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Volume 100 (1986)
- Issue 6 Pages 1403-
- Issue 5 Pages 1109-
- Issue 4 Pages 825-
- Issue 3 Pages 521-
- Issue 2 Pages 259-
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Volume 100, Issue 6
Displaying 1-32 of 32 articles from this issue
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Russ B. ALTMAN, Oleg JARDETZKY1986 Volume 100 Issue 6 Pages 1403-1423
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNon-crystallographic approaches to the determination of protein structure must solve the problem of insufficient and low information content experimental data. Most successful methods augment experimentation with theoretical constraints (for example, potential energy functions or optimization error metrics). We believe it is important to separate the contributions of experimentation and theory in the construction of protein structure. The PROTEAN system defines protein topology on the basis of experimental data alone. Its performance on three data sets, derived from the lac-repressor headpiece of E. coli, sperm whale myoglobin, and domain 1 of bacteriophage T4 lysozyme, indicates that there may be families of related conformations that are consistent with the experimental data. These conformations provide insight into the strengths and weaknesses in the data sets. They also provide a set of structures with which to begin theoretical refinements. We outline here a strategy which maintains a clear distinction between refinements based on theory and those based on experiment, and thus allows a careful analysis of the properties of such refinement methodsView full abstractDownload PDF (2369K) -
Mary Louise OGILVIE, Michael E. DOCKTER, Laura WENZ, T. Kent GARTNER1986 Volume 100 Issue 6 Pages 1425-1431
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo lectins have been isolated: one from the venom of Lachesis muta (bushmaster lectin) and one from Dendroaspis jarnesonii venom (Jameson's mamba lectin). The lectin from bushmaster venom (BML) is similar to the lactose-binding lectins previously isolated from snake venoms (Gartner et al. (1980) FEBS Lett. 117, 13-16; Gartner & Ogilvie (1984) Biochem. J. 224, 301-307) in that it is calcium-dependent, lactose inhibitable, and is a dimer of molecular weight 28, 000. In contrast, the lactose-blockable lectin from Jameson's mamba venom (JML) has an apparent molecular weight of 26, 000 and agglutinates erythrocytes in the presence of EDTA. The absorption spectra of BML were affected by the binding of calcium, or calcium and lactose to the lectin. However, JML spectra were not affected by these conditions. While the hemagglutination activity of each of the previously described lactose-binding snake venom lectins is inhibited by reducing agent, the activities of BML and JML are not affected by reducing agent. Antiserum against bushmaster lectin cross-reacts with thrombolectin, cottonmouth lectin (CML), rattlesnake lectin (RSL), and copperhead lectin (CuHL) but not lectin from Jameson's mamba venom. This evidence plus a comparison of atomic absorption spectra, isoelectric points and amino acid analyses of the lectins demonstrate that JML and BML are different from thrombolectin, CML, RSL, and CuHL.View full abstractDownload PDF (1796K) -
Hirofumi ONISHI, Tetsuo MAITA, Takayuki MIYANISHI, Shizuo WATANABE, Ge ...1986 Volume 100 Issue 6 Pages 1433-1447
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA fluorescent fragment of Mr=23, 800 was obtained by the papain digestion of N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylene diamine (abbreviated as IAEDANS)-modified chicken gizzard myosin. The fragment was isolated by gel filtration on a Sephadex G-100 column in the presence of 5M guanidine-HCI followed by anion exchange chromatography on a QAE Sephadex A-50 column. This fragment contained 203 amino acid residues which could be assigned as a COOH-terminal part of the S-1 heavy chain based on the homology with the known sequence of rabbit skeletal myosin fragment. The amino acid sequence was K-G-M-F-R-T-V-
20 40
G -Q- L-Y- K -E-Q-L-T-K -L- M-T-T- L-R-N-T-N-P-N-F-V-R-C-I-I-P-N-H-E-K-R-A-
60
G-K-L-D-A-H-L-V-L-E-Q-L-R-C-N-G-V-L-E-G-l-R-I-C-R-Q-G-F-P-N-R-I-V-F-Q-
80 100
E-F-R-Q-R-Y-E-I-L-A-A-N-A-I-P-K-G-F-M-D-G-K-Q-A-C-1-L-M-I-K-A-L-E-L-
120 140
D-P-N-L-Y-R-1-G-Q-S-K-1-F-F-R-T-G-V-L-A-H-L-E-E-E-R-D-L-K-I-T-D-V-I-1-A-
160
F-Q-A-Q-C-R-G-Y-L-A-R-K-A-F-A-K-R-Q-Q-Q-L-T-A-M-K-V-I-Q-R-N-C-A-A-
180 200
Y-L-K-L-R-N-W-Q-W-W-R-L-F-T-K-V-K-P-L-L-Q-V-T-R. The cysteine residue which was modified with IAEDANS was of the SH1 type (Cys-65). Pro-197 was suggested to be the NH2-terminal boundary of the alpha-helical coiled-coil rod sequence of gizzard myosin, based on the homology with the nematode sequence reported by MacLachlan and Karn (Proc. Natl. Acad. Sci. U. S. 80, 4253-4257 (1983)). Three different COOH-terminal peptides (Val-Lys-Pro-Leu-Leu-Gln-Val-Thr-Arg, Val-Lys-Pro-Leu-Leu-Gln, and Val-Lys-Pro-Leu-Leu) were isolated from the tryptic digest of this fragment, suggesting that three close peptide bonds in the myosin neck (Leu 198-Gin 199, Gin 199-Val 200, and Arg 203-) are susceptible to papain digestion. The COOH-terminal 53-residue sequence of this fragment, constituting the neck part of myosin, contained 13 positively charged residues but no negatively charged residue. The possible role of the positive charges in the binding of the heavy chain to the regulatory light chain is discussed.View full abstractDownload PDF (1026K) -
Satoru YAMAMOTO, Emi KUSUNOSE, Masatoshi KAKU, Kosuke ICHIHARA, Masami ...1986 Volume 100 Issue 6 Pages 1449-1455
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEarlier, we reported the isolation of a cytochrome P-450 highly active in prostaglandin A (PGA) ω-hydroxylation (PGA ω-hydroxylase) from rabbit kidney cortex, small intestine, and colon microsomes. In the present studies, the effects of peroxisomal proliferating agents on the PGA ω-hydroxylase have been examined. Administration of clofibrate or di(2-ethylhexyl)phthalate (DEHP) resulted in a significant increase in the PGA1, ω-hydroxylase activity of kidney cortex, liver, and small intestine microsomes. Similar findings were also obtained for laurate hydroxylase activity in kidney and liver microsomes. Kidney PGA ω-hydroxylase (designated cytochrome P-450ka) was isolated and highly purified from clofibrate-or DEHP-treated rabbits, with a yield 3 times higher than that from untreated, or phenobarbital- or 3-methylcholanthrene-treated rabbits. Cytochrome P-450ka from clofibrate- or DEHP-treated rabbits exhibited the same properties as those from untreated rabbits. Guinea pig antiserum against cytochrome P-450ka strongly inhibited the ω-hydroxylation of PGA1, by kidney cortex microsomes from clofibrate-treated rabbits. The PGA1, ω-hydroxylase activity of clofibrate-treated liver microsomes was also inhibited by this antiserum, suggesting that a PGA ω-hydroxylase immunochemically related to cytochrome P-450ka exists in liver microsomes.View full abstractDownload PDF (544K) -
Misako TANIGUCHI, Masayasu INOUE1986 Volume 100 Issue 6 Pages 1457-1463
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChanges in the level of glutathione (GSH), the turnover rate, and γ-glutamyltrans-ferase (GGT) activity were examined in newborn, weanling, and adult male Wistar rats, the objective being to elucidate the mechanisms which control the hepatic GSH level during maturation as well as under conditions of different degrees of protein ingestion. The hepatic GGT activity in the newborn rats was high at birth, decreased within a few days to I to 2% of the initial level, and remained unchanged thereafter, when these rats were fed a normal diet after 3 weeks of age. In contrast, the hepatic GSH level increased 3-4-fold while total GGT activity in the kidney increased 6-8-fold. When weanling rats were fed a low protein diet (containing 10% soy protein) for 3 weeks, the hepatic GSH level decreased markedly while the GGT activity increased 5-6-fold. The turnover rate of hepatic GSH also increased, as determined by the use of buthionine sulfoximine, a specific inhibitor of GSH synthesis; a value of 2.1h was obtained in comparison with 3.5h for that of rats fed the normal laboratory chow (CRF-1). On the other hand, feeding adult rats on the low protein diet resulted in a marked decrease in hepatic GSH level with no effect on either hepatic or renal GGT activity. These results together with other observations may suggest that GSH translocated out of liver cells in the newborn rats is degraded mainly by these cells, while the tripeptide secreted by hepatocytes of adult rats is metabolized predominantly in extrahepatic tissues, such as the kidney. Feeding weanling rats on the low protein diet altered the ontogenic maturation of interorgan metabolism and transport of GSH.View full abstractDownload PDF (521K) -
Nobuhito SONE1986 Volume 100 Issue 6 Pages 1465-1470
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt is possible to prepare liposomal vesicles by solubilization of total bacterial membranes with n-heptyl β-D-thioglucoside followed by reconstitution into proteoliposomes by a freeze-thaw-sonication procedure with soybean phospholipids. The resulting proteoliposomes from total membrane fraction of sufficiently aerated cells of the thermophilic bacterium PS3 containing cytochrome aa3 showed a reasonable H+ pumping activity upon addition of reduced cytochrome c. On the other hand, the proteoliposomes reconstituted from air-limited PS3 cells containing cytochrome o and those from Nitrobacter agilis cells containing cytochrome aa3 did not show H+ pumping upon addition of reduced cytochrome c, although the vesicles showed “respiratory control”; 3-4-fold stimulation of oxygen consumption took place upon addition of an uncoupler. In proteoliposomes prepared from PS3 membranes by this method, H+-translocating ATPase (F0•F1) was successfully reconstituted as well, suggesting that this method has wide applicability for investigation of enzymes catalyzing transmembrane processes.View full abstractDownload PDF (472K) -
Tsutomu ARAKAWA1986 Volume 100 Issue 6 Pages 1471-1475
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method for calculating the isopotential partial specific volumes of proteins in concentrated salt, sugar, and amino acid solutions has been developed. It is based on the finding that the preferential hydration of the protein in these solutions is relatively independent of the concentration of the additive and is proportional to the specific surface area of the proteins, i.e., to the ratio of the total accessible surface area to molecular weight. Agreement between the calculated and experimental values was satisfactory, indicating the reliability of the proposed method. These calculations show that the isopotential partial specific volume increases greatly with the concentration of the additive, in particular in the case of Na2SO4, (NH4)2SO4, and sucrose, and for smaller proteins.View full abstractDownload PDF (372K) -
Koh IBA, Ken-ichiro TAKAMIYA, Mitsuo NISHIMURA, Shigeru ITOH1986 Volume 100 Issue 6 Pages 1477-1480
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe EPR spectra of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides were measured at liquid helium temperature. The purified cytochrome b-562 gives a high spin signal at g=6.0. Anaerobic titration of this signal confirmed the presence of two redox components with Em=40 and -110mV at pH7.5. These values are consistent with the published ones, Em=55 and - 100mV at pH7.0, that were optically estimated for the same type of preparation (Iba et al. (1985) FEBS Left. 183, 151-154). The power saturation behavior of the g=6.0 signal at different redox potentials indicated a direct spin-spin interaction between these two redox centers.View full abstractDownload PDF (276K) -
Satoshi B. SATO, Yasushi SAKO, Shohei YAMASHINA, Shun-ichi OHNISHI1986 Volume 100 Issue 6 Pages 1481-1492
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have developed a novel method for isolating specific endocytic vesicles using magnetic ligands and high-gradient magnetic separation. Ligands were prepared by coating extremely fine ferrite particles (10-20nm) with bovine serum albumin and then conjugating asialoglycopeptides. These ligands were introduced into rat liver by perfusion at 16 or 37°C, or by injection through the tail vein. The ligand particles were observed as electron-dense small grains in membrane-bound vesicles in Kupffer as well as parenchymal cells by electron microscopy. Livers were taken out, homogenized and lightly centrifuged. The supernatant was pumped into a separator glass tube filled with very fine ferritic stainless steel fibers and placed in a magnetic field of 0.9-2T. Vesicles containing ferrite particles were collected with a high efficiency (ca. 70% of endocytosed magnetic ligands). About 70% of uptake appeared to be mediated by the asialoglycoprotein receptors. The captured vesicles were practically free from marker enzymes for plasma membranes, endoplasmic reticulum, and Golgi apparatus. Lysosomal enzyme activity of the vesicles increased with the time of perfusion at 37°C but not at 16°C. Protein composition of the captured vesicles was analyzed by one- and two-dimensional gel electrophoresis. The composition changed characteristically with time on perfusion at 16 and 37°C. The present method provides a powerful tool to collect prelysosomal endocytic vesicles containing specific ligands and lysosomes fused with these specific endocytic vesicles.View full abstractDownload PDF (3921K) -
Masakazu MURAKAMI, Michio NAKAMURA, Shigeki MINAKAMI1986 Volume 100 Issue 6 Pages 1493-1497
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe 2, 6-dichlorophenolindophenol (DCIP)-reducing activity of the phagocytosis-associated NADPH oxidase was investigated using homogenates and a membrane fraction (F2) of elicited guinea pig peritoneal macrophages stimulated by phorbol myristate acetate. Essentially all of the stimulation-specific DCIP reduction under aerobic conditions could be inhibited when high concentrations of superoxide dismutase (SOD), about 10 times those usually used to inhibit the superoxide (O-2)-mediated cytochrome c reduction, were used. SOD inhibited the DCIP reduction by chemically generated O2- in the same manner as the stimulation-specific DCIP reduction by the macrophage F2, and the concentration of SOD necessary for 50% inhibition was about 10 times that for the reduction of cytochrome c. Under anaerobic conditions, however, the NADPH oxidase could reduce DCIP, though the rate was slow because we could not use a sufficiently high DCIP concentration. The observations indicate that the NADPH oxidase preferentially reduces oxygen under aerobic conditions, though the oxidase can reduce DCIP in the anaerobic state.View full abstractDownload PDF (432K) -
Hideo NAKAGAWA, Kazuhiko SAKATA1986 Volume 100 Issue 6 Pages 1499-1506
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGelatinase has been partially purified from exudate in the acute phase of carrageenin-induced inflammation in rats. The enzyme occurs in a latent form that can be activated with 4-aminophenylmercuric acetate (APMA). The latent gelatinase was separated into an active gelatinase and a protein fraction by zinc-chelating Sepharose 6B column chromatography in the final step of purification, suggesting that the latent gelatinase is an enzyme-inhibitor complex. The pH optimum of the active gelatinase is about 7.5 and no reactivity toward native type I collagen or α-casein was detected. The molecular weights of the latent and active gelatinases were about 245, 000 and about 185, 000, respectively, as determined by gel filtration on Sephadex G-200. On the other hand, both latent and active gelatinases occurred in multiple forms in SDS-substrate polyacrylamide gel electrophoresis; the latent gelatinase showed two bands with molecular weights of 105, 000 and 69, 000, and two additional bands of 88, 000 and 83, 000 appeared when the latent gelatinase was activated with APMA, while the active gelatinase showed all four species. The active gelatinase was inhibited by metallo-proteinase inhibitors, but not by serineor cysteine-proteinase inhibitors, suggesting that the exudate gelatinase is a metalloproteinase The active gelatinase was also inhibited by serum proteins such as albumin and γ-globulin, suggesting that gelatinase does not remain in an active form in the inflammatory lesion, where the vascular permeability is increased.View full abstractDownload PDF (1387K) -
Akira SHIMADA, Eiii ITO1986 Volume 100 Issue 6 Pages 1507-1521
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA glucosyltransferase, extracted from the membranes of Bacillus cereus AHU 1030 with Tris-HC1 buffer containing 0.1% Triton X-100 at pH 9.5, was separated from an endogenous glucosyl acceptor by chromatography on DEAE-Sepharose CL-6B subsequent to chromatography on Sepharose 6B. Structural analysis data showed that the glucosyl acceptor was a glycerol phosphate polymer linked to β-gentiobiosyl diglyceride. The enzyme catalyzed the transfer of glucosyl residues from UDP-glucose to C-2 of the glycerol residues of repeating units of the acceptor. On the other hand, a lipoteichoic acid which contained 0.3 D-alanine residue per phosphorus was isolated from the cells by phenol treatment at pH 4.6. Except for the presence of D-alanine, this lipoteichoic acid had the same structure as the glucosyl acceptor. The rate of glucosylation observed with the n-alanine-containing lipoteichoic acid as the substrate was less than 40% of that observed with the D-alanine-free lipoteichoic acid, indicating that the ester-linked n-alanine in the lipoteichoic acid interferes with the action of the glucosyltransferase. The enzyme also catalyzed glucosylation of poly(glycerol phosphate) which was synthesized in the reaction of a separate enzyme fraction with CDP-glycerol. Thus, it is likely that the glucosyl-transferase functions in the synthesis of cell wall teichoic acid.View full abstractDownload PDF (1262K) -
Hideaki NISHINO, Akio ITO1986 Volume 100 Issue 6 Pages 1523-1531
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTissue, cellular, and subcellular distributions of OM cytochrome b-mediated NADH-semidehydroascorbate (SDA) reductase activity were investigated in rat. NADH-SDA reductase activity was found in the post-nuclear particulate fractions of liver, kidney, adrenal gland, heart, brain, lung, and spleen of rat. Liver, kidney, and adrenal gland had higher NADH-SDA reductase activity than other tissues, and OM cytochrome b-dependent activity was 60-70% of the total activity. On the other hand, almost all of the reductase activity of heart and brain cells was mediated by OM cytochrome b. The ratio of the OM cytochrome b-mediated activities of NADH-SDA reductase to rotenone-insensitive NADH-cytochrome c reductase varied among these tissues. OM cytochrome b-mediated NADH-SDA reductase and rotenone-insensitive NADH-cytochrome c reductase activities were mainly present in the parenchymal cells of rat liver. The localization of the cytochrome-mediated reductase activities in the outer mitochondrial membrane was confirmed by subfractionation of liver mitochondria. Among the submicrosomal fractions, OM cytochrome b-mediated NADH-SDA reductase activity was highest in the cis-Golgi membrane fraction, in which monoamine oxidase activity was also highest. On the other hand, OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase activity showed a slightly different distribution pattern from the NADH-SDA reductase activity. Thenoyltrifluoroacetone (TTFA), a metal chelator, effectively inhibited the NADH-SDA reductase activity, though other metal chelators did not affect the activity. TTFA failed to inhibit rotenone-insensitive NADH-cytochrome c reductase activity at the concentration which gave complete inhibition of NADH-SDA reductase activity.View full abstractDownload PDF (698K) -
Tetsuya OIDA1986 Volume 100 Issue 6 Pages 1533-1542
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBinding of free fatty acid (FFA) to human serum albumin (HSA) was studied by 1H-NMR spectroscopy. Addition of FFA to defatted HSA at a mole ratio (FFA/HSA) up to 4 caused a small change in the NMR spectrum of HSA. The integrated intensity of sharp signals of the histidine Cz proton region of HSA decreased as the mole ratio was increased from 0 to 4 for both medium chain (lauric acid) and long chain (palmitic acid, stearic acid, and oleic acid) FFA's. By contrast, when the mole ratio was increased above 4, several histidine C2 proton signals coalesced and sharpened. Therefore, the HSA molecule appears to have a different conformation on binding with more than 4 FFA molecules, which allows increased local motions of HSA. By analyzing the NMR difference spectra of HSA with various amounts of FFA, the conformational change of HSA was investigated in more detail. The difference spectrum between [HSA+2FFA] and [HSA+FFA] was almost the same as the difference spectrum between [HSA+FFA] and [HSA], which suggests that one primary site binds a pair of FFA molecules. These results are consistent with those of a spectroscopic study with polyene fatty acids (Berde, C. B. et al. (1979) J. Biol. Chem. 254, 391-400). The existence of a bimolecular complex of FFA molecules in aqueous solution may facilitate this type of binding. Similarly, it was found that the third and fourth FFA molecules were bound to a secondary site on HSA, because the difference spectrum between [HSA+4FFA] and [HSA+ 3FFA] was nearly equal to the difference spectrum between [HSA+3FFA] and [HSA+2FFA]. Further addition of FFA resulted in a drastic spectral change of HSA. The NMR difference spectrum between HSA solutions with perdeuterated FFA and those with undeuterated FFA gave the 1H-NMR spectra of FFA molecules bound to HSA. Titration of FFA revealed that, in the binding to the primary site of HSA, the carboxyl group of FFA is tightly bound to the protein, whereas the methyl group is not so firmly bound. In contrast, in the binding to low affinity sites, the methyl group is bound to HSA as tightly as other portions of the molecule.View full abstractDownload PDF (810K) -
Kaoru SUGASAWA, Yukio ISHIMI, Fumio HANAOKA, Masa-atsu YAMADA1986 Volume 100 Issue 6 Pages 1543-1550
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA chemical cross-linking reagent, dithio-bis (succinimidyl propionate), is known to be capable of cross-linking histones in nucleosomes so as to give one major product with the molecular weight of about 100, 000. Because the product has been supposed to represent the cross-linked histone octamer, the reaction has been used for studying the movements of core histones in nucleosomes. However, the precise protein composition of the product has not been determined thus far, so that the use of the reaction was limited. We report here that the 100 kilodalton product is composed of the core histones, and does not contain significant amounts of any other proteins. Moreover, quantitative analysis of the content of each core histone confirmed that the four types of core histones participate in the product with an equal molar ratio. As one can specifically observe the behaviors of histone octamers with this reaction, it should be useful for research in various fields related to the dynamics and functions of the nucleosome.View full abstractDownload PDF (2411K) -
Naoko ODA, Masayuki YOSHIDA, Shuji TANAKA, Hiroshi KJHARA, Motonori OH ...1986 Volume 100 Issue 6 Pages 1551-1560
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInteractions of dimeric Trimeresiiras flavoviridis (the Habu snake) phospholipase A2 (PLA2), des-octapeptide(l-8)-PLA2 (L-fragment) (14% of PLA2 activity), and p-bromophenacyl bromide (BPB)-inactivated PLA2 (BP-PLA2) with dyes, namely, proflavine, 1-anilinonaphthalene-8-sulfonate (Ans), and 2-toluidinylnaphthalene-6-sulfonate (Tns), were investigated. All dyes were bound in a 1:1 molar ratio to the subunit of the proteins. Proflavine was bound most strongly to PLA2 and Ans and Tns were bound to the three proteins with comparable affinities. Capabilities of the dyes for inhibiting alkylation of His-47 of PLA2 with BPB were in the following order: Ans>proflavine>Tns. Fluorescences of Ans and Tns that were increased in the presence of PLA2 were further greatly enhanced upon the addition of Ca2+, with concomitant formation of the ternary complexes. Ca2+, however, inhibited, competitively or noncompetitively, the bindings of the dyes to PLA., All dyes were bound to the active site of PLA2 but with different orientations. Inactivation of L-fragment with BPB was inhibited by the dyes in the following order: Tns> proflavine_??_Ans. Addition of Ca2+ to the binary complexes formed from L-frag-ment and Ans or Tns caused no additional enhancement of fluorescence in spite of the formation of the ternary complexes. The active site structures are different between PLA2 and L-fragment, and the N-terminal octapeptide moiety of PLA2 possibly plays a role in maintaining the optimally arranged active site structure of the molecule. Comparison of the data suggests that the N-terminal moieties of PLA2s from snakes of an elapid family and from mammalain pancreas are essential for catalysis of a micellar substrate, whereas those of PLA2s from snakes of a viperid family, such as T. flavoviridis, are not. BP-PLA2 bound Ca2+and was similar to L-fragment in terms of the fluorescence measurements. It appears that the active site of PLA2 has a space large enough to accommodate p-bromophenacyl, Ans or Tns, and Ca2+ together. Comparison of the emission maxima of Ans and Tns complexed with the three proteins indicated that Tns could be a useful fluorescent probe informing us of the state (disorder) of the active site of PLA2.View full abstractDownload PDF (806K) -
Takashi IDE, Michihiro SUGANO1986 Volume 100 Issue 6 Pages 1561-1568
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe metabolic fate of the geometrical isomers of 6-, 9-, and 11-octadecenoic acids was studied in isolated perfused rat livers. Although the ketogenecities of these monounsaturated fatty acids generally decreased as the double bond was moved away from the carboxyl group, a dependence on the geometrical difference was found only for 9-octadecenoate, the rate being significantly lower in the cis-isomer. Very low density lipoprotein lipid secretion was distinctly and specifically decreased when trans-9-octadecenoic acid was perfused, but no such difference was observed with other isomers. Various traps-isomers were actively incorporated into the hepatic lipids and secreted apparently at the expense of preexisting endogenous cis-octadecenoate. The trans-isomers were all incorporated exclusively at the 1-position of hepatic phosphatidylcholine. Concentrations of the cis-octadecenoate in the glycerolipids secreted and remaining in the liver were characteristically modified depending on the location of the ethylenic bond of the cis-octadecenoate. Thus, both the location of the double bond in the acyl-chain and the geometrical configuration specifically influence the rate of oxidation and esterification of octadecenoic acid in perfused rat liver.View full abstractDownload PDF (507K) -
Shigenori KAWAHARA, Yutaka KIRINO, Seiji NAGAO, Yoshinori NOZAWA1986 Volume 100 Issue 6 Pages 1569-1573
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn ion channel in ciliary membrane vesicles isolated from Tetrahymnena pyriformis NT-1 was characterized with a single-channel recording technique applied to the membrane reconstituted into a planar lipid bilayer made of soybean phospholipid. The gating of this channel is independent of membrane potential and it spends most of the time in its open-channel state. It is selective for metal cations over gluconate anion although it little distinguishes cations, Ca2+, Ba2+, Mg2+, and K+. This channel seems to be identical to the monovalent cation channel in Tetrahymena thermophila BII cilia, which was recently reported by Oosawa and Sokabe (Am. J. Physiol. 249, C177-0179, 1985). The channel conductance saturates with increasingion concentration. The maximum value (in pS) for the single-channel conductance obtained in symmetric solutions of sufficiently high concentration is in the order of K+(212) > Ba2+(41) > Ca2+ (22) > Mg2+ (20). On the other hand, the affinity of the channel for these ions is in the reverse order.View full abstractDownload PDF (396K) -
Mieko OSHIMA, Mitsuko HASHIGUCHI, Noboru SHINDO, Seiichi SHIBATA1986 Volume 100 Issue 6 Pages 1575-1582
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to study the toxicity of aminoglycoside, human skin fibroblasts were used as a model for basic studies, since they are known to have a specific aminoglycoside-binding site and to translocate the drug into the cells. Following the exposure of fibroblasts to gentamicin for 3 days, the cells formed many osmitophilic lamellar materials (myeloid bodies) in the lysosomes, while the other cellular structures appeared to remain normal. Although gentamicin was intensively accumulated within the lysosomes, intralysosomal pH, determined by the fluorescence intensity ratio method using fluorescein-isothiocyanate-labeled dextran, did not alter. Among the lysosomal enzymes, the activities of six different glycosidases were unchanged. On the other hand, sphingomyelinase and acid lipase activities were greatly decreased, while phospholipase A activity was increased. These results indicate that the lipid metabolism of fibroblasts is altered by gentamicin treatment, and that perturbation of intralysosomal pH can not be the cause of the changes observed in cell lysosomal enzyme activities.View full abstractDownload PDF (2138K) -
Akira WADA1986 Volume 100 Issue 6 Pages 1583-1594
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSKaltschmidt and Wittmann's two dimensional gel electrophoresis was improved in the following points. Preruns using radical scavengers were carried out to eliminate free radicals remaining in gels. Gelation of sample solutions was not performed to avoid immobilization of proteins in the sample gels. Instead, for preparing sample gels, prior to the first dimension (1-D) electrophoresis, another electrophoresis was performed to charge proteins into gel pieces polymerized previously. Proteins migrated together with charged reductants to avoid formation of artificial disulfide bridges during migration. The second dimension (2-D) electrophoresis was carried out at a more acidic pH, 3.6, to get better separation of very small and basic proteins. With these modifications, quantitative yield and reproducibility became better, and many faint spots disappeared not only at the high molecular weight side but also in the region containing primary spots of ribosomal proteins. The proportionality of the migration distance to the logarithm of molecular weight was also increased. On the improved 2-D electrophoretogram of Escherichia coli ribosomal proteins, four new spots, called protein A, B, C, and D, were found in the basic region. Proteins A, B, and C belong to 50S subunits but D to 30S. Their molecular weights were determined electrophoretically as 6, 400, 4, 900, 8, 200, and 5, 900, respectively. Their copy numbers in crude ribosomes were estimated to be 0.6, 0.4, 0.3, and 0.1, respectively, by using 14C-labeling.View full abstractDownload PDF (1951K) -
Akira WADA1986 Volume 100 Issue 6 Pages 1595-1605
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour new proteins, A, B, C, and D, found in Escherichia coli ribosomes by an improved two dimensional gel electrophoresis were characterized by oxidation, reduction, and carboxymethylation of cysteine residues, and CsC1 fractionation. The cysteine contents of proteins A, B, C, and D were determined to be 1 ± 0, 3 ± 1, 5±1, and 0 ± 0 by carboxymethylation with iodoacetic acid. The components of protein complexes, which formed numerously under non-reducing conditions, were analyzed. Including protein A, B, and C, every ribosomal protein (r-protein) having cysteine residue(s) except unconfirmed S1 was proved to form such complexes with various combinations. The cysteine residue in protein A, in particular, was highly reactive to make intermolecular S-S bridges so that spot A almost disappeared on the second dimension gel under the non-reducing conditions. Proteins B and C shifted their spots by reduction towards upper left side as do all known r-proteins having plural cysteine residues except S1. This suggests that proteins B and C change their conformation by intramolecular S-S bridges. The CsCI density gradient centrifugation of high salt washed 70S ribosomes showed that protein A belonged to the insoluble split proteins, proteins B and C to the core particles, and protein D and a small population of B to the soluble split proteins. The electrophoretic behaviors, CsCI fractionation and stoichiometry of the four new proteins suggested strongly that they were intrinsic ribosomal constituents different from known ribosomal proteins or factors.View full abstractDownload PDF (2973K) -
Keiko MITSUNAGA, Yukio FUJINO, Ikuo YASUMASU1986 Volume 100 Issue 6 Pages 1607-1615
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn sea urchin embryos, primary mesenchyme cells, descendants from micromeres produced at the 16-cell stage, form spicules or CaCO3 deposits in their skeletal vacuoles, at the post-gastrula stage. Micromeres isolated at the 16-cell stage also differentiate into spicule-forming cells during their culture at the same time schedule as in the embryos. The present study was planned to observe change in the activity of Cl-, HCO3--ATPase, which was expected to contribute to the carbonate supply for CaCO3 deposition, during development. ATP-hydrolysis in the microsome fraction, obtained from embryos of the sea urchin, Hemicentrotus pulcherrimus, and from micromere-derived cells in culture was stimulated by Cl- and HCO3- in the presence of ouabain and EGTA. The ATP-hydrolysis was inhibited by ethacrynic acid, an inhibitor of Cl-, HCO3--ATPase. The activity of Cl-, HCO3--ATPase in embryos and in micromere-derived cells increased during development, keeping pace with the rate of calcium deposition in spicules. Formation of calcified spicules in the cultured micromere-derived cells was inhibited by ethacrynic acid. These results indicate that Cl-, HC03--ATPase plays an important role in the mechanism of CaCO3 deposition in the primary mesenchyme cells.View full abstractDownload PDF (721K) -
Masaki HAMANO, Katsutoshi NITTA, Kunihiro KUWAJIMA, Shintaro SUGAI1986 Volume 100 Issue 6 Pages 1617-1622
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA Ca2+-sensitive electrode was used for determination of the binding strength of Ca2+ to bovine α-lactalbumin in 60mM Tris buffer (pH 7.8-8.5) in the presence of various concentrations of NaCl. The dependence of the apparent binding constant on the concentration of NaCI was consistent with competitive binding of Ca2+ and Na+, and the binding constants of Ca2+ and Na+ were found to be 2.2 (±0.5) × 107M-1 and 99 (± 33)M-1, respectively, at 37°C and pH 8.0. The temperature dependence of the binding constant of Ca2+ was examined between 30 and 45°C; extrapolation of the dependence led to a binding constant of _??_1 × 108M-1 at pH 8.4 and 25°C. The electrostatic contribution and conformational effect of the protein were also taken into consideration, and the intrinsic binding constant of Ca2+ to native α-lactalbumin was calculated to be (1.2-1.5) × 1010M-1 at 37°C and pH 8.0.View full abstractDownload PDF (450K) -
Ryotaro TSUJIMURA1986 Volume 100 Issue 6 Pages 1623-1629
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn understanding of the mechanism of kainic acid toxicity to neurons could provide important clues to pathogenesis of Huntington's chorea. The existence of high-affinity binding sites for kainate, a foreign compound, is suggestive of the existence of kainate-like substances in the brain. In addition to such neurotoxic kainate-like substances, and endogenous inhibitor of kainate binding may also exist in the brain to allow the synaptic function to operate normally. Based on this idea, the existence of molecules which inhibit [3H]kainate binding to synaptic membranes was examined in rat brain. An endogenous inhibitor of [3H]kainate binding to synaptic membranes was found in the supernatant obtained from synaptic membranes of rat brain. The inhibitor is a thermostable, basic protein with a relatively low molecular weight.View full abstractDownload PDF (522K) -
Terutaka TSUDA, Yasuo HAMAMORI, Yasuo FUKUMOTO, Kozo KAIBUCHI, Yoshimi ...1986 Volume 100 Issue 6 Pages 1631-1635
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIncubation of quiescent cultures of Swiss 3T3 cells with epidermal growth factor (EGF) caused an increase in c-myc mRNA. Under these conditions, EGF did not induce phosphoinositide turnover, formation of diacylglycerol, formation of inositol tris-, his-, and monophosphates, protein kinase C activation, or Ca2+ mobilization. Although it has been reported that both protein kinase C and Ca2+ may be responsible for the platelet-derived growth factor- and fibroblast growth factor-induced increases in c-myc mRNA in Swiss 3T3 cells (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., & Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192), these results indicate that neither protein kinase C nor Ca2+ is involved in the EGF-induced increase in c-myc mRNA, and that an unidentified system may be involved in this reactionView full abstractDownload PDF (368K) -
Yasuo TSUNOGAE, Isao TANAKA, Takashi YAMANE, Jun-ichi KIKKAWA, Tamaich ...1986 Volume 100 Issue 6 Pages 1637-1646
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of the complex formed by bovine trypsin and Bowman-Birk type protease inhibitor AB-I extracted from azuki beans (Vigna angularis) ‘Takara’ has been analyzed. The structure was solved by the application of the phase combination of single isomorphous phases and trypsin model phases, followed by phase improvement using the iterative Fourier technique. From the resulting electron density map, a three-dimensional atomic model of the trypsin binding domain of AB-I has been built. The peptide chain at the trypsin reactive site turns back sharply at Pro29 and forms a 9-residue ring (Cys24-Cys32). The ‘front side’ of this ring, consisting of the reactive site (Cys24-Met28), interacts with trypsin in a similar manner to other families of inhibitors and forms a stable complex, which seems to be maintained by the interactions with the ‘back side’ of this ring (Pro29-Cys34). The similar spatial arrangements of the ‘back side’ of this inhibitor and the ‘secondary contact region’ of the other inhibitors with respect to the reactive site suggest an important common role of these regions in exhibiting inhibitory activity.View full abstractDownload PDF (2712K) -
Kichisuke NISHIMOTO, Hiroo FUKUNAGA, Kunio YAGI1986 Volume 100 Issue 6 Pages 1647-1653
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of hydrogen bonding at hetero atoms of oxidized flavin on its electron acceptability was studied by the ab initio molecular orbital method. The calculations were carried out for all possible lumiflavin-H2O complexes and for some lumiflavin-formamide complexes. Calculated data showed that the magnitudes of hydrogen bonding energy at the hetero atoms are in the order of N(3)H > N(5) > 0(12) > N(l) > O(14). It was found that the atomic orbital coefficient of the lowest unoccupied molecular orbital is the largest at N(5) and that hydrogen bonding at N(1), N(5), O(12), and O(14) increases the electron acceptability of the oxidized flavin at N(5), while hydrogen bonding at N(3)H decreases it.View full abstractDownload PDF (432K) -
Keizo TESHIMA, Yutaka KITAGAWA, Yuji SAMEJIMA, Saju KAWAUCHI, Kiyoshi ...1986 Volume 100 Issue 6 Pages 1655-1662
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe hydrolysis of 1, 2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the phospholipase A2 from the venom of Agkistrodon halys blomhoffii, was studied at 25°C and the ionic strength of 0.1 in the presence of 3-33.3mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micelle concentration (cmc), were analyzed according to the Michaelis-Menten equation. The pH-dependence curve of the Km value exhibited only one transition below pH 8. The analytical results indicated that the pK value of 6.30 of an ionizable group changed to 6.54 on the binding of the monodispersed substrate. This ionizable group was assigned as the α-amino group on the basis of its pK value, which had been determined from the pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) (Ikeda and Samejima (1981) J. Biochem. 90, 799-804, and Haruki et al. (1986) J. Biochem. 99, 99-109). The pH-dependence curve of the kcat value exhibited two transitions, below pH 6.5 and above pH 9.5. The analytical results indicated the participation of two ionizable groups with pK values of 5.55 and 10.50. Deprotonation of the former and protonation of the latter group were found to be essential for the catalysis. The former ionizable group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Ikeda et al. (1981) J. Biochem. 90, 1125-1130). The latter group was tentatively assigned as the invariant Tyr 52, which is located in close proximity to the imidazole ring of His 48 (Dijkstra et al. (1983) J. Mod. Biol. 168, 163-179). These findings were compatible with the results of our previous study on the cobra (Naja naja atra) venom phospholipase A2, although in the case of the cobra enzyme the additional minor participation of the α-amino group was observed.View full abstractDownload PDF (629K) -
Masataka ISHINAGA, Yasuko OKITA, Akiko ITO1986 Volume 100 Issue 6 Pages 1663-1668
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of propylgallate (PrG, an antioxidant) on the thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) was studied by means of differential scanning calorimetry. A DPPC/PrG mixture displayed distinctive thermotropic behavior that was significantly different from that of a DPPC/cholesterol or DPPC/ vitamin E mixture. Although the enthalpy of the phase transition (ΔH) for DPPC decreased at a low concentration of the PrG and the transition peak became broadened, ΔH increased again and the peak became sharper on the addition of more PrG. The same was observed for DPPC/methylgallate and DPPC/ethylgallate mixtures, but not for a DPPC/butylgallate mixture. On the other hand, the transition temperature (Tm) of the DPPC/gallate derivative mixtures decreased with an increase in the chain length of the acyl moiety of the gallate derivatives. The pretransition and subtransition of the DPPC/PrG mixture were eliminated on the addition of a PrG, and Tm of the DPPC/PrG mixture approached about 26°C. These results suggested that the chain length of the acyl moiety must be Cl to C3 for the unique effect of the gallate derivatives described above, and that DPPC forms a complex with PrG as a pure component.View full abstractDownload PDF (419K) -
Kimimitsu ODA, Koichiro MIKI, Shinichi HIROSE, Noboru TAKAMI, Yoshio M ...1986 Volume 100 Issue 6 Pages 1669-1675
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing an immunoblotting technique, we have studied the processing of plasma proteins in subcellular fractions of rat liver including rough and smooth microsomes and the Golgi subfractions. Each subcellular fraction was directly subjected to SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies against α1-protease inhibitor, haptoglobin, and the third component of complement (C3) in combination with 125I-protein A or 125I-rabbit anti-(goat IgG)-IgG. The results demonstrated that proteolytic processing of precursors of complement C3 and haptoglobin occurs in different compartments along the secretory pathway; conversion of prohaptoglobin takes place in the endoplasmic reticulum, while that of pro-C3 occurs in the Golgi complex. The processing in oligosaccharide chains of glycoproteins was also analyzed. The Golgi fraction was characterized by the presence of the mature 56 kDa α1-protease inhibitor, which was indistinguishable from the serum α1-protease inhibitor in SDS-polyacrylamide gel electrophoresis. In contrast, the immature 51 kDa form was the only form of α1-protease inhibitor found in the microsomal fraction. Similar results were obtained for the β subunit of haptoglobin; the immature 33 kDa form was detected in the microsomal fraction, while the mature 36 kDa form was found in the Golgi fraction. Taken together, these results identified the intracellular sites where these plasma proteins are modified by selective proteolysis and/or glycosylation.View full abstractDownload PDF (2860K) -
Youichi MIYAMOTO, Masaaki KURODA, Eisuke MUNEKATA, Tomoh MASAKI1986 Volume 100 Issue 6 Pages 1677-1680
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSonication of F-actin in the absence of added ATP in the solvent induces denaturation of the actin. When phalloidin is added to actin at the molar ratio of 1:2, the denaturation is completely inhibited. More directly, pelleting experiments have indicated that the binding of phalloidin to actin subunits is saturated at the same molar ratio. The protection of F-actin from heat denaturation or depolymerization with 0.6M KI is also complete with one molecule of the toxin over two actin subunits. Therefore, it is concluded that the binding ratio of phalloidin and the actin subunit is not 1:1 but 1:2.View full abstractDownload PDF (303K) -
Hiromi TAKANO-OHMURO, Kazuhiro KOHAMA1986 Volume 100 Issue 6 Pages 1681-1684
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGizzard smooth muscle myosin, the 20, 000 Mr light chain (L20) of which had been phosphorylated in vitro with a calmodulin-myosin light chain kinase system, was separated into 5 isolated bands in a pyrophosphate polyacrylamide gel. Their mobilities were in the following order: myosin with 2 unphosphorylated L20 (GM)<myosin with 1 unphosphorylated and 1 mono-phosphorylated L20 (GMP1)<myosin with 2 mono-phosphorylated L20 (GMP2)<myosin with 1 mono-phosphorylated and 1 di-phosphorylated L20 (GMP3)<myosin with 2 di-phosphorylated L20 (GMP4). We used this pyrophosphate polyacrylamide gel electrophoresis to analyze the phosphorylated state of taenia coli smooth muscle during K+-induced contraction. During the initial 2 min contraction, phosphorylated forms corresponding to GMP1 and GMP2 were detected in addition to the unphosphorylated form.View full abstractDownload PDF (1863K)
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