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N Katunuma

Publications and source records attributed to N Katunuma.

At least 19 recordsLinked to original sources
PubMed1992

Effects of selective inhibition of cathepsin B and general inhibition of cysteine proteinases on lysosomal proteolysis in rat liver in vivo and in vitro.

Intraperitoneal administration of N-(L-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prolin e (CA-074) to rats at a dose of 4 mg/100 g greatly inhibited cathepsin-B activity in both liver and kidney for at least 4 h. Its inhibitory effect was selective for cathepsin-B activity in the liver but not in the kidney. The effects of selective inhibition of cathepsin-B activity by CA-074 treatment, and general inhibition of cysteine proteinases by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl-3-methylbutylamid e (E-64-c) on the degradation of fluorescein isothiocyanate (FITC)-labeled asialofetuin in liver lysosomes, were examined in vivo. Undegraded or partially degraded FITC-labeled asialofetuin and its FITC-labeled degradation products were both found in the lysosomes and were easily separated by Sephadex G-25' column chromatography. The FITC-labeled degradation products were mainly lysine with an FITC-labeled epsilon-amino group. Accumulation of undegraded or partially degraded FITC-labeled asialofetuin in the lysosomes was marked after E-64-c treatment, but slight after CA-074 treatment. Under the marked inhibition of general lysosomal cysteine-proteinase activity by E-64-c or marked selective inhibition of cathepsin-B activity by CA-074 in vitro, degradation of FITC-labeled asialofetuin by disrupted lysosomes was analyzed on the basis of measurement of FITC-labeled degradation products by Sephadex G-25 column chromatography. It was suppressed markedly but incompletely by E-64-c as well as by CA-074, but more weakly than by E-64-c. These results shows that E-64-sensitive cysteine proteinases are important in lysosomal protein degradation, but cathepsin B has only a role in part and that an E-64-resistant proteinase(s) may also be important.

Animals↗
PubMed1992

Phosphorylated cystatin alpha is a natural substrate of epidermal transglutaminase for formation of skin cornified envelope.

Both keratohyalin granules (KHG) and cornified envelopes were stained histochemically in an indirect immunofluorescent study by antiphosphorylated cystatin alpha antibody, indicating that phosphorylated cystatin alpha is a component of the cornified envelope proteins. When phosphorylated cystatin alpha (P-cystatin alpha) was incubated with epidermal transglutaminase (TGase) and Ca2+ ions, polymerized protein was produced by formation of epsilon-(gamma-glutamyl)lysine cross-linking peptide bonds between lysine residues of cystatin alpha and glutamine residues of suitable protein(s) in the enzyme preparation. However, phosphorylated and non-phosphorylated cystatins were polymerized to similar extents by the TGase. Immunofluorescent and immunoelectron microscopic observations revealed that P-cystatin alpha could be detected in vivo in the KHG and cornified envelopes. Treatment of sphingosine, a specific inhibitor of protein kinase C, markedly suppressed the incorporation of cystatin alpha into KHG. Thus phosphorylation of cystatin alpha by protein kinase C may play an important role in targeting cystatin alpha into KHG.

Animals↗
PubMed1992

Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein.

A novel trypsin-like protease associated with rat bronchiolar epithelial Clara cells, named Tryptase Clara, was purified to homogeneity from rat lung by a series of standard chromatographic procedures. The enzyme has apparent molecular masses of 180 +/- 16 kDa on gel filtration and 30 +/- 1.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Its isoelectric point is pH 4.75. Studies with model peptide substrates showed that the enzyme preferentially recognizes a single arginine cleavage site, cleaving Boc-Gln-Ala-Arg-4-methylcoumaryl-7-amide most efficiently and having a pH optimum of 7.5 with this substrate. The enzyme is strongly inhibited by aprotinin, diisopropylfluorophosphate, antipain, leupeptin, and Kunitz-type soybean trypsin inhibitor, but inhibited only slightly by Bowman-Birk soybean trypsin inhibitor, benzamidine, and alpha 1-antitrypsin. Immunohistochemical studies indicated that the enzyme is located exclusively in the bronchiolar epithelial Clara cells and colocalized with surfactant. An immunoreactive protein with a molecular mass of 28.5 kDa was also detected in airway secretions by Western blotting analyses, suggesting that the 30-kDa protease in Clara cells is processed before or after its secretion. Proteolytic cleavage of the hemagglutinin of influenza virus is a prerequisite for the virus to become infectious. Tryptase Clara was shown to cleave the hemagglutinin and activate infectivity of influenza A virus in a dose-dependent way. These results suggest that the enzyme is a possible activator of inactive viral fusion glycoprotein in the respiratory tract and thus responsible for pneumopathogenicity of the virus.

Amino Acid Sequence↗
PubMed1992

Analysis of degradation of proteins labeled with fluorescein isothiocyanate by Sephadex G-25 affinity chromatography.

Fluorescein isothiocyanate (FITC) has strong affinity to Sephadex G-25 gel. Amino acids labeled with FITC were found to have affinity to the gel, their elution from a Sephadex G-25 column being markedly retarded. On the other hand, proteins labeled with FITC had no affinity to the gel and were eluted in the void volume of the column. The affinity of FITC-labeled compounds to a Sephadex G-25 column decreased with increase in their molecular mass. On the basis of these findings, the degradations of various FITC-labeled proteins by disrupted lysosomes in vitro were analyzed using a small Sephadex G-25 column. FITC-labeled degradation products were easily separated from the substrate proteins, and their production was shown to increase with the incubation time and to be suppressed by the proteinase inhibitor leupeptin. This procedure is a simple method for assay of protein degradation and should be useful in analyses of the roles of proteinases in protein degradation.

Animals↗
PubMed1992

Incorporation and degradation by kidney lysosomes of cystatin alpha injected intravenously.

Cystatin alpha was purified from Escherichia coli transfected with a recombinant cystatin alpha gene and injected into the tail vein of rats. Its fate was then followed using a sensitive enzyme immunoassay for cystatin alpha. Results showed that it was rapidly removed from the blood and taken up by the kidney. Percoll density-gradient analysis showed that it was rapidly incorporated into lysosomes in the kidney. Its level in the kidney was maximal 30 min after its injection then rapidly decreased. The activity of cathepsin H in the kidney lysosomal fraction was markedly decreased 30 min after injection of cystatin alpha but recovered rapidly. Anti-(cystatin alpha) antibody precipitated cathepsin H and anti-(cathepsin H) antibody precipitated cystatin alpha in an extract of the lysosome-rich fraction of the kidney 30 min after injection of cystatin alpha. These results indicate that cystatin alpha was taken up by kidney lysosomes, formed a complex with cathepsin H and initially decreased the activity of cathepsin H, but that later the level of cystatin alpha in the kidney rapidly decreased.

Animals↗
PubMed1992

A membrane-bound metallo-endopeptidase from rat kidney. Characteristics of its hydrolysis of peptide hormones and neuropeptides.

A membrane-bound metallo-endopeptidase that hydrolyzes human parathyroid hormone (1-84) and reduced hen egg lysozyme between hydrophilic amino acid residues was isolated from rat kidney [Yamaguchi et al. (1991) Eur. J. Biochem. 200, 563-571]. In this study, the hydrolyses of various peptide hormones and neuropeptides by the metallo-endopeptidase were examined using an automated gas-phase protein sequencer. The purified enzyme hydrolyzed the oxidized insulin B chain and substance P most rapidly, followed by big endothelin 1, neurotensin, angiotensin 1, endothelin 1, rat alpha-atrial natriuretic peptide and bradykinin, in this order. The enzyme mainly cleaved these peptides at bonds involving a hydrophilic amino acid residue. However, it cleaved bonds between less hydrophilic amino acid pairs in several short peptides, e.g. at the His5-Leu6 bond in oxidized insulin B chain, the Ile28-Val29 bond in big endothelin-1 and the Ile5-His6 and Phe8-His9 bonds in angiotensin 1. The enzyme cleavage sites of oxidized insulin B chain and angiotensin 1 were different from the reported sites cleaved by meprin and by endopeptidase 2, respectively. Kinetic determination of bradykinin hydrolysis by the purified enzyme yielded values of Km = 18.1 microM and kcat = 0.473 s-1, giving a ratio of kcat/Km = 2.62 x 10(4) s-1.M-1. The Km value was about 20-fold lower than that reported for meprin and endopeptidase 2. These results indicate that the membrane-bound metallo-endopeptidase from rat kidney is distinguished from meprin and endopeptidase 2 in its substrate specificity and is not parathyroid hormone specific, but has potential capacities to inactivate various biologically active peptide hormones and neuropeptides in vivo.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of cathepsin J from rat liver.

Cathepsin J has been partially purified [Liao, J. C. R. & Lenney, J. F. (1984) Biochem. Biophys. Res. Commun. 124, 909-916], but its detailed properties are still unknown. In this study, we have purified cathepsin J completely and characterized it. It was purified to homogeneity from the mitochondrial-lysosomal fraction of rat liver by acid treatment, followed by ammonium sulfate precipitation (20-65%), and chromatographies on S-Sepharose, ConA-Sepharose, Affi-gel 501, HPLC DEAE-5PW and HPLC TSK G3000SW. Cathepsin J was found to be a lysosomal high-molecular-mass cysteine protease of about 160 kDa consisted of two different subunits. One subunit (alpha subunit) was a glycoprotein with a molecular mass of 19-24 kDa which was reduced to 19 kDa by treatment with endoglycosidase F. It has the amino acid sequence LPESWDWRNVR at its N-terminus, which was very similar to those at the N-termini of rat cathepsins B, H and L. The other subunit (beta subunit) was a glycoprotein with a molecular mass of 17 kDa, which was reduced to 14 kDa by treatment with endoglycosidase F. It had DTPANETYPDLLG at its N-terminus, which had no similarity with the N-terminal sequences of other cathepsins. Cathepsin J showed strong affinity for synthetic substrates such as N-benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-coumaryl-7-amide and glycyl-arginine beta-naphthylamide. It was activated by thiol reagents and chloride ion and was inhibited by cysteine protease inhibitors. However, its initial inhibition constant Ki(initial) by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3- methylbutylamide (E-64-c) was 1800 nM, which was 100-500 times those of cathepsins B and L. Many properties of cathepsin J were similar to those of cathepsin C (dipeptidylaminopeptidase I) reported as a lysosomal cysteine protease with dipeptidyl-aminopeptidase activity [McDonald, J. K., Reilly, T. J. & Ellis, S. (1964) Biochem. Biophys. Res. Commun. 16, 135-140]. Furthermore, antiserum against rat liver cathepsin C reacted with rat liver cathepsin J. These findings suggested that cathepsin J is identical with cathepsin C.

Amino Acid Sequence↗
PubMed1992

Biomodulators of glucocorticoid: amplifiers and suppressors of glucocorticoid action.

We have found several compounds that specifically modulate the action of glucocorticoid in vivo and in vitro without themselves having any glucocorticoid-like action and have proposed the concept of "Glucocorticoid Action Biomodulators". These biomodulators consist of "Glucocorticoid Sensitivity Amplifier" (GSA), "Glucocorticoid Potency Amplifiers" (GPAs), and suppressors of glucocorticoid action. GSA increased the incorporation of glucocorticoid into the liver and its binding to cytosol receptor without changing the total receptor concentration in liver cytosol and the equilibrium constant of the glucocorticoid binding reaction. GPAs, potent activators of protein kinase C, markedly enhanced the glucocorticoid action and the glucocorticoid action was inhibited by the inhibitors of protein kinase C. H-7, an inhibitor of protein kinase C, inhibited the translocation of glucocorticoid-receptor complex into nuclei without affecting the extent of phosphorylation of glucocorticoid receptor. These findings suggest that GPA(s) and the suppressors modulate some protein(s) which regulates the translocation of glucocorticoid receptor into nuclei.

Animals↗
PubMed1991

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization.

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.

Amino Acid Sequence↗
PubMed1991

Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C.

The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins.

A selective inhibitor of cathepsin B, a derivative of E-64 (compound CA-074), and pepstatin-asialofetuin, a potent inhibitor of cathepsin D, were used for an in vivo study of the selective role of these proteinases in lysosomal proteolysis. Administration of compound CA-074 or pepstatinasialofetuin to rats caused only a slight shift of the lysosomal density and no increase in sequestered enzymes in the autolysosomal fraction, although cathepsin B or D activity in the liver was markedly inhibited. These treatments also had little effect on the inhibition of the degradation of endocytosed FITC-labeled asialofetuin. In contrast, leupeptin treatment caused marked inhibition of lysosomal degradation of endogenous and exogenous proteins. These results suggest a small contribution of cathepsins B and D to the initiation of lysosomal proteolysis.

Animals↗
PubMed1991

Tryptase TL2 in the membrane of human T4+ lymphocytes is a novel binding protein of the V3 domain of HIV-1 envelope glycoprotein gp 120.

A novel membrane-bound serine esterase in cultured human T4+ lymphocytes, recently purified and named tryptase TL2, binds specifically to the external envelope protein gp 120 of HIV-1, interacting with its V3 domain. This binding was selectively blocked by inhibitors of tryptase TL2 with a GPCR sequence in their reactive site, synthetic peptides corresponding with the sequences of the V3 domains of various HIV-1 strains with the GPGR sequence, and antibody against tryptase TL2, or neutralizing antibody against the V3 domain of HTLV-IIIB. These findings suggest that tryptase TL2 is a binding protein of the V3 domain of HIV-1 envelope glycoprotein.

Amino Acid Sequence↗
PubMed1991

The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity.

From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two-chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X-ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha-carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha-carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy-terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this "occluding loop' provide positively charged anchors for the C-terminal carboxylate group of such polypeptide substrates. These structural features explain the well-known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2-side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo- and exopeptidase activity. The "occluding loop' does not allow cystatin-like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.

Amino Acid Sequence↗
PubMed1991

Identification of endoprotease activity in the trans Golgi membranes of rat liver cells that specifically processes in vitro the fusion glycoprotein precursor of virulent Newcastle disease virus.

A ubiquitous host endoprotease(s) responsible for activation of the fusion glycoprotein precursor (F0) of virulent Newcastle disease virus (NDV) is an important determinant for its spreading and organ tropism in the host. To characterize the virus-activating protease (VAP), we isolated endoprotease activity from the trans Golgi membranes of rat liver cells by using F0-containing NDV particles grown in a lymphoid cell line NALM6 as substrate. The enzyme cleaved in vitro only the F0 protein of virulent NDV but not that of an avirulent strain, suggesting that it specifically recognizes pairs of basic residues at the cleavage site. Furthermore, the enzyme was found to be membrane-bound, calcium ion-dependent, and active over a broad pH range, from 6 to 8. The inhibitor spectrum of the protease together with the enzyme properties described above indicates that it is a KEX2-like enzyme. Experiments using monensin, A23187, and chloroquine indicate that the F0 cleavage of virulent NDV occurs normally in rat primary hepatocytes at or before the trans Golgi and is a calcium-dependent process. The correspondence between the characteristics of the cleavage in rat hepatocytes and those of the rat protease in vitro indicates that the endoprotease is a strong candidate for the VAP that determines the pantropic nature of virulent NDV.

Animals↗
PubMed1991

H-7 reduces the nuclear binding of [3H]dexamethasone in rat liver slices but does not affect the phosphorylation of glucocorticoid receptor.

The effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, on the nuclear binding of [3H]dexamethasone and on the phosphorylation of glucocorticoid receptor was studied in rat liver slices to ascertain the role of protein kinase C in the expression of glucocorticoid action. H-7 reduces the nuclear binding of [3H]dexamethasone in rat liver slices. It does not affect the extent of phosphorylation of glucocorticoid receptor both in the absence or in the presence of glucocorticoid. These findings indicate that protein kinase C may be involved in the nuclear binding of glucocorticoid receptor but does not directly influence the receptor phosphorylation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

A novel membrane-bound serine esterase in human T4(+)-lymphocytes is a binding protein of envelope glycoprotein gp120 of HIV-1.

A novel membrane-bound serine esterase, named tryptase TL2, which is immunologically reactive with the antibody inhibiting induction of syncytia by human immunodeficiency virus-1 (HIV-1) (HATTORI, T., KOITO, A., TAKATSUKI, K., KIDO, H., and KATUNUMA, N., 1989, FEBS Lett., 248, 48-52), has been purified from a human T4+ lymphocyte clone. The enzyme has a molecular mass of 198 +/- 15 kDa, and is composed of two subunits of 32 kDa and four subunits of 28 kDa. The enzyme was strongly inhibited by the envelope glycoprotein gp120 of HIV-1, by synthetic peptides of V3 domains of gp120 s with the sequence GPGR in their center, which correspond to the principal neutralizing epitopes of the gp120s of various HIV-1 strains, by Kunitz-type inhibitors with the sequence GPCR in their active site, such as trypstatin, H130, and [Arg15, Glu52] aprotinin and by the microbial inhibitors leupeptin and antipain. This enzyme was specifically bound to the inhibitor V3 domain of gp120 of HIV-1, and this binding was blocked by the inhibitors of tryptase TL2, with a central motif GPCR or GPGR sequence in their center, but not by leupeptin and antipain without the motif. These findings suggest that tryptase TL2 is important in target site recognition and binding of HIV-1 in co-operation with CD4 receptor in the initial process of HIV-1 infection.

Alpha-Globulins↗
PubMed1991

Gene structures of rat cathepsins H and L.

The gene structures of rat cathepsins H and L have been determined. Cathepsin H gene spans more than 21.5 Kbp and comprises more than 12 exons. On the other hand, cathepsin L gene spans 8.5 Kbp and comprises 8 exons. In both genes, two intron insertion positions are conserved at the amino acid level. Both enzymes, therefore, diverged from a common ancestral gene. In the 5'-upstream region of the cathepsin L gene, no TATA box, one CAAT box and some SP-1 binding sites exist, common with other lysosomal enzyme genes. Furthermore, two AP-2 binding sites exist: a promoter under the control by the tumor promoter (TPA) and cAMP, and a cAMP response element (CRE). These results suggest that the cathepsin L gene expression is controlled by these factors.

Amino Acid Sequence↗