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Volume 106 (1989)
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Volume 106, Issue 6
Displaying 1-35 of 35 articles from this issue
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Michio Iwai, Masakazu Kobayashi, Kouichi Tamura, Yoshinori Ishii, Hisa ...1989 Volume 106 Issue 6 Pages 949-951
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe primary structure of human IGF-I, except for the disulfide bond system, has been reported by Rinderknecht and Humbel. IGF-I afforded the corresponding characteristic peptide fragment on V8 protease digestion, which contained Cys6, Cys47, Cys48, and Cys52. Two possible fragments, Type I with Cys6-Cys47 and Cys48-Cys52, and Type II with Cys6-Cys48 and Cys47-Cys52, were synthesized. The disulfide bond systm of IGF-I was unequivocally determined to be the Type II form along with Cys18-Cys61. Interestingly, the Type I system was included in the disulfide bond isomer produced as the main by-product in the refolding step on IGF-I synthesis by the recombinant DNA method.View full abstractDownload PDF (668K) -
Sumiko Kimura, Koscak Maruyama1989 Volume 106 Issue 6 Pages 952-954
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSα-Connectin (also called titin 1) has been isolated from rabbit back muscle. Myofibrils were well washed with 5mM NaHCO3 and then extracted with 0.2M sodium phosphate, pH 7.0. The extract was dialyzed against 0.1M potassium phosphate, pH 7.0, to sediment myosin. The supernatant, adjusted to 0.18M potassium phosphate, pH 7.0, and 4M urea, was subjected to DEAE Toyopearl column chromatography.β-Connectin was eluted in the flow-through fraction and α-connectin was eluted at around 0.1M NaCl, when a 0 to 0.25M NaCl gradient was applied. The separated α-connectin was dialyzed against 0.2M potassium phosphate, pH 7.0. The resultant α-connectin showed the same mobility as that in an SDS extract of rabbit back muscle on SDS gel electrophoresis using 1.8% polyacrylamide gels. A monoclonal antibody against chicken breast muscle β-connectin reacted with the α-connectin isolated from rabbit back muscle.View full abstractDownload PDF (1044K) -
Tsuyoshi Okagaki, Sugie Higashi-Fujime, Kazuhiro Kohama1989 Volume 106 Issue 6 Pages 955-957
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe actin-activated ATPase activity of Physarum myosin has been shown to be inhibited by μM levels of Ca2+, the mode of which is in contrast to the activating. effect of Ca2+ on scallop myosin (Kohama, K.(1987) Adv. Biophys. 23, 149-182 for a review). To determine if Ca2+ regulates ATP-dependent sliding between actin and the myosins, fluorescent actin-filaments were allowed to move on the myosins fixed to a glass surface. The movement on Physarum and scallop myosins was inhibited and activated, respectively, by Ca2+. For this myosin-linked regulation to occur for Physarum myosin, myosin phosphorylation was shown to be a prerequisite.View full abstractDownload PDF (709K) -
Hideyuki Yoshimura, Shigeru Endo, Mutsuo Matsumoto, Kuniaki Nagayama, ...1989 Volume 106 Issue 6 Pages 958-960
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe highly dissociable α3β3 subunit complex (Mr=319, 582) of thermophilic ATP synthase was crystallized on a mercury surface under oxygen. The two-dimensional crystal was compared with that of TF1 (Mr=385, 351, α3β3δε subunit complex) by means of computer image processing. The crystals showed the same hexagonal lattice (a=b=10nm), despite the difference in their molecular weights. The color images of the two protein molecules were also hexagonal. However, there was an open hole in the image of the α3β3 complex, where small subunits (γ, δ, and ε) of TF1 may have been located. The structure of this heterohexamer is consistent with that deduced from other physical parameters.View full abstractDownload PDF (1644K) -
Yasuhiro Yamashita, Yasuyuki Imai, Toshiaki Osawa1989 Volume 106 Issue 6 Pages 961-965
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESST cells from enlarged lymph nodes of MRL/MpJ-lpr/lpr (lpr) mice were found to express more binding sites for strongly hemagglutinating Phaseolus vulgaris agglutinin (PHA-E4) and fewer binding sites for Ricinus communis agglutinin (RCA) than those from normal MRL/MpJ-+/+ (+/+) mouse lymph node. We found that high-molecular-weight (180K-220K) glycoproteins on lpr T cells were strongly stained with these lectins on Westernblotting. These glycoproteins were found to belong to the CD45 family, by absorption with monoclonal anti-CD45 antibody. We also found that the other glycoproteins (105K and 120K glycoproteins on lpr T cells and a 105K glycoprotein on +/+ T cells) were strongly stained with the lectins which preferentially bind to mucin-type (O-linked) sugar chains on the cell surface. These glycoproteins were found to be leukosialins, by absorption with antileukosialin serum. From the results of the lectin-binding to these glycoproteins after sialidase treatment, CD45 antigens and leukosialin molecules on lpr T cells were found to have many more terminal α2, 3-linked sialic acids than those on +/+ T cells, and this fact explains why lpr T cells have more binding sites for PHA-E4 but fewer binding sites for RCA.View full abstractDownload PDF (2557K) -
Hiroyuki Kogaki, Seiji Inoue, Kiyoshi Ikeda, Yuji Samejima, Tamotsu Om ...1989 Volume 106 Issue 6 Pages 966-971
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSPhospholipase A2 inhibitor was purified from the blood plasma of Habu, Trimeresurus flavoviridis, by Sephadex G-200 gel filtration, DEAE-cellulose chromatography, and Blue-Sepharose CL-6B column chromatography. The purified inhibitor was shown to be a glycoprotein with a molecular weight of about 100K. It was found to consist of four subunits whose molecular weights were around 20-24K. In order to examine the inhibition mechanism of the inhibitor, the interaction of the inhibitor with a phospholipase A2 from T. flavoviridis venom was examined by Sephadex G-100 gel filtration. One inhibitor molecule was found to bind directly to one phospholipase A2 molecule in both the presence and absence of Ca2+. The inhibitor inhibited the phospholipase A2 from T. flavoviridis venom with an apparent dissociation constant, K1, of 1.7×10-10M, but not the porcine pancreas enzyme or the Agkistrodon halys blomhoffii enzyme belonging to the same family, Crotalidae, as T. flavoviridis, or the phospholipase C from Bacillus cereus.View full abstractDownload PDF (2081K) -
Yoko Watanabe, Sachiko Abe, Shigeko Araki, Toshiro Kumanishi, Mei Sata ...1989 Volume 106 Issue 6 Pages 972-976
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSA phosphonoglycosphingolipid, designated as FGL-IIb, was first identified in nerve fibers of Aplysia kurodai by two-dimensional TLC (Abe, S. et al. (1986) Biomed. Res. 7, 47-51), and its chemical structure has been determined to be 3, 4-O-(1-carboxyethylidene) Galβ1→ 3GalNAcα1→3 (Fucα1→ 2)(2-aminoethylphosphonyl→6) Galβ1→4Glcβ1→1cemmide (Araki, S. et al., submitted). Cryostat and paraffin sections of the nervous tissue and skin of Aplysia were examined immunohistochemically with antiserum against FGL-IIb. With this antiserum, only nerve bundles were stained distinctly: nerve cells in ganglia and in subcutaneous and muscular tissues and other cell elements were not stained. From histochemical findings in cryostat sections pretreated with chloroform-methanol (2: 1, v/ v) and from the results of Western blot analysis of the nervous tissue, the staining was concluded to be due to glycolipid antigens. The antiserum reacted with FGL-IIb and other phosphonoglycosphingolipids named FGL-I, FGL-IIa, FGL-V, and F-9 on TLC plates. This reactivity of FGL-IIb was abolished by mild acid-methanol treatment, and the lost reactivity was recovered by alkaline hydrolysis. These findings suggest that the free carboxyl group of the pyruvic acid of FGL-IIb is essential for the immunological reaction and that all the glycolipids listed above have the same epitope as that of FGL-IIb. Immunohistochemical findings indicated that these glycolipids including FGL-IIb are localized specifically in nerve bundles of Aplysia.View full abstractDownload PDF (3312K) -
Keiichi Watanabe, Yukiko Suemasu, Gunki Funatsu1989 Volume 106 Issue 6 Pages 977-981
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSLuffin-a, aribosome-inactivating protein from the seeds of sponge gourd (Luffa cylindrica), was modified with 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) at pH8.0 and 20°C. Theinhibitory activity of the modified luffin-a on protein synthesis using rabbit reticulocytelysate was lost rapidly at a rate compatible with that of the modification of a single highlyreactive amino group in the initial stage of the reaction. By cation-exchange FPLC of theproducts of 5-min modification, TNP-luffin-a containing one modified amino group wasobtained and shown to have only 6.7% of the activity of native luffin-a without any grossconformational change. The amino acid composition and sequence of the TNP-peptide, isolated by reverse-phase HPLC of the tryptic digest of the TNP-luffin-a, unambiguouslydemonstrate the trinitrophenylation of lysine residue at position 231. From these results, it was concluded that Lys231 of luffin-a is highly reactive to TNBS and is located at or nearthe active site of luffin-a.View full abstractDownload PDF (1380K) -
Nucleotide Sequence Analysis of the 5' Flanking Region of the Gene1Shuichi Yanagisawa, Katsura Izui1989 Volume 106 Issue 6 Pages 982-987
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSTo clone the genomic DNA fragment containing the putative promoter region of thegene for phosphoenolpyruvate carboxylase [EC 4.1.1.31] involved in C4 photosynthesis (C4-type PEPC), maize genomic libraries were screened. On probing with a 384-bp fragment from the N-terminal coding region of the maize cDNA for C4-type PEPC, four EcoRI-fragments differing in the restriction map were cloned, reflecting the presence of a small gene family. Southern blot analyses were carried out on the genomic DNA and the cloned DNA fragments using several segments of the cDNA for C4-type PEPC as probes. The results indicated that the C4-type PEPC is encoded by a single gene and the cloned 7.0-kb fragment was derived from this gene. The transcription start site, as determined in a primer extension experiment, was located at about 4 kb downstream of the 5' end of the cloned fragment. The nucleotide sequence was determined for the region which extended about 1 kb upstream from the transcription start site and possible signal sequences related to gene expression were found, including four classes of direct repeats. The sequences of the corresponding regions of the other two cloned fragments (8.9 and 12.9kb) which strongly hybridized with the 384-bp probe were very similar to each other, but they were quite different in the 5' upstream region from the sequence of the gene for C4-type PEPC.View full abstractDownload PDF (2490K) -
Eisaku Katayama1989 Volume 106 Issue 6 Pages 988-993
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSCaldesmon is universally associated with smooth muscle thin filaments, and reportedly interacts with actin, calmodulin, tropomyosin, and myosin. I attempted to determine the positions of the chymotryptic fragments which have been used to study the sites of such interactions in its primary structure. Such assignment, combined with the accumulated data of fragment studies, made it possible to clarify the functional domain organization of the oaldesmon molecule. Using a specific cleavage method involving nitrothiocyanobenzoate, I also determined the number and locations of cysteinyl residues in the amino-acid sequence. Two cysteinyl groups thus determined were located close to the ends and almost at the same distance apart from the N- and C-termini of the whole molecule, respectively. Taking these locations and the extraordinary sensitivity to oxidation into consideration, I could reasonably elucidate the origin of the controversial data and the interpretation so far reported from various laboratories, concerning the length and conformation of the native molecule. Caldesmon might be folded in solution and its contour length could be almost double the accepted value, which was hydrodynamically estimated.View full abstractDownload PDF (2714K) -
Masachika Irie, Kazuko Ohgi, Reiko Nitta, Miyuki Ikeda, Motoko Ueno1989 Volume 106 Issue 6 Pages 994-997
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe difference spectra obtained upon the addition of nucleotides to bovine kidney RNase, which shows 40% sequence homology with bovine pancreatic RNase, are markedly different from those of bovine pancreatic RNase. As one of the factors which possibly contribute to this difference, we examined the effect of the substitution of Phe120 in bovine pancreatic RNase by Leu in RNase K2 on the difference spectra.View full abstractDownload PDF (924K) -
Masaki Hosoya, Jun-Ichi Miyazaki, Tamio Hirabayashi1989 Volume 106 Issue 6 Pages 998-1002
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSSmooth muscle of chicken embryonic gizzards has been shown to contain 9 tropomyosin isoforms (E1, E2, E3, E4, E5, E6, E7, E8, and E9) in addition to α and β isoforms (Hosoya et al.(1989) J. Biochem. 105, 712-717). At the early stages of development, the amount of these isoforms was larger than those of α and β isoforms. However, they gradually decreased at later stages and finally disappeared completely after hatching. By using two-dimensional gel electrophoresis and an image analyzing system, we examined the process of tropomyosin accumulation in gizzard smooth muscle development. The accumulation patterns of tropomyosin isoforms and their relative molar ratios to actin in embryonic development were different from those in the stages after hatching. The relative molar ratio of tropomyosin to actin in the thin filament preparation of embryonic gizzards was lower than that of adult, and it gradually increased in the course of embryonic development.View full abstractDownload PDF (2427K) -
Hisanori Nozawa, Hiroshi Yamagata, Yasuo Aizono, Mituyoshi Yoshikawa, ...1989 Volume 106 Issue 6 Pages 1003-1008
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe complete amino acid sequence of a major molecular form of subtilisin inhibitor from adzuki beans (Vigna angularis) was established by manual analysis using 4-N, Ndimethylaminoazobenzene-4'-isothiocyanate (DABITC). Sequencing was performed on the peptides which were derived by digesting the inhibitor with lysyl-endopeptidase and Staphylococcus aureus V8-protease. The inhibitor consisted of 92 amino acid residues and the molecular weight was calculated to be 10, 800. A minor form of subtilisin inhibitor was found, which lacked the amino-terminal 19 residues of the major one. Comparison of amino acid sequences revealed that the adzuki bean subtilisin inhibitors were 29-68% homologous in sequence to the inhibitors of so-called “potato inhibitor I family.”View full abstractDownload PDF (1293K) -
Phenotype Modulation and Restoration of Smooth Muscle Characteristics in Cells in CultureYasuharu Sasaki, Takashi Uchida, Yasuo Sasaki1989 Volume 106 Issue 6 Pages 1009-1018
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSWe examined the relationship between growth arrest of smooth muscle cells and structural changes in microfilament bundles, and also that between the structural changes and the actions of contractile agonist using a multipassagable variant cell line (SM-3) derived from rabbit aortic smooth muscle cells. The content of smooth muscle type α-actin increased with density-dependent growth arrest of the SM-3 cells, but was attenuated in the logarithmically growing cultures. As assessed cytochemically, the growth-arrested cells contained longitudinally oriented bundles of actin-containing microfilament and myosin-based filaments visualized with rhodamine-phalloidin and antibody against myosin light chain 20, respectively, whereas both actin-and myosin-containing structures in logarithmically growing cells showed slight, shortened, or diffused patterns. Electron microscopic examination of the growth-arrested cells revealed that the cells contained numerous and conspicuous microfilament bundles associated with many compact electron-dense bodies. In addition, pinocytotic vesicles were often found near the plasma membrane in the growth-arrested cells. SM-3 cells in the growth-arrested phase responded to prostaglandin F2α (3-30μM) and rat endothelin (0.1-1.0μM) with a reversible contractile response, in association with monophosphorylation and/or diphosphorylation of the myosin light chain 20. However, the influence of the contractile agonists was greatly reduced during logarithmic growth. These results suggest that in the SM-3 cells in the growth-arrested phase, there is a restoration of the contractile architecture and the myosin light chain phosphorylation system. Thus, this SM-3 cell line is expected to serve as a useful model for examining biochemical and physiological phenomena of smooth muscle.View full abstractDownload PDF (7560K) -
Yun Soo Bae, Hyoungman Kim1989 Volume 106 Issue 6 Pages 1019-1025
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe interactions of human apolipoprotein A-I (apo A-I) with dipalmitoylphosphatidylcholine (DPPC) in vesicular complexes at low protein concentrations and in micellar complexes at high protein concentrations are compared. The C-terminal segment of this protein, with a relative molecular weight (Mr) of about 11, 000, is protected on trypsin treatment of apo A-I-vesicle complexes. A segment within the sequence from Leu-189 to Arg-215 of apo A-I penetrates the hydrophobic interior of the membrane, as found in a hydrophobic labeling experiment involving 3-(trifluoromethyl)-3-(m-[125I] iodophenyl)- diazirine ([125I] TID). No appreciable stretch of apo A-I in micellar complexes was found to be protected from the tryptic digestion. This indicates that the interactions of apo A-I with lipids in the vesicular and micellar complexes are different. The binding equilibrium of apo A-I as to DPPC vesicles at low protein concentrations, as studied by hydrophobic labeling of the bilayer-penetrating segment, is reached within about 1h, while the formation of micellar complexes at high protein concentrations takes about 24h at 42°C. Time-dependent labeling studies involving photoreactive phosphatidylcholine (PC) with high apo A-I concentrations suggested an initial interaction with the head group region of the bilayer followed by interaction with the tail ends of the acyl chains of the lipid. A possible mechanism for the micellization process is discussed.View full abstractDownload PDF (2292K) -
Kazutoshi Yanagibashi, Yuji Ohno, Noboru Nakamichi, Takashi Matsui, Ke ...1989 Volume 106 Issue 6 Pages 1026-1029
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSIn an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.View full abstractDownload PDF (1046K) -
Miki Shimada, Kiyoshi Nagata, Norie Murayama, Yasushi Yamazoe, Ryuichi ...1989 Volume 106 Issue 6 Pages 1030-1034
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe role of growth hormone in the expression of two forms of hepatic cytochrome P-450 (P-450), P-4506β-1 (6β-3) , and P-4506β-4, was investigated using RNA blots. The level of P-4506β-1 (6β-3) mRNA was twenty times higher than that of P-4506β-4 mRNA in untreated male rat livers. The levels of P-4506β-1 (6β-3) and P-4506β-4 mRNAs were increased two fold and three fold, respectively, by hypophysectomy of adult male rats. By intermittent injection of human growth hormone (hGH) into hypophysectomized male rats, both mRNAs were decreased to the level of normal rats, and almost disappeared after continuous infusion of hGH. In female rats, these two mRNAs were not detected, but were increased remarkably by hypophysectomy. The increases in these mRNAs were almost abolished after continuous infusion of hGH in hypophysectomized female rats. The effect of hGH on PB-mediated induction of P-4506β-1 (6β-3) and P-4506β-4 mRNAs was also examined. The PB-mediated increases in P-4506β-1 (6β-3) and P-4506β-4 mRNAs were higher in hypophysectomized male rats (2.5-fold and 10.9-fold, respectively) than in normal male rats (1.5-fold and 5.2-fold, respectively). Thus, the levels of P-4506β-1 (6β-3) and P-4506β-4 mRNAs were 4.1-fold and 7.3-fold, respectively, higher in PB-induced hypophysectomized rats than in normal male rats. Concerning the postnatal developmental profiles, P-4506β-1 (6β-3) mRNA was detectable at neonate and reached a maximal level at around 17 days of age. The mRNA level, however, abruptly diminished only in female rats at puberty. The change in the mRNA level was largely correlated with that in P-4506β-1 protein level in normal rats as reported previously (Y. Yamazoe et al.(1988) J. Biochem. 104, 785-790). P-4506β-4 mRNA was detectable in the neonate. The mRNA was detected in livers of male and female rats up to 20 days of age, but was undetectable in adult female rats. In an experiment using different aged rats, the extent of PB-induced increases in P-4506β-1 (6β-3) and P-4506β-4 mRNAs was higher in 20-day-old than in 9-week-old male and female rats. These results suggest that pituitary growth hormone suppresses the constitutive and PB-induced levels of expression of two different forms of hepatic testosterone 6β-hydroxylases, P-4506β-1 (6β-3) and P-4506β-4, mainly at the pretranslational level.View full abstractDownload PDF (2600K) -
Yumiko Saito, Seiichi Kawashima1989 Volume 106 Issue 6 Pages 1035-1040
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSWe report here the possible involvement of a new protease in neurite initiation by PC12h cells. Addition of a leupeptin analogue (Ac-Leu-Leu-Nle-al, ALLNal) to PC12h cells on culture plates coated with collagen type I caused de novo neurite outgrowth. Other protease inhibitors (Ac-Leu-Leu-Met-al, leupeptin, E64c, E64d, soybean trypsin inhibitor, hirudin, aprotinin, diisofluorophosphate, 6-aminocapric acid, and pepstatin A) could not mimic this neurite-initiating action. ALLNal induced the initiation of one or two long neurites from the cell body, and increased the cellular level of acetylcholinesterase to an extent similar to nerve growth factor (NGF). However, ALLNal-induced neuritogenesis is different from that induced by NGF, in which many neurites are induced from a single cell body. In addition, in contrast to neurons induced by NGF, which survive for a long time, ALLNal-induced differentiation was transient, and after 48h percentage of cells bearing neurites started to decrease. After about 120h exposure to ALLNal, neurites had mostly disappeared and the acetylcholinesterase activity level was not as great as that produced by NGF. These results provide evidence that ALLNal and NGF elicit neurite initiation by different mechanisms, and suggest the existence of a regulatory system of neuronal differentiation through specific protease-protease inhibitor interaction.View full abstractDownload PDF (2915K) -
Eikichi Hashimoto, Hirohei Yamamura1989 Volume 106 Issue 6 Pages 1041-1048
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESScAMP and Ca2+-independent histone kinase was generated from rat liver plasma membrane in an ionic strength-dependent manner by the action of an endogenous trypsin-like protease (Hashimoto, E. et al.(1986) FEBS Lett. 200, 63-66). In addition to the effect of ionic strength, this proteolytic activation of protein kinase proceeded faster at alkaline pH. In an attempt to identify the activated kinase as the protease-activated form of protein kinase C (protein kinase M), the active enzyme released from plasma membrane was highly purified and characterized. Various properties including Mg2+ requirement in histone phosphorylation, substrate specificity, effects of protein kinase activators, and inhibitors and comparison of catalytic properties by peptide map analysis were compatible with those of protein kinase M reported earlier. Immunoblot analyses also supported the idea that the protein kinase subjected to proteolytic activation was protein kinase C. The subtype of protein kinase C detected in this study was identified as type III enzyme encoding α-type sequence from the elution profile from hydroxyapatite column. These results suggest that type III protein kinase C bound to rat liver plasma membrane has an ability to be activated by endogenous trypsin-like protease dependently on the alteration of ionic strength and pH around the plasma membrane.View full abstractDownload PDF (2089K) -
Keiko Kawamoto, Isao Horibe, Kiyohisa Uchida1989 Volume 106 Issue 6 Pages 1049-1053
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSA new hydrolase for conjugated bile acids, tentatively named chenodeoxycholyltaurine hydrolase, was purified to homogeneity from Bacteroides vulgatus. This enzyme hydrolyzed taurine-conjugated bile acids but showed no activity toward glycine conjugates. Among the taurine conjugates, taurochenodeoxycholic acid was most effectively hydrolyzed, tauro-β-muricholic and ursodeoxycholic acids were moderately well hydrolyzed, and cholic and 7β-cholic acids were hardly hydrolyzed, suggesting that this enzyme has a specificity for not only the amino acid moiety but also the steroidal moiety. The molecular weight of the enzyme was estimated to be approximately 140, 000 by Sephacryl S-300 gel filtration and the subunit molecular weight of the enzyme was 36, 000 by SDS-polyacrylamide gel electrophoresis. The optimum pH was in the range of 5.6 to 6.4. The NH2-terminal amino acid sequence of the enzyme was Met-Glu-Arg-Thr-Ile-Thr-Ile-Gln-Gln-Ile-Lys-Asp-Ala-Ala-Gln. The enzyme was activated by dithiothreitol, but inhibited by sulfhydryl inhibitors, p-hydroxymercuribenzoate, N-ethylmaleimide, and dithiodipyridine.View full abstractDownload PDF (1699K) -
Takuji Kohzuma, Naoko Oda, Hiroshi Kihara, Motonori Ohno1989 Volume 106 Issue 6 Pages 1054-1058
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSWhen Trimeresurus flavoviridis phospholipase A2 was reacted with methyl p-nitrobenzenesulfonate, its activity decreased following first-order kinetics. The pH dependence of the rate constants of inactivation showed that His-48 with an apparent pKa of 6.5 controls the reaction. In the pH region below 6.5, N1-methylhistidine was predominantly formed. On the other hand, N1, N3-dimethylhistidine was almost exclusively produced in the pH region above 6.5. No N3-methylhistidine was detected at any pH tested. Such observations suggested that the first methylation occurred at the N1-position of the imidazole ring followed by a second methylation at the N3-position, and that His-48 couples the carboxylate of Asp-99 at the N3-position of the imidazole ring, in accord with the interaction observed in the crystal structure of homologous Crotalus atrox phospholipase A2. As it has been reported that, in the reaction of chymotrypsin with methyl p-nitrobenzenesulfonate at pH 7.8, only monomethylation occurred at the N1-position of the His-57 imidazole group (Nakagawa, Y. & Bender, M. L.(1970) Biochemistry 9, 259-267), the nature of the active site histidine-aspartate couple of T. flavoviridis phospholipase A2 seems not to be identical with that of chymotrypsin.View full abstractDownload PDF (1094K) -
Norihisa Kikuchi, Kiyoshi Nagata, Masaru Shin, Kenji Mitsushima, Hiros ...1989 Volume 106 Issue 6 Pages 1059-1063
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSArg-42 or Lys-43 or Arg-44 of human pancreatic secretory trypsin inhibitor (PSTI) was replaced by Thr or Ser by site-directed mutagenesis, and the inactivation rates of the mutants after mixing with human trypsin were compared with that of the natural form. The inactivation rate decreased for one mutant (Arg-44→Ser), whereas no change was observed for another (Arg-42→Thr) and an increase was observed for a third (Lys-43→Thr). Kinetic studies on the interactions between human trypsin and synthetic peptides, comprising the regions of Phe39-Ser47 of the respective PSTI species, showed that human trypsin cleaved the Arg42-Lys43 bond preferentially to the Arg44-Gln45 bond. However, it is cleavage of the latter bond that is thought to cause inactivation of human PSTI. These results suggest that the Arg44-Gln45 bond of human PSTI is responsible for its inhibitory activity, and inactivation of human PSTI is probably caused by deletion of the dipeptide Lys43-Arg44.View full abstractDownload PDF (1396K) -
Nobuo Okabe, Kana Goto1989 Volume 106 Issue 6 Pages 1064-1067
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe nuclear thyroid hormone binding protein (NTHB) with the molecular weight of 57kDa was obtained from rat liver nuclear extracts by using HPLC and DEAE-Sephadex A-25 ion exchange chromatography methods. Fluorescein isothiocyanate-labeled 3, 5, 3'-triiodo-L-thyronine (F-T3) was used as a fluorescent probe to identify the hormone binding protein. Purified NTHB has a single binding site for T3 with the apparent binding constant of (3.3±0.7)×108M-1. NTHB is an acidic protein with a pI of 5.0. The secondary structure of NTHB is characterized by about 42% helical and 18%β-structure from CD measurements.View full abstractDownload PDF (1004K) -
Ken-Ichi Furukawa, Yuko Tawada-Iwata, Munekazu Shigekawa1989 Volume 106 Issue 6 Pages 1068-1073
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSWe examined the effect of membrane potential (Em) on the activity of the plasma membrane Ca2+ pump in cultured rat aortic smooth muscle cells (VSMCs). Inside-negative K+ diffusion potential higher or lower than the resting Em (-46mV) was artificially imposed on VSMCs with various concentrations of extracellular K+ (K+o) and 1μM valinomycin. We found that the recovery phase of the intracellular Ca2+ transient elicited with 1μM ionomycin was accelerated by depolarizing Em, whereas it was retarded by hyperpolarizing Em. The rate of extracellular Na+ (Na+o)-independent 45Ca2+ efflux from VSMCs stimulated with 1μM ionomycin increased almost linearly with a change in Em from -98 to -3mV. This effect of Em was abolished by extracellularly added LaCl3 or a combination of high pH (pH 8.8) and high Mg2+ (20mM), conditions that presumably inhibit the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., & Shigekawa, M.(1988) J. Biol. Chem. 263, 8058-8065). Intracellular contents of Na+ and K+ and intracellular pH, on the other hand, were not influenced by the change in Em under the conditions used. These results indicate that alteration in Em can modulate the intracellular Ca2+ concentration in intact VSMCs by changing the rate of Ca2+ extrusion by the plasma membrane Ca2+ pump. The data strongly suggest that the plasma membrane Ca2+ pump in VSMCs is electrogenic.View full abstractDownload PDF (1865K) -
Shinji Asano, Yoshiaki Tabuchi, Noriaki Takeguchi1989 Volume 106 Issue 6 Pages 1074-1079
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSA monoclonal antibody (designated as HK4001) was prepared against hog gastric H+, K+-ATPase. It dose-dependently inhibited the H+, K+-ATPase activity, formation of the K+-sensitive phosphoenzyme, and proton uptake into gastric, vesicles. The H+, K+-ATPase activity was completely inhibited by addition of the antibody at a molar ratio of 1: 2 (antibody/catalytic subunit) at pH 7.8. The maximal inhibition decreased with decrease in pH of the medium (7.8>7.4>6.2). The Fab fragment obtained by digestion of the antibody with papain was also inhibitory. The antibody did not inhibit the K+-dependent pnitrophenylphosphatase or the labeling of the enzyme with fluorescein isothiocyanate. It inhibited gastric H+, K+-ATPase from rabbits and rats, but did not cross-react with related cation-transport ATPases (Na+, K+-ATPase or Ca2+-ATPase) or H+-ATPase in the multivesicular body. From these and related findings, the antibody was suggested to recognize a highly specific site on the cytosolic surface of H+, K+-ATPase. The conformation of the epitope was conserved after treatment with Triton X-100, but not sodium dodecyl sulfate. In addition, judging from the stoichiometry of inactivation of H+, K+-ATPase by this antibody, the functional unit of H+, K+-ATPase was suggested to be a dimer or a tetramer (not a trimer) of the catalytic unit.View full abstractDownload PDF (2105K) -
Takeo Yamaguchi, Hideo Kawamura, Eiji Kimoto, Mitsuru Tanaka1989 Volume 106 Issue 6 Pages 1080-1085
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe release of hemoglobin from human erythrocytes hemolyzed beforehand by hydrostatic pressure, osmotic pressure, and freeze-thaw methods was examined as a function of temperature (0-45°C) and pH (5.5-8.8) at atmospheric pressure. Only in the case of high pressure (2, 000 bar) did the release of hemoglobin increase significantly with decreasing temperature and pH. Maleimide spin label studies showed that the temperature and pH dependences of hemoglobin release were qualitatively explicable in terms of those of the conformational changes of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins showed the diminution of band intensities corresponding to spectrin, ankyrin, and actin in the erythrocytes hemolyzed by high pressure. Cross-linking of cytoskeletal proteins by diamide stabilized the membrane structure against high pressure and suppressed hemoglobin release. These results indicate that the disruption of cytoskeletal apparatus by high pressure makes the membrane more leaky.View full abstractDownload PDF (2253K) -
Yasuhiro Ohta, Yoji Tsukada, Tsunetake Sugimori1989 Volume 106 Issue 6 Pages 1086-1089
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSAn Arthrobacter ureafaciens mutant (M1057) capable of producing neuraminidase constitutively was isolated by NTG mutagenesis from A. ureafaciens KMS 3663. Four molecular species (L, M1, M2, and S) of neuraminidase isozymes were homogeneously purified from the mutant and parent strains by means of DEAE-cellulose, affinity chromatography, ammonium sulfate precipitation, chromatofocusing, and Ultrogel AcA44 gel filtration. The molecular weights of L, M1, M2, and S isozymes were shown to be approximately 88, 000, 66, 000, 66, 000, and 52, 000, respectively. The optimal pHs and Km values of these isozymes for N-acetylneuraminosyl-α, (2-6)-lactose were 4.5-5.5 and 0.6-0.8mM. Neuraminidase L, M1, M2, and S were able to hydrolyze oligosaccharides, glycoproteins and gangliosides containing α, (2-3)-, α, (2-6)-, and α, (2-8)-linked N-acetylneuraminic acid. Among these isozymes isolated, isozyme S was most active on colominic acid.View full abstractDownload PDF (1516K) -
K. K. Pun1989 Volume 106 Issue 6 Pages 1090-1093
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSLoss of bone substance is a common manifestation of hyperparathyroidism. This suggests that parathyroid hormone (PTH) plays an important role as to bone mass. To investigate the mechanism underlying this change in bone mass, I studied the effects of PTH on collagen synthesis and mitogenesis of UMR-106 rat osteoblastic osteosarcoma cells. PTH inhibits the mitogenesis of UMR-106 rat osteosarcoma cells, the half-maximal concentration being 10-8 to 10-7M, which is similar to the EC50 for cyclic AMP accumulation. Cyclic AMP, whose intracellular concentration was increased by PTH, plays a role in the modulation of mitogenesis, as shown by the comparable inhibitory effects of 8-bromoadenosine- 3', 5'-cyclic AMP (10-4M), forskolin (10-7M), and the phosphodiesterase inhibitor, IBMX (10-5M). PTH, in a similar concentration range, directly inhibited collagen synthesis. Concurrent with the suppression of collagen synthesis, the amounts of a1 (I) and a2 (I) collagen mRNA decreased proportionately. The results show that PTH modulates collagen synthesis at the transcriptional level. I concluded that parathyroid hormone inhibits the mitogenesis of osteoblasts as well as collagen synthesis by these cells. The decreases in the number of osteoblasts and the amount of collagen synthesis contribute to the loss of bone substance in hyperparathyroidism.View full abstractDownload PDF (1039K) -
María Dolores Pérez, Conchita Díaz de Villegas, L ...1989 Volume 106 Issue 6 Pages 1094-1097
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSβ-Lactoglobulin isolated from milk of cow, sheep, and goat had about 0.5mol of fatty acids bound per mol of monomer protein. Fatty acids, mainly palmitic and oleic acids, were the major components (about 75% of total lipids). Albumin isolated from the same samples had about 4.5mol of fatty acids bound per mol of protein. These two proteins were the only whey proteins able to bind labeled fatty acids in vitro. Interaction of β-lactoglobulin and albumin with insolubilized fatty acids showed some differences, suggesting different structures of the respective fatty acid binding sites.View full abstractDownload PDF (1633K) -
Naoto Shibuya, Kiyoshi Tazaki, Zhiwei Song, George E. Tarr, Irwin J. G ...1989 Volume 106 Issue 6 Pages 1098-1103
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThree elderberry lectins isolated from the bark of three different species of the genus Sambucus which are native to Europe (S. nigra), North America (S. canadensis), and Japan (S. sieboldiana) were studied comparatively with regard to their carbohydrate bindingproperties and some structural features. All three lectins contained two identical carbo-hydrate binding sites per molecule and showed a very high specificity for the Neu5Ac (α2-6)-Gal/GalNAc sequence. However, relative affinities for various oligosaccharides weresignificantly different among them, suggesting differences in the detailed structure of thecarbohydrate binding sites of these lectins. The three lectins were immunologically related, but not identical, and all were composed of hydrophobic and hydrophilic subunit regions, although the molecular sizes of these subunits were slightly different among the threelectins. N-terminal sequence analysis of the subunits of these lectins suggested that theyhave a very similar structure in this region but also indicated the occurrence of N-terminalprocessing such as the deletion of several amino acid residues at the N-termini for bothhydrophobic and hydrophilic subunits of all three lectins. Tryptic peptide mapping of thethree lectins showed a similar pattern for all of them but also showed the presence of someunique peptides for each lectin.View full abstractDownload PDF (1869K) -
Makoto Nakagawa, Kazuya Matsuura, Akira Hara, Hideo Sawada, Yasuo Buna ...1989 Volume 106 Issue 6 Pages 1104-1109
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSChemical cross-linking and NADPH binding studies suggested that the native dihydrodiol dehydrogenase from monkey kidney is a basic dimer having a molecular weight of 78, 000 and one active site per the subunit. The enzyme oxidized specifically trans-dihydrodiols of benzene and naphthalene, whereas it catalyzed the reduction of dihydroxyacetone and dihydroxyacetone phosphate at a physiological pH, 7.4. The Km and kcat values for dihydroxyacetone phosphate were 5.0 mM and 4.3s-1, respectively. The enzyme transferred the 4-pro-R hydrogen atom of NADPH to the carbonyl substrate. Immunochemical experiments using an antibody against the dimeric enzyme revealed the specific distribution of the enzyme in the kidney of this animal. By immunohistochemical staining with the specific antibody, the immunoreactivity was found in proximal and distal tubules of the cortex, and in the loop of Henle of the medulla.View full abstractDownload PDF (2817K) -
Assignment of Tyrosyl Residues NitratedRyuji Kobayashi, Akio Kanatani, Tadashi Yoshimoto, Daisuke Tsuru1989 Volume 106 Issue 6 Pages 1110-1113
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSA neutral protease from Bacillus subtilis var. amylosacchariticus was modified with tetranitromethane (TNM) at pH 8.0 for 1h at 25°C, by which treatment the proteolytic activity toward casein was markedly reduced, whereas activity changes toward N-blocked peptide substrates were variable depending upon the substrate used. The modified enzyme was digested with a Staphylococcus aureus V8 protease at pH 7.9 and the resultant peptides were separated by HPLC. Two peptides which contain nitrotyrosyl residue (s) were purified. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the neutral protease, and Tyr-158 was identified as PTH-nitrotyrosine. The other one was the amino-terminal peptide of residue Nos. 1-22, and Tyr-21 was shown to be nitrated. From a comparison with the active site structure of thermolysin, which is a zinc metalloprotease with a high sequence homology to B. subtilis neutral proteases, nitration of Tyr-158 was inferred to be closely related to the activity changes of the neutral protease from B. subtilis var. amylosacchariticus.View full abstractDownload PDF (884K) -
Kazuo Yamasaki, Taibo Yamamoto1989 Volume 106 Issue 6 Pages 1114-1120
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe functional significance of the molecular interaction of Ca2+-ATPase in the sarcoplasmic reticulum (SR) membrane was examined using intermolecular cross-linking of Ca2+-ATPase with N, N'-(1, 4-phenylene) bismaleimide (PBM). When SR vesicles were allowed to react with 1mM PBM at pH 7 and 23°C for various intervals and subjected to SDS-PAGE, the amount of the major band of monomeric ATPase decreased with a half life of about 20min. Higher orders of oligomers were concurrently formed without accumulation of any particular species of oligomer. When SR vesicles were allowed to react with 1 mM PBM in the presence of 1 mM adenyl-5'-imidodiphosphate (AMP-PNP), the rate of oligomerization was markedly reduced and the amount of dimeric Ca2+-ATPase increased with time. After 1h, more than 40% of the Ca2+-ATPase had accumulated in the dimeric form. When 1 mol of fluorescein isothiocyanate (FITC) was bound per mol of ATPase, the effects of AMP-PNP on the cross-linking with PBM were completely abolished. When SR vesicles were treated with PBM in the presence of 0.1 mM vanadate in Ca2+ free medium, the oligomerization of the Ca2+-ATPase by PBM was strongly inhibited. The vanadate effect on the cross-link formation was completely removed by the presence of Ca2+ and AMP-PNP in the reaction medium. When SR vesicles were pretreated with PBM in the presence of AMP-PNP and digested with trypsin for a short time, the dimeric ATPase was degraded to a peptide with an apparent molecular mass of about 170kDa. Further digestion resulted in degradation to a 130 kDa peptide. As they were phosphorylated by [γ-32P] ATP in the presence of Ca2+, these peptides contained the subfragment A. In addition, the SDS-PAGE pattern of the dimeric ATPase after the prolonged digestion lacked subfragment A1, suggesting that ATPase molecules were cross-linked by PBM through an SH group in the A1 region of this enzyme.View full abstractDownload PDF (3927K) -
Hisanori Yamamoto, Yasutada Imamura, Mitsuo Tagaya, Toshio Fukui, Masa ...1989 Volume 106 Issue 6 Pages 1121-1125
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSAdenosinetriphosphopyridoxal (AP3PL) specifically modifies Lys684 of Ca2+-ATPase of sarcoplasmic reticulum (SR-ATPase) in the presence of Ca2+, leading to its inactivation (Yamamoto, H. et al.(1988) J. Biochem. 103, 452-457). We have now investigated the effects of AP3PL on SR-ATPase in the absence of Ca2+. Similarly to its action in the presence of Ca2+, AP3PL inhibited the Ca2+-transporting activity in a dose-dependent manner in the absence of Ca2+ as well. ATP and ADP protected SR-ATPase against inactivation by this reagent. One mole of AP3PL was bound per mol of SR-ATPase with concomitant loss of the Ca2+-transporting activity. Binding of AP3PL to SR-ATPase was prevented by ATP. AP3PL-labeled SR membranes were digested with thermolysin and labeled thermolytic peptides were purified through C18 reversed-phase HPLC. Two major AP3PL-labeled peptides were obtained in approximately 1: 1 ratio; one was an octapeptide corresponding to 679-ValGluProSerHisLysSerLys-686, and the other, a nonapeptide corresponding to 487-PheSerArgAspSerLysArgMetSer-495 (Lys indicates a labeled Lys residue) of SRATPase. Lys684 in the former turned out to be the same as the highly specific target of AP3PL in the presence of Ca2+ which was identified previously. The target site specificity of AP3PL thus changed significantly but not entirely on binding of Ca2+ to SR-ATPase. This indicates that the spatial arrangement around the γ-phosphoryl group of the bound ATP is affected by Ca2+ ions bound at the transport site. It is also likely that Lys492 and Lys684 are situated close together in the ATP binding site of SR-ATPase.View full abstractDownload PDF (1483K) -
Yukio Fujiki, Makoto Tsuneoka, Yutaka Tashiro1989 Volume 106 Issue 6 Pages 1126-1131
Published: 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe biosynthesis of nonspecific lipid transfer protein (nsLTP) was investigated. Total RNA of rat liver was translated in a rabbit reticulocyte lysate cell-free protein-synthesizing system with [35S] methionine as label. The immunoprecipitation of translation products with affinity-purified anti-nsLTP antibody yielded 14.5- and 60-kDa [35S] polypeptides. The molecular mass of the former polypeptide was -1.5kDa larger than that of the purified mature nsLTP (13kDa). The site of synthesis of nsLTP was studied by in vitro translation of free and membrane-bound polyribosomal RNAs followed by immunoprecipitation. mRNA for both the 14.5- and 60-kDa polypeptides were found predominantly in the free polyribosomal fraction in both normal and clofibrate-treated rats. Clofibrate, a hypolipidemic drug that proliferates peroxisomes, did not increase the relative amount of nsLTP mRNA in rat liver. Pulse-chase experiments in rat hepatoma H-35 cells suggested that nsLTP was synthesized as a larger precursor of 14.5kDa and converted to a mature form of 13kDa. We have recently shown that nsLTP is highly concentrated in peroxisomes in rat hepatocytes [Tsuneoka et al.(1988) J. Biochem. 104, 560-564]. Taken together, these results suggest that nsLTP is synthesized as a larger precursor of 14.5kDa on cytoplasmic free polyribosomes, then post-translationally transported to peroxisomes, where the precursor is presumably proteolytically processed to its mature form of 13kDa. The relationship between the 13-kDa nsLTP and the 60-kDa polypeptide is also discussed.View full abstractDownload PDF (3427K)
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