All issues
Volume 108 (1990)
- Issue 6 Pages 893-
- Issue 5 Pages 695-
- Issue 4 Pages 505-
- Issue 3 Pages 341-
- Issue 2 Pages 145-
- Issue 1 Pages 1-
Volume 108, Issue 3
Displaying 1-26 of 26 articles from this issue
- |<
- <
- 1
- >
- >|
-
Akira Yamada, Naokata Ishii, Keiichi Takahashi1990 Volume 108 Issue 3 Pages 341-343
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe active movement of fluorescence-labeled actin filaments along thick filaments isolated from molluscan smooth muscle was observed. Along a single thick filament, actin filaments moved toward the center of the thick filament at the speed of 1.19±0.38 μm s-1 (mean±SD, n=42) and detached themselves from it upon reaching the central zone. Movement of actin also occurred in the opposite direction, i.e., away from the center, albeit at a much lower velocity (0.09±0.07 μm s-1, n=17). Thus, the thick filament shows functional bipolarity in terms of velocity but does not determine the direction of the movement.View full abstractDownload PDF (2851K) -
Yasuyuki Suzuki, Kazuhiko Yamamoto, Hyogo Sinohara1990 Volume 108 Issue 3 Pages 344-346
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour overlapping cDNA clones encoding contrapsin were isolated from a mouse liver eDNA library constructed in the expression vector, λgtll. M13 vector sequence analysis revealed that contrapsin cDNA contained an open reading frame of 1, 254 bases encoding 418 amino acids. The N-terminal amino acid sequence of the isolated contrapsin matched residues 30 to 48 of the sequence deduced on nucleotide analysis. One clone, which had the longest 3' untranslated region, contained two sets of tandem polyadenylation signals, AATACA and AATAAA, which were located 497 bases apart, while the remaining three clones terminated at the first signal. The entire reading frame sequence of contrapsin cDNA showed 64% homology with that of human α-1-antichymotrypsin.View full abstractDownload PDF (322K) -
Masashi Suzuki, Masayo Sugiura, Setsuro Ebashi1990 Volume 108 Issue 3 Pages 347-355
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protease activity specific to spermatogenous histones was found in the egg extract of sea urchin. The enzyme responsible for this activity, named SPKK protease because of its substrate specificity, was purified as a monomeric 28 kDa protein. SPKK protease activity is inhibited by leupeptin and is specific to the repeat of sequences like Ser-Pro-Lys-Lys (the SPKK motif) [Suzuki, M. (1989), EMBO J. 8, 797-804]. The DNA-binding sites of sea urchin spermatogenous histones H1 and H2B, which protect the linker DNA of chromatin, are made up of sequences rich in the SPKK motif. SPKK protease may contribute not only to the unpacking of sperm chromatin but also to transcription activation of the male origin gene at fertilisation. SPKK protease resembles another protease activity on nucleolin [Burger et al. (1982) Eur. J. Biochem. 128 475-480] in its characteristics.View full abstractDownload PDF (4243K) -
Masashi Suzuki, Hitoshi Sohma, Michio Yazawa, Kohichi Yagi, Setsuro Eb ...1990 Volume 108 Issue 3 Pages 356-364
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protein kinase phosphorylating sea urchin spermatogenous histones, H1 and H2B, was found in sea urchin egg homogenate and purified. The kinase is activated by cAMP and is composed of two different types of subunits with molecular masses 41 and 46 kDa. The kinase phosphorylates a peptide, Ser-Pro-Arg-Lys-Ser-Pro-Arg-Lys, which is a double repeat of the DNA-binding SPKK motif [Suzuki M., (1989) EMBO J. 8, 797-804]. We name this kinase SPkinase because it exclusively phosphorylates H1 and H2B, the only histones containing SPKK motifs. Phosphorylation of H1 by SPkinase decreases the DNA-binding ability of HI. This paper is the first to report purification of a kinase which affects the DNA-binding ability of a gene regulatory protein.View full abstractDownload PDF (2096K) -
Senya Matsufuji, Youichi Miyazaki, Ryuhei Kanamoto, Takaaki Kameji, Ya ...1990 Volume 108 Issue 3 Pages 365-371
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOrnithine decarboxylase antizyme is a unique inhibitory protein induced by polyamines and involved in the regulation of ornithine decarboxylase. A cDNA was isolated from a rat liver cDNA library by the screening with monoclonal antibodies to rat liver antizyme as probes. The expression products of the cDNA in bacterial systems inhibited rat ornithine decarboxylase activity in a manner characteristic of antizyme and rabbit antisera raised against its direct expression product reacted to rat liver antizyme, confirming the authen-ticity of the cDNA. On RNA blot analysis with the cDNA probe, an antizyme mRNA band of 1.3 kb was detected in rat tissues. Antizyme mRNA did not increase upon administration of putrescine, an inducer of antizyme, and its half-life after actinomycin D treatment was as long as 12 h in rat liver, suggesting that antizyme mRNA is constitutively expressed and antizyme synthesis is regulated at the translational level. Similar-sized mRNAs hybridiza-ble to the cDNA were also found in various mammalian and non-mammalian vertebrate tissues under physiological conditions. In addition, chicken and frog antizymes showed immunocrossreactivity with rat antizyme. The ubiquitous presence and the evolutionally conserved structure of antizyme in vertebrate tissues suggest that it has an important function.View full abstractDownload PDF (4296K) -
Hiroshi Takagi, Hideki Narumi, Kenzo Nakamura, Takuji Sasaki1990 Volume 108 Issue 3 Pages 372-378
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA clone of silkworm (Bombyx mori) larval hemolymph antitrypsin (sw-AT) has been isolated from a fat body cDNA library. The cDNA has an open reading frame which codes a 392-amino acid residue polypeptide comprising a 16-residue signal peptide and a 376-residue mature sw-AT of Mr 41, 805. The reactive site of sw-AT for inhibition of bovine trypsin [Sasaki, T. et al. (1987) J. Biochem. 102, 433-441] was identified as Lys343-Va1344. Alignment of the sw-AT amino acid sequence with those of 11 members of the serpin superfamily of proteins clearly confirmed the homology of sw-AT with serpins. The amino acid sequence of sw-AT is 56% identical with that of the proteinase inhibitor from a lepidopteron, Manduca sexta [Kanost, M.R. et al. (1989) J. Biol. Chem. 264, 965-972], but the sequence around the reactive site shows no homology and the inhibitory specificity for proteinases is very different.View full abstractDownload PDF (2383K) -
Atsuo Suzuki, Takashi Yamane, Yoshiaki Ito, Toshihiro Nishio, Hideki F ...1990 Volume 108 Issue 3 Pages 379-381
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCrystallization of bacterial a-amylases has been achieved by the hanging-drop vapor diffusion method. The crystals of Bacillus licheniformis and B. licheniformis 584 amylases are isomorphous to each other. The crystals of B. licheniformis amylase belong to the tetragonal system, space group P42212 with cell dimensions of a=119.3 and c=85.4 Å. The asymmetric unit contains one molecule of amylase, with a volume per molecular mass, Vm, of 2.75 A3/Da. The crystals of B. licheniformis and B. licheniformis 584 amylases diffract beyond 2.5 A resolution and are suitable for X-ray diffraction analysis. The crystals of B. amyloliquefaciens amylase are orthorhombic, and have space group C2221 with cell dimensions of a=154, b=298, and c=90 Å. The asymmetric unit contains three to five molecules. In the crystallization of B. licheniformis and B. licheniformis 584 amylases, the addition of EDTA was indispensable to obtain large single crystals, while it had an adverse effect on the crystallization of B. amyloliquefaciens amylase, producing a large amount of small crystals.View full abstractDownload PDF (1564K) -
Teruko Sugo, Keisuke Watanabe, Toru Naraki, Michio Matsuda1990 Volume 108 Issue 3 Pages 382-387
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChemical modification of γ-carboxyglutamic acid (Gla) residues in human prothrombin toγ-methyleneglutamic acid (γ-MG1u) residues elicited a conformation similar, if not identical, to that of des-γ-carboxy prothrombin or PIVKA-II, i.e, prothrombin molecules induced by vitamin K antagonists or vitamin K deficiency states. The reaction seems to proceed sequentially by preferentially modifying a Gla at residue 32 that is located innermost among 10 Gla residues of human prothrombin. The initial modification resulted in nearly 50% losses of barium salt adsorption, the procoagulant activity and thrombin generation by the prothrombinase complex. The subsequent modification of two Gla residues at positions 6 and 16 gave rise to the immunoreactivity to an established monoclonal antibody that specifically recognizes the des-γ-carboxy prothrombin. Further modification of Gla residues increased the reactivity to the antibody, indicating that the conformation recognized by the antibody was stabilized so as to more readily fit the recognition site of the antibody. The appearance of the immunoreactivity was obviously related to the modification of Gla residues in prothrombin, since all other similarly treated derivatives of prothrombin lacking the Gla-domain failed to react with the antibody. Such chemically modified prothrombins may serve as models for studying abnormal des-γ-carboxy prothrombin produced in vitamin K deficiency states.View full abstractDownload PDF (1644K) -
Naohiro Yoshigi, Hajime Taniguchi, Takashi Sasaki1990 Volume 108 Issue 3 Pages 388-392
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn endo-cellulase cDNA has been screened from a Robillarda sp. Y-20 cDNA expression library using polyclonal antibodies (immunoscreening). Western blot analysis showed that recombinant CMCase I fused to β-galactosidase with the molecular weight of approximate-ly 150, 000 was expressed in Escherichia coli Y1090. Sequence analysis of the cloned cDNA revealed that it consisted of 1, 136 nucleotides. By comparison of the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of the purified protein (residues 1 to 18, determined by protein sequencing), the cDNA was found to lack 44 nucleotides at its 5' end corresponding to residues 1 to 15. Therefore, the mature cellulase (CMCase I) was deduced to be composed of 375 amino acid residues and the molecular weight of its protein was calculated to be 41, 004. Yaguchi et al.reported that the N-terminal amino acid sequence of an endo-β-1, 4-glucanase (endo-cellulase)from Schizophyllum commune was homologous with the active site of various hen egg-white type lysozymes, and the homology offered evidence for a lysozyme-type mechanism in enzymatic hydrolysis of cellulase [Biochem. Biophys. Res. Commun. (1983) 116, 408-411]. Pentillä et al. also pointed out that some amino acid homology was found between endo-glucanase I from Trichoderma reesei and the lysozyme of the phage T4 [Gene (1986) 45, 253-263]. From the result of sequence alignment of the endo-cellulase from Robillarda sp. Y-20 and four kinds of lysozymes, there was a possibility that the endo-cellulase from Robillarda sp. Y-20 also hydrolyzes carboxymethyl cellulose by a lysozyme-type mechanism.View full abstractDownload PDF (1756K) -
Toshihiro Yasuda, Shuichi Awazu, Wataru Sato, Reiko Iida, Yukie Tanaka ...1990 Volume 108 Issue 3 Pages 393-398
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA deoxyribonuclease I was purified from the urine of a 46-year-old male (a single individu-al) by using a series of column chromatographies to a homogeneous state as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was found to be a glycoprotein, containing 1 fucose, 7 galactose, 10 mannose, 6 glucosamine, and 2 sialic acid residues per molecule. The N-terminal amino acid sequence up to the 27th residue of the enzyme was similar to that of pancreatic deoxyribonuclease I from bovine and other species. The catalytic properties of the enzyme derived from a single individual closely resembled those of deoxyribonuclease I purified from human urine collected from several volunteers [Ito, K. et al. (1984) J. Biochem. 95, 1399-1406]. The purified enzyme was found to consist of multiple forms with different pI values. These findings are compatible with the existence of genetic polymorphism of deoxyribonuclease I in human urine previously reported [Kishi, K. et al. (1989) Hum. Genet. 81, 295-297]. This multiplicity of the urine enzyme might be due to variations in the primary structure and/or differences in the content of sialic acid.View full abstractDownload PDF (3241K) -
Akila Mayeda, Yoji Hayase, Hideo Inoue, Eiko Ohtsuka, Yasumi Ohshima1990 Volume 108 Issue 3 Pages 399-405
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe chemically synthesized antisense 12 mer 2'-O-methylribonucleotides and surveyed a scanning (signal-tracking) process as well as sequences within a β-globin transcript acting in the splicing reaction in vitro. The pre-mRNA transcript contained the sequences of the first exon, first intron, and a major part of the second exon of the human β-globin gene. We found that the antisense 2'-O-methylribonucleotides could anneal effectively to the target site in the pre-mRNA during the splicing reaction. A 2'-O-methylribonucleotide comple-mentary to the donor (5') splice site completely inhibited authentic splicing and activated an upstream cryptic donor site. A 2'-O-methylribonucleotide complementary to the branch site inhibited normal branch formation and greatly reduced subsequent generation of the spliced product. Six other 2'-O-methylribonucleotides complementary to loci in the exons or the intronic region between the donor and branch sites had no significant effect on the splicing reaction. These observations suggest that an extensive scanning of the present pre-mRNA across the six regions tested is not essential for the splicing reaction. We propose that a short antisense 2'-O-methylribonucleotide provides a practical and conve-nient method to examine cis-acting sequences of RNA. The advantages of this method in comparison with site-directed mutagenesis or deletion are discussed.View full abstractDownload PDF (7063K) -
Kyoko Momoi, Kiyoshi Fukui, Masazumi Tada, Yoshihiro Miyake1990 Volume 108 Issue 3 Pages 406-413
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlthough D-amino acid oxidase (DAO) [EC 1.4.3.3] activity in rabbit kidney extract was undetectable, protein immunoreactive toward rabbit anti-pig kidney DAO antiserum and RNAs that hybridized with fragments of human and pig DAO cDNAs were detected distinctly in the rabbit kidney. A cDNA clone, RD22, was isolated from the rabbit kidney eDNA library by hybridization with a fragment of human DAO cDNA. Analysis of the nucleotide sequence revealed a 2, 018 nucleotide sequence encoding a protein consisted of 347 amino acids. The number of amino acid residues was identical with those of human and pig DAOs, and the amino acid sequence showed 80 and 83% identity with pig and human DAOs, respectively. RNAs that hybridized with RD22 DNA fragment also existed in rabbit kidney, and their sizes were the same as those of the RNAs detected with the human and pig DAO cDNA fragments. RD22-derived protein was hardly synthesized by an in vitro expression system. However, a eDNA fragment lacking most of the 5'-untranslated region and its mutants containing base changes around the initiation codon did direct protein synthesis. Moreover, the protein derived from the partial cDNA fragment containing a large part of the coding region sequence showed immunoreactivity toward anti-pig DAO antiserum. The results suggest that one of the causes of the very poor synthesis of DAO protein in rabbit kidney is translational suppression in the synthetic process.View full abstractDownload PDF (3816K) -
Tatsuya Moriyama, Hiroshi Narita, Manabu Oki, Takao Matsuura, Makoto K ...1990 Volume 108 Issue 3 Pages 414-419
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo types of cytosolic phospholipase C specific for phosphoinositides were purified from human platelets. The molecular masses of the purified enzymes were 440 and 290 kDa. These enzymes were concluded to be respectively a trimer and a dimer of homologous 146 kDa polypeptides. The 146 kDa polypeptide may be an immunologically novel isozyme among the 140-150 kDa PLC isozymes. Both enzymes hydrolyzed phosphatidylinositol and phosphatidylinositol 4, 5-bisphosphate in a Ca2+-dependent manner.View full abstractDownload PDF (1653K) -
Hisami Yamada, Shuji Muramatsu, Takeshi Mizuno1990 Volume 108 Issue 3 Pages 420-425
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe attempted to find Escherichia coli proteins which preferentially bind to a curved DNA sequence even in the presence of an excess amount of a non-curved DNA sequence as a competitor, mainly by means of a DNA-binding gel retardation assay. Since the two sequences used had nearly the same nucleotide compositions, including consecutive dA5 stretches, we reasoned that this strategy would allow us to identify proteins which preferentially recognize an overall DNA curvature. We purified such a protein from E. coli. Its preferential binding to the curved DNA was found to be inhibited by distamycin, which removes the curvature from appropriate DNA sequences. The purified protein was identified as the E. coli nucleoid protein, H-NS.View full abstractDownload PDF (4249K) -
Fumie Hashimoto, Hidenori Hayashi1990 Volume 108 Issue 3 Pages 426-431
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo clarify the significance of catalase in peroxisomes, we have examined the effect of aminotriazole treatment of rats on the activity of β-hydroxybutyryl-CoA dehydrogenase in liver peroxisomes. When the effect of H2O2 on the dehydrogenase activity was examined using an extract of liver peroxisomes from aminotriazole-treated rats, the acetoacetyl-CoA-dependent oxidation of NADH was found to increase considerably on the addition of dilute H2O2. Such an effect of H2O2 was not seen on the β-hydroxybutyryl-CoA-dependent reduction of NAD nor with extracts from untreated animals. We then noticed that similar NADH oxidation was caused non-enzymatically by a mixture of acetoacetyl-CoA and H2O2. The oxidation was dependent on both acetoacetyl-CoA and H2O2, and was blocked by scavengers of oxyradicals such as ascorbate and ethanol. Degradation products formed during the reaction of acetoacetyl-CoA with H2O2 had no NADH oxidizing activity, indicat-ing that effective oxidant(s) were generated during the reaction of H2O2 with acetoacetyl-CoA. No other fatty acyl-CoA so far examined nor acetoacetate could replace acetoacetyl-CoA in this reaction. Therefore, if H2O2 were to be accumulated in peroxisomes, it would decrease both NADH and acetoacetyl-CoA, thus affecting the fatty acyl-CoA β-oxidation system. These results, together with our previous finding that peroxisomal thiolase was significantly inactivated by H2O2 [Hashimoto, F. & Hayashi, H. (1987) Biochim. Biophys. Acta 921, 142-150] suggest that the role of catalase in peroxisomes is at least in part to protect the fatty acyl-CoA β-oxidation system from the deleterious action of H2O2.View full abstractDownload PDF (654K) -
Hidenori Yamada, Taizo Yamashita, Hideki Domoto, Taiji Imoto1990 Volume 108 Issue 3 Pages 432-440
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe found that the reaction of hen egg-white lysozyme with an equimolar amount of tetranitromethane (TNM) at pH 8.0 and room temperature yielded derivatives in which the N-C bond of Gly104 is oxidatively cleaved, and a mono-nitrotyrosine lysozyme in which Tyr23 is nitrated. This bond cleavage occurred more predominantly with a decrease in the nitration of Tyr23, when the reaction was carried out under more dilute conditions. A possible mechanism in which a phenoxyl radical of Tyr 23 (an intermediate of nitration) is involved was proposed for this oxidative bond cleavage. When lysozyme was reacted with a 10 times molar excess of TNM, in addition to a mono-nitrotyrosine lysozyme in which only Try23 is nitrated, a di-nitrotyrosine lysozyme in which Tyr20 and Tyr23 are both nitrated and a tri-nitrotyrosine lysozyme in which Tyr20, Tyr23, and Tyr53 are all nitrated were obtained. However, no other possible mono- or di-nitrotyrosine lysozymes could be isolated. Thus, it is concluded that the three tyrosine residues in lysozyme are essentially nitrated sequentially with TNM in the order of Tyr23, Tyr20, and Tyr53. Since the derivatives obtained here were all active, none of the three tyrosine residues or the residues around Gly104 are considered to be very important for the lysozyme activity.View full abstractDownload PDF (1018K) -
C. Tyler Burt, H.-M. Cheng, Scott Gabel, Robert E. London1990 Volume 108 Issue 3 Pages 441-448
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe difference between the 31P NMR resonance position of the β-peak of ADP and the γ-peak of ATP at a fixed monovalent ion concentration level and pH is shown in vitro to depend only on free magnesium concentration. This difference can vary by more than 1 ppm depending on the pH of the solution and free magnesium concentration. Using 31P NMR and ion-selective electrodes we have constructed experimental curves to show how calibration of experimental results can be achieved. Theoretical calculations can be used to predict the functional dependence of the chemical shift difference on the above parameters. However, using the reported ion dissociation constants the fit was not exact and the discrepancy increased at higher pH values. To demonstrate that this technique can yield valid in vivo results and to analyze a previously unreported system where free magnesium levels vary, sample spectra from lenses and enucleated eyes are given. The data show a disagreement between the present methods and the more conventional ATP method. The reason for this difference is not known although some possible reasons are suggested.View full abstractDownload PDF (851K) -
Tohru Yamada, Nobuko Akutsu, Kentaro Miyazaki, Katsumi Kakinuma, Masas ...1990 Volume 108 Issue 3 Pages 449-456
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThreo-Ds-3-isopropylmalate dehydrogenase coded by the leuB gene from an extreme thermophile, Thermus thermophilus strain HB8, was expressed in Escherichia coli carrying a recombinant plasmid. The thermostable enzyme thus produced was extracted from the E. coli cells, purified, and crystallized. The enzyme was shown to be a dimer of identical subunits of molecular weight (4.0±0.5)×104. The Km for threo-Ds-3-isopropylmalate was estimated to be 8.0×10-5 M and that for NAD 6.3×10-4 M. The optimum pH at 75°C in the presence of 1.2M KCl was around 7.2. The presence of Mg2+ or Mn2+ was essential for the enzyme action. The enzyme was activated about 30-fold by the addition of 1 M KCl or RbCl. The high salt concentration decelerated the thermal unfolding of the enzyme, and accelerated the aggregation of the unfolded protein. Based on these effects, the molecular mechanism of the unusual stability of the enzyme is discussed.View full abstractDownload PDF (1485K) -
Masao Miki1990 Volume 108 Issue 3 Pages 457-461
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn unsplitable analogue of ATP (adenylyl imidodiphosphate; AMPPNP) was incorporated into F-actin [Cooke, R. (1975) Biochemistry 14, 3250-3256]. The resulting polymers (F-actin-AMPPNP) activated the ATPase activity of myosin subfragment-1 (S1) as efficiently as normal F-actin; neithr the maximum velocity at infinite actin concentration (Vmax) nor the affinity of actin to S1 in the presence of ATP (1/KATPase) changed, which indicates that the terminal phosphate of the bound nucleotide at the cleft region between the two domains of the actin molecule [Kabsch, W., Mannherz, H. G., & Suck, D. (1985) EMBO J. 4, 2113-2118] is not directly involved in a myosin binding site. However, the interaction of F-actin with troponin-tropomyosin was strongly modulated by the replacement of ADP with AMPPNP. The troponin-tropomyosin complex strongly enhanced the activation of S1-ATPase activity by F-actin-AMPPNP in the presence of Ca2+, although it has no effect on the activation by normal F-actin-ADP. KATPase was enhanced about threefold by troponin-tropomyosin in the presence of Ca2+, while Vmax was not markedly changed. F-actin-AMPPNP is highly potentiated by troponin-tropomyosin even with low S1 to actin ratios and at high ATP conditions. In the absence of Ca2+, the activation by F-actin-AMPPNP was inhibited normally by troponin-tropomyosin. The results suggest that the terminal β-phosphate of the bound nucleotide in F-actin is located in a region which is important for regulation of the interaction with myosin.View full abstractDownload PDF (621K) -
Ming Li, Teruo Ishibashi1990 Volume 108 Issue 3 Pages 462-465
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA rat liver glutathione S-transferase isozyme (GST 3-3) has been purified to apparent electrophoretic homogeneity by procedures including Sephadex G-100, S-hexylglutathione-linked Sepharose, and CM-cellulose column chromatographies. The present study provides evidence for the existence of endogenous fatty acids in the purified GST 3-3 by gas-liquid chromatographic and mass-spectrometric analyses. The liver GST isozyme associated long chain fatty acids such as 14:0, 16:0, 18:0, and 18:1 and the molar ratio of fatty acids to the protein was estimated to be around 3.2. When the enzyme preparation was freed of fatty acids by a mild delipidization technique using Lipidex, the GST activity was significantly decreased. Computer analysis of the circular dichroism spectra revealed that rat liver GST 3-3 contained approximately 49% α-helix and 24% random coil. By delipidizing, the enzyme's α-helix was decreased to 4% and the random coil was in turn increased to 62%. The enzymatic and structural properties of the delipidized enzyme, however, could be restored to nearly the original levels by recombining with the fatty acids. These findings suggest that weakly bound fatty acids are responsible for the functional capacity of the GST by virtue of changing the protein conformation.View full abstractDownload PDF (1792K) -
Per Falk, Lansing C. Hoskins, Göran Larson1990 Volume 108 Issue 3 Pages 466-474
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFive strains of human fecal bacteria, of the Ruminococcus and Bifidobacterium genera, produce extracellular α- and β-glycosidases that degrade intestinal mucin oligosaccha-rides and glycosphingolipids of the lacto-series type 1 chain. We have tested the activities and substrate specificities of these enzymes using para-nitrophenyl glycosides and glycosphingolipids of different core chains (lacto, neolacto, globo, isoglobo, galabio, and ganglio), carrying different blood group determinants (A, H, X, Y, Forssman, and para-Forssman), and with different degrees of sialylation (mono- to tetra-sialo). Lactotetraosyl-ceramide and neolactotetraosylceramide were the only core glycosphingolipids degraded by enzymes from these strains, resulting in lactosylceramide and glucosylceramide as the major end products. R. gnavus strain VI-268 did not degrade lactotetraosylceramide but only neolactotetraosylceramide yielding lactotriaosylceramide and lactosylceramide as the major end products. All strains but R. gnavus VI-268 also produced lactosylceramide from a bi-antennary 10-sugar glycosphingolipid with two blood group H determinants based on a lactotetraosylceramide core. Apart from strain specific blood group A-degrading (R. torques strain VIII-239 and IX-70, R. gnavus strain VI-268 and B. infantis VIII-240) and Forssman-degrading (R. torques VIII-239 and IX-70) activities, all strains also degraded the II-5, X-5, and Y-6 glycosphingolipids. All strains released N-acetylneur-aminic acid from the gangliosides sialosyl-neolactotetraosylceramide, GD3, GD1a, GD1b, GT1b, and GQ1b, corresponding to 2, 3-α- and 2, 8-α-N-acetylneuraminidase activities. The R. torques strains VIII-239 and IX-70 also partially desialylated GM1 to lactotetraosyl-ceramide. The para-nitrophenyl glycoside degradations were often incompatible with the data from the glycosphingolipids degradations. We conclude that the high degree of substrate specificities of these bacterial glycosidases is primarily directed towards the main oligosaccharides structures produced in the human gastrointestinal epithelium, i.e. lactoseries type 1 and type 2 chains. Their degradation may, through the production of nutrients, growth factors and receptors be important for establishment and maintainance of the intestinal microbiota.View full abstractDownload PDF (2284K) -
Kazuhiko Saeki, Yumi Suetsugu, Yoshio Yao, Takekazu Horio, Barry L. Ma ...1990 Volume 108 Issue 3 Pages 475-482
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo distinct ferredoxins were purified from Rhodobacter capsulatus SB1003. Their complete amino acid sequences were determined by a combination of protease digestion, BrCN cleavage and Edman degradation. Ferredoxins I and II were composed of 64 and 111 amino acids, respectively, with molecular weights of 6, 728 and 12, 549 excluding iron and sulfur atoms. Both contained two Cys clusters in their amino acid sequences. The first cluster of ferredoxin I and the second cluster of ferredoxin II had a sequence, CxxCxxCxxx-CP, in common with the ferredoxins found in Clostridia. The second cluster of ferredoxin I had a sequence, CxxCxxxxxxxxCxxxCM, with extra amino acids between the second and third Cys, which has been reported for other photosynthetic bacterial ferredoxins and putative ferredoxins (nif-gene products) from nitrogen-fixing bacteria, and with a unique occurrence of Met. The first cluster of ferredoxin II had a CxxCxxxxCxxxCP sequence, with two additional amino acids between the second and third Cys, a characteristic feature of Azotobacter- [3Fe-4S] [4Fe-4S] -ferredoxin. Ferredoxin II was also similar to Azoto-bacter-type ferredoxins with an extended carboxyl (C-) terminal sequence compared to the common Clostridium-type. The evolutionary relatinship of the two together with a putative one recently found to be encoded in nitENXQ region in this bacterium [Moreno-Vivian et al. (1989) J. Bacteriol. 171, 2591-2598] is discussed.View full abstractDownload PDF (822K) -
Kengo Kanamaru, Hirofumi Aiba, Takeshi Mizuno1990 Volume 108 Issue 3 Pages 483-487
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, the transfer of a phosphoryl group between the EnvZ and OmpR proteins, which are involved in expression of the ompF and ompC genes in response to the medium osmolarity, was demonstrated in vitro. In this study, the histidine (His) residue at position 243 of the EnvZ protein, and the aspartate (Asp) residues at positions 12 and 55 of the OmpR protein were changed, respectively, by means of site-directed mutagenesis. We characterized the mutant proteins in terms of not only their in vitro phosphotransfer reactions but also their in vivo osmoregulatory phenotypes. The mutant EnvZ protein was defective in its in vitro ability not only as to EnvZ-autophosphorylation but also OmpR-phosphorylation and OmpR-dephosphorylation. This particular mutant EnvZ protein seemed to exhibit null functions as to the in vivo osmoregulatory phenotype. The mutant OmpR protein with the amino acid change at position 12 was clearly phosphorylated in vitro, but at a very low rate as compared with the wild-type OmpR protein. in vitro phosphorylation of the mutant OmpR protein with the amino acid change at position 55 was more severely affected. This mutant OmpR protein appeared to exhibit null functions as to the in vivo osmoregulatory phenotype. These results suggest that the histidine residue at position 243 of the EnvZ protein and the aspartate residues at positions 12 and 55 of the OmpR protein are deeply involved in the phosphotransfer between the EnvZ and OmpR proteins.View full abstractDownload PDF (3418K) -
Shin-ichi Tokishita, Hisami Yamada, Hirofumi Aiba, Takeshi Mizuno1990 Volume 108 Issue 3 Pages 488-493
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe EnvZ protein is presumably a membrane-located osmotic sensor, which specifically phosphorylates the activator protein, OmpR, involved in expression of the ompF and ompC genes in Escherichia coli. In this study, we developed an in vitro system for analyzing the intact form of the EnvZ protein located in the isolated cytoplasmic membrane. This particular form of the EnvZ protein exhibited its in vitro ability not only as to OmpR-phosphorylation but also OmpR-dephosphorylation. It was found that when a high concentration of a mono-cation (K+, Na+, or Li+) was present during the in vitro reactions, OmpR-dephosphorylation was preferentially inhibited and consequently the phospho-rylated form of the OmpR protein was accumulated under the in vitro conditions used, although the K+ ion appears to be essential for the OmpR-phosphorylation reaction. Procaine, a local anesthetic, is known to affect the osmotic regulation of the ompF and ompC genes in vivo. In this study, procaine was also found to preferentially inhibit OmpR-dephosphorylation mediated by the EnvZ protein in vitro.View full abstractDownload PDF (2030K) -
Hideo Nakagawa, Michinori Hirata, Kazuya Hoshino, Kazuhiko Sakata, Sin ...1990 Volume 108 Issue 3 Pages 494-498
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGelatinase was extracted at 60°C from the collagen fiber-rich fraction of granulation tissue induced by carrageenin in rats. A large part of the extracted gelatinase was unbound to Zn-chelating Sepharose. The unbound gelatinase gave a single band corresponding to a molecular mass of 57 kDa on SDS-substrate PAGE, but showed a much higher molecular mass (>200 kDa) on Sephadex G-150 gel filtration. In addition, that the unbound fraction contained gelatin fragments was revealed by SDS-PAGE. When the unbound fraction of Zn-chelating Sepharose was incubated at 37°C, the gelatin fragments disappeared and the apparent molecular mass of gelatinase in gel filtration decreased. This gelatin degradation of the unbound fraction was enhanced by treatment with 4-aminophenylmercuric acetate (APMA). The results suggest that the gelatinase is bound to gelatin fragments in the unbound fraction. After the treatment with APMA, the gelatinase was purified to homogene-ity; the purified gelatinase gave a single band corresponding to a molecular mass of 57 or 67 kDa on SDS-PAGE under nonreducing or reducing conditions, respectively. The purified gelatinase is a metalloproteinase, and extensively degraded gelatin, but showed no proteolytic activity toward α-casein or types I and IV collagens. The results suggest that the 67-kDa active gelatinase is bound to collagen fibers and plays an important role in a rapid degradation of collagen fibers in granulation tissue.View full abstractDownload PDF (4064K) -
Masumi Eto, Rie Suzuki, Fumi Morita, Hideto Kuwayama, Norio Nishi, Sei ...1990 Volume 108 Issue 3 Pages 499-504
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe heptapeptide Ile-Arg-Ile-Cys-Arg-Lys-Gly-OEt is the analog of the S-site, one of the actin-binding sites in myosin [ Suzuki et al. (1987) J. Biol. Chem. 262, 11410-11412]. Various substituted heptapeptides were synthesized, and the dissociation constant of each acto-heptapeptide complex was measured. Comparison of the dissociation constants indicated that the hydrophobic side chain of Ile-1 was critical for the binding with F-actin, but not that of Ile-3. The positive charge and the side chain length of Arg-2 were also important. The presence of a sulfur atom in the Cys-4 was also necessary. The affinity of the N-terminal Ile-Arg-Ile part for F-actin was influenced by the kind of residues in the C-terminal tetrapeptide part. Based on these results, the side chains of Ile(702), Arg(703), and Cys(SH1)(705) in myosin subfragment-1 heavy chain were assigned to be critical for the binding with F- actin. The amino acid sequence of S-1 heavy chain containing these critical residues for the S-site from residue number 700 to 717 can be predicted as an analogue of the segment B of the ATP-binding site [Walker et al. (1982) EMBO J. 1, 945-951]. The actin-binding S-site possibly shares a part of the ATP-binding site in myosin. We discuss the possibility that the S-site is an inhibitory site of myosin ATPase and the so-called actin-activation of myosin ATPase is a deinhibition induced by transient binding of F-actin to the S-site.View full abstractDownload PDF (664K)
- |<
- <
- 1
- >
- >|