity-based Stokes radius than with the translational frictional coefficient-based Stokes radius. This means that the effect of the hydrodynamic flow of dissolved molecules during gel chromatography should be considered. The ratio of transport of solute by bulk flow as compared with that by net diffusion (i.e., Brownian motion) is large under some conditions. On the other hand, we consider that the distribution coefficient obtained in static equilibrium experiments should be determined by the translational frictional coefficient-based Stokes radius, since the solvent does not flow. On this basis, we discuss the meaning of the Stokes radius and the separation mechanism of macromolecules by gel filtration.View full abstractDownload PDF (523K) -
Yasuyuki ISHII, Toshiharu HASE, Yoshihiro FUKUMORI, Hiroshi MATSUBARA, ...1983 Volume 93 Issue 1 Pages 107-119
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe methylamine dehydrogenase from Pseudomonas AM1 is a tetramer composed of two subunits, light(L)- and heavy-subunits. The amino acid sequence of the L-subunit was determined by sequence analyses of trypsin, chymotrypsin, staphylococcal protease, and thermolysin peptides of Cm-protein. The subunit consisted of a single polypeptide chain of 129 amino acid residues, with alanine and serine at the amino(N)- and carboxyl(C)-terminus, respectively. Yellow-colored peptides containing a prosthetic group were composed of two polypeptide chains and the prosthetic group was covalently bound to two residues at positions 55 and 106, which could not be identified yet. The molecular weight of the subunit was 13, 500 excluding the binding residues and the prosthetic group. Various structural features are discussed.View full abstractDownload PDF (841K) -
Atsushi IKAI, Toshihiro KITAMOTO, Masaaki NISHIGAI1983 Volume 93 Issue 1 Pages 121-127
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA high molecular weight protease inhibitor was purified from the egg white of Cuban crocodile (Crocodylus rhombifer). It inhibited the casein hydrolyzing activity of trypsin, subtilisin and papain. Its native molecular weight was 730, 000 and it consisted of four subunits of equal molecular weight, each pair of which were disul-fide bonded. The amino acid composition, circular dichroic spectrum and electron micrographs of this protein are also presented. Upon incubation with trypsin this protein yielded a fragment of Mr=80, 000, similar in size to the one known to originate from α2-macroglobulin under the same conditions.
The molecular parameters of this protein and the broad inhibitory activity towards thiol and serine proteases with different substrate specificities suggest that it is a protein closely related to a macroglobulin in mammalian serum. From its native molecular weight and amino acid composition we believe that this protein is also a reptilian counterpart of the avian ovomacroglobulin described by Miller and Feeney (3).View full abstractDownload PDF (1357K) -
Toyopearl HW-65C: Ammonium Sulfate as a New Column Chromatographic Adsorbent for Enzyme PurificationNaoko SAKIHAMA, Hideki OHMORI, Nobuko SUGIMOTO, Yohsuke YAMASAKI, Reik ...1983 Volume 93 Issue 1 Pages 129-134
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe found that Toyopearl HW-65C gel matrix adsorbed ferredoxin and ferredoxin-NADP+ reductase in the presence of concentrated ammonium sulfate. Ferredoxin was strongly adsorbed on the gel in 80% saturated ammonium sulfate, and ferre-doxin-NADP+ reductase was adsorbed in 40% saturated ammonium sulfate. The phenomenon was utilized for purification of ferredoxin and the reductase on a Toyopearl HW-65C: ammonium sulfate column. The technique greatly simplified the early stage of purification of ferredoxin and the reductase. The improved purification methods further involved column treatments with DEAE-Toyopearl 650M and Matrex Red A. The effectiveness of the columns is reported.
Since a number of other proteins such as cytochrome c, myoglobin, chymotryp-sinogen A, ovalbumin, and glucose oxidase were also adsorbed well in an appro-priately concentrated ammonium sulfate solution, the method may be of general use in enzyme purification.View full abstractDownload PDF (373K) -
Katsuko YAMASHITA, Yoko TACHIBANA, Hitoshi SHICHI, Akira KOBATA1983 Volume 93 Issue 1 Pages 135-147
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe carbohydrate moieties of γ-glutamyltranspeptidase (γ-GTP) purified from bovine kidney were chemically released as oligosaccharides. The oligosaccharide mixture was fractionated into a neutral fraction and three groups of acidic fractions containing one, two, and three sialic acid residues. Both the neutral fraction and the neutral oligosaccharide mixture obtained from the pooled sample of all acidic fractions by sialidase treatment were separated into seven components by Bio-Gel P-4 column chromatography. Structural studies of these components by sequential exoglyco-sidase digestion in combination with methylation analysis indicated that the γ-GTP contains at least twelve neutral sugar chains which are either the high mannose type, or the bi- and triantennary complex type, and thirteen acidic sugar chains of the bi-, tri-, and tetraantennary complex type.
The characteristic feature of the sugar chains of the γ-GTP is that most of the complex type sugar chains contain an N-acetylglucosamine residue at the C-4 position of the β-mannosyl residue of their trimannosyl core, and their outer chain moieties are enriched in non-reducing terminal β-N-acetylglucosamine residues.View full abstractDownload PDF (786K) -
Shigeo NAKAJO, Kumiko HAYASHI, Kazuyasu NAKAYA, Yasuharu NAKAMURA1983 Volume 93 Issue 1 Pages 149-157
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have demonstrated the identity of calmodulin tightly bound to the particulate fractions of AH-66 hepatoma cells and normal liver with authentic soluble calmodulin and have compared the particulate calmodulin content of AH-66 cells with that of normal liver. Calmodulin bound to the particulate fractions of the hepatoma and normal liver cells was purified to electrophoretic homogeneity by a simple procedure which involves solubilization of the particulate fractions with LIS, extraction of the solubilized solution with 37.5% phenol, gel filtration, and affinity chromatography on Fluphenazine-Sepharose. There were no detectable differences between the particulate calmodulin thus purified and authentic soluble calmodulin of rat brain. The particulate calmodulin in the hepatoma and normal liver cells was assayed based on its ability to activate calmodulin-deficient phosphodiesterase after partial purification of calmodulin from the particulate fractions by solubilization with LIS and extraction with phenol as described above. In addition, the particulate calmodulin content in the hepatoma and normal liver cells was also measured after solubilization of the particulate fractions with Lubrol PX. The results obtained by these two different procedures indicate that calmodulin content in the particulate fraction as well as in the soluble fraction of the hepatoma is significantly higher than that in the corresponding fractions of normal liver.View full abstractDownload PDF (1169K) -
Koichiro TANAKA, Tomisaburo KAKUNO, Jinpei YAMASHITA, Takekazu HORIO1983 Volume 93 Issue 1 Pages 159-167
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The chlorophyllase [EC 3.1.1.14] purified from greened rye seedlings hydrolyzed the bacteriochlorophyll isolated from Rhodospirillum rubrum, but not the pigment bound to the membrane of chromatophores or spheroplasts from the bacterium.
2. Acetone, if added at such concentrations that the bound bacteriochlorophyll would not be solubilized, enabled the enzyme to hydrolyze the bound pigment. The acetone concentrations required for half the maximum hydrolysis rates were 16% with chromatophores and 7% with spheroplasts.
3. The enzymic hydrolysis of the bound bacteriochlorophyll in the presence of acetone removed bacteriochlorophyllide from the membrane, leaving its esterifying alcohol, possibly all-trans-geranylgeraniol, in situ.
4. Washing of chromatophores with 30% o acetone removed about 10% of the bound bacteriochlorophyll. The bound pigment remaining after washing was not hydrolyzed by the enzyme unless acetone was added.
5. It seems possible that light-harvesting bacteriochlorophyll was mostly, if not all, bound to the inner surface of chromatophores (the outer surface of spheroplasts), having its esterifying alcohol residue buried in the membrane and its porphyrin residue emerging from the membrane into the inside solution; thus, chlorophyllase could not make contact with the ester linkage between the esterifying alcohol and porphyrin moieties of the pigment unless the esterifying alcohol residue was partly exposed.View full abstractDownload PDF (1161K) -
Hirofumi SHOUN, Kei ARIMA, Teruhiko BEPPU1983 Volume 93 Issue 1 Pages 169-176
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCertain anions were found to inhibit p-hydroxybenzoate hydroxylase from Pseudomonas desmolytica. The inhibition was of competitive or mixed type with respect to NADPH (apparent K1=4-30mm). Among the anions, monovalent anions such as halogen ions and azide inhibited ionization of the phenolic hydroxyl group of the substrate (p-hydroxybenzoate) on binding with the enzyme•substrate complex of p-hydroxybenzoate hydroxylase, without dissociating the substrate from the enzyme. On the other hand, multivalent anions (anions of polybasic acids), such as inorganic phosphate, borate, and sulfate, did not inhibit the ionization. Halogen ions induced remarkable spectral changes in the FAD moiety of the enzyme on binding, while the change due to inorganic phosphate was only slight. Chloride inhibited the binding of NADH with the enzyme as well as that of NADPH, whereas borate inhibited the binding of only NADPH. These results indicate that the monovalent and multivalent anions probably bind to the sites in the enzyme which interact, respectively, with the pyrophosphate and 2'-phosphate moieties of NADPH. The results provide strong support for the catalytic mechanism in which the phenolate anion of p-hydroxybenzoate participates in the process of substrate hydroxylation by C (4 a) peroxyflavin. The results also suggest that repeated ionization/ neutralization of the phenolic hydroxyl group of the substrate may occur during one cycle of the catalytic turnover.View full abstractDownload PDF (461K) -
Kazuko HORI, Masayuki KANDA, Setsuko MIURA, Yoko YAMADA, Yoshitaka SAI ...1983 Volume 93 Issue 1 Pages 177-188
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe transfer of phenylalanine from gramicidin S synthetase 1 (GS 1) to gramicidin S synthetase 2 (GS 2) was studied by the use of combinations of wild-type GS 1 with various GS 2s from a wild strain and gramicidin S non-producing mutant strains of Bacillus brevis Nagano. The combinations of mutant GS 2 s lacking 4'-phosphopantetheine (from BI-4, C-3, E-1, and E-2) did not transfer D-phenyl-alanine from GS 1, although they could activate all the constituent amino acids. Other mutant GS 2 s containing 4'-phosphopantetheine, except GS 2 from BII-3 (proline-activation lacking) accepted D-phenylalanine from intact GS 1.
To ascertain more directly whether 4'-phosphopantetheine is involved in the transfer of D-phenylalanine from GS 1 to GS 2, pepsin digests of GS 2 that accepted [14C]phenylalanine were analyzed by Sephadex G-50 column chromatography and thin-layer chromatography (TLC). Radioactivity of [14C]phenylalanine was always associated with a peptide containing 4'-phosphopantetheine. Furthermore, the position of radioactivity was distinct from the position of 4'-phosphopantetheine on TLC after alkaline treatment or performic acid oxidation of the digests.View full abstractDownload PDF (2433K) -
Shigeru FUJII, Tadao HASHIMOTO, Yukuo YOSHIDA, Retsu MIURA, Toshio YAM ...1983 Volume 93 Issue 1 Pages 189-196
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ATPase inhibitor from yeast, Saccharomyces cerevisiae, was found to strongly inhibit the ATPase activity when it was preincubated in an acidic pH region. This paper describes the conformational changes of the inhibitor in the acidic pH region, as studied by 1H NMR spectroscopy. In the pH range from 7.43 to 4.48, two conformations were detected. With lowering of the pH in this pH region, one conformation increased at the expense of the other. In the two conformations, His 39 had different pKa values which were determined at 23°C to be 6.08±0.04 and 5.91±0.03 from the pH-titration curves of His 39. The exchange between the two conformations was reversible with pH and the rate of the conformational change was estimated to be slower than 22 s-1 at 23°C as estimated by the line-widths (7.0 Hz) of the peaks from the C2-proton of His 39 in the two conformations. The equilibrium constant between the two conformations was dependent on the temperature. From the equilibrium constants in the temperature range from 23 to 45°C, the apparent ΔHa' of the conformational change was calculated to be 14 kcal/mol at pH 6.95. It was confirmed by 1H NMR spectra that at pH 6.95 the structure of the inhibitor was reversible with temperature at least below 80°C. The reversibility is consistent with the high thermal stability of the protein.View full abstractDownload PDF (495K) -
Mieko OGURO, Hiroshi NAGANO1983 Volume 93 Issue 1 Pages 197-203
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe succeeded in reconstituting the endogenous nuclear DNA synthesis of the sea urchin. Endogenous DNA synthesis of isolated nuclei was reconstituted by mixing the salt-treated nuclei (chromatin exhibiting essentially no endogenous DNA synthesis) and the salt extract containing DNA polymerase-a. DNA synthesis in this reconstitution system showed a level of activity and a mode of inhibition by aphidicolin similar to those of the original isolated nuclei (noncompetitive with respect to dCTP). On the other hand, the inhibitory mode was competitive with respect to dCTP in DNA synthesis in the reconstituted system obtained from the chromatin and purified DNA polymerase-a, indicating that some other factor(s) in addition to DNA polymerase-a is necessary for the reconstitution with reference to the inhibitory mode of aphidicolin.
We also studied the template activity of the chromatin. When chromatin was used as a template, inhibition by aphidicolin of DNA polymerase-a was noncom-petitive and uncompetitive with respect to the template at high and low concentrations, respectively. Treatment of chromatin with 5M urea gave urea-treated chromatin (nonhistone protein-deprived chromatin) and the extract (mainly nonhistone protein fraction). Inhibition by aphidicolin of DNA polymerase-a was uncompetitive with respect to the urea-treated chromatin. However, when chromatin reconstituted from the urea-treated chromatin and the extract was used as a template, the inhibitory mode by aphidicolin was similar to that with original chromatin, indicating that the nonhistone protein fraction contained factor(s) which modified the inhibitory mode of aphidicolin. Thus, the inhibitory mode of aphidicolin is a useful parameter for monitoring the resolution and reconstitution of endogenous DNA synthesis of isolated nuclei.View full abstractDownload PDF (413K) -
Satoshi OGIHARA, Mitsuo IKEBE, Koui TAKAHASHI, Yuji TONOMURA1983 Volume 93 Issue 1 Pages 205-223
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn an attempt to elucidate the Ca2+-regulated mechanism of motility in Physarum plasmodia, we improved the preparation method for myosin B and pure myosin. The obtained results are as follows:
1. We obtained two types of myosin B which are distinguishable from each other with respect to their sensitivity to Ca2+. The inactive type of myosin B had low superprecipitation activities both in the presence and in the absence of Ca2+. The active type showed very high superprecipitation activity in EGTA, and the activity was conspicuously inhibited by Ca2+. The active type was converted into the inactive type by treatment with potato acid phosphatase. Also the inactive type or the phosphatase-treated active type was converted into the active type upon reacting with ATP-γ-S.
2. In the reaction with ATP-γ-S, only the myosin HC of myosin B was phospho-rylated. The phosphorylation was independent of Ca2+ and calmodulin, and the extent was about 1 mol/mol HC.
3. The Ca2+ sensitivity in the superprecipitation of the active type was not decreased by adding an excess amount of F-actin. Besides, the actin-activated Mg2+-ATPase activity of purified phosphorylated myosin was not Ca2+-Sensitive. Therefore, presence of a Ca2+-dependent inhibitory factor(s) that could bind to myosin was suggested.
4. The Mg2+-ATPase activity of purified phosphorylated myosin was 7-8 times enhanced by F-actin, but that of dephosphorylated myosin was hardly activated at all.
5. In a gel filtration in 0.5M KCl, phosphorylated myosin was eluted behind dephosphorylated myosin. Electron microscopy applying the rotary-shadow method showed significant difference in flexibility in the tail between phosphorylated and dephosphorylated myosin molecules.
6. In 40mM KCl and 5-10mM MgCl2, phosphorylated myosin formed thick filaments, but dephosphorylated myosin did not, whether there was ATP or not.
The above results clearly show that the phosphorylation of myosin HC is indispensable to ATP-induced superprecipitation, the actin-activated Mg2+-ATPase activity, and the formation of thick filaments of myosin. A myosin-linked factor(s) that inhibits an actin-myosin interaction in a Ca2+-dependent manner may exist.View full abstractDownload PDF (2985K) -
Mieko OSHIMA, Yumiko OSAWA1983 Volume 93 Issue 1 Pages 225-234
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOuter and inner (cytoplasmic) membranes were partially purified from the gram negative extremely thermophilic bacteria, Thermus thermophilus HB-8 by sucrose density gradient centrifugation. In spite of our efforts to separate them, the inner membrane fraction contained some outer membrane components as determined by enzyme assay and electrophoresis.
When studied by 5DS spin labeling, the outer membranes showed a larger 2T11 value (lower fluidity) than the inner membranes, although the fatty acid compositions were similar. The inner membranes of the cells cultured at higher temperature showed a larger 2T11 value than the cells cultured at lower temperature. A similar phenomenon was observed with the TEMPO parameter of liposomal membranes. The upper break point (Th) of the inner membranes observed by spin labeling was slightly lower than the culture temperature of the cells, and the lower break point (T1) corresponded well to the lowest temperature limit of growth.
The calorimetric heating curve of the inner membranes had a broader temperature range of transition than that of the liposomal membranes. The transition temperature observed by calorimetry seems to reflect the melting properties of the membrane lipids, while fatty acid spin probe probably reports the local environment of the membrane, which is more directly related to its bilogical function.View full abstractDownload PDF (1364K) -
Yoshiyuki KIKUNO, Hidenobu TAKAHASHI, Tomoji SUZUKI1983 Volume 93 Issue 1 Pages 235-241
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPlasma prekallikrein, a precursor protein of kallikrein [EC 3. 4. 21. 8], was highly purified from porcine plasma by chromatography on a DEAF-Sephadex A-50 column, followed by rechromatography on a DEAE-Sephadex A-50 column, chromatography on a CM-Sephadex C-50 column and affinity chromatography on a p-aminobenzamidine-ε-aminocaproic acid-Sepharose 4B column. By this procedure, 3.3mg of purified material was obtained from 1.6 liters of porcine plasma and about 240-fold purification was achieved from the first DEAE-Sephadex A-50 chromatography. The purified protein was found to give a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel disc electrophoresis. This preparation did not contain kallikrein, Factor XII (Hageman factor) of the blood coagulation system, high molecular weight (HMW) kininogen or plasma kininase. Thus, the material is presumed to be functionally pure.
The molecular weight of prekallikrein was estimated to be about 88, 000 by SDS-polyacrylamide gel electrophoresis, and prekallikrein consists of a single polypeptide chain. Activation of prekallikrein by trypsin [EC 3. 4. 21. 4] was found to involve the cleavage of a single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation.
This trypsin-activated kallikrein released kinin rapidly from bovine HMW kininogen. However, liberation of kinin was extremely slow from bovine low molecular weight (LMW) kininogen. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI) and Trasylolt, but not by Polybrene® or egg-white trypsin inhibitor (EWTI).View full abstractDownload PDF (1073K) -
Masaaki KODAMA, Tamao NOGUCHI, Junichi MARUYAMA, Takehiko OGATA, Kaneh ...1983 Volume 93 Issue 1 Pages 243-247
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA nontoxic high-molecular fraction was separated from the extracts of toxic liver of a puffer, Takifugu poecilonotus, by Sephadex G-50 gel filtration. This fraction became toxic when digested with RNase T2. The toxins were partially purified by activated charcoal treatment, followed by chromatography on Bio-Gel P-2 and Bio-Rex 70, and were analyzed by TLC and electrophoresis. The results showed that most of the toxicity is accounted for by tetrodotoxin, and the remainder by saxitoxin and other unidentified toxins. The corresponding high-molecular fraction separated from nontoxic liver of another puffer, T. rubripes, did not release any toxin on RNase digestion.View full abstractDownload PDF (276K) -
Receptor-Mediated Introduction of Pepstatin-Asialofetuin Conjugate into Lysosomes of Rat HepatocytesKoji FURUNO, Nahoko MIWA, Keitaro KATO1983 Volume 93 Issue 1 Pages 249-256
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.View full abstractDownload PDF (497K) -
Akira HACHIMORI, Toshihiro FUJII, Kosuke OHKI, Eisaku IIZUKA1983 Volume 93 Issue 1 Pages 257-264
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInorganic pyrophosphatase [EC 3. 6. 1. 1] was purified from porcine brain to an electrophoretically homogeneous state. The molecular weight of the enzyme was estimated to be 62, 000 by gel filtration and that of the subunit to be 33, 000 by gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the enzyme consists of two identical subunits. The stability of the purified enzyme was dependent on its protein concentration. The enzyme was stable above 50 μg/ml at 20°C, but it was gradually inactivated below this concentration, even at 0°C unless other proteins such as bovine serum albumin, calmodulin, etc. were present. Those added proteins not only protected the enzyme from inactivation, but also completely reactivated the enzyme after it had been once inactivated. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate but not that of other phosphate esters. Only Mg2+ was required as an activating cation, and other divalent cations inhibited the activity to some degree. The addition of sulfhydryl reagents prevented the inhibition of activity by divalent cations.View full abstractDownload PDF (958K) -
Saori TAKAHASHI, Tamiko OHSAWA, Retsu MIURA, Yoshihiro MIYAKE1983 Volume 93 Issue 1 Pages 265-274
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe high molecular weight (HMW) renin was purified from porcine kidney by a procedure involving extraction with a buffer system containing protease inhibitors, ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B column chromatography, gel filtration on Ultrogel AcA 44 and aminohexyl-Sepharose 4B column chromatography. The resulting preparation showed a single band on isoelectric focusing, exhibiting an isoelectric point at pH 5.25, and was stable on storage at -80°C for 4 months. The specific activity was 3.97mg of angiotensin I formed/mg of protein per h at 37°C and at pH 6.5 with porcine angiotensinogen as the substrate. When the HMW renin was exposed to acid, renin activity increased by about 5-fold and the free form of fully active renin was recovered from the acidified HMW renin, leaving an insoluble aggregate of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the HMW renin showed two protein bands, of which one was identified as renin from the electrophoretic mobility and the other was the protein, assigned as renin binding protein (RnBP), that was insolubilized by acidification. The purified HMW renin is a complex of renin with RnBP, and the molecular weights of RnBP and renin in the HMW renin were estimated to be 39, 000 and 32, 000, respectively, by gel permeation liquid chromatography in 6M guanidine-HCl. A modified rapid method for purification of renin is also presented.View full abstractDownload PDF (2104K) -
Yoshinori HAMAI, Sigeru KUNO1983 Volume 93 Issue 1 Pages 275-279
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA kinetic analysis of cyclic 3', 5'-adenosine monophosphate (cAMP) synthesis in an adenine auxotroph of Escherichia coli 3000 was made by assaying the incorporation of [3H]adenine into cAMP during exponential growth. The rate of increase in intracellular [3H]cAMP was very slow (0.1-0.2 pmol/min/DU660). The steady state level was attained at about 40-min incubation after the addition of [3H]adenine, and was estimated to be 5 to 7 pmol/DU660. The rate and level of intracellular cAMP were scarcely affected by growth conditions, such as change of carbon source, whereas the excretion of cAMP into the medium began immediately after the addition of [3H]adenine, and continued at a rate of 5 to 7 pmol/min/DU660 in the glycerol medium. The excretion rate decreased to 1.4 pmol/min/DU660 in the presence of glucose. These results are inconsistent with the view that the excretion rate is dependent on the intracellular concentration of cAMP. Although the decreased rate of cAMP synthesis in the presence of glucose accounts for the permanent catabolite repression of inducible enzyme systems, no immediate depression in cAMP synthesis, which might account for the transient repression, was found after the addition of glucose.View full abstractDownload PDF (301K) -
Ayanori YAMAKAWAI, Sigeru KUNO1983 Volume 93 Issue 1 Pages 281-286
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCatabolite repression of β-galactosidase synthesis in E. coli 3000A1 (adenine-) was studied under a variety of growth conditions. The differential rate of induced β-galactosidase synthesis was maximal at the growth rate of 0.75 division per h, irrespective of whether growth conditions were aerobic or anaerobic. The addition of cyclic AMP (CAMP) to the medium partly restored the repressed synthesis of β-galactosidase under some growth conditions, but showed little or no effect on the enzyme synthesis under other conditions. Although growth rate and profile of β-galactosidase synthesis in glucose-grown cells were similar to those in arabinosegrown cells, the acceleration of β-galactosidase synthesis upon the addition of cAMP was found only in glucose-grown cells. The cells aerobically grown in the presence of glycerol, xylose, or arabinose showed a high synthetic rate of cAMP and were insensitive to exogenously supplied cAMP as regards β-galactosidase synthesis. Although the cells grown with glucose showed similar rates of cAMP synthesis under aerobic and anaerobic conditions, the differential rate of β-galactosidase synthesis was much higher in the anaerobic state than in the aerobic state. These findings support the idea that catabolite repression found in the strain is caused through two mechanisms, i.e., cAMP-mediated and cAMP-independent ones.View full abstractDownload PDF (362K) -
Miwako NISHIZAWA, Kazushi TANABE, Akio MATSUKAGE, Hiromu NAKAMURA, Tat ...1983 Volume 93 Issue 1 Pages 287-290
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA DNA polymerase which was active with unprimed poly(dC) as a template was detected in the DNA polymerase u fraction from rat thymoma tissue. The activity was dependent on rGTP and associated with ribonucleotide polymerizing activity. The activity was partially sensitive to aphidicolin and dideoxy GTP and resistant to a-amanitin. Thus, this is a rat “replicase” which is very similar to that found in mouse cells. The replicase activity with poly(dT) or phage Id single stranded circular DNA was greatly enhanced by the mouse replicase-stimulating factor beyond the species difference.View full abstractDownload PDF (211K) -
Mitsushi INOMATA, Masami HAYASHI, Megumi NAKAMURA, Kazutomo IMAHORI, S ...1983 Volume 93 Issue 1 Pages 291-294
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOne form of calcium-activated neutral protease (CANP) highly sensitive to calcium ions was purified by column chromatographic procedures to homogeneity. The purified enzyme required μM order Ca2+ (μCANP), and the half-maximum activity was attained at 50 μM Ca2+. The electrophoretic mobility in a non-denaturing buffer showed that this enzyme is less acidic than another CANP which required mM order Ca2+ (mCANP). On SDS-polyacrylamide gel electrophoresis, the enzyme separated into two components with molecular weights of 79, 000 and 28, 000, respectively. Of these, the former was slightly larger than the counterpart of mCANP (Mr 76, 000). Thus, pCANP cannot be derived from mCANP by limited autolysis.View full abstractDownload PDF (1369K) -
Kazuho OZAKI, Hiroaki SUGINO, Takayuki HASEGAWA, Sho TAKAHASHI, Sadash ...1983 Volume 93 Issue 1 Pages 295-298
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protein that functionally resembles mammalian and Acanthamoeba profilins, has been purified from Physarum plasmodia. Physarum profilin consists of a single polypeptide with a molecular weight of 11, 000-13, 000. It has an isoelectric point of 5.35-5.40 under denaturing conditions. The amino acid composition of this protein is similar to those of profilins isolated from other sources. Physarum profilin prolongs the process of actin polymerization in a concentration-dependent fashion. This effect is much stronger for Physarum G-actin than for muscle G-actin.View full abstractDownload PDF (902K) -
Kiwako SAKABE, Kyoyu SASAKI, Noriyoshi SAKABE, Hiroshi KONDO, Takehiro ...1983 Volume 93 Issue 1 Pages 299-302
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structure of the chicken gizzard G-actin•DNase I complex has been determined at 5Å resolution by an X-ray diffraction method. Protein phases were computed by the multiple isomorphous replacement method using four heavy atom derivatives. The mean figure of merit was 0.65. Dimensions of the three molecular species, the complex, G-actin and DNase I, were determined based on the “cypress wood” models derived from the electron density map. The natures of the heavy atom binding sites are discussed in relation to the distinction between the two component molecules. The pattern of successive contacts between actin molecules observed in the present crystal seems unrelated to that found in F-actin.View full abstractDownload PDF (1538K) -
Tadaomi TAKENAWA, Tomoh MASAKI, Katsutoshi GOTO1983 Volume 93 Issue 1 Pages 303-306
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSurgical denervation of rat vas deferens causes supersensitivity in that the tissue sensitivity and the maximum response to a variety of agonists increase. To understand the molecular mechanism of supersensitivity in smooth muscle, norepinephrine(NE)-induced alteration in phospholipid metabolism was studied using control and denervated vasa deferentia. When the tissue was stimulated by NE, only [32P]P1 incorporation into phosphatidic acid(PA) was increased in proportion to the increase in NE concentration without any significant effect on that into other phospholipids. This PA labeling was significantly accelerated by denervation. In the denervated tissue, PA labeling was stimulated by lower concentrations of NE and the maximum response to NE was increased compared to the control. The breakdown of phos-phatidylinositol 4-monophosphate(DPI) and phosphatidylinositol 4, 5-diphosphate (TPI) was also accelerated by NE. But the influence of denervation on this NE-induced DPI and TPI was not marked. Therefore, it is likely that denervation clearly enhanced NE-induced PA labeling without an appreciable effect on that of the other phospholipids. Furthermore, the absolute amount of PA was also increased by NE, and this increase was exaggerated by denervation. Considering that PA can behave as a Ca2+ ionophore in the plasma membrane, these results suggest that the stimulated accumulation of PA plays an important role in receptor-linked supersensitivity in smooth muscle.View full abstractDownload PDF (809K) -
Eragam Shyam Prasad REDDY, Damodaran VIJAYARANGAM, Pushpa Mittra BHARG ...1983 Volume 93 Issue 1 Pages 307-310
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing RNAase SPL, a recently described nuclease from bovine seminal plasma, that can recognize certain Mg2+-dependent structural features in native naturally occurring RNAs, it is shown that these features are wholly or partially destroyed by 8M urea, 1mM spermine, 0.35M KCl or NaCl, and 10mM EDTA, urea and spermine being the most effective and EDTA the least; these features are not destroyed by heating and rapid cooling unless a medium with a high ionic strength is used. Since the buffer used so far for reconstitution of ribosomes contains 0.35M KCl, it seems possible that a partial opening up of ribosomal RNAs may be necessary for ribosome assembly.View full abstractDownload PDF (266K) -
Kanefusa KATO, Fujiko SUZUKI, Takashi NAKAJIMA1983 Volume 93 Issue 1 Pages 311-313
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSoluble S-100 protein levels in epididymal adipose tissue of male rats (1.3±0.1 μg/mg protein) were markedly decreased (to 0.15±0.06μg/mg protein) by serial injection of epinephrine (0.1mg/day for 9 days). But injection of insulin in a similar manner (4 U/day for 9 days) had little effect on the adipose S-100 protein levels (1.1±0.2 μg/mg protein). The S-100 protein levels in cerebrum, spleen, and adrenal gland were not affected by epinephrine or insulin administration. These results strongly suggested that S-100 protein levels in adipose tissue are regulated hormonally.View full abstractDownload PDF (139K) -
Shizuo HANDA, Yasunori KUSHI, Hideki KAMBARA, Kenichi SHIZUKUISHI1983 Volume 93 Issue 1 Pages 315-318
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSecondary ion mass spectra of underivatized neutral sphingoglycolipids are presented. In the spectra of mono- and di-glycosylceramide, ions (M+H)+ and (M+H-H2O)+ were observed as relatively intense quasimolecular ions, whereas in the spectra of higher glycolipids, the quasimolecular ion species were predominantly (M+Na)+. Ions due to the ceramide moiety were observed as intense peaks comparable to quasimolecular ions. Ions derived from the fragments cleaved at the glycosidic linkages were hardly detected due to their low intensities. In general, secondary ion mass spectrometry provides good stable spectra for a long time during analysis.View full abstractDownload PDF (180K) -
Masanobu ARITA, Masao IWAMORI, Tetsuo HIGUCHI, Yoshitaka NAGAI1983 Volume 93 Issue 1 Pages 319-322
Published: January 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnalysis of gangliosides from bovine brain was successfully performed by a newly developed method of fast atom bombardment mass spectrometry (FAB-MS). The use of triethanolamine with a few drops of 1, 1, 3, 3-tetramethylurea as the matrix solution gave intense molecular ions of intact gangliosides tested, namely, GM1 and GD1a, corresponding to (M+Na)+ and (M+2Na-H)+. Glycerol, that is usually used as a matrix solution for FAB-mass spectrometry, was not suitable for the analysis of gangliosides. Along with the molecular ion species, the ions pertaining to the carbohydrate sequence also appeared on the spectrum. The method was found to be useful for structural analysis using intact molecules of gangliosides and neutral glycosphingolipids.View full abstractDownload PDF (187K)
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