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Volume 109, Issue 1
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Ko Onodera, Hideaki Moriyama, Akio Takenaka, Nobuo Tanaka, Nobuko Akut ...1991 Volume 109 Issue 1 Pages 1-2
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA chimeric gene was constructed by fusing the Bacillus subtilis and Thermus thermophilus genes coding for 3-isopropylmalate dehydrogenase, and expressed in Escherichia coli. The chimeric enzyme was crystallized in a size suitable for X-ray structure analysis. The crystal has a space group of P3121 or P3221, a=b=77.1 Å and c=158.3 Å, which is isomorphous with that of the native enzyme from T. thermophilus.View full abstractDownload PDF (365K) -
Takashi Igarashi, Toshiya Tsuchiya, Tetsuo Satoh1991 Volume 109 Issue 1 Pages 3-5
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA glutathione S-transferase isozyme which is absent in normal rat liver has been isolated from the hereditary hyperbilirubinuria rat liver cytosol. The enzyme was purified to apparent homogeneity by GSH-affinity chromatography and HPLC on CM-Sepharose CL-6B. It is a heterodimer of two non-identical subunits, i.e., subunit 2 and a previously uncharacterized subunit referred to here as subunit Yx. Immunoblot analysis indicated that GST 2-Yx belongs to the alpha class. GST 2-Yx is characterized by its 4-fold higher activity towards 4-hydroxy-non-2-enal, compared to that of GST 2-2.View full abstractDownload PDF (875K) -
Shohei Koide, So Iwata, Hiroshi Matsuzawa, Takahisa Ohta1991 Volume 109 Issue 1 Pages 6-7
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Lactate dehydrogenase of Thermus caldophilus GK24 was purified from Escherichia coli containing an overexpression plasmid. The enzyme was crystallized from polyethylene glycol 6000 solutions without ligands by the hanging drop vapor diffusion method. Two forms of crystals were obtained. The crystals grown at pH 6.0 were characterized by means of an X-ray diffraction experiment, while those grown at pH 6.5 and 7.0 did not give detectable diffraction spots. The crystals grown at pH 6. 0 belonged to monoclinic space group P21 the cell dimensions being a=54.8 Å, b=138.2Å, c=86.1 Å, and β=93.3°. These crystals diffract to beyond 2.5 Å spacing and are stable on X-ray irradiation.View full abstractDownload PDF (552K) -
Osamu Asami, Izumi Ihara, Norihide Shimidzu, Shin Shimizu, Yumiko Tomi ...1991 Volume 109 Issue 1 Pages 8-13
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA hepatocyte growth factor (HGF)-like substance that strongly stimulated DNA synthesis of adult rat hepatocytes in primary culture was found to increase markedly in liver of rats treated with carbon tetrachloride (CCl4). This increase of HGF-like activity was time- and dose-dependent, and 36 h after a dose of CCl4 of 0.2ml per 100g body weight the activity was about 20-times the normal level. The extent of induction of HGF-like factor correlated well with the extent of liver damage. The HGF-like factor was purified to homogeneity from the liver of CCl4-treated rats by a four-step procedure. The purified HGF-like factor had a molecular weight of 82-85 kDa, as estimated by SDS-PAGE, and was a heterodimer composed of a large subunit of about 69 kDa and a small subunit of 34 kDa linked by disulfide bridges. This factor had similar biological and chemical properties to HGF purified from rat platelets. Moreover, the N-terminal amino acid sequence of its 34-kDa subunit was identical to that of the small subunit of rat HGF. These findings indicate that the HGF-like factor in damaged liver of CCl4-treated rats is HGF and that liver itself can produce HGF when injured.View full abstractDownload PDF (1577K) -
Ichiro Shimizu, Akira Ichihara, Toshikazu Nakamura1991 Volume 109 Issue 1 Pages 14-18
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHepatocyte growth factor (HGF) stimulating DNA synthesis of adult rat hepatocytes in primary culture was found in the ascites and plasma from patients with liver cirrhosis, but not in those from patients without cirrhosis. HGF was purified about 400-fold in 10% yield from cirrhotic ascites by ultrafiltration, cation-exchange chromatography on a S-Sepharose column, and affinity chromatography on a heparin-Sepharose CL-6B column. The partially purified factor was a heat- and acid-labile cationic protein with a molecular weight of 100, 000-150, 000. Its effect was half-maximal at 3.8μg/ml, and was additive with those of insulin and epidermal growth factor. HGF in ascites from patients with cirrhosis had the same properties as HGF purified and characterized from rat platelets. These findings suggest that HGF is secreted into the ascites from the plasma or liver of patients with cirrhosis and may increase in the plasma with the development of hepatic impairment and act in repair of the damaged liver of patients with chronic liver disease.View full abstractDownload PDF (648K) -
Naoya Suzuki, Koshin Mihashi1991 Volume 109 Issue 1 Pages 19-23
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd=5.0nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd=230nM) were created, while the high-affinity binding site was reserved (Kd=3.4nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd=3.9nM) plus two low-affinity binding sites (Kd=550nM); (2) the Ca2+-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.View full abstractDownload PDF (677K) -
Yumi Tamao, Kazuo Noguchi, Yuki Sakai-Tomita, Hirnkn Hama, Tadashi Shi ...1991 Volume 109 Issue 1 Pages 24-29
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus is unique in requiring Cl- for activity. We cloned the nutA gene encoding the 5'-nucleotidase and sequenced it. It contained an open reading frame consisting of 1, 680 nucleotides capable of encoding a protein of 560 amino acid residues. The first 21 amino acid residues of the N-terminal portion of this protein seem to be a signal peptide. The rest of the polypeptide (539 residues) is hydrophilic, and its molecular weight was calculated to be 60, 008, which is in good agreement with the value of 63 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the 5'-nucleotidase derived from the cloned nutA gene. We tried to determine the amino acid sequence of the N-terminal portion of the purified enzyme. However, the N-terminal residue seemed to be blocked. As this 5'-nucleotidase can be solubilized from membrane vesicles with detergent, it may be a lipoprotein. The amino acid sequence around the possible cleavage site of the 5'-nucleotidase had homology with the sequences of the cleavage sites of the lipoproteins of Escherichia coli and other bacteria. The amino acid sequence had high (about 60%) homology with the sequence of periplasmic 5'-nucleotidase (uridine diphosphate sugar hydrolase, the product of the ushA gene) of E. coli. It also contained regions that showed some homology with the nucleotide binding sites of many nucleotide binding proteins.View full abstractDownload PDF (665K) -
Yasuyuki Ishizuka, Akihito Shoda, Shigeki Yoshida, Yukio Kawamura, Kaz ...1991 Volume 109 Issue 1 Pages 30-35
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecombinant human prorenin (rh-prorenin) was purified from supernatants of Chinese hamster ovary (CHO) cell line transfected with the cDNA for rh-prorenin by employing a simple two-step procedure which consisted of ammonium sulfate precipitation and immunoaffinity chromatography using a monoclonal antibody specific for the profragment of human prorenin. About 100-fold purification with 35% recovery was achieved after the two steps. Purified rh-prorenin migrated as a single protein band with apparent molecular weights of 46, 000-47, 000 and about 50, 000 on SDS-PAGE and gel filtration (HPLC), respectively, although it consisted of multiple components (pI values, 5.6-6.4) that could be resolved by isoelectric focusing (IEF). The treatment of rh-prorenin with endo-β-N-acetylglucosaminidase converted the rather broad protein band to a sharp band on SDS-PAGE and reduced the number of multiple pI peaks on IEF. Amino-terminal sequence analysis of both the purified rh-prorenin and rh-renin revealed Leu-Pro-Thr-Asp- and Leu-Thr-Leu-Gly-, respectively, which agreed with those predicted from the base sequences of their cDNA. These data suggested that microheterogeneity of rh-prorenin is due to the carbohydrate moiety, but not to the protein moiety. Purified rh-prorenin was almost inactive, but was cleaved at the carboxyl end of a dibasic pair Lys-2-Arg-1 by trypsin and converted to active renin. However, at the early stage during trypsin activation, new intermediate forms between rh-prorenin and rh-renin were formed, suggesting multiple activation steps of rh-prorenin in addition to the one step activation.View full abstractDownload PDF (1323K) -
Kaoru Morishita, Katsuichi Sakano, Kazumi Takeda, Susumu Maeda1991 Volume 109 Issue 1 Pages 36-44
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe v-sis oncogene of simian sarcoma virus encodes a protein which is homologous to the human platelet-derived growth factor B-chain. The v-sis protein undergoes a series of processing steps including dimer formation and proteolytic digestion to generate several molecular sizes of the protein. Two of these v-sis proteins were expressed alone or as polyhedrin-sis fusion proteins using the Bombyx mori nuclear polyhedrosis virus vector. The polyhedrin-sis fusion proteins contained a collagenase-sensitive site at the junction. The expression levels of the fusion proteins whose polyhedrin portions consisted of only 8 amino-terminal amino acids were 3-4 times higher than those of non-fusion proteins. One of these fusion proteins was expressed in silkworm larvae and the v-sis protein was isolated from the fusion protein by collagenolysis followed by chromatography. Because the purified v-sis protein exhibited the same molecular size on SDS-polyacrylamide gels under reducing and non-reducing conditions, it was concluded to be monomeric in structure. It possessed chemotactic activity but lacked mitogenic activity. In addition, a small amount (approximately 1%) of monomeric v-sis protein was converted in vitro to the mitogenically active v-sis protein, which could be a homo-dimer.View full abstractDownload PDF (1949K) -
Dong-Hyun Kim, Kyoichi Kobashi1991 Volume 109 Issue 1 Pages 45-48
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel sulfotransferase purified from a human intestinal bacterium stoichiometrically catalyzed the transfer of a sulfate group of phenylsulfate esters to phenolic compounds. Vmax values of the enzyme reaction were measured with various concentrations of a sulfate donor substrate, p-nitrophenylsulfate, and of a sulfate acceptor substrate, tyramine. Double reciprocal plots of the acceptor concentration and Vmax showed a linear correlation. One of the reaction products, tyramine O-sulfate, competitively inhibited the enzyme as to a donor substrate, p-nitrophenylsulfate (PNS), but the other reaction product, p-nitrophenol (PNP), noncompetitively inhibited it as to PNS. These kinetic data suggest that the sulfate transfer reaction proceeds according to a ping pong bi bi mechanism. The enzyme was activated by Mg2+ and inhibited by EDTA, which suggests that it is a metalloenzyme.View full abstractDownload PDF (316K) -
Kazutoyo Terada, Takuya Murata, Katsura Izui1991 Volume 109 Issue 1 Pages 49-54
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhosphoenolpyruvate carboxylases (PEPC) [EC 4.1.1.31] from a wide variety of organisms contain a unique and highly conserved sequence, 578THGRGGSIGRGGAP591 (coordinates for the Escherichia coli enzyme), which has been presumed to participate in the binding of phosphoenolpyruvate (PEP). Since previous chemical modification studies had suggested the importance of His579 for the catalytic activity, the role of His579 was investigated by constructing variants of E. coli PEPC, in which this residue was substituted to Asn (H579N) or Pro (H579P). Kinetic studies with partially purified enzymes revealed the following: (1) The apparent maximal velocities in the presence of acetyl-CoA (CoASAc, one of the allosteric activators) were 29% and 5.4% of the wild-type enzyme, for H579N and H579P, respectively. (2) The half-saturation concentration for PEP was increased about 40-fold by the substitutions, while those for another substrate (HCO3-) and the metal cofactor (Mg2+) were increased only 2- to 4-fold. (3) The half-saturation concentrations of four kinds of allosteric activators and of dioxane, an artificial activator, were also changed to various extents. Among them the most remarkable increase was observed for CoASAc (28-fold). (4) The concentration of an allosteric inhibitor, aspartate, required for 50% inhibition remained substantially unchanged. It was concluded that the imidazole group of His579 is not obligatory for the enzyme catalysis, but plays important roles in catalytic and regulatory functions.View full abstractDownload PDF (1110K) -
Shigeru Fujii, Kiyo Kuroda, Sabu Kasai, Retsu Miura1991 Volume 109 Issue 1 Pages 55-60
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe brewer's yeast old yellow enzyme (OYE) was reconstituted with 8-fluoro-8-demethyl FMN (8F-FMN). The reconstituted enzyme exhibited absorption maxima at 355 and 450 nm in the visible region. This reconstituted enzyme underwent no further spectral changes, showing no evidence of modification in the flavin moiety. However, when the reconstituted enzyme was subjected to specific limited proteolysis with bovine α-chymotrypsin, gradual spectral changes were observed with disappearance of the 355- and 450-nm bands ac-companied by the appearance of a new band at 496 nm. Identical spectral changes were observed when the proteolytically cleaved OYE (nicked OYE) was reconstituted with 8F-FMN. The process associated with these spectral changes was found to be unimolecular by kinetic analysis. Reverse-phase HPLC analysis revealed that these spectral changes resulted from covalent bond formation between 8F-FMN and the protein moiety after the proteolytic cleavage of the protein into 14K and 34K fragments. The reverse-phase HPLC monitored at 490nm showed that the chromophore with 496nm absorption maximum was covalently attached to the 14K fragment. The amino acid sequence analysis of the flavinylat-ed 14K fragment together with that of the 14K fragment of native OYE indicated that the N-terminal leucine of the 14K fragment is the site of flavinylation. These findings imply that the amino group of the N-terminal leucine of the 14K fragment became available as the result of proteolysis and that this amino group nucleophilically attacked the 8-position of 8F-FMN, forming a covalent bond between the flavin moiety and the 14K fragment. Hence the 14K domain of OYE is the flavin-binding domain and the xylene portion of FMN is located near the N-terminal region of the 14K domain.View full abstractDownload PDF (1100K) -
Toshiharu Yagi, Haruo Misono, Katsuyuki Tanizawa, Tohru Yoshimura, Ken ...1991 Volume 109 Issue 1 Pages 61-65
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSignificant differences were found in the reaction rate, and the substrate and reaction specificities between the half reactions and the overall reactions catalyzed by L-lysine: 2-oxoglutarate 6-aminotransferase. The half reactions between an amino donor and the enzyme-bound pyridoxal 5'-phosphate, and also between an amino acceptor and the bound pyridoxamine 5'-phosphate followed first order reaction kinetics. The extrapolated first order rate constants and dissociation constants of the substrates were determined for the half reactions: lysine, 0.87 min-1 and 5.5mM; glutamate, 1.1 min-1 and 10.5mM; alanine, 0.66 min-1 and 6.6 mM; 6-aminohexanoate, 0.43 min-1 and 13.3 mM; and 2-oxoglutarate, 0.33 min-1 and 2.5mM. As compared with the values reported for the overall reactions [ Soda, K., Misono, H., & Yamamoto, T. (1968) Biochemistry 7, 4102-4109], the reactivity of the inherent substrates was lower by over 4 orders in the half reaction than that in the overall reaction, and the reactivity of alanine with the bound pyridoxal 5' -phosphate was reduced to 10% of that in the overall reaction. The substrate specificity in the half reaction was much lower than that in the overall reaction, which was re-examined in a reaction system containing the same concentration of the enzyme as that for the half reactions. Lysine 6-aminotransferase catalyzes the transfer of only the terminal amino group of lysine to 2-oxoglutarate in the overall reaction. However, in the half reaction, the 2-amino group as well as the terminal one was transferred to the bound pyridoxal 5'-phosphate. The ratio of reactivity of the 2-amino group to that of the 6-amino group was considerably influenced by the pH of the reaction mixture. These results show that 2-oxoglutarate and lysine bound to the enzyme probably affect the rates of the half reactions between amino donors and the enzyme-bound pyridoxal 5'-phosphate, and of those between amino acceptors and the enzyme-bound pyridoxamine 5' -phosphate, respectively.View full abstractDownload PDF (567K) -
Ryoko Iwamoto, Yujiro Imanaga1991 Volume 109 Issue 1 Pages 66-69
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPseudomonas fluorescens (Migula) (IFO 14808) has both a membrane-bound PQQ-depen-dent D-glucose (D-Glc) dehydrogenase [EC 1.1.99.17] [which also acts on D-glucosamine (D-G1cN)] and a PLP-dependent D-glucosaminate (D-G1cNA) dehydratase [EC 4.2.1.26]. Further, these two enzymes were induced when D-G1cN was added to the culture medium. However, D-glucosamine-6-phosphate (D-G1cN-6-P) isomerase [EC 5.3.1.10], another enzyme involved in the metabolism of D-G1cN, was only present at a low level in this bacterium. The bacterium was able to grow in a minimal medium containing D-G1cN or D-G1cNA as the sole source of carbon and nitrogen. Intact cells of P. fluorescens (Migula) converted D-GlcN to D-G1cNA and then to 2-keto-3-deoxy-D-gluconate (KDGA). These results demonstrate that D-G1cN is metabolized via D-G1cNA to KDGA in P. fluorescens (Migula) (Entner-Doudoroff pathway). In contrast, Enterobacter cloacae(IFO 13535) and Agrobacterium radiobacter (IAM 1526) have significant amounts of D-G1cN-6-P isomerase with low levels of the D-Glc dehydrogenase and D-G1cNA dehydratase. Further, only the isomerase activity was induced on the addition of D-G1cN to the culture medium. These results demonstrate that there is a new route (Entner-Doudoroff pathway), i. e., in addition to the known one (Embden-Meyerhof pathway), for the metabolism of D-G1cN in bacteria and one of the two routes is predominant in the each of bacteria examined.View full abstractDownload PDF (843K) -
Haruo Homareda, Yoshine Nagano, Hideo Matsui1991 Volume 109 Issue 1 Pages 70-77
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Kd for ouabain-sensitive K+ or Rb+ binding to Na+, K+-ATPase was determined by the centrifugation method with radioactive K+ and Rb+ in the presence of various combinations of Na+, ATP, adenylylimidodiphosphate (AMPPNP), adenyly1-(β, γ-methylene)diphos-phonate (AMPPCP), P1 and Mg2+. From the results of the K+ binding experiments, Kd for Na+ was estimated by using an equation describing the competitive inhibition between the K+ and Na+ binding. 1) The Kd for K+ binding was 1.9μM when no ligand was present. Addition of 2mM Mg2+ increased the Kd to 15-17μM. In the presence of 2mM Mg2+, addition of 3mM AMPPCP with or without 3mM Na+ increased the Kd to 1, 000 or 26μM, respective-ly. These Kds correspond to those for K+ of Na•E1 •AMPPCPMg or E1•AMPPCPMg, respec-tively. 2) Addition of 4 mM ATP with or without 3mM Na+ decreased the Kd from 15-17μM to 5 or 0.8μM, respectively. Because the phosphorylated intermediate was observed but ATPase activity was scarcely observed in the K+ binding medium containing 3mM ATP and 2mM Mg2+ in the absence of Na+ as well as in the presence of Na+ at 0°C, it is suggested that Kt+ binds to E2-P•Mg under these ligand conditions. 3) The Kd for Na+ of the enzyme in the presence of 3 mM AMPPCP or 4mM ATP with Mg2+ was estimated to be 80 or 570μM, respectively. The affinity ratio of K+ to Na+ of the enzyme (Kd for Na+ /Kd for K+) showed a large increase (240-fold) from 3 to 710 on changing the nucleotide used from AMPPCP to ATP. Consequently, it is presumed that the transition from E1•ATPMg to E2-P•Mg induces nearly 240-fold increase in the affinity ratio. 4) Addition of 4mM P1 in the presence of 2mM Mg2+ increased the Kd for K+ from 17μM to 40μM. Addition of 3mM Na+ in the presence of P1 and Mg2+ slightly increased the Kd from 40 to 49μM, contrary to the result in the absence of Mg2+. The Kd for Na+ in the presence of P1 increased from 0.29 to 13mM on addition of 2mM Mg2+. Therefore, the K+-insensitive E2-P•Mg formed from Mg2+ and P1 [Post et al. (1975) J. Biol. Chem. 250, 691-701] was shown to have low affinity not only for K+ but also for Na+.View full abstractDownload PDF (926K) -
Masanobu Kawakami, Yukio Kondo, Yasuo Imai, Mitsuko Hashiguchi, Hiroko ...1991 Volume 109 Issue 1 Pages 78-82
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProduction of a cachexia-inducing factor(s) by the SEKI melanoma cell line, established from a human melanoma, has been well documented. Conditioned medium from cultures of this melanoma cell line contains a factor(s) that inhibits the activity of lipoprotein lipase (LPL) in fully differentiated 3T3-L1 adipocytes. The mode of inhibition of this enzyme by the factor, i. e. its dose-dependency and time course, is very similar to that of LPL-inhibition by a macrophage-derived cachexia-inducing factor, cachectin/tumor necrosis factor (cachectin/TNF). However, the conditioned medium of SEKI melanoma cells does not contain any immuno-reactive substances reactive in enzyme-linked immunosorbent assay (ELISA) with anti-cachectin/TNF antibody, or with anti-interleukin 1 α or β antibodies. This LPL-suppression factor present in the conditioned medium seems to be a peptide because of its heat-lability and apparent molecular weight of more than 25, 000. The conditioned media from cultures of four other different cell lines were found to show no significant suppression of LPL activity. These results imply that SEKI melanoma cells produce a cachexia-inducing factor(s) similar to cachectin/TNF but that the molecule involved is different.View full abstractDownload PDF (679K) -
Atsuko Hayashi-Nagai, Haruko Kitagaki-Ogawa, Isamu Matsumoto, Masao Ha ...1991 Volume 109 Issue 1 Pages 83-88
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine plasma fibronectin as well as human plasma fibronectin was adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0 M, indicating that both fibro-nectins have very high surface hydrophobicities. On digestion with thermolysin, porcine plasma fibronectin yielded five fragments (140-150, 43, 25, 17, and 14 kDa) similar to those reported for human fibronectin, although porcine fibronectin was more resistant to the digestion than human fibronectin. The three heparin-binding fragments were found to have a wide range of surface hydrophobicities, the 140-150 kDa fragment having the lowest, the 25 kDa fragment a higher, and the 14 kDa fragment the highest among all the fragments. The 43 kDa collagen-binding and 17 kDa fragments had surface hydrophobicities as high as that of fibronectin. It is noteworthy that the 43 kDa collagen-binding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbo-hydrates.View full abstractDownload PDF (1988K) -
Shigeki Tazawa, Mamoru Unuma, Naoki Tondokoro, Yoji Asano, Tomoya Ohsu ...1991 Volume 109 Issue 1 Pages 89-98
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA membrane protein fraction was obtained from rat liver rough microsomes by affinity chromatography on a concanavalin A-Sepharose column and then a chelating-Sepharose column. This protein fraction comprised about 2% of the total membrane proteins of rough microsomes and the ribosome-binding activity of ribosome-stripped rough microsomes was predominantly found in this protein fraction, as determined with a liposome assay system. To identify the essential components responsible for the ribosome binding, two approaches were employed. Trypsin treatment of liposomes reconstituted with this protein fraction resulted in the loss of the ribosome-binding activity in parallel with the loss of a dominant band, estimated Mr 34, 000, in SDS-polyacrylamide gels. Next, the direct interac-tion between the binding sites on the membrane of reconstituted liposomes and 60S ribosomal subunits was investigated by photocrosslinking using sulfosuccinimidyl 2- (m-azido-o-nitrobenzamido)-ethy1-1, 3'-dithiopropionate (SAND). The photocrosslinked com-plex was formed between 60S ribosomal subunits pretreated with SAND and binding-site proteins on the membrane of the liposomes. Then, after the liposomes were solubilized, the complex was isolated by sucrose gradient centrifugation of the binding mixture. The crosslinked proteins were released from 60S ribosomal subunits by cleavage of crosslinks with β-ME and analyzed by SDS-polyacrylamide gel electrophoresis and 125I-autoradio-graphy. The 34-kDa protein (p34) was the predominant component that crosslinked to the 60S ribosomal subunits and was found in proportion to the amount of 60S ribosomal subunits added to the system. The p34 was distinguishable by immunoblot analysis from urate oxidase, which is the 34-kDa protein of peroxisomal cores contaminating rough microsomes. These results suggest that the present p34 is a likely candidate molecule for the ribosome-binding activity of rough microsomes.View full abstractDownload PDF (5190K) -
Nobuaki Tominaga, Ryuzo Sakakibara, Masahito Shimojo, Masatsune Ishigu ...1991 Volume 109 Issue 1 Pages 99-105
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAs we previously reported [ Sakakibara et al. (1986) Biochem. Biophys. Res. Commun. 137, 443-452; and Tominaga et al. (1989) J. Biochem. 105, 992-997], subunits of human chorionic gonadotropin (hCG) containing immature N-linked sugar chains (immature subunits), i. e., the 21kDa form of α-subunit and the 23 and 19kDa forms of β-subunit, are present predominantly in first trimester placental cells. The molecular mass of intracel-lular hCG consisting of these subunits, based on gel filtration, was approximately 200kDa, suggesting homo- or hetero-oligomerization of intracellular hCG. In the present study, we purified the 21 kDa form of α-subunit as well as the 23 and 19kDa forms of β-subunit from fresh normal first trimester placental tissues by gel filtration and reverse-phase high-performance liquid chromatography. Purified subunits were hydrolyzed (with a decrease in their molecular weighs) by endoglycosidase H and α-mannosidase but not by sialidase or sialidase followed by O-glycanase, indicating that those forms have presumably only high-mannose-type N-linked sugar chains but not 0-linked sugar chains of the type present in mature β-subunit. Fifteen cycles of Edman degradation of the purified forms of the subunits were performed. Only one phenylthiohydantoin amino acid, which was the same amino acid as in the urinary β-subunit, was detected at each step for the mixture of 23 and 19 kDa forms of β-subunit, indicating that the protein backbones of both forms are identical to each other as well as to the urinary β-subunit. Thus, it is estimated that the 23 and 19 kDa forms bind two and one high-mannose sugar chains, respectively. The N-terminal sequence of the 21 kDa form of α-subunit was also the same as that of the urinary α-subunit. The purified immature subunits were able to combine with each other. hCG, reconstituted with immature subunits, stimulated testosterone production in rat Leydig cells with an activity of about 10% that of urinary hCG. When hCG, reconstituted with immature subunits, was filtered by a column of YMC pack 200 diol, three peaks of hCG were obtained. The major peak was obtained at a similar elution position to urinary hCG but the other fractions were obtained at positions of much higher molecular weight, estimated as 100 and 200 kDa. These results indicate that immature hCG tends to aggregate and may exist as a homo-oligomer in the cells.View full abstractDownload PDF (2063K) -
Koh Asano, Minoru Suzuki, Shizuo Hands, Mieko Oshima1991 Volume 109 Issue 1 Pages 106-112
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSix major acidic glycosphingolipids were isolated from human amnion using DEAE Sephadex A-25 and silica beads column chromatography. The structures of these glyco-sphingolipids were determined by methylation analysis, TLC immunostaining and/or negative ion FAB-MS, and were concluded to be II3α NeuAcLacCer(GM3), IV3 αNeuAcnLc4Cer (sialyl [α2-3] paragloboside), IV6 α NeuAcnLc4Cer (sialyl [α2-6] paragloboside), IV3 α NeuAcIII4α FucLc4Cer (sialyl Lea), VI3α NeuAcnLc6Cer (i-ganglioside) and II3α (NeuAcα 2→8NeuAc)LacCer (GD3). In addition, several minor glycosphingolipids were detected with specific monoclonal antibodies, including glycolipids with NeuAcα2-3Galβ1-4G1cNAc-β1- or NeuAcα2-6Ga1β1-4G1cNAcβ1- determinant. Our results show that the glycosphin-golipids of human amnion are characterized by having mainly type II chain analogues and onto-fetal antigens.View full abstractDownload PDF (3619K) -
Yuko Ohara-Nemoto, Takayuki Nemoto, Minoru Ota1991 Volume 109 Issue 1 Pages 113-119
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTransformed and bacterially expressed glucocorticoid receptors free from Mr 90, 000 heat shock protein (hsp90) have a 100-fold lower steroid-binding affinity than the hsp90-bound nontransformed receptor, suggesting that hsp90 is needed for high-affinity steroid binding [Nemoto, T., Ohara-Nemoto, Y., Denis, M., & Gustafsson, J.-Å. (1990) Biochemistry 29, 1880-1886]. To investigate whether or not this phenomenon is common to all steroid receptors, we investigated the steroid-binding affinities of bacterially expressed and transformed androgen receptors. The C-terminal portion of the rat androgen receptor containing the putative steroid-binding domain was expressed as a fusion protein of protein A in Escherichia coli. The recombinant protein bound a synthetic androgen, [3H] - R1881, with high affinity (Kd=0.8±0.3nM). Glycerol gradient analysis revealed that the recombinant protein sedimented at around the 3S region irrespective of the presence of molybdate, indicating that the receptor is present in mon
omeric form. The steroid-free transformed androgen receptor was obtained by exposure of rat submandibular gland cytosol to 0.4 M NaCl in the absence of steroid. High-performance ion-exchange liquid chromatography analysis showed that the transformed androgen receptor bound to [3H] - R1881 with high affinity. Thus these observations indicate that, in contrast to the glucocorticoid receptor, hsp90 is not required for the high-affinity steroid binding of the androgen receptor. In addition, the hsp90-free androgen receptor prebound with radioinert R1881 was efficiently relabeled with [3H]R1881, while the triamcinolone acetonide-bound, transformed glucocorticoid receptor failed in ligand exchange. The inability to achieve ligand exchange probably reflects the low steroid-binding affinity of this entity.View full abstractDownload PDF (2132K) -
Sachio Morimoto1991 Volume 109 Issue 1 Pages 120-126
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+ binding to fast skeletal muscle troponin C reincorporated into troponin C-depleted (CDTA-treated) myofibrils has been measured directly by using 45Ca and indirectly by using a fluorescent probe. Direct Ca2+ -binding measurements have shown that the Ca2+affinity of the low-affinity sites is enhanced in the absence of ATP and conversely reduced when myosin is selectively extracted from myofibrils, compared to the Ca2+ affinity in the presence of ATP. Fluorescence intensity changes of a dansylaziridine label at the Met-25 residue of troponin C have shown the same Ca2+-sensitivity whether or not ATP is present, while much lower Ca2+-sensitivity is seen in the myosin-extracted myofibrils. Since the Met-25 residue is in the amino terminal side α-helix of Ca2+-binding site I and far from Ca2+-binding site II in the primary structure, Ca2+- binding to site II has been evaluated by assuming that the fluorescence change monitors Ca2+ binding to site I alone. Ca2+ binding to site H thus estimated has shown high positive cooperativity only in the presence of ATP and has been found to be nearly proportional to the activation of myofibrillar ATPase, suggesting that Ca2+-binding site II is directly involved in the activation of myofibrillar ATPase activity. On the other hand, Ca2+-binding site I has been suggested to regulate the interaction of weakly binding cross-bridges with the thin filament, since the fluorescence change in the presence of ATP is saturated at the free Ca2+ concentration required for the activation of myofibrillar ATPase.View full abstractDownload PDF (1241K) -
Hideki Fukata, Akemi Mochida, Naomi Maruyama, Hirosuke Fukasawa1991 Volume 109 Issue 1 Pages 127-131
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn ATP-independent DNA topoisomerase has been isolated from chloroplasts of cauliflower leaves (Brassica oleracea var. botrytis) through DEAE-cellulose, AF-blue Toyo-pearl, and hydroxyapatite column chromatography. The sedimentation coeffient and Stokes radius of this enzyme are 3.6S and 3.6 nm, respectively, and the molecular weight of native enzyme is estimated to be 54, 000. This enzyme changes the linking number in steps of one. The enzyme activity is stimulated by MgCl2, and this enzyme shows optimum activity at 30°C in the range of 3mM MgCl2 +100mM KCl-10mM MgCl2 +50mM KCl. The enzyme activity was reduced remarkably by N-ethylmaleimide, indicating that a free sulfhydryl group is important for the activity; heparin and ellipticine also reduced the activity. Both cauliflower chloroplast topoisomerase and spinach chloroplast topoisomerase can relax positive supercoils as well as negative supercoils. From these properties, cauliflower chloroplast topoisomerase can be classified as a eukaryotic type I DNA topoisomerase.View full abstractDownload PDF (2232K) -
Mari Kono, Michiko Sekine, Kyoko Nakamura, Yasuhiro Hashimoto, Yousuke ...1991 Volume 109 Issue 1 Pages 132-136
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have reported that WHT/Ht mice express neither GM2(NeuGc) nor GM1(NeuGc) in the liver or erythrocytes due to a defect on the Ggm-2 gene, which was demonstrated to control the activity of UDP-GaINAc:GM3(NeuGc) N-acetylgalactosaminyltransferase in mouse liver, and, in addition, WHT/Ht mice do not express a detectable amount of GM2(NeuGc) but do express GM1(NeuGc) in tissues other than the liver and erythrocytes, such as the spleen, thymus, heart, lung, kidney, and testis [Nakamura et al. (1988) J. Biochem. 103, 201-208]. In order to determine whether the phenotype of WHT/Ht mice exhibiting an undetectable amount of GM2(NeuGc) in these tissues is genetically controlled or not, we analyzed the expression of gangliosides in the progeny obtained on backcross mating between (BALB/ c X WHT/Ht)F1 and WHT/Ht mice, and in a GM2(NeuGc) congenic mouse, WHT.C. Concern-ing the expression of GM2(NeuGc) in the liver, lung, and kidney, 102 backcross mice could be segregated into two types. One type expressed a detectable amount of GM2(NeuGc) in the liver, lung, and kidney, and the other type did not. The ratio of the numbers of mice exhibiting these two types was 42 : 60, indicating that the two phenotypes were genetically determined by the involvement of a single autosomal gene. Recombination as to GM2(Neu-Gc) expression in the liver, lung, and kidney was not detected among the 102 backcross mice. Analysis of the GM2(NeuGc) congenic mouse indicated that a detectable amount of GM2(NeuGc) was expressed in the liver, erythrocytes, lung, kidney, heart, spleen, and small intestine. The GM2(NeuGc) congenic mouse is considered to carry all the chromo-somes of the WHT/Ht mouse, except for an about 15 centimorgan long chromosome segment derived from the BALB/c mouse and responsible for the positive expression of GM2(NeuGc) in the liver. These results, therefore, indicate that Ggm-2 itself, or a gene or genes closely linked to it, is responsible for the expression of a detectable amount of GM2(NeuGc) in tissues other than the liver and erythrocytes. On the basis of these results, we suggest that there are at least two pathways for the biosynthesis of GM2(NeuGc), and that WHT/Ht mice can biosynthesize GM2(NeuGc) and GM1(NeuGc), even if GM2(NeuGc) is not detected by the method we used, under the involvement of autosomal genes independent of Ggm-2 in the tissues other than the liver and erythrocytes.View full abstractDownload PDF (897K) -
Shunji Ohsako, Hitoshi Nakazawa, Shun-ichi Sekihara, Atushi Ikai, Taka ...1991 Volume 109 Issue 1 Pages 137-143
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo confirm directly the role of Thr-286 as the autophosphorylation site responsible for the appearance of Ca2+-independent activity of Ca2+/calmodulin-dependent protein kinase II α subunit, we constructed two mutated cDNAs of Thr-286 to Pro or Ala using site-directed mutagenesis and introduced into Chinese hamster ovary cells. The mutant enzymes expressed in stable cell lines were partially purified and their catalytic properties were confirmed to be similar to those of wild-type kinase, except that the mutant kinase which were deprived of Thr-286 as an autophosphorylation site could not be converted to Ca2+-independent forms upon autophosphorylation. Other autophosphorylation sites of the mutants were essentially unchanged from those of the wild-type kinase and phosphoryla-tion of such sites did not convert them to Ca2+-independent forms. The results indicate that Thr-286 is the only indispensable autophosphorylation site for the appearance of Ca2+-independent activity of calmodulin-dependent protein kinase II α subunit.View full abstractDownload PDF (3620K) -
Shigeru Fujii, Yasuki Nonaka, Mitsuhiro Okamoto, Retsu Miura1991 Volume 109 Issue 1 Pages 144-149
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction between 2', 5'-ADP and NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria was examined by titrating the enzyme with 2', 5'-ADP, while the 31P-signals of 2', 5'-ADP were being monitored by 31P-NMR. From the titration profile, the dissociation constant for the complex of the enzyme with 2', 5'-ADP was estimated to be 0.22±0.05 0.05mM. Adrenodoxin reductase was reconstituted with 13C-enriched FADs. The 13C-enriched FADs used were [2-13C]-, [4, 10a- 13C2]-, and [4a- 13C] FAD. The 13C-NMR spectra of these reconstituted enzyme preparations showed 13C-resonance peaks corresponding to the enriched carbon atoms at 160.6, 165.1, 136.6, and 152.4ppm (2-, 4-, 4a-, and 10a-13C atoms, respectively). When 2', 5'-ADP was bound to the reconstituted enzyme, these 13C-resonance peaks did not shift appreciably from those of the unbound enzyme, whereas in the complex of the reconstituted enzyme with NADP+, the signals for 4- and 10a-13C shifted to higher fields by 2.1 and 0.7ppm, respectively and the 4a-13C signal shifted to a lower field by 1.4ppm. These results suggest that in the complex of the enzyme with NADP+ the pyridine moiety is located in the vicinity of C(4a)-C(4) region and that the π-electron density of the 4a-position of flavin is decreased in the enzyme-NADP+ complex. This argues in favor of the electron transfer from the dihydropyridine moiety of NADPH to the electron-deficient N(5)=C(4a) region of flavin.View full abstractDownload PDF (732K) -
Fuminori Tokunaga, Hiroshi Nakajima, Sadaaki Iwanaga1991 Volume 109 Issue 1 Pages 150-157
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123, 000), amino acid composition (1, 011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by α-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor_??_showed α-thrombin-like specificity toward synthetic tripeptide substrates. This factor_??_was also strongly inhibited by an α-thrombin inhibitor, n-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor_??_are similar to those of mammalian α-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiat-ed by trypsin-like enzymes.View full abstractDownload PDF (1497K) -
Akio Saito, Hyogo Sinohara1991 Volume 109 Issue 1 Pages 158-162
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit liver cDNA coding for α-1-antiproteinase F has been isolated and sequenced. The protein sequence deduced from the nucleotide sequence consists of a 24 amino acid signal peptide and 389 amino acids of the mature polypeptide. Rabbit α-1-antiproteinase F showed 74 and 64% homology to human α-l-antiproteinase at the nucleotide and amino acid levels, respectively, but the N-terminal five amino acids are lacking in the rabbit protein. The sequences of α-1-antiproteinase F of rabbit, human, baboon, sheep, rat, and mouse show about 40% identity, and the reactive site (Met-Ser) is conserved. On the other hand, variable regions are located in the second half to the C-terminal as well as in the N-terminal region.View full abstractDownload PDF (625K) -
Toshiyuki Takasage, Toshiaki Imagawa, Ken-ichi Furukawa, Tarou Ogurusu ...1991 Volume 109 Issue 1 Pages 163-170
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphoryla-tion of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1mM [γ-32P] ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maxi-mum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22±5, 17±4, and 15±9% (average±SD, n=4-5), respectively. In contrast, incubation of microsomes with 5 μM CaM alone and 5 μM CaM plus 1mM ATP decreased [3H]ryanodine binding by 38±14 and 53±15% (average±SD, n=6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phospho-rylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.View full abstractDownload PDF (4388K) -
Motoshige Miyano, Kiyoshi Fukui, Fusao Watanabe, Saori Takahashi, Masa ...1991 Volume 109 Issue 1 Pages 171-177
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo recombinant mutants of porcine kidney D-amino acid oxidase [EC 1.4.3.3, DAO], in which Tyr(228) and His(307) are replaced with Phe and Leu, respectively, have been expressed in Escherichia coli and purified to apparent homogeneity. The molecular size and amino-terminal sequence of the two mutants were the same as those of the native DAO. Kinetic analysis revealed that the Michaelis constants of the Phe-228 and Leu-307 mutants for D-alanine were 71- and 10-fold and the inhibition constants for benzoate, a potent competitive inhibitor, were 1, 189- and 18-fold greater than those of the native DAO, respectively. The maximum velocities of the Phe-228 and Leu-307 mutants were 66 and 58% that of the native DAO. The kinetically estimated dissociation constant of the Leu-307 mutant for FAD was 28-fold greater than that of the native DAO, whereas the value of the Phe-228 mutant was comparable to that of the native DAO. The Leu-307 mutant and the recombinant wild-type DAO were inactivated by D-propargylglycine (D-PG), a suicide substrate. However, the Phe-228 mutant was resistant to the inactivation. Absorption peaks of the Phe-228 mutant were blue-shifted about 10 nm from the corresponding peaks of the wild-type DAO, and the oxidized form was fully reduced by D-alanine without appearance of the purple intermediate. The results show that the enzymatic properties of this enzyme change remarkably upon substitution of Tyr(228) with Phe and moderately upon substitution of His(307) with Leu, a crucial role of Tyr(228) and a subsidiary role of His(307) in the catalytic reaction of the native DAO being suggested.View full abstractDownload PDF (2402K) -
Yuko Kamada, Hisako Muramatsu, Yuko Arita, Toshihide Yamada, Takashi M ...1991 Volume 109 Issue 1 Pages 178-183
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycoproteins which bound to Dolichos biflorus agglutinin (DBA) were isolated from the small intestine of 129/Sv mice. Among oligosaccharides released from the carbohydrate moieties of the glycoproteins by endo-β-galactosidase, the major one with N-acetylgalacto-samine at the non-reducing end was isolated by QAE-Sephadex A-25 column chromatography. The structure of the oligosaccharide was elucidated to be GalNAcβ1→4(NeuAcα2→3)Galβ1→4GlcNAcβ1→3Gal by compositional analysis, methylation analysis before and after mild acid hydrolysis, sequential glycosidase digestion, secondary ion mass spectrometry (SIMS), and nuclear magnetic resonance spectroscopy. The SIMS signal of m/z 1, 071 was consistent with the presence of the branched sequence, Ga1NAc(NeuAc)GalGlcNAc, and the signal was also detected in the high-molecular-weight fraction obtained after endo-β-galactosidase digestion. The pentasaccharide identified here has the terminal structure of ganglioside GM2, and an apparently identical one has been identified as the epitope of blood group Sda and the DBA binding site in human T-H urinary glycoprotein. Thus, the present result has extended our knowledge of the biological meaning of the oligosaccharide structure and has established that GalNAcβ1→4(NeuAcα2→3)Galβ1→4GlcNAc is a DBA binding site in the small intestine of the mouse.View full abstractDownload PDF (903K) -
Urara Tomita, Atsushi Inanobe, Ichiro Kobayashi, Katsunobu Takahashi, ...1991 Volume 109 Issue 1 Pages 184-189
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of mastoparan and compound 48/80 on the activities of αβγ-trimeric GTP-binding proteins (G proteins) were studied with purified G0 and G1-1 which had been reconstituted into phospholipid vesicles. Pertussis toxin-catalyzed ADP-ribosylation of G0 or G1-1, was inhibited by mastoparan or compound 48/80, suggesting that the G proteins were dissociated into their constituent α- and βγ-subunits in the presence of these compounds. The steady-state rate of GTP hydrolysis catalyzed by G0 or G1-1 was stimulated by the two compounds. Both the stimulations were due to increases in the rate of the GDP-GTP exchange reaction occurring on the G proteins. However, the modes stimulation of the GTPase activity depended on the type of G protein used, and the stimulations caused by the two compounds were differently affected by pertussis toxin-catalyzed ADP-ribo-sylation of G proteins. Moreover, the mastoparan-induced stimulation of the GTPase activity was partially inhibited by compound 48/80. Thus, the two histamine secretaG0gues mastoparan and compound 48/80 appear to activate G proteins differently, though they interact with the signal-transducing proteins, at least partly, at a common binding site.View full abstractDownload PDF (735K) -
Isao Matsuura, Kazushi Ishihara, Yoriko Nakai, Michio Yazawa, Hiroko T ...1991 Volume 109 Issue 1 Pages 190-197
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA site-directed mutagenesis study was carried out in order to understand the regulatory mechanism of calmodulin. We started from the yeast (Saccharomyces cerevisiae) cal-modulin gene since it has many differences in amino acid sequence and inferior functional properties compared with the vertebrate calmodulin. Recombinant yeast calmodulins were generated in Eschelichia coli transformed by constructed expression plasmids. Three recombinant calmodulins were obtained. The first two were YCM61G, in which the Ca2+-binding site 2 (the four Ca2+ -binding EF-hand structures in calmodulin were number-ed from the N-terminus) was converted to the same as that in vertebrate calmodulin, and YCMΔ132-148, in which the C-terminal half sequence of site 4 was deleted. These two recombinant calmodulins had the same maximum Ca2+ binding (3 mol/mol) as yeast calmodulin, which indicates that site 4 of yeast calmodulin was the one losing Ca2+ binding capacity. YCMΔ132-148 could not activate target enzymes, whereas its Ca2+ binding profile was similar to those of yeast calmodulin and YCM61G. Therefore, the structure in site 4 which cannot bind Ca2+ is indispensable for the regulatory function of yeast calmodulin. The complete regulatory function of vertebrate calmodulin can be attained by the combina-tion of 4 Ca2+ binding structures. The negative charge cluster in the central α-helix region is suggested to stabilize the active conformation of calmodulin, since the third yeast calmodulin mutant, YCM83E, which had the negative charge cluster, increased the maximum activation of myosin light chain kinase.View full abstractDownload PDF (2209K)
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