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Volume 104 (1988)
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- Issue 2 Pages 159-
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Volume 104, Issue 2
Displaying 1-32 of 32 articles from this issue
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Kimimitsu Oda, Yukio Ikehara1988 Volume 104 Issue 2 Pages 159-161
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have found a proteolytic activity in Golgi membranes which efficiently converts [35S]methionine-labeled proalbumin, isolated from pulse-labeled rat hepatocytes in culture, to serum albumin in an in vitro assay system. The proalbumin-convertingactivity was dependent on Ca2+ and the maximum activity was observed at pH 5.5-6.0. Since the enzyme activity was found to be resistant not only to both leupeptin and E-64 but also to thiol-blocking reagents, it is unlikely that cathepsinB is involved in the proteolytic conversion of proalbumin occurring in the Golgi complex.View full abstractDownload PDF (1373K) -
Hiromitsu Okano, Toshivasu Endo, Satoshi Shiokawa, Yasuyo Kyoden, Yuki ...1988 Volume 104 Issue 2 Pages 162-164
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe present evidence for the continuous erythroid differentiation of a cell line, KU-812-F, without the addition of an inducer. Erythroid differentiation of these cells wasconfirmed according to the following findings: 1) erythroid morphology; 2) existence of glycophorin A and carbonic anhydrase I as a membrane marker protein and an enzyme characteristic of erythroid cells, respectively; 3) synthesis of adult type (HbA and HbA2) and fetal type (HbF) hemoglobins, as detected on isoelectric focusing; and 4) transcription of the α-, β-, and γ-globin genes, as detected on Northern blot analysis.View full abstractDownload PDF (1358K) -
Kazutoshi Nakazawa, Takao Ando, Teruyuki Kimura, Hisashi Narimatsu1988 Volume 104 Issue 2 Pages 165-168
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA full-length cDNA clone for mouse N-acetylglucosamine (β1-4)galactosyltransferase (β1-4GT) [EC 2.4.1.90] and several clones diverged from the β1-4GT cDNA were isolated from a mouse F9 cDNA library and then sequenced. The β1-4GT cDNA has an open reading frame consisting of 399 amino acids. The homology at the amino acid level is 80 and 91% as to the partial sequences of bovine and human milk β1-4GT, respectively. The general enzyme structure of the β1-4GT seems to be similar to that of a rat β-galactoside (α2-6) sialyltransferase. Junctions of the common and divergent regions of cDNA have dinucleotides, AG, suggesting that the variety of cDNA clones is generated through alternative splicing.View full abstractDownload PDF (1556K) -
Peng Wang, Satoshi Toyoshima, Toshiaki Osawa1988 Volume 104 Issue 2 Pages 169-172
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConcanavalin A (Con A) stimulation resulted in the rapid redistribution of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes. Thischange in GTP-binding activity was dependent on the Con A concentration. To investigate the relationship between this redistribution and phospholipase C (PLC) activity, the effect of GTPγS on the cytosol PLC activity was also examined, and it was found that GTPγS enhanced the phosphatidylinositol 4, 5-bisphosphate (PIP2) hydrolysis activity in the cytosol of Con A-stimulated thymocytes more than in that of unstimulated thymocytes. This enhancement by GTPγS was also dependent on the Con A concentration. The results suggest that in murine thymocytes, the GTP-binding protein (G-protein) involved in the regulation of PLC activity may be translocated from the membrane to the cytosol upon Con A stimulation. Besides, the dose dependence curve for the change in the GTPγS-binding activity was similar to that for inositol phosphates formation in Con A-stimulated thymocytes, suggesting that the translocation of the G-protein is closely related to PLC activation. Furthermore, the effects of cytosol fractions containing the 38-43 and 23-28 kDa GTP-binding subunits of G-proteins on the PIP2 hydrolysis activity of partially purified PLC were examined. The fraction containing the 23-28 kDa subunit evidently enhanced the PLC activity but that containing the 38-43 kDa subunit enhanced the activity to a much lower extent. Moreover, the 23-28 kDa subunit fraction of Con A-stimulated thymocytes was more effective as to enhancement of the PLC activity than that of unstimulated thymocytes. These results suggest that the GTP-binding subunit with an approximate molecular weight of 23-28 kDa may be translocated from the membrane to the cytosol and then Pn h n et. the evtosolic PLC activity upon Con A stimulation.View full abstractDownload PDF (504K) -
Yuji Ohyama, Ken-ichi Kasai1988 Volume 104 Issue 2 Pages 173-177
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVertebrate endogenous lectins have been implicated in cellular interactions that contribute to embryonic development. We have isolated a cloned segment of the gene for chick 14K type β-galactoside-binding lectin from a genomic DNA library. Analysis of the structure of the cloned gene as well as the results of genomic Southern blot hybridization revealed that the gene is unique and that the mRNA for the lectin is encoded by four exons separated by three introns. The whole sequence spans 3.1 kilobases in the gene. The first exon encodes only two amino acid residues of the N-terminus of the mature protein and the other three exons encode, respectively, one of the three repeating sequences found in this lectin. These facts strongly support the idea that gene duplications have occurred during the evolution of this lectin. The previous study (Y. Ohyama et al. (1986) Biochem. Biophys. Res. Commun. 134, 51-56) suggested that this lectin is not synthesized as a precursor molecule with a cleavable signal sequence at its amino terminus, although it is known to be secreted into the extracellular matrix. Sequence determination of the upstream region of the mRNAindicated that the ATG located just before the codon for the N-terminal amino acid of the mature protein is the actual translation initiator. Thus it was proved that this lectin is synthesized without an N-terminal cleavable signal sequence, as suggested before.View full abstractDownload PDF (1593K) -
Tsuneo Oda, Masanobu Tokushige1988 Volume 104 Issue 2 Pages 178-183
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTryptophanase purified from Escherichia coli B/1t7-A was irreversibly inactivated by chloramine T (sodium N-chloro-p-toluenesulfonamide). The mode of inactivation was rather complex and did not follow pseudo-first-order kinetics. The inactivation of the apoenzyme was much faster than that of the holoenzyme. The Km value for the synthetic substrate S-o-nitrophenyl-L-cysteine (SOPC) increased concomitantly with the modification. In contrast, the Km value for the coenzyme, pyridoxal 5'-phosphate (PLP), was not altered. L-Serine, another substrate, and L-alanine, a competitive inhibitor, protected the enzyme from inactivation. Determination of SH groups in the enzyme protein with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) showed that modification of two SH groups per enzyme subunit resulted in a complete inactivation. When the enzyme was subjected to chloramine T-modification following the SH group modification with DTNB, further inactivation was still observed, even after the addition of dithiothreitol. The SH-blocked enzyme preparation thus obtained, however, exhibited less pH dependency of inactivation by chloramine T than that of the native enzyme. The amino acid analysis of the chloramine T-modified enzyme showed that modification of four or five methionine residues among the 16 residues per subunit proceeded concomitantly with the complete inactivation. Modification of the enzyme with chloramine T quenched the absorption peak near 500 nm, characteristic of a quinoidal structure formed by labilization of the α-proton. These results suggest the possibility that chloramine T modifies not only the SH groups, but also methionine residues important for the catalytic activity of the enzyme.View full abstractDownload PDF (695K) -
Akihiro Yokoyama, Michio Murata, Yasukatsu Oshima, Takashi Iwashita, T ...1988 Volume 104 Issue 2 Pages 184-187
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMaitotoxion, a putative Ca2+ channel agonist, was isolated from cultures of the marine dinoflagellate Gambierdiscus toxicus as a colorless amorphous solid. The toxin reacted positively to Dragendorff's reagent but not to ninhydrin reagent. The mouse lethality of maitotoxin determined by intraperitoneal injection was 0.13μ/kg. Chemical features of the toxin were elucidated mainly by various spectroscopic measurements. The molecular weight of maitotoxin as a disodium salt was estimated to be 3, 424.5±0.5 from the negative fast atom bombardment (FAB) mass spectrum. The presence of two sulfate ester groups in the molecule was apparent from the IR and mass spectra, and from analyses of solvolysis products. Despite of its large size, maitotoxin seems to have no known repeating units, such as amino acids and sugars, no carbonyl groups, no side chains other than methyls or an exomethylenes, and no carbocycles.View full abstractDownload PDF (440K) -
Suguru Kawato, Fumiko Mitani, Tetsutaro Iizuka, Yuzuru Ishimura1988 Volume 104 Issue 2 Pages 188-191
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRotational diffusion of the total cytochrome P-450 (P-450scc plus P-45011β.) in bovine adrenocortical mitochondria was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the hemo • CO complex by a vertically polarized laser flash. Analysis of r (t) was based on a “rotation-about-membrane normal” model. The measurements were used to investigate intermolecular interactions of cytochrome P-450 with other membrane proteins. The absorption anisotropy decayed within 1 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the presence and absence of deoxycorticosterone (DOC), a substrate for cytochrome P-450 11β. The observed value for the normalized time-independent anisotropy r(∞)/ r (∞) and the average rotational relaxa-tion time φ are r(∞)/ r(0)=0.88 and Φ=233 μ s when DOC is absent, and r(∞)/ r(0)=0.65 and φ=350μS when DOC is present. Judging from the φ value, rotating P-450 is not a monomeric molecule, but would be a small microaggregate with an average diameter of about 120 Å. A significantly high value of r(∞)/r (0) implies co-existence of mobile and immobile populations of cytochrome P-450. Based on the assumption that the heme angle tilts 55° from the membrane plane (Gut et al. (1983) J. Biol. Chem. 258, 8588-8594), 65% (when DOC is present) or 88% (when DOC is absent) of cytochrome P-450 in mitochondria is immobilized within the experimental time range of 2 ms due to the presence of immobile protein microaggregates. The present observation suggests that binding of DOC to cyto-chrome P-45011β induces a significant mobilization of cytochrome P-450 molecules, pos-sibly due to changes in protein-protein interactions with its redox partners i.e. adre-nodoxin and adrenodoxin reductase.View full abstractDownload PDF (548K) -
Mitsuyoshi Motizuki, Hideki Kohno, Kunio Tsurugi1988 Volume 104 Issue 2 Pages 192-195
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing mutant strain ABYS1 of Saccharomyces cerevisiae lacking four main vacuolar proteinases, proteinase A, proteinase B, carboxypeptidase Y, and carboxypeptidase S, we examined the identities of chromatin-associated proteinases, ruling out possible contamination of the chromatin fraction by them. The chromatin of strain ABYS1 showed three peaks of proteolytic activity at pH 4, 7, and 11, and these activities were found to be derived from three species of proteinases, the aspartic, serine neutral, and serine alkaline ones. As these chromatin-associated proteinases of strain ABYS1 were identical in both quality And quantity to those of wild-type strain of yeast, we suggest that the yeast chromatin contains three species of specific proteinases as essential components.View full abstractDownload PDF (495K) -
Naoko Okawara, Mari Ogata, Tatsuhiko Yagi, Sadao Wakabayashi, Hiroshi ...1988 Volume 104 Issue 2 Pages 196-199
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of ferredoxin (Fd) I, purified from Desulfovibrio vulgaris Miyazaki, has been established. Fd I is strikingly similar to Fd III ofD. africanus Benghazi with 84% homology. Both have the sequence, -Cys-x-x-Asp-x-x-Cys-x-x-x-Cys-Pro- in the N-terminal half, and the sequence, -Cys-x-x-Cys-x-x-Cys-x-x-x-Cys-Glu- in the C-terminal half of the molecule, instead of the common sequences for ligation to the usual [4Fe-4S] clusters. Fd I has 76% homology to Fd I of D. desulfuricans Norway.View full abstractDownload PDF (420K) -
Tatsuya Sueyoshi, Akihiro Hara, Toshio Shimada, Michio Kimura, Takashi ...1988 Volume 104 Issue 2 Pages 200-206
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular interaction of bovine kininogen and its derivatives with papain was investigated. High-molecular-weight kininogen (HMWK) or low-molecular-weight kininogen (LMWK) and inactive papain treated with N-[N-(L-3- trans-carboxyoxiran-2-carbonyl)-L-leucyl]agmatine (E-64) formed, respectively, a complex, which was dissocia-ble on sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE). The densitometric determination of the bands separated on SDS-PAGE and amino acid analysis of the samples extracted from the electrophoresis gel revealed that the complex between kininogen and papain is formed in a molar ratio of one to one. Moreover, analysis of the inhibition of the caseinolytic activity of papain by these kininogens indicated that HMWK, LMWK, and kinin-free derivatives obtained from both kininogens inhibit active papain with a stoichiometry of 1:1. On the other hand, the papain activity was inhibited by two kinds of cyanogen bromide fragments isolated from the heavy chain of HMWK. These two fragments with K1, values of 38 and 0.64 nM corresponded, respectively, to residue Nos. 47 intact HMWK and LMWK, one of the two potential reactive sites interacts with papain to form a complex and that the other reactive site becomes active only after separation of the two sites.View full abstractDownload PDF (2098K) -
Takao Ojima, Kiyoyoshi Nishita1988 Volume 104 Issue 2 Pages 207-210
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDepartment of Chemistry, Faculty of Fisheries, Hokkaido University Some biochemical properties of the Mr 52, 000 component of Akazara scallop striated adductor troponin, which had been tentatively identified as troponin-I, were compared with those of rabbit troponin-I. Both the Mr 52, 000 component and rabbit troponin-I together with rabbit tropomyosin inhibited the Mg-ATPase activity of rabbit reconstituted actomyosin to 1/10 of the original activity. The inhibition was neutralized by the addition of Akazara scallop and rabbit troponin-C or Patinopecten scallop calmodulin. The Mr 52, 000 component and rabbit troponin-I were insoluble below 0.15 M KC1, but were solubilized by complexing with an equimolar amount of troponin-C or calmodulin. On alkaline urea-polyacrylamide gel electrophoresis, the Mr 52, 000 component as well as rabbit troponin-I was found to form a stable complex with troponin-C or calmodulin in the presence of Ca2+.View full abstractDownload PDF (2586K) -
Shigeru Hokari, Masahiro Hasegawa, Masahiko Tanaka, Yoshikatsu Sakagis ...1988 Volume 104 Issue 2 Pages 211-214
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe distribution of deoxyuridine triphosphate nucleotidohydrolase (dUTPase) [EC 3.6.1. 23] in the cytosol of various rat tissues was investigated by measuring the enzyme activity and by immunochemical analyses. Among nine rat tissues, thymus, and spleen showed the highest activities of the enzyme per gram of tissue, while intestine, stomach, lung and liver showed very low levels. Rabbit antibodies directed against purified dUTPase of anemic rat spleen showed reactivity with partially purified dUTPases from other rat tissues such as thymus, testis, or regenerating liver. Immunotitration and immunoblot experiments also indicated that the dUTPases in various rat tissues had very similar antigenicity and apparently the same subunit molecular size (Mr=19, 500), suggesting that the enzyme lacks organ-specificity. Immunoblot analysis of dUTPase protein with crude extracts from various rat tissues showed a similar distribution to that of the enzyme activity. No immuno-reactive band corresponding to the dUTPase was detected in intestine, although intestinal mucosa has been recognized as an actively proliferating tissue.View full abstractDownload PDF (1726K) -
Naomi Rodrig, Taka Osanai, Masao Iwamori, Yoshitaka Nagai1988 Volume 104 Issue 2 Pages 215-219
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCultured MDCK cells were treated with various gangliosides and sialylated compounds and their effects on the intracellular level of cAMP were compared to those of arginine vasopressin (AVP) and other substances which elevate cAMP. Since all those agents could stimulate dome formation, its correlation with cAMP production is discussed. Most gangliosides increased intracellular cAMP 3-4-fold, the increase being dose-dependent up to 25 μM ganglioside. AVP and cAMP analogs increased intracellular cAMP 3-40-fold. A unique feature of the ganglioside-induced cAMP increase was its extremely long time course (70 h), as compared to that induced by other agents which show much faster and less prolonged effects in other biological systems. This might indicate that gangliosides differ from AVP and other agents in the mechanisms by which they stimulate intracellular cAMP increase. The time course and the level of cAMP increase induced by GM3 or AVP did not correlate with those of dome formation. Furthermore, the ability of some gangliosides and other agents to induce dome formation did not correspond to their ability to elevate cAMP. It is suggested that although the remarkable dome-stimulating activity of gangliosides may be induced in part by a cAMP-dependent mechanism, gangliosides also act directly on the cellular components influencing dome formation, without involving changes in intracel-lular cAMP.View full abstractDownload PDF (598K) -
Sachiko Abe, Yoko Watanabe, Shigeko Araki, Toshiro Kumanishi, Mei Sata ...1988 Volume 104 Issue 2 Pages 220-226
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAntiserum was raised against 3-O-MeGalβ1→3Ga1NAcαl→3[6'-O-(2-aminoethylphos. phonyl)Galαl-2(2-aminoethylphosphonyl→6)Galβl→4G1cβ1→1Ceramide(SGL-II)isolated from the skin of a mollusc, Aplysia kurodai. This antiserum reacted with SGL-II and other phosphonoglycosphingolipids of Aplysia such as SGL-I', F-21, and some minor glycolipids on TLC plates, but it did not react with ganglioside or globoside. The sugars recognized were 3-O-methylgalactose at the non-reducing end and galactose at the branched chain of the glycolipids. One membrane glycoprotein (Mr 280, 000) reacted strongly, and some other proteins reacted weakly with the antiserum. Immunohistochemical examina-tion of the nervous tissues revealed distinct staining in the periganglionic tissue of the ganglia, and the perineural sheath of the proximal portion of the peripheral nerves. The neuropil and satellite cells were also stained. In the skin, subcutaneous connective tissues were moderately stained, and the cytoplasm of small mononuclear cells and foamy cells was also stained. The staining patterns were essentially the same in paraffin and cryostat sections. From the findings with sections pretreated with chloroform-methanol (2:1, v/v), it was suggested that the periganglionic and perineural stainings were due to glycoproteins, including an SDS-soluble glycoprotein of Mr 280, 000, while those of the other regions were due to SGL-II and glycolipids immunologically related to SGL-II. The stainings in the skin sections were largely due to glycoproteins.View full abstractDownload PDF (5775K) -
Yasuzo Nishina, Hiromasa Tojo, Kiyoshi Shiga1988 Volume 104 Issue 2 Pages 227-231
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSResonance Raman (RR) spectra of the complex of anionic semiquinoid D-amino acid oxidase (DAO) with picolinate in H2O and D2O were observed in the 300-1, 750cm-1 region. RR spectra were also measured for the complex of the semiquinoid enzyme reconstituted with isotopically labeled FAD's, i. e., [4a-13C]-, [4, 10a-13C2]-, [2-13C]-, [5-15N]-, and [1, 3-15N2]-FAD. On the basis of the isotope effects, tentative assignments of the observed bands of the anionic semiquinoid flavin were made. The spectra differ from those of oxidized, neutral semiquinoid, and anionic reduced flavins previously reported. The 1, 602 cm-'band was not shifted for any FAD labeled in ring II and/or ring III and was assigned to a ring I mode. The 1, 516 cm-1 band underwent an isotopic shift upon [4a-13C]- or [4, 10a-13C2]-labeling. The band was assigned to the mode containing C(4a)-C(10a) stretching. The 1, 331 and 1, 292 cm-1 bands shifted upon [4a-13C]- or [5-15N]-labeling and were assigned to the modes containing C(4a)-N(5) stretching. The 1, 217 and 1, 188 cm-1 bands were assigned to the skeletal vibrations of ring III coupled with the N(3)-H bending mode. The RR spectrum of the complex of anionic semiquinoid DAO with α-iminopropionate or N-methyl-a-iminopropionate was essentially identical with that of the complex with picolinate.View full abstractDownload PDF (485K) -
Masao Miki, Cristobal G. dos Remedios1988 Volume 104 Issue 2 Pages 232-235
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe resonance energy transfer between fluorescein-5-isothiocyanate (FITC) attached to Lys-61 and Co2+ bound to the high-affinity metal binding site was measured. The distance between FITC and Co2+ on the actin molecule was calculated to be either 1.9nm, using the absorption spectrum of Co-EDTA or 2.8 nm, using the absorption spectrum of Co2+ bound to carboxypeptidase as a model spectrum of Co2+ bound to actin, respectively. The effects of the polymerization of actin and of the interaction of actin with myosin subfragment-1 (S1) on the solvent accessibility of the fluorescein molecule attached to Lys-61 or Cys-374 were measured. The accessibility of the probe at Lys-61 was reduced following polymerization and also appreciably reduced by interaction with Sl. The accessibility of the probe attached to Cys-374 was affected to only a small degree. These results indicate that the Lys-61 residue is located close to an actin-actin contact region as well as being close to an S1 binding site, although it is not directly involved [Miki, M. (1987) Eur. J. Biochem. 164, 228-235]. The accessibility of the probe at Lys-61 was also decreased by the addition of the tropomyosin-troponin complex, although the accessibility of the probe at Cys-374 was not affected at all. Thus, Lys-61 appears to be involved in the binding site of the regulatory proteins.View full abstractDownload PDF (517K) -
Yuji Kuwabara, Masafumi Maruyama, Yasuo Watanabe, Seiji Tanaka, Masayo ...1988 Volume 104 Issue 2 Pages 236-241
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembrane-bound phospholipase B was purified to a homogeneous state from Torulaspora delbrueckii cell homogenate. Cell homogenate was extracted with Triton X-100, and the enzyme was precipitated with acetone. The acetone powder was washed repeatedly with Tris-HCl buffer (pH 8.0) until no phospholipae B activity was detected in the soluble fraction. The enzyme was extracted with Triton X-100 from the final residue and purified about 1, 390-fold by sequential chromatofocusing, Sepharose 6B, and DEAE-Sephadex A-50 column chromatography. The final preparation showed a single broad protein band on SDS-polyacrylamide gel electrophoresis when stained with silver stain reagent and PAS-reagent. The molecular weight of phospholipase B was about 390, 000 and 140, 000-190, 000 as estimated by gel filtration on Sepharose 613 and SDS-polyacrylamide gel electrophoresis, respectively, suggesting that phospholipase B is an oligomeric protein. The isoelectric point was at pH 4.5. Phospholipase B has two pH optima, one acidic (pH 2.5-3.0) and the other alkaline (pH 7.2-8.0). At acidic pH the phospholipase B activity was greatly increased in the presence of divalent metal ions, although metal ions are not a factor for enzyme activity. On the other hand, at alkaline pH the enzyme required Ca2+ or Mn2+ for activity. The pH- and thermal-stabilities at both pHs were similar. The phospholipase B hydrolyzed all diacylphospholipids tested at acidic pH, but hydrolyzed only phosphatidyl-choline at alkaline pH. The hydrolysis rates of lysophospholipids were much higher (about 10-fold) than those of diacylphospholipids at both pHs.View full abstractDownload PDF (1563K) -
Kazuhiko Saeki, Sadao Wakabayashi, Walter G. Zumft, Hiroshi Matsubara1988 Volume 104 Issue 2 Pages 242-246
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete primary structure of Pseudomonas stutzeri strain ZoBell ferredoxin was determined by a combination of protease digestion, Edman degradation, and carboxypep- tidase digestion and was: TFV VTDNCIKCKYTDCVEVCPVDCFYEGPNFLVIHPDECIDCALCEPECPAQAIFSEDEVPEDQQEFIELNADLAEVWPNITE K K D A L A D A E E W D G V K D K L Q Y L E R. The calculated molecular weight was 12, 110 excluding iron and sulfur atoms. The amino acid sequence was highly homologous to those of Azotobacter vinelandii and Pseudomonas ovalis ferredoxins. It showed, like the other two, a Tyr-Thr insertion between the second and third Cys, and extra Cys at position 24 and, compared to Clostridium- and Bacillus-type ferredoxins, an extended C-terminal sequence.View full abstractDownload PDF (512K) -
Akira Iwata, Michiaki Kohno, Yoshihiro Sokawa1988 Volume 104 Issue 2 Pages 247-250
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen serum-starved HeLa S3 cells were stimulated to proliferate by addition of fetal calf serum (FCS), (2'-5')oligoadenylate synthetase (2-5A synthetase) activity was induced. Although no interferon (IFN) activity was detectable in the HeLa S3 cell-conditioned culture medium after growth stimulation, addition of anti -IFN-β monoclonal antibody inhibited both the expression of the 2-5A synthetase gene and the production of the enzyme, suggesting that endogenous IFN-β was involved in 2-5A synthetase induction. Purified preparations of three growth factors, epidermal growth factor, platelet-derived growth factor, and insulin, also induced 2-5A synthetase through IFN-β. When serum-starved HeLa S3 cells were treated with FCS, DNA synthesis was initiated synchronously, with peaks after 12 and 32 h, although the level of 2-5A synthetase reached a maximum after the first peak of DNA synthesis. Inhibition of 2-5A synthetase induction by anti -IFN-β antibody enhanced the second, but not the first cycle of DNA synthesis. These results suggested that in HeLa S3 cells, after stimulation with growth factors the IFN/2-5A synthetase system played a role in cell growth negative regulatory mechanisms.View full abstractDownload PDF (1652K) -
Keiichi Yamamoto, Takamitsu Sekine, Kazuo Sutoh1988 Volume 104 Issue 2 Pages 251-254
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the interaction of calmodulin with calmodulin binding proteins, we studied the location of the interaction sites on calmodulin by using a chemical cross-linking reagent. Calmodulin prepared from wheat germ was cross-linked to myosin light chain kinase and troponin-I with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linked products were cleaved partially with cyanogen bromide and cross-linked sites were determined by peptide mapping analysis using SDS-urea polyacrylamide gel electrophoresis. Peptides which contain the cross-linked site were displaced from their position because of the attached fragments of myosin light chain kinase or troponin I. The peptide of calmodulin from the N-terminal to Met-73 in the cross-linked product with myosin light chain kinase had the same mobility as that of uncross-linked calmodulin on the map though the amount of the peptide was decreased in the cross-linked product. The peptide from the N-terminal to Met-110 in the cross-linked product was displaced from its position. Similar change in the mobility of the calmodulin peptides was also observed in the cross-linked products with troponin I. It was concluded, therefore, that at least one cross-linked site for myosin light chain kinase and one for troponin I were located between Met-73 and Met-110 of the wheat germ calmodulin.View full abstractDownload PDF (1749K) -
Fumio Nanjo, Kazuo Sakai, Taichi Usui1988 Volume 104 Issue 2 Pages 255-258
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSp-Nitrophenyl β-glycosides of N-acetylchitooligosaccharides (PNP-(G1cNAc)n n=3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-β-D-glucosaminide (PNP-G1cNAc) from each substrate. Furthermore, the initial rate of PNP-GleNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-β-chitopentaoside (PNP-(G1cNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-β-chitotrioside (PNP-(GleNAc)3) and p-nitrophenyl tetra-N-acetyl-β-chitotetraoside (PNP-(G1cNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(G1cNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving β-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 μg of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(G1cNAc)5 as a substrate was shown to be useful for lysozyme assay.View full abstractDownload PDF (471K) -
Yoshiharu Shirasu, Kazuta Takemura, Hideaki Yoshida, Yuko Sato, Hirosh ...1988 Volume 104 Issue 2 Pages 259-264
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endo-glycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.View full abstractDownload PDF (1672K) -
Kouji Nakamura, Yoshiki Itoh, Kunio Yamane1988 Volume 104 Issue 2 Pages 265-269
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPrediction of the secondary structure, a sequence of 33 amino acids, for the Bacillus subtilis α-amylase signal peptide suggested the presence of a β-turn structure in it. Through substitution of G1y27 and/or Pro28 with Ala residues by means of the site-directed mutagenesis method, the secondary structure was predicted to consist of an α-helix conformation throughout the signal peptide containing a small region of random coil. The effect of the structural modification upon the secretion of proteins was analyzed as to the production of β-lactamase after the DNA regions encoding the parental and modified signal peptides had been fused in frame to the DNA fragment for the β-lactamase using HindIII linker DNA. The production of β-lactamase increased to 4 to 6 times in B. subtilis cells and to 1.5 to 2.0 times in Escherichia coli with the modified signal peptides. The modification of the signal peptide affected the transcription level of the fused genes.View full abstractDownload PDF (556K) -
Shigeo Murase, Kazuyo Yamada, Yukio Naito, Junzo Hirose, Harumi Okuyam ...1988 Volume 104 Issue 2 Pages 270-275
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lysophosphoinositide-specific phospholipase C (lysoPI-PLase C) in porcine platelet membranes had an optimal pH of 9.2 and the activity at a physiological pH of 7.3 was 20% of the maximum in the absence of added divalent metals (Murase, S. et al. (1985) J. Biol. Chem. 260, 262). The activity was completely inhibited by 1 mM EGTA in the assay mixture but was restored by addition of excess Ca2+ or Mn2+, indicating that this is a metalloenzyme. However, membranes pretreated with 1 mM EGTA and washed with buffer retained full activity at a free Ca2+ concentration of 5 nM and no stimulation was observed by added Ca2+ at pH 9.2. In contrast to the results obtained at pH 9.2, addition of Ca2+ stimulated lysoPI-PLase C activity severalfold at pH 7.3, apparently by shifting down the optimal pH and broadening the pH profile. The effect of Ca2+ at pH 7.3 was to enhance Vmax with no significant change in Km value. The stimulatory effect of Ca2+ at pH 7.3 alone did not appear to be of physiological significance since millimolar concentrations of Ca2+ were necessary to reach the maximum activity. However, a shift in pH had a profound effect on the Ca2+-dependency of the activity. A rise in 2 pH units increased the apparent affinity for Ca2+ 10, 000-fold. These results indicate that the alkalinization and the rise in free Ca2+ concentration known to occur in stimulated platelets could synergistically provide conditions under which the lysoPI-PLase C exerts its activity when the substrate lysoPI is generated by phospholipase A.View full abstractDownload PDF (665K) -
Akihiko Tsuji, Kiyoshi Omura, Yoshiyuki Suzuki1988 Volume 104 Issue 2 Pages 276-278
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIntracellular transport of two lysosomal enzymes, acid α-glucosidase and, β-hexosaminidase, was analyzed in human fibroblasts. The precursors of β-hexosaminidase in normal fibroblasts were released from the membrane fraction by treatment with mannose 6-phosphate, but the precursor of α-glucosidase was not. Percoll density gradient centrifugation revealed a normal amount of acid α-glucosidase activity in heavy lysosomes in I-cell disease fibroblasts despite impaired maturation and defective phosphorylation, and β-hexosaminidase activity was markedly reduced in lysosomes. It was concluded that the membrane-bound precursor of acid α-glucosidase is transported to lysosomes by a phosphomannosyl receptor-independent system although the enzyme lacks the recognition marker for the phosphomannosyl receptor and processing of an intermediate form to mature forms does not occur in this disease.View full abstractDownload PDF (1676K) -
Shigeru Tatsumi, Mikio Suzuno, Takahisa Taguchi, Michiki Kasai1988 Volume 104 Issue 2 Pages 279-284
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAg+-induced Ca2+ release in isolated sarcoplasmic reticulum (SR) was studied by the stopped flow method monitoring chlortetracycline fluorescence change. After improving the experimental procedure, the initial rate of Ca2+ release could be determined more precisely than before. Micromolar concentrations of Ag+ specifically enhanced Ca2+ efflux from heavy fraction of SR vesicles (HSR). This specific effect was referred to as Ag+-induced calcium release. The Ag+-induced Ca2+ efflux was activated by caffeine and ATP, but was inhibited by Mg2+ and procaine. Further, Ag+ enhanced the Ca2+-induced Ca2+ release over the whole range of Ca2+ concentrations, similarly to ATP. Parallel to Ca2+ efflux, Mg2+ efflux, measured by the same method, was also activated by Ag+. Choline permeability determined by the light scattering method was also activated by Ag+. The results suggest that Ag+ binds to the activation site of the Ca2+-induced Ca2+ release channel and opens the channel. The Ag+ binding site is different from the Ca2+ binding site but similar to the ATP binding site.View full abstractDownload PDF (671K) -
Tetsu Hozumi1988 Volume 104 Issue 2 Pages 285-288
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTrypsin was used as a probe of F-actin conformation. F-actin is known to be refractory to proteolysis [Jacobson, G. R. and Rosenbusch, J. P. (1976) Proc. Natl. Acad. Sci. U. S. 73, 2742-2746]. However, here it was found that F-actin could also be digested by trypsin to a 33-kDa fragment (like G-actin) when free MgADP is present in the medium. The amounts of degradation of F-actin depended on the ADP concentration; saturation occurred at about 0.5 mM. Elimination of divalent cations from the medium completely suppressed the effect of ADP on the digestion of F-actin. Other nucleotides were also examined. The effect decreased in the order ADP>ATP_??_IDP>GDP=UDP. Adenine, adenosine, AMP, and PP, had no effect at all. -ADP had the effect, and its fluorescence was changed on the addition of F-actin. The intrinsic tryptophan fluorescence spectrum of F-actin was ADP-dependent. These results suggest the presence of a second nucleotide interacting site on actin and that ADP interaction at this site induces conformational changes in monomeric actin molecule in F-actin filaments.View full abstractDownload PDF (1601K) -
Masachika Irie, Reiko Nitta, Kazuko Ohgi, Yasushi Niwata, Hideaki Wata ...1988 Volume 104 Issue 2 Pages 289-296
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe primary structure of a non-secretory ribonuclease from bovine kidney (RNase K2) was determined. The sequence determined was VPKGLTKARWFEIQHIQPRLLQCNKAMSGV NNYTQHCKPENTFLHNVFQDVTAVCDMPNIICKNGRIINCHQSPKPVNLTQCNFIAGRYPDC RYHDDAQYKFFIVACDPPQKTDPPYHLVPVHLDKYF. The sequence homology with human non-secretory RNase, bovine pancreatic RNase, and human secretory RNase are 46, 34.6, and 32.3%, respectively. The bovine kidney RNase has two inserted sequences, a tripeptide at the N-terminus and a heptapeptide between the 113th and 114th position of bovine pacreatic RNase; on the other hand, it is deleted of the hexapeptide consisting of the 17th to the 22nd amino acid residue of RNase A. The amino acid residues assumed to be the constituents of the bovine pancreatic RNase active site are all conserved except F120 (L in RNase K2).View full abstractDownload PDF (874K) -
Masato Okada, Hachiro Nakagawa1988 Volume 104 Issue 2 Pages 297-305
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing angiotensin I as a substrate, the activity of protein tyrosine kinase was determined in various rat tissues, and its developmental change in rat brain was investigated. The specific activity was shown to be the highest in the brain among the tissues examined in neonatal rats, while it was the highest in the spleen in adult rats. In the brain, the activity varied during development and was the highest in the first postnatal week. To identify the protein tyrosine kinase and examine its relationship with pp60c-src, which is known to be highly expressed in neuronal cells, we attempted to characterize the enzyme from neonatal and adult rat brain, using poly(Glu, Tyr) as a substrate. Neonatal brain was found to express two types of pp60c-srcand a novel protein tyrosine kinase to almost the same level, while adult brain expressed pp60 c-src predominantly. The neonatal type of pp60 c-src and the novel enzyme were designated as pp60 c-srcand N-PTK in the present study, respectively. pp60 c-src, pp60 nc-src and N-PTK were purified about 660-. 370-, and 260-fold from crude homogenate of neonatal brain, respectively, by procedures including sequential column chromatography on DEAE cellulose, hydroxylapatite, Ultrogel AcA44, and poly(Glu, Tyr) Sepharose. N-PTK behaved as a molecule with apparent Mr =50, 000 on Ultrogel AcA44 gel filtration chromatography. It was not immunoprecipitated by anti-pp60 c-src antiserum and did not phosphorylate IgG heavy chain of anti-pp60 c-src antibody. It required mainly Mn2+ for activity and phosphorylated tyrosine-containing polyamino acids and synthetic peptides such as angiotensin II and RR-SRC peptide. The substrate specificity, Km values for poly(Glu, Tyr) and ATP, requirement of metal ions and inhibition by quercetin were apparently different from those of pp60 c-src.View full abstractDownload PDF (2196K) -
Takayoshi Iio, Takuhiro Nishio, Hiroshi Kondo1988 Volume 104 Issue 2 Pages 306-311
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe midpoint pCa value of the fluorescence titration curve of the complex of 2-((4'- iodoacetamido)anilino)-naphthalene-6-sulfonic acid-labeled troponin (IAANS-Tn) and nonpolymerizable tropomyosin (NPTm) was much larger than that for the complex of Tn containing dansylaziridine-labeled troponin C (DANZ-TnC) and NPTm. The midpoint was pCa 8.25 for the former protein and 6.80 for the latter protein in 0.1 M KC1, 50 mM Na-cacodylate-HC1 (pH 7.0); and pCa 7.90 for the former protein and 6.70 for the latter protein in the presence of 3 mM MgCl2 in the same solvent system. The time course of the fluorescence intensity change of the protein complex subsequent to rapid decrease of free Ca2+ concentration of the solution was measured with a stopped-flow spectrophotometer: The process was exponential and its rate constant was 9.9 s-1 for IAANS-Tn-NPTm at pCa 8.95 and 26.6 s-1 for Tn(DANZ-TnC)-NPTm at pCa 8.99 in the absence of MgCl2 in the same solvent system as in the fluorescence titration experiment. IAANS binds to Cys-133 of TnI and DANZ to Met-25 in the low affinity Ca2+ -binding sites of TnC. These results suggest that IAANS bound to Cys-133 of TnI does not directly detect the Ca2+ -binding to the low affinity Ca2+ -binding site of TnC, but does detect the conformational change of the Tn-NPTm complex induced by the Ca2+ -binding. Analysis of our data shows that IAANS bound to Cys-133 of TnI detects the conformational change between the inhibitory state and the activation state of the regulatory protein system (Tn-NPTm complex), and that DANZ bound to Met-25 of TnC detects the Ca2+ -binding to both of the low affinity Ca2+-binding sites of TnC.View full abstractDownload PDF (664K) -
Masao Miki1988 Volume 104 Issue 2 Pages 312-315
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe carbethoxylation of histidine residues in G-actin impairs actin polymerization. The histidine residue essential for polymerization was identified as histidine-40 [Hegyi, G., Premecz, G., Sain, B., & Mühlrad, A. (1974) Eur. J. Biochem. 44, 7-12]. Non-polymerizable actin was separated from the polymerizable fraction after partial carbethoxylation. The non-polymerizable actin recovered the ability to polymerize following addition of phalloidin. Taking into account the evidence that phalloidin does not bind to G-actin in the absence of salt, the results indicate that the actin monomer undergoes a conformational change and subsequently binds phalloidin before polymerization. The resulting polymers activated S1 ATPase activity as effectively as control F-actin. In the presence of tropomyosin and troponin, a strong inhibition of actin-activated ATPase activity was observed in the absence of Ca2+, although no inhibition was observed in the presence of Ca2+. These results indicate that His-40 is not directly involved in a myosin binding site nor in a tropomyosin-troponin binding site.View full abstractDownload PDF (441K)
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