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Volume 98, Issue 1
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Akiko KUMAGAI, Koji OKAMOTO1985 Volume 98 Issue 1 Pages 1-7
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy the use of a shake culture system, we have previously shown (Oyama, M., Okamoto, K., & Takeuchi, I. (1982) J. Cell Sci. 56, 223-232) that both cAMP and cAMP-dependent cell contact are required for prespore differentiation in Dictyostelium discoideum. The present study was undertaken to examine changes of the plasma membrane proteins during prespore differentiation in the shake culture system. Rabbit antibodies prepared against the plasma membrane fraction of the differentiated cells inhibited the reaggregation of the differentiated cells but not that of aggregation-competent cells. This result indicates that new contact sites are formed in the differentiated cells. By the combined use of the antibody-conjugated immunoadsorbent with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, changes of membrane proteins were analyzed with the cells incubated under various conditions. Three proteins were found to be present specifically in the differentiated cells only in the presence of cAMP, one of which (105 K protein) appeared when cells became adhesive, but before prespore specific proteins were detected. Two others (80 K and 58 K proteins) appeared during prespore differentiation after cells formed agglomerates.View full abstractDownload PDF (1433K) -
Yoshihiro OZEKI, Katsuhiro SAKANO, Atsushi KOMAMINE, Yoshiyuki TANAKA, ...1985 Volume 98 Issue 1 Pages 9-17
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChalcone synthase was purified to homogeneity by polyacrylamide gel electrophoresis from cell suspension cultures of carrot in which anthocyanin synthesis was induced by transferring the cells from a medium containing 2, 4-dichlorophenoxyacetic acid (2, 4-D) to one lacking it. A molecular weight of 80, 000-85, 000 for the enzyme was determined by gel filtration and disc-gel polyacrylamide electrophoresis, and one of about 40, 600 for the subunit by SDS slab-gel electrophoresis. The primary reaction product was chalcone and the pH optimum of the reaction was 8.0. The Km values for 4-coumaroyl-CoA and malonyl-CoA were 5.7 μm and 18 μM, respectively. These properties of carrot chalcone synthase were discussed in comparison to those of that from cell cultures of parsley reported previously.
Antiserum against chalcone synthase from carrot was obtained from mice bred under specific pathogen free conditions. Crossreactivity was examined by Westernblotting, and the high specificity of the antiserum against chalcone synthase was demonstrated.View full abstractDownload PDF (2086K) -
Yuko GIGA, Atsushi IKAI1985 Volume 98 Issue 1 Pages 19-26
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe most abundant glycoprotein in the coelomic fluid of sea urchin Anthocidaris crassispina was purified and its subunit structure, molecular form in the native state, amino acid composition, and electron micrographic image were studied. The results showed that the protein in its native state was basically a tetramer with a total molecular weight of about 700, 000, which was in equilibrium with a high molecular weight form corresponding to an octamer. The electron micrograph of the tetramer showed two ellipsoidal units aligned in parallel with a wide gap in between. The subunits all had the same molecular weight of 180, 000±10, 000 and were disulfide bonded in pairs. The carbohydrate content was about 16% with mannose and fucose as the two most abundant sugars.
Although this protein accounted for 70% of the total protein in the coelomic fluid, it did not take part in the known activities of the fluid, namely hemagglutination and coagulation. Despite its structural similarity to the mammalian alpha-2-macroglobulin or reptilian and avian ovomacroglobulins it did not interact with bovine trypsin or chymotrypsin.
This protein showed immunological cross reactivity with 23 S glycoprotein purified from sea urchin eggs which, we believe, corresponds to the previously described 22-27 S protein particles in eggs.View full abstractDownload PDF (2266K) -
Mutsumi SUGITA, Hiromi NAKAE, Takeshi YAMAMURA, Yoshiko TAKAMIYA, Osam ...1985 Volume 98 Issue 1 Pages 27-34
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTotal neutral and acidic glycosphingolipids were prepared from whole tissues of the sea-water bivalve, Meretrix lusoria, and the former preparation was further fractionated into subgroups by silicic acid column chromatography. The fractions obtained as mono-(ceramide monosaccharide, CMS), di-(CDS) and triglycosylceramides (CTS) were characterized by thin-layer chromatography, partial hydrolysis with exoglycosidases, methylation studies, CrO3 oxidation, and GLC analysis of the component sugars, fatty acids and long-chain bases. The following structures are proposed: Gal-Cer and Glc-Cer for CMS, Gal (β1→4) Glc-Cer and Man (β1→4) Glc-Cer (MlOse2Cer) for CDS, Man (α1→3) Man (β1→4) Glc-Cer (MlOse3Cer) and Gal (α1→3) Man (β1→4) Glc-Cer (II3αGal-MlOse2Cer) for CTS. To our knowledge II3αGal-MIOse2Cer has not previously been reported. The fatty acid composition of CMS, CDS, and CTS consisted almost entirely of saturated C16-C24 acids with large amounts of 2-hydroxypalmitic acid and 2-hydroxystearic acid. The long-chain bases consisted of 4-sphingenine and 4, 8-sphingadienine.
More complex neutral glycolipids than CTS, as well as an acidic glycolipid, were examined by TLC and GLC of the constituent sugars, and an immunochemical technique. They contained tetra- to nonasaccharides as sugar moieties and the structures are suggested to be as follows: GIcNAc (β1→2) Man (α1→3) Man (β1→4) Glc-Cer (Lipid I, MlOse4Cer); GlcNAc (β1→2) Man (α1→3) [Xyl (β1→2)] Man (β1→4) Glc-Cer (Lipid II, MIXOse5Cer); Fuc3Me (α1→2) Xyl 3 Me(β1→4) [GalNAc 3 Me (α1→3)] Fuc (α1→4) GlcNAc (β1→2) Man (α1→3) [Xyl (β1→2)] Man (β1→4) Glc-Cer (Lipid III, V3αGalNAc3Me, V4βXyl 3 Me, VI2αFuc 3 Me-MlXOse6Cer); and GlcA 4 Me(β1→4) [GalNAc 3 Me (α1→3)] Fuc (α1→4) GleNAc (β1→2) Man (α1→3) [Xyl (β1→2)] Man (β1→4) Glc-Cer (Lipid IV, V3αGalNAc 3 Me, V4βGlcA 4 Me-MlXOse6Cer).
The latter four complex glycolipids were earlier encountered in fresh-water bivalves (Hori et al. (1977) J. Biochem. 81, 107-114; 82, 1281-1285; (1981) 90, 1529-1535; (1981) J. Biol. Chem. 256, 10979-10985; (1983) 258, 2239-2245), and their occurrence also in this sea-water bivalve, M. lusoria, suggests that such a glycolipid pattern may be widespread in related families.View full abstractDownload PDF (1253K) -
Nobuo IDA, Masanobu TOKUSHIGE1985 Volume 98 Issue 1 Pages 35-39
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDuring the catalysis of the fumarate amination reaction, aspartase was markedly activated by the product, L-aspartate, as shown by a steep increase in the reaction rate. When NH4+ was replaced by NH2OH, the hydroxylamination reaction proceeded without any acceleration, and was activated upon addition of L-aspartate. The activation required the Mg2+ ion and the alkaline pH, and the half-saturation concentration of L-aspartate for activation was as low as 0.07mM, which was far lower than the Km value for catalysis. Fumarate showed no activating effect in contrast to L-aspartate, and L-aspartate lowered the Km value for fumarate instead of acting as a competitive inhibitor. Besides L-aspartate, α-methyl-DL-aspartate exhibited an activating effect without serving as a substrate. These results suggest that the activation is mediated by an indirect action of L-aspartate which is bound to a site distinct from the catalytic site.View full abstractDownload PDF (407K) -
Yoshihiro ASAKURA, Yoshiko KIKUCHI, Mitsuhiro YANAGIDA1985 Volume 98 Issue 1 Pages 41-47
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn S 1-hypersensitive site was found at the 60 bp direct repeats of the cis-acting, stability and/or copy number control region of the yeast 2 μ DNA in the supercoiled hybrid plasmid pDB 248'. It was retained in a different plasmid, pYK 2121, consisting of pBR 322 and the 300 bp long repeated DNA. Analyses of 5'-end-labeled fragments and nucleotide sequence determination showed that the S 1-cleavage site was at the central part of an AT-rich 19 bp palindrome present in the repeats. Two other homologous palindromes (21 and 15 bp) containing the 12 by consensus sequences were not cleaved. The nucleotide sequences at the base of the stem and/or loop may determine the efficiency of the cruciform extrusion.View full abstractDownload PDF (3628K) -
Yusei YAMAMOTO, Kouji FUJIMOTO, Takafumi OHMURA, Shuichiro MAEDA, Kazu ...1985 Volume 98 Issue 1 Pages 49-56
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInduction of interleukin 2 (IL 2) mRNA synthesis in human tonsillar lymphocytes was studied by quantifying the relative levels of IL 2 mRNA in the lymphocytes stimulated under various conditions by the dot hybridization method. A remarkable increase of IL 2 mRNA was induced by stimulation with phytohemagglutinin (PHA) in the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The kinetic study revealed that the IL 2 mRNA level of the lymphocytes increased from 2 h of culture, reached a maximal level at 12 h, maintained a relatively high level until 48 h and then sharply decreased by 72 h after the stimulation. Inhibition experiments with actinomycin D showed that the increase was due to a transient synthesis of the mRNA after the stimulation, which almost stopped by 12-16 h. DNA synthesis and cell division were not necessary for the induction of IL 2 mRNA production but the induction was inhibited by dexamethasone, showing that the production was mainly associated with the G1 phase of the cell cycle. Two-step culture experiments showed that prior exposure of the lymphocytes to TPA for 1 h at 37°C resulted in a remarkable increase of IL 2 mRNA on subsequent stimulation with PHA. This suggests that TPA induces certain changes in the biochemical pathway of signal transduction so that the cells can be triggered to express IL 2 gene by subsequent stimulation with mitogen.View full abstractDownload PDF (1489K) -
Toshiko SHIBAYAMA, Kazuyasu NAKAYA, Yasuharu NAKAMURA1985 Volume 98 Issue 1 Pages 57-62
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Cap 42 (b), a Ca2+-dependent F-actin capping phosphoprotein of 42, 000 daltons, was shown to be localized in the cytosol of Physarum polycephalum by measurements of phosphorylatability in the absence of Ca2+. The phosphorylation of Cap 42 (b) in the cytosol changed during the cell cycle: it was high in the S and G 2 phase, and low in the M phase and boundary phase between S and G 2 phase. When the isolated Cap 42 (b) was added to M phase cytosol, the phosphorylation of Cap 42 (b) was significantly increased by at least 6-fold. Compared with this result, about 2-fold increase in the phosphorylation of Cap 42 (b) was observed when the Cap 42 (b) kinase was added to M phase cytosol. Therefore, it is likely that the low level of Cap 42 (b) phosphorylation in M phase cytosol is mostly due to the decreased amount of phosphorylatable Cap 42 (b) and to a lesser extent due to a low level of the Cap 42 (b) kinase activity.View full abstractDownload PDF (2566K) -
Akira TAKAHASHI, Mihoko USAMI, Tomoko KADOTA, Ken KADOTA1985 Volume 98 Issue 1 Pages 63-68
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCoated vesicles prepared from bovine brain contained cyclic nucleotides- and Ca2+-calmodulin-independent protein kinases which in the presence of Mg2+ catalyzed the phosphorylation of an endogenous 48, 000 Mr protein of coated vesicles (C-48), phosvitin and troponin T. Phosvitin was phosphorylated either in the presence of ATP or GTP. The phosphorylation of C-48, on the other hand, was specific for ATP. Heparin inhibited the phosphorylation of phosvitin but not that of C-48. Mn2+ inhibited the phosphorylation of phosvitin, while Mn2+ substituted for Mg2+ in the phosphorylation of C-48. When the coated vesicles were prepared in the presence of NaF, C-48 contained 2.5-2.8 mol of phosphate/mol. On incubation with Mg2+ and ATP, C-48 incorporated 1.2-1.6 mol of phosphate/mol. With C-48 as a substrate, the value of its apparent Km for ATP was 6 μM. With phosvitin as a substrate, the value of its apparent Km was 20 μm. The phosphorylated amino acid residues in the phosvitin were identified as serine and threonine. Phosphothreonine was detected in C-48. These results suggest that brain coated vesicles possess two different classes of protein kinase, a casein kinase II and C-48 kinase.View full abstractDownload PDF (1591K) -
Hidehiro SAKURAI, Anthony SAN PIETRO1985 Volume 98 Issue 1 Pages 69-76
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTreatment of photosystem I particles from spinach (Spinacia oleracea) with dodecyl sulfate destroyed the protein-bound Fe-S centers and converted some of the acidlabile sulfide to zero-valence sulfur which remained covalently bound to the proteins. When the proteins were resolved by gel-permeation chromatography or by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate, a considerable amount of zero-valence sulfur was associated with the large molecular weight polypeptide (s) (63, 000 and 59, 000). The results strongly suggest that an intact twopeptide P 700 chlorophyll α-protein is an Fe-S protein.View full abstractDownload PDF (1589K) -
Masanori YASUI, Shigeharu HARADA, Yasushi KAI, Nobutami KASAI1985 Volume 98 Issue 1 Pages 77-80
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structure of a ferricytochrome c' extracted from Rhodospirillum rubrum has been determined at 6 Å resolution by the X-ray crystallographic method. The crystals, obtained by dialyzing the protein solution against polyethylene glycol 4000, belong to the hexagonal space group P61. Two heavy atom derivatives were obtained by soaking the native crystals in K2PtCl6 and CH3HgCl solution. The phases calculated by the multiple isomorphous replacement method gave an overall figure of merit of 0.90 at 6 Å resolution. The resulting electron density map showed the molecular boundary clearly, and gave molecular dimensions of 50×25×30 Å for a monomer molecule. From visual examination of this map, the cytochrome c' from Rhodospirillum rubrum has a similar chain-folding pattern to the cytochrome c' from Rhodospirillum molischianum, the structure determination of which has already been carried out.View full abstractDownload PDF (1126K) -
Hirofumi ONISHI1985 Volume 98 Issue 1 Pages 81-86
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, we (Suzuki et al. (1978) J. Biochem. 84, 1529) reported that the sedimentation constant of chicken gizzard myosin in the presence of ATP was approximately 10 S in 0.15M or 0.2M KCl and approximately 6 S in 0.3M or higher concentrations of KCl. The 10 S-myosin and 6 S-myosin were considerably different in conformation from each other. I now report the finding that the transformation of 6 S-myosin to the 10 S conformation results in a drastic change in the reactivity of thiol groups of gizzard myosin with N-iodoacetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine (abbreviated as IAEDANS). The so-called SH1-type thiol groups (Sekine et al. (1962) J. Biol. Chem. 237, 2769) were present on 68 kilodalton fragments (produced by tryptic digestion) of gizzard myosin. The reactivity of the thiol groups with IAEDANS was greatly decreased by the 6 S to 10 S transformation of gizzard myosin molecules. Two other findings were obtained. (a) Blocking the SH1-type thiol groups made the Mg-ATPase activities (in the presence of gizzard native tropomyosin) of gizzard myosin and of acto-gizzard myosin insensitive to calcium and to phosphorylation of regulatory light chains, although calcium-dependent phosphorylation of the IAEDANS-modified myosin could still occur. (b) It also made gizzard myosin filaments resistant to the disassembly action of ATP.View full abstractDownload PDF (523K) -
Eiki KOMINAMI, Toshifumi TSUKAHARA, Yoshiaki BANDO, Nobuhiko KATUNUMA1985 Volume 98 Issue 1 Pages 87-93
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe concentrations of cathepsins B and H in various tissues and peripheral blood cells of rats were determined by means of sensitive immunoassays. The minimum detectable amounts of cathepsins B and H were 30 pg and 20 pg/assay, respectively, and the presence of endogenous thiol proteinase inhibitors did not interfere with the immunoassays. Cathepsin B was found at high levels in the kidney, vagina, spleen, and adrenal gland, and cathepsin H at high levels in the kidney, vagina, liver, lung, and spleen. Low levels of cathepsins B and H were present in the heart, skeletal muscle, and testis. The ratios of cathepsins B and H in various organs were different: the brain and adrenal gland contained much higher levels of cathepsin B than of cathepsin H, whereas the lung and liver contained higher levels of cathepsin H than of cathepsin B. In several organs such as the kidney, spleen, liver, and lungs, the level of cathepsins B plus H was much higher than that of thiol proteinase inhibitors (TPI-α+TPI-β), whereas in tissues containing large amounts of TPI-α, such as the skin, esophagus and stomach, the level of inhibitors was higher than that of cathepsins B plus H. Of the peripheral blood cells tested, macrophages had the highest contents of cathepsins B and H, and so their level of cathepsins B plus H was much higher than that of TPI-α plus TPI-β, whereas lymphocytes and neutrophils contained comparable amounts of proteinases and inhibitors. The level of cathepsin B in macrophages obtained from rats injected with sodium caseinate was 6 times that in resident macrophages, whereas there was less than two-fold difference in the levels of cathepsin H in resident and elicited macrophages.View full abstractDownload PDF (1333K) -
Hayato IHARA, Takuji SASAKI, Akio TSUBOI, Hideo YAMAGATA, Norihiro TSU ...1985 Volume 98 Issue 1 Pages 95-103
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleotide sequence of a thermophilic, liquefying α-amylase gene cloned from B. stearothermophilus was determined. The NH2-terminal amino acid sequence analysis of the B. stearothermophilus α-amylase confirmed that the reading frame of the gene consisted of 1, 644 base pairs (548 amino acids). The B. stearothermophilus α-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E. coli. The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation. Comparison of the amino acid sequence inferred from the B. stearothermophilus α-amylase gene with those inferred from other bacterial liquefying α-amylase genes and with the amino acid sequences of eukaryotic α-amylases showed three homologous sequences in the enzymatically functional regions.View full abstractDownload PDF (717K) -
Takashi KAWAMURA, Naoshi ICHIHARA, Shigeki SUGIYAMA, Hajime YOKOTA, No ...1985 Volume 98 Issue 1 Pages 105-116
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe occurrence and formation of UDP-N-acetyl-D-glucosaminuronic acid (UDPGIcNAcA) and UDP-N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA) were studied in Micrococcus luteus ATCC 4698. UDP-N-acetylhexosaminuronic acid separated from D-cycloserine-inhibited cells was shown to be a mixture of UDPGlcNAcA and UDP-ManNAcA in the ratio of 87:13, whereas that obtained from untreated cells was a 96:4 mixture of these two nucleotides. Crude enzyme preparations obtained from the supernatant fraction of cells catalyzed the NAD+-dependent conversion of UDP-GlcNAc into UDP-GIcNAcA and UDP-ManNAcA. Studies on the partial separation and properties of enzymes revealed that UDP-GlcNAcA is synthesized directly from UDP-GlcNAc by the action of UDP-GlcNAc dehydrogenase and that UDP-ManNAcA is synthesized from UDP-GlcNAc through the successive actions of UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase. However, enzymatic conversion of UDP-GlcNAcA to UDP-ManNAcA was not detected. Ammonium sulfate protects both dehydrogenases from inactivation during storage and incubation. Partially purified UDP-GlcNAc dehydrogenase required dithiothreitol and the particulate fraction for its full activity. The apparent Km values of UDP-GlcNAc dehydrogenase for UDP-GlcNAc and NAD+ were 0.28 and 1.43mM, respectively. The optimum pH of this enzyme was higher than 9 in Tris-HCl buffer. p-Chloromercuribenzoate at 27 μM as well as 10mM ethanol almost completely inhibited the UDP-GlcNAc dehydrogenase reaction.View full abstractDownload PDF (1192K) -
Takashi IGARASHI, Tetsuo SATOH, Keiko IWASHITA, Satoshi ONO, Koichi UE ...1985 Volume 98 Issue 1 Pages 117-123
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activities of hepatic cytosolic glutathione S-transferases (GSTs) towards 1, 2-dichloro-4-nitrobenzene in male rats were higher than those in females, however, the enzyme activities towards 1-chloro-2, 4-dinitrobenzene were not significantly different between the two sexes. SDS-PAGE analysis of GSTs purified from male and female rat hepatic cytosols by affinity column chromatography showed that there was a significant difference in the subunit composition between the two sexes. With regard to the several isozymes of GSTs in male and female rats, isozymes with basic and neutral/acidic isoelectric points were separated into seven molecular species by chromatofocusing. These sex differences in the quantitative proportions of GST isozymes were also confirmed by imrnunotitration using anti-GST-BL and -AC antibodies.
On the other hand, glutathione peroxidase (GSH-Px) activities in rat hepatic cytosol towards hydrogen peroxide and cumene hydroperoxide were markedly higher in females than in males. Of the two types of GSH-Px, selenoenzyme (Se-GSH-Px) and the Se-independent enzyme (non-Se-GSH-Px), the former was found to be mainly responsible for the sex difference in the enzyme activities. Moreover, the GSH-Px activity of GSTs, non-Se-GSH-Px, was also higher in females than that in males. Since GST isozymes of the BL type are known to possess GSH-Px activity towards cumene hydroperoxide, the increased activities of non-Se-GSH-Px in the female hepatic cytosol seemed to be mainly due to the increased transferase activities of the isozymes, GST-L2 and -BL.View full abstractDownload PDF (1249K) -
Akihiro SANDA, Yoshio TAKIZAWA, Masanori IWAMA, Masachika IRIE1985 Volume 98 Issue 1 Pages 125-132
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl) carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at least the modification of two carboxyl groups seemed to induce the loss in enzymatic activity.
2. In the presence of 1M cytidine, RNase Rh activity was protected from the CMC-modification. Under conditions in which the enzyme was inactivated to 20% activity, about 70% of the enzymatic activity was retained in the presence of cytidine. The inactivation of the RNase Rh pre-treated with CMC in the presence of cytidine with [14C] CMC indicated that the RNase Rh lost its enzymatic activity with the incorporation of about one [14C] CMC. Therefore, it could be concluded that one carboxyl group is involved in the active site of RNase Rh.
3. The binding of the CMC-modified RNase Rh with 2'-AMP was studied spectrophotometrically. The affinity of the modified RNase Rh towards 2'-AMP decreased markedly upon CMC modification.View full abstractDownload PDF (713K) -
Hisanori MINAMI, Ken-ichi MIYAMOTO, Yasuhiro FUJII, Yukihiro NAKABOU, ...1985 Volume 98 Issue 1 Pages 133-139
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIntestinal and hepatic ornithine decarboxylase (ODC) activities increased to a preak 4 h after administration of a diet containing casein or an amino acid mixture simulating that of casein to rats starved for 12 h. All amino acids except cysteine with a two or three carbon skeleton, including those with a D-configuration, and α-aminoisobutyric acid (AIB) strongly induced intestinal ODC when given in the diet or administered intragastrically. Amino acids with a four carbon skeleton were far less effective as inducers and other amino acids did not induce intestinal ODC at all.
The amino acids that induced hepatic ODC showed no particular structural characteristics: glycine and cysteine were very effective, threonine, tryptophan, methionine, and phenylalanine were less effective, and serine, valine, isoleucine, and histidine were only slightly effective.
Elevation of ODC activity after amino acid administration was not due to stabilization of the enzyme protein with the amino acids.
Intestinal ODC was induced by intragastric but not intraperitoneal injection of glycine, although these treatments resulted in similar increases in the tissue concentration of glycine. On the contrary, hepatic ODC was induced by glycine regardless of the administration route.
Intestinal ODC was also induced only in the segment of the intestine perfused with a solution of an amino acid with which the activity increased in the feeding experiment.
These results suggest that the accumulation of an amino acid per se is not a trigger for induction of intestinal ODC and that an amino acid must act on the mucosal surface to induce the enzyme.View full abstractDownload PDF (590K) -
Kunihiko KONNO, Shizuo WATANABE1985 Volume 98 Issue 1 Pages 141-148
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChymotryptic digestion of scallop myosin yielded two different preparations of subfragment-1, having the following features. 1. The major product from chymotryptic digestion of scallop myosin was subfragment-1 (S 1) either in Ca-medium or in EDTA-medium. However, the S 1 preparations obtained from the digestion in Ca-medium, abbreviated as Ca-S 1 (CT), had both types of light chain subunits (regulatory light chains (R-LC) and essential light chains (SH-LC)), and 100 Kdaltons (Kd) heavy chain subfragments (HCs), whereas the S 1 preparations obtained from the digestion in EDTA-medium, ED-S 1 (CT), had no R-LC, partially fragmented SH-LC (SH-LC'), and 90 Kd HCs. 2. On the other hand, Ca-S 1 (CT) and ED-S 1 (CT) were practically identical with each other in ATPase activity and in actin-binding ability. The two S 1 preparations were also identical in that the Mg-ATPase activity of both S 1 and acto-S 1 was insensitive to calcium ions. 3. Ca-S 1 (CT), which contained both R-LC and SH-LC in a stoichiometric amount, was further digested with trypsin, which is known to cleave rabbit skeletal myosin not only at the head-tail junction but also in the head. The tryptic digestion of Ca-S 1 (CT) appeared, in terms of the SDS-gel electrophoretic pattern, to occur at a much faster rate in Ca-medium than in EDTA-medium, and with a different digestion profile. It is therefore suggested that association of R-LC induces changes in the heavy chain conformation which result in an increase in the proteolytic digestability of heavy chains and in an alteration of the site of proteolytic cleavage on heavy chains.View full abstractDownload PDF (2419K) -
Shin-ichi KAMIYA, Bunji YOSHITOMI, Kunihiko KONNO, Shizuo WATANABE1985 Volume 98 Issue 1 Pages 149-156
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHeavy meromyosin (HMM) and subfragment-1 (S 1) were obtained from squid mantle myosin by tryptic digestion and chymotryptic digestion, respectively. Squid HMM (T) and S 1 (CT) preparations contained stoichiometric amounts of the two types of light chain subunit; regulatory light chain, LC-2, and essential light chain, LC-1.
1. No difference was detected in the chymotryptic digestabilities of squid mantle myosin in Ca-medium and in EDTA-medium. This is in contrast to the digestability of scallop adductor myosin. 2. The Mg-ATPase activity of HMM (T) alone and that of acto-HMM (T) were both sensitive to calcium ions. In contrast, the activity of S 1 (CT) alone and that of acto-S 1 (CT) were both insensitive to calcium ions. 3. The affinity of HMM (T) for actin was not affected by calcium ions, but the amount of HMM (T) bound to actin was increased by calcium ions from 20% to 60% of the total amount of HMM (T). On the other hand, the actin affinity of S 1 (CT) and the amount of S 1 (CT) bound to actin were both unaffected by calcium ions. The role of calcium ions in the regulation of contraction in molluscan muscles is discussed.View full abstractDownload PDF (2500K) -
II. Interaction of Phospholipase A Inhibitor with Phospholipase A and Its SpecificityMasao MIWA, Toru ICHIHASHI, Hisaya MOTOJIMA, Ikuyo ONODERA-KUBOTA, Mak ...1985 Volume 98 Issue 1 Pages 157-165
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNon-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed unless the inhibitor was preincubated with the enzyme. The inhibition seemed to be due to the formation of the enzyme-inhibitor complex, which was identified by immunoelectrophoresis. The enzyme-inhibitor interaction was observed maximally on incubation at physiological pH, but not below pH 5. The inhibitor was inactivated by trypsin digestion and heat treatment. It suppressed the phospholipase A2 activities of rat blood plasma as well as of the snake venom and porcine pancreas, but not the enzyme activities such as those of phospholipase C of Bacillus cereus, lipase of porcine pancreas, trypsin, and papain. The inhibitor also showed the ability to decrease membrane-bound phospholipase A1 and A2 activities in intracellular organelles such as plasma membranes, mitochondria, lysosomes, and microsomes. In view of these facts, it was concluded that the plasma inhibitor is specific for phospholipase A.View full abstractDownload PDF (1520K) -
Toshiyuki SAKAKI, Kenji OEDA, Mitsuko MIYOSHI, Hideo OHKAWA1985 Volume 98 Issue 1 Pages 167-175
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat cytochrome P-450MC cDNA was expressed in Saccharomyces cerevisiae AH 22, SHY 3 and NA 87-11 A cells under the control of the yeast ADH 1 promoter and terminator. Although the three yeast strains transformed with the constructed expression plasmid, pAMC 1, contained approximately three copies of the plasmid, the levels of both P-450MC mRNA and the corresponding protein in the AH 22 cells carrying plasmid pAMC 1 were 1.4- to 1.7-fold and 2-fold higher than in the other two strains, respectively.
The P-450MC protein was purified from the microsomal fraction of AH 22 cells carrying pAMC 1 by a rapid purification method. The apparent molecular weight, chromatographic behavior, spectral properties, substrate specificity and immunochemical properties of the purified P-450MC protein were indistinguishable from those of rat liver P-450MC.I and P-450MC-II (Sasaki, T., et al. (1984) J. Biochem. 96, 117-126). The NH2-terminal amino acid sequence of the purified protein up to 10 residues was the same as those of P-450MC-I and P-450MC-II. In addition, HPLC analysis of the microsomal fraction of AH 22 cells containing pAMC 1 indicated that the synthesized P-450MC protein corresponds to P-450MC-II, but not P-450MC-I.
With another purification method, we obtained the cleaved P-450MC protein which lacked the NH2-terminal 30 amino acids of intact P-450MC. The spectral properties and monooxygenase activities towards benzo (a) pyrene and 7-ethoxycoumarin of the cleaved P-450MC were nearly the same as those of intact P-450MC.View full abstractDownload PDF (1237K) -
Makoto KAWAKAMI, Koji NISHIO1985 Volume 98 Issue 1 Pages 177-186
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLarge amounts of glycyl-tRNA synthetase were purified from the posterior silk glands of Bombyx mori. The synthetase was estimated to be a dimer with a molecular weight of 180, 000. When the enzyme solution was diluted, the dimer dissociated into monomers which were inactive in tRNA aminoacylation. The aminoacylation was investigated with two isoaccepting tRNAsGly isolated from the posterior silk glands. Transfer RNA1Gly was aminoacylated 2-fold faster than tRNA2Gly. Transfer RNA-binding experiments revealed that tRNA1Gly binds with the enzyme in a molar ratio of 2:1, whereas tRNA2Gly formed a 1:1 complex with the enzyme. Based on these experimental results, we proposed that the Bornbyx mori glycyl-tRNA synthetase has two active sites for tRNA aminoacylation and that the number of tRNA molecules bound on the synthetase closely correlates with the velocity of aminoacylation.View full abstractDownload PDF (2924K) -
Umeji MURAKAMI, Koki UCHIDA1985 Volume 98 Issue 1 Pages 187-197
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe in situ contents of myosin, actin, α-actinin, tropomyosin, troponin, and desmin were estimated in dog cardiac, rabbit skeletal, and chicken smooth muscles. Whole muscle tissues were dissolved with 8M guanidine hydrochloride and subjected to twodimensional gel electrophoresis, which is a nonequilibrium pH gradient electrophoresis (Murakami, U. & Uchida, K. (1984) J. Biochem. 95, 1577-1584) with some modification. The amount of protein in a spot on a slab gel was determined by quantification of the extracted dye. Dye binding capacity of individual myofibrillar proteins was determined by using the purified protein.
Myosin contents were 82±7 pmol/mg wet weight in cardiac muscle, 105±10 pmol/mg wet weight in skeletal muscle, and 45±4 pmol/mg wet weight in smooth muscle. Actin contents were 339±15 pmol/mg wet weight in cardiac muscle, 625±27 pmol/mg wet weight in skeletal muscle, and 742±13 pmol/mg wet weight in smooth muscle. The subunit stoichiometry of myosin in the three types of muscles was two heavy chains and four light chains, and there was one light chain 2 for every heavy chain. The molar ratio of actin to tropomyosin was 7/1 in the three types of muscles. Striking differences were seen in the molar ratio of myosin to actin: 1.0/4.1 in cardiac muscle, 1.0/6.0 in skeletal muscle, and 1.0/16.5 in smooth muscle.View full abstractDownload PDF (2154K) -
Toshihiko HIRANO, Haruki YAMADA, Toshio MIYAZAKI1985 Volume 98 Issue 1 Pages 199-208
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the cell adhesion of aggregation-competent Dictyostelium cells, the requirement for the carbohydrate moiety of the glycoprotein appeared to be indirect in that it acts to protect the protein moiety from proteolytic degradation; however, the effect was limited to the tunicamycin (TM)-sensitive carbohydrate moiety (Hirano, T., et al. (1983) J. Biochem. 93, 1249-1257). In the present study, we showed that the EDTA-stable adhesion of aggregation-competent Dictyostelium cells was abolished by the treatment of intact cells with jack bean α-mannosidase, whereas neuraminidase, β-galactosidase, β-N-acetylhexosaminidase, or α-L-fucosidase had no effect. The EDTA-stable cohesiveness of TM-treated cells in the presence of leupeptin (TM/LP cells) was also abolished by the treatment of the cells with α-mannosidase. The effect of α-mannosidase was not prevented in the presence of LP. The N-glycoside-deficient contact site A (an adhesion-mediating glycoprotein) was obtained from TM/LP cells and was shown to have a molecular weight of 70, 000. This protein (p 70) was shown to still have carbohydrates as detected by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) and subsequent staining of the gel with periodic acid-silver stain. Moreover, p 70 reacted with anti-gp 68, which has a specificity against α-mannosyl residues of carbohydrate chains. However, p 70 treated with α-mannosidase showed decreased reactivity with anti-gp 68. The monovalent antibody fragment of anti-contact site A or anti-p 70 inhibited EDTA-stable cell adhesion of both control and TM/LP cells. These results indicated that TM-resistant mannosyl residues of contact site A are directly involved in EDTA-stable adhesion of aggregation-competent cells. This is the first report of the direct involvement of the carbohydrate moiety in cell adhesion of aggregation-competent Dictyostelium cells. A schematic model is presented of the role of the carbohydrate moiety in EDTA-stable cell adhesion, including the direct effect of carbohydrates.View full abstractDownload PDF (908K) -
Osamu URAYAMA, Hideaki NAGAMUNE, Makoto NAKAO, Yukichi HARA, Hiroyuki ...1985 Volume 98 Issue 1 Pages 209-217
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA hybridoma cell line progucing mouse monoclonal antibody against pig kidney Na, K-ATPase was established. The antiboky, named 38 (mAb 38, IgG 1), was purified from mouse ascites fluid by chromatography on a protein A-Sepharose column. Antigens immobilized on microplate wells with p-benzoquinone were used from titer assays. mAb 38 xross-reacted with both dodecyloctaethyleneglycol monoether (C12E8)-solubilized enzyme and membranous sodium dodecyl sulfate (SDS)-treated enzyme from kidney with gigh affinity (50)% binding=1.6 nM). However, the antibody bound to neither α- nor β-subunit separatd by preparative SDS-polyacrylamide gel electrophoreses (PAGE). The stoichiometry of antibody binding to the purified enzyme was estimated to be about 0.86 mol of IgG per mol of αβ-protomer. Na, K-ATPase proteins were recovered from a xolumn of mAb 38-coupled Affi-Gel by elution with pH 3 buffer when C12E8-solubilized kidney enzyme or detergent extracts of brain microsomes were applied to it, confirming that the mAb is directed to Na, K-ATPase. mAb 38 at saturation level concxentrations had erythrocytes. In an immunofluorescence study, the antibody bound to intact erythantiody binding to inside-out vesicles under hypotonic conditions was lower than that of the control. Most of the antibody binding activity remained when the kidney enzyme was treated with sialidase. These results suggest that this mAb 38 was raised against an intact conformation of a cell-surface-exposed site of Na, K-ATPase.View full abstractDownload PDF (1657K) -
Shoshi OTA, Eiko MUTA, Yasuko KATAHIRA, Yoshiko OKAMOTO1985 Volume 98 Issue 1 Pages 219-228
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo check whether crude stem and fruit bromelains can be fractionated further or not, systematic separation procedures were applied to both enzymes. Six proteolytically active components, which were designated as SBB 1-5 and SBA, were fractionated from crude stem bromelain by successive use of gel filtration on Sephadex G-75, and chromatographies on CM-Sephadex and DEAE-Sephacel. One main and one minor active components, designated as FBA and FBB, respectively, were also separated from crude fruit bromelain by chromatographies on DEAESephacel and then CM-Sephadex. Some of the physico-chemical and enzymatic properties of these eight components were compared. Each component migrated as a single band on SDS-polyacrylamide gel electrophoresis. Molecular weights determined by the same electrophoresis were about 27, 000 for SBB 1-3 and FBB, and about 23, 000 for the other four components. In terms of amino acid composition, FBB resembled SBB 1-3, which were remarkably similar to each other. FBA was also similar to SBA in amino acid composition, and contained much less basic amino acids than SBB 1 through 5. The principal amino-terminal residues determined by the cyanate method were valine in SBB 1-5 and SBA, and alanine in FBA and FBB. The principal carboxyl-terminal residues determined by the hydrazinolysis method were glycine in SBB 1-3, SBA and FBA, and serine in SBB 4-5 and FBB. However, fractional amounts of a few other amino- and carboxylterminal residues were also detected. As regards enzymatic activities, FBA and SBB 4 and 5 were much more active than the other five components against casein and some synthetic substrates [Bz-Arg-amide (at pH 6.1), Z-Gly-X, and Z-Ala-X (at pH 3.5)] with the notable exception that FBA was much less active than SBB 4 and 5 toward tripeptides (X-Gly-Gly).View full abstractDownload PDF (1324K) -
Tadashi MATSUDA, Shigeharu NAGASAWA, Takehiko KOIDE, Jiro KOYAMA1985 Volume 98 Issue 1 Pages 229-236
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have investigated the limited proteolysis of the third component of complement, C 3, by a human leukocyte protease, cathepsin G, by using a chemically modified C 3, which was prepared by treatment of C 3 with methylamine and a fluorescent thiol reagent, N-(dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and was thus named DACM-C 3 me. Although native C 3 was hardly cleaved by cathepsin G, DACM-C 3 me was cleaved by cathepsin G into three major fragments, which were termed C 3 c-G (150, 000 daltons, 150 kd), C 3 d-G (25 kd), and C 3 a-G (10 kd). C 3 c-G was composed of four disulfide-linked polypeptide chains of 75 kd, 35 kd, and two 25 kd. C 3 d-G and C 3 a-G were single-chain fragments derived from the α chain. The N-terminal sequence of C 3 d-G was determined as Thr-Glu-Asp-Ala-Val-, suggesting that cathepsin G released C 3 d-G by cleaving a Met-Thr peptide bond which is located at 19 residues toward the N-terminal from the cysteinyl residue forming an internal thiolester linkage in native C 3.
C 3 d-G, like C 3 d-K (a C 3 d fragment produced by the action of plasma kallikrein), was found to have bioactivities such as leukocytosis-inducing and immunosuppressive activities.View full abstractDownload PDF (2929K) -
Yuko GIGA, Atsushi IKAI1985 Volume 98 Issue 1 Pages 237-243
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA large glycoprotein with a sedimentation coefficient, s020, W, of 23.3 S was purified to homogeneity from sea urchin eggs (Anthocidaris crassispina) by gel filtration on Sepharose CL-4 B and ion-exchange chromatography on DEAE-cellulose. The molecular weight of the protein was 700, 000 as determined by sedimentation equilibrium. On polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS) it showed a single band with an apparent molecular weight of 180, 000 or 360, 000 in the presence or absence of 2-mercaptoethanol, respectively. The protein consisted of four polypeptides of equal molecular weight, which were disulfide bonded in pairs. Its carbohydrate content as determined by the phenol-sulfuric acid method was 20% of the total weight. The amino acid and carbohydrate compositions, circular dichroic spectrum and electron microscopic image are also presented.
The protein showed many structural similarities with the previously purified major glycoprotein (MCP) in the coelomic fluid of the same animal in addition to being immunologically cross reactive with it. However, the two proteins were distinct glycoproteins. Their biological functions have not been identified.View full abstractDownload PDF (2364K) -
Akira WADA, Taira OHNISHI, Yasuki NONAKA, Mitsuhiro OKAMOTO, Toshio YA ...1985 Volume 98 Issue 1 Pages 245-256
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen corticosterone was incubated with cytochrome P-45011 β, purified from bovine adrenocortical mitochondria in the presence of adrenodoxin, NADPH-adrenodoxin reductase and an NADPH generating system, aldosterone as well as 18-hydroxycorticosterone were formed with turnover numbers of 0.23 and 1.1 nmol/min/nmol P-450, respectively. Phospholipids extracted from adrenocortical mitochondria remarkably enhanced the activity of aldosterone formation by the cytochrome P-45011 β-reconstituted system. The apparent Km and turnover number were estimated to be 6.9 μM and 2.0 nmol/min/nmol P-450 for aldosterone formation in the presence of the lipidic extract. When 18-hydroxycorticosterone was tested as a substrate, cytochrome P-45011 β showed catalytic activity for aldosterone synthesis with an apparent Km and turnover number of 325 μM and 5.3 nmol/min/nmol P-450, respectively. Carbon monoxide and metyrapone inhibited the production of aldosterone from corticosterone and that from 18-hydroxycorticosterone. These results suggest that conversion of corticosterone and of 18-hydroxycorticosterone to aldosterone occurs through P-45011 β-catalyzed reaction.View full abstractDownload PDF (1044K) -
Yasuki NONAKA, Sigeo AIBARA, Toshihiro SUGIYAMA, Toshio YAMANO, Yuhei ...1985 Volume 98 Issue 1 Pages 257-260
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSingle crystals of NADPH-adrenodoxin oxidoreductase were grown in 50mM potassium phosphate (pH 7.4) containing 5% glycerol and ammonium sulfate. The crystals are monoclinic, belong to space group P 21 and have dimensions of a=83.4 Å, b=62.6 Å, c=59.3 Å, α=γ=90°, and β=107.1°. There is one molecule per asymmetric unit.View full abstractDownload PDF (1314K) -
Takayoshi IIO1985 Volume 98 Issue 1 Pages 261-263
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe skeletal muscle troponin complex, the troponin T subunit of which was labeled with 2-((4'-iodoacetamido) anilino) naphthalene-6-sulfonic acid, showed a fluorescence titration curve with a midpoint of around pCa 6.75. Addition of 2mM MgCl2 had no effect on the fluorescence titration curve. Therefore, we concluded that Ca2+ binding to the low affinity Ca2+-binding sites of troponin C induces a conformational change of troponin T, but Ca2+ binding to the high affinity Ca2+-binding sites does not.View full abstractDownload PDF (219K) -
Yasunori KUSHI, Shizuo HANDA1985 Volume 98 Issue 1 Pages 265-268
Published: July 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and rapid method for the analysis of lipids on a thin layer chromatography (TLC) plate by matrix-assisted secondary ion mass spectrometry (SI-MS) is reported. Analysis was performed without elution of the sample from the TLC plate. Mass spectra obtained by this method are free from interference due to the TLC plate absorbent and reagents used for the detection of the spots. About 1 μg of lipids applied on a TLC plate can be analyzed by this method. On scanning the plate, mass chromatograms of each lipid were obtained based on its migration distance along the plate.View full abstractDownload PDF (909K)
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