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Volume 130 (2001)
- Issue 6 Pages 721-
- Issue 5 Pages 575-
- Issue 4 Pages 471-
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- Issue 2 Pages 169-
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Volume 130, Issue 4
Displaying 1-13 of 13 articles from this issue
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Shuichi Kojima, Hideyuki Yanai, Kin-ichiro Miura2001 Volume 130 Issue 4 Pages 471-474
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe propeptide of subtilisin BPN', which functions as an intramolecular chaperone and a temporary inhibitor of subtilisin, is unique in that it acquires its three-dimensional structure by formation of a complex with the cognate protease. We previously showed that the successive amino acid replacements Ala47→Phe, G1y13→Ile, and Va165→lle in the propeptide to increase its hydrophobicity resulted in formation of a tertiary struc-ture, accompanied by increased ability to bind to the protease and increased resistance to proteolysis. In this study, we examined the effects of these tertiary-structure-forming mutations on the intramolecular chaperone activity of the propeptide. The successive amino acid replacements mentioned above were introduced into pro-subtilisin*, possessing a Ser221→Ala mutation in the catalytic residue. Refolding experiments were started by rapid dilution of the denatured pro-subtilisin*, and formation of tertiary structure in subtilisin was monitored kinetically by increase in tryptophan fluores-cence. The wild-type pro-subtilisin* was found to refold with a rate constant of 4.8 × 10-3 s-1 in the equation describing an intramolecular process. The Ala47→Phe replacement in the propeptide resulted in a 1.2-fold increase in the rate constant of subtilisin refolding. When the additional replacement G1y13→Ile was introduced, refolding of subtilisin was substantially accelerated, and its kinetics could be fitted to a double exponential pro-cess composed of a fast phase with a rate constant of 2.1 × 10-2 s-1 and a slow phase with a rate constant of 4.5 × 10-3 s-1. The rate constant of the fast phase was increased slightly by a further replacement, Va165→Ile. Since the slow phase is considered to correspond to proline isomerization, we concluded that tertiary-structure-forming mutations in the propeptide produce positive effects on its intramolecular chaperone activity through acceleration of the propeptide-induced formation of the tertiary structure of subtilisin BPN'.View full abstractDownload PDF (405K) -
Kenji Soejima, Noriko Mimura, Masaki Hirashima, Hiroaki Maeda, Takayos ...2001 Volume 130 Issue 4 Pages 475-480
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe identified a novel metalloprotease, which could be responsible for cleaving the Tyr842-Met843 peptide bond of von Willebrand factor (vWF). This metalloprotease was purified from Cohn Fraction-I precipitate of human pooled plasma by the combination of gel filtration, DEAE chromatography, and preparative polyacrylamide gel electrophoresis in the presence of SDS. The NH2-terminal amino acid sequence of the isolated protein was: AAGGILHLELLVAVGPDVFQAHQEDTRRY. Based on this sequence, we searched human genomic and EST databases, and identified compatible nucleotide sequences. These results suggested that this protein is a novel metalloprotease, a member of the family of a disintegrin and metalloprotease with thrombospondin type-1 motifs (ADAMTS), and its genomic DNA was mapped to human chromosome 9q34. Multi-ple human tissue northern blotting analysis indicated that the mRNA encoding this protease spanned approximately 5 kilobases and was uniquely expressed in the liver. Furthermore, we determined the cDNA sequence encoding this protease, and found that this protease was comprised of a signal peptide, a proregion followed by the putative furin cleavage site, a reprolysin-type zinc-metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (TSP1) motif, a cysteine-rich region, a spacer domain, and COOH-terminal TSP1 motif repeats.View full abstractDownload PDF (994K) -
Shigenobu Kimura, Hirokazu Nishida, Takashi Iyanagi2001 Volume 130 Issue 4 Pages 481-490
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPorcine NADH-cytochrome b5 reductase catalytic domain (Pb5R) has the RXY(T/S)+(T/S) flavin-binding motif that is highly conserved among the structurally related family of flavoprotein reductases. Mutations were introduced that alter the Arg63, Tyr65, and Ser99 residues within this motif. The mutation of Tyr65 to either alanine or phenylalanine destabilized the protein, produced an accelerated release of FAD in the presence of 1.5M guanidine hydrochloride, and decreased the kcat values of the enzyme. These results indicate that Tyr65 contributes to the stability of the protein and is important in the elec-tron transfer from NADH to FAD. The mutation of Ser99 to either alanine or valine, and of Arg63 to either alanine or glutamine increased both the Km values for NADH (KmNADH) and the dissociation constant for NAD+(KdNAD+). However, the mutation of Ser99 to threonine and of Arg63 to lysine had very little effect on the KmNADH and KdNAD+ values, and resulted in small changes in the absorption and circular dichroism spectra. These results suggest that the hydroxyl group of Ser99 and the positive charge of Arg63 contribute to the maintenance of the properties of FAD and to the effective binding of Pb5R to both NADH and NAD+. In addition, the mutation of Arg63 to either alanine or glutamine increased the apparent Km values for porcine cytochrome b5 (Pb5), while changing Arg63 to lysine did not. The positive charge of Arg63 may regulate the electron transfer through the electrostatic interaction with Pb5. These results substantiate the important role of the flavin-binding motif in Pb5R.View full abstractDownload PDF (894K) -
Tadashi Ueda, Makiko Nagata, Taiji Imoto2001 Volume 130 Issue 4 Pages 491-496
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined the effects of osmolytes, sucrose and trehalose, on the deterioration of hen lysozyme as a model protein. Sucrose and trehalose depressed the aggregation of lysozyme molecules caused by heating at 100°C at pH 6. Since lysozyme was fully denatured under these conditions, the effects of sucrose and trehalose on the denatured state of lysozyme were investigated using reduced S-alkylated lysozyme, a model of denatured hen lysozyme. From analyses of circular dichroism spectra and fluorescence spectra, sucrose and trehalose were found to induce α-helical conformations and some tertiary structures around tryptophan residues in the reduced S-alkylated lysozyme. Moreover, these compounds also depressed chemical reactions such as deamidation and racemiza-tion, which often cause the deterioration of proteins, on the reduced S-alkylated lysozyme. Therefore, the data suggest that sucrose and trehalose have a propensity to depress such deterioration as the aggregation of protein molecules or chemical reactions in proteins by inducing some tertiary structures (including α-helical structures) in the polypeptide chain.View full abstractDownload PDF (499K) -
Katsuhiko Kojima, Eiki Yamasaki, Sakae Kitada, Tadashi Ogishima, Akio ...2001 Volume 130 Issue 4 Pages 497-502
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondrial processing peptidase (MPP) specifically cleaves off the N-terminal presequence of the mitochondrial protein precursor. Previous studies demonstrated that Arg at position -2 from the cleavage site, which is found among many precursors, plays a critical role in recognition by MPP. We analyzed the structural elements of bovine cytochrome P450 side-chain cleavage enzyme precursor [pre-P450(SCC)], which has Ala at position -2, for recognition by MPP. Replacement of Ala position -2 of pre-P450(SCC) with Arg resulted in an increase in the cleavage rate. Replacement with Gly caused a reduction in the cleavage rate and the appearance of an additional cleavage site downstream of the authentic site. A pre-P450 (SCC) mutant with Met at position -2 retained cleavage efficiency equal to that of the wild type. These results indicate that -2 Ala of pre-P450(SCC) is recognized by MPP as a determinant for precise cleavage, and that the amino acid at -2 is required to have a straight methylene chain for interaction with the S2 site. The preference for distal basic residues, a hydrophobic residue at +1, and hydroxyl residues at +2 and +3, was almost the same as those of the precursors with Arg at -2, indicating that the recognition mechanism of pre-P450(SCC) by MPP is essentially the same as that of the precursors with Arg at position -2.View full abstractDownload PDF (1617K) -
Sachiko Okuno, Takako Kitani, Hitoshi Fujisawa2001 Volume 130 Issue 4 Pages 503-513
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr177 and Thr196, respectively, by the upstream Ca2+/calmodulin-dependent protein kinases CaM-kinase kinase α and β, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase α is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase α by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase α toward PKIV peptide, which contains the sequence surrounding Thr196 of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca2+/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca2+/calmodulin. Six phosphorylation sites on CaM-kinase kinase α, Ser24 for autophosphorylation, and Ser52, Ser74, Thr108, Ser458, and Ser
for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca2+/calmodulin suppresses phosphorylation on Ser52, Ser74, Thr108, and Ser458 by PKA, but accelerates phosphorylation on Ser475. The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.View full abstractDownload PDF (1362K) -
Takako Kitani, Sachiko Okuno, Hitoshi Fujisawa2001 Volume 130 Issue 4 Pages 515-525
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported that rat brain Ca2+/lcalmodulin-dependent protein kinase (CaM-kinase) IV is inactivated by cAMP-dependent protein kinase (PKA) [Kameshita, I. and Fujisawa, H. (1991) Biochem.Biophys. Res. Commun. 180, 191-196]. In the preceding paper, we demonstrated that changes in the activity of CaM-kinase IV by PKA results from the phosphorylation of CaM-kinase kinase a by PKA and identified six phosphory-lation sites, Ser24 for autophosphorylation, and Ser52, Ser74, Thr108, Ser458, and Ser475 for phosphorylation by PKA. In the present study, a causal relationship between the phos-phorylation and change in the activity toward PKIV peptide has been studied using mutant enzymes with amino acid substitutions at the six phosphorylation sites. The fol-lowing conclusions can be drawn from the experimental results: (i) Phosphorylation of Ser74 and/or unidentified sites causes an increase in activity; (ii) phosphorylation of Thr108 or Ser458 causes a decrease in the activity; (iii) the inhibitory effect of the phospho-rylation of Thr108 is canceled by the stimulatory effect of the phosphorylation, but that of Ser458 is not; and (iv) the inhibitory effects of Thr108 and Ser458 are synergistic. In contrast to the activity toward PKIV peptide, the activity toward CaM-kinase IV appears to be decreased by the phosphorylation of Thr108 but not significantly affected by the phos-phorylation of Ser458.View full abstractDownload PDF (1516K) -
Kaeko Tozawa, Hiromasa Yagi, Kumiko Hisamatsu, Kiyoshi Ozawa, Masasuke ...2001 Volume 130 Issue 4 Pages 527-533
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe C2 proton signals of all (twelve) histidine residues of the TF1 β subunit in the 1H-NMR spectrum have been identified and assigned by means of pH change experimentsand site-directed substitution of histidines by glutamines. pH and ligand titration experiments were carried out for these signals. Furthermore, the ATPase activity of the reconstituted α3β3γ complex was examined for the twelve mutant β subunits. Two of threeconserved histidines, namely, His-119 and 324, were found to be important for expression of the ATPase activity. The former fixes the N-terminal domain to the central domain. His-324 is involved in the formation of the interface essential for the α3β3γ complex assembly. The other conserved residue, His-363, showed a very low pKa, suggestingthat it is involved in the tertiary structure formation. On the binding of a nucleotide, only the signals of His-173, 179, 200, and 324 shifted. These histidines are located in thehinge region, and its proximity, of the β subunit. This observation provided further sup-port for the conformational change of the β monomer from the open to the closed formon the binding of a nucleotide proposed by us [Yagi et al. (1999) Biophys. J. 77, 21762183]. This conformational change should be one of the essential driving forces in therotation of the α3β3γ complex.View full abstractDownload PDF (1027K) -
Tatsuya Umeda, Zen Kouchi, Hiroyuki Kawahara, Shigeo Tomioka, Noboru S ...2001 Volume 130 Issue 4 Pages 535-542
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembers of the caspase family have been implicated as key mediators of apoptosis in mammalian cells. However, few of their substrates are known to have physiological significance in the apoptotic process. We focused our screening for caspase substrates on cytoskeletal proteins We found that an actin binding protein, filamin, was cleaved from280 kDa to 170, 150, and 120 kDa major N-terminal fragments, and 135, 120, and 110 kDa major C-terminal fragments when apoptosis was induced by etoposide in both the human monoblastic leukemia cell line U937, and the human T lymphoblastic cell line Jur-kat. The cleavage of filamin was blocked by a cell permeable inhibitor of caspase-3-likeprotease, Ac-DEVD-cho, but not by an inhibitor of caspase-1-like protease, Ac-YVAD-cho. These results suggest that filamin is cleaved by a caspase-3-like protease. To examinewhether caspase-3 cleaves filamin in vitro, we prepared a recombinant active form ofcaspase-3 directly using a Pichia pastoris overexpression system. When we applied recombinant active caspase-3 to the cell lysate of U937 and Jurkat cells, filamin wascleaved into the same fragments seen in apoptosis-induced cells in vivo. Platelet filamin was also cleaved directly from 280 kDa to 170, 150, and 120 kDa N-terminal fragments, and the cleavage pattern was the same as observed in apoptotic human cells in vivo. These results suggest that filamin is an in vivo substrate of caspase-3.View full abstractDownload PDF (1944K) -
Hiroyuki Wakabayashi, Shunji Natsuka, Motoyasu Honda, Masaaki Naotsuka ...2001 Volume 130 Issue 4 Pages 543-552
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe sugar chains of human urinary thrombomodulin were studied. N- and O-linked sugar chains were simultaneously liberated by hydrazinolysis followed by N-acetylation and were tagged with 2-aminopyridine. Then the structures of the N- and O-linked pyridylamino (PA-) sugar chains were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion. The major N-linked sugar chains of human urinary thrombomodulin were found to be monosialo- and disialofucosylbiantennary chains, while the major Olinked sugar chain was ±Siaα2-3Galβ1-3 (±Siaα2-6)GalNAc. Thrombomodulin also contained the reported structure SO4-3GlcAβ1-3Galβ1-3(±Siaα2-6)Galβ1-4Xyl [H. Wakabayashi, S. Natsuka, T. Mega, N. Otsuki, M. Isaji, M. Naotsuka, S. Koyama, T. Kanamori, K. Sakai, and S. Hase (1999) J. Biol. Chem. 274, 5436-5442]. In addition to these sugar chains, a single Glc was linked to Ser 287.View full abstractDownload PDF (622K) -
Jie Lilo, Jurunei Zhou, Wei Zou, Ping Shen2001 Volume 130 Issue 4 Pages 553-559
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interactions between adenylate kinase (AK) and a monoclonal antibody against AK (McAb3D3) were examined by means of optical biosensor technology, and the sen-sograms were fitted to four models using numerical integration algorithms. The interaction of a solution of McAb3D3 with immobilized AK follows a double exponential function and the data fitted well to an inhomogeneous ligand model. The interaction of a solution AK with immobilized McAb3D3 follows a single exponential function and the data fitted well to a pseudo-first order reaction model. The true association constants of AK binding to McAb3D3 in solution were obtained from competition BlAcore measurements. The difference in results obtained with solid-phase BlAcore and competition BlAcore may be due to rebinding of the dissociated analyte to the immobilized surface. The results obtained with BlAcore are compared to those obtained by ELISA methods. We suggest that the best method for analysis of BlAcore data is direct, global fitting of sensorgrams to numerical integration algorithms corresponding to the different possible models for binding.View full abstractDownload PDF (560K) -
Miguel Bussière, Jean E. Vance, Robert B. Campenot, Dennis E. V ...2001 Volume 130 Issue 4 Pages 561-568
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo determine the relative contribution of cell bodies and distal axons to the production of acetylcholine, we used retinoic acid to induce a cholinergic phenotype in compart-mented cultures of rat sympathetic neurons. When [3H]choline was given to cell bodies/ proximal axons for 24 h, 98% of the radiolabel was recovered as choline, phosphocholine, CDP-choline and phosphatidylcholine, whereas only 1 to 2% of the radiolabel was incorporated into acetylcholine. Choline taken up by cell bodies and transported to axons is poorly utilized for acetylcholine biosynthesis. In contrast, when distal axons were supplied with [3H]choline, 11% of the radiolabel was recovered in acetylcholine after 24 h, the remainder being incorporated into precursors/metabolites of phosphatidylcholine. The lack of acetylcholine synthesis in cell bodies/proximal axons could not be ascribed to an absence of choline acetyltransferase activity in this region of the neurons, since the specific activity of this enzyme was similar in cell bodies/proximal axons and distal axons. The rate of choline uptake by distal axons (15.3±4.4 nmol/5 min/mg protein) was _??_10-fold greater than by cell bodies/proximal axons (1.6±0.8 nmol/5 min/mg protein). Moreover, choline uptake into distal axons was inhibited by 74.5% by hemicho-linium-3, and by 80.1% by removal of Na+ from the medium. In contrast, choline uptake by cell bodies/proximal axons was not significantly inhibited by hemicholiniurn-3 or Na+ removal. These results suggest that the majority of axonal acetylcholine is synthesized in distal axons/axon terminals from choline taken up by a high-affinity choline transporter in distal axons.View full abstractDownload PDF (668K) -
Tian-Ren Lee, Hsing-Ping Hsu, Gwo-Chyuan Shaw2001 Volume 130 Issue 4 Pages 569-574
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe adjacent yrhI and yrhJ genes were identified by the Bacillus subtilis genome sequencing project. We now report that yrhJ (renamed CYP102A3) encodes a cytochrome P450 and that yrhI (renamed bscR) encodes a repressor that negatively regulates the transcription of the bscR-CYP102A3 operon. The transcriptional initiation site of bscR has been mapped by primer extension analysis. An 18-bp perfect palindromic sequence centered 65. 5 by downstream from the transcriptional initiation site of bscR has been identified as the binding site for BscR by gel mobility shift assays. Base substi-tutions in the 18-bp inverted repeat resulted in derepression of the bscR-xylE transcriptional fusion in vivo. bscR-xylE fusion studies and Northern blot analysis revealed that oleic acid and palmitate could induce the expression of the bscR-CYP102A3 operon to a considerable extent. However, only oleic acid was capable of preventing the binding of BscR to its operator DNA in vitro, suggesting that the induction of CYP102A3 expression by oleic acid and palmitate in B. subtilis might be mediated through different mechanisms.View full abstractDownload PDF (3411K)
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