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Biomedical subjects

N Katunuma

Publications and source records attributed to N Katunuma.

At least 109 records · Page 6Linked to original sources
PubMed1990

Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins.

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of alpha-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B less than calpain II congruent to calpain I less than cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (Ki) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (Ki, 36 nM) and calpain II (Ki, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (Ki, 0.5 nM); acetyl-Leu-Leu-Met-H for cathepsin B (Ki, 100 nM).

Aldehydes↗
PubMed1989

Gene structure and 5'-upstream sequence of rat cathepsin L.

The structure of rat cathepsin L gene has been determined. The gene spans 8.5 kilobase pairs comprising 8 exons, and has an intron located near the active site cysteine residue. The gene structure does not correspond well to the functional units of the proteinase. These characteristics are found to be in common with the cysteine proteinase gene family. In the 5'-upstream region, one CAAT-box and four SP-1 binding sites, together with two AP-2 binding sites and CRE, but no typical TATA-box are found. Further, SP-1 and AP-2 binding sites and an octamer motif are also found in the 1st intron, suggesting a complex regulatory mechanism for the expression of the cathepsin L gene.

Amino Acid Sequence↗
PubMed1989

Studies on the reactive site of the cystatin superfamily using recombinant cystatin A mutants. Evidence that the QVVAG region is not essential for cysteine proteinase inhibitory activities.

For study of the inhibition mechanism of the cystatin superfamily, cystatin A artificial mutants were obtained in which a well-conserved QVVAG region in the cystatin superfamily was changed to KVVAG or QVTAG and these mutants were then expressed in E. coli. For this, genes with these sequences were synthesized enzymatically from 11 oligodeoxynucleotides and expressed under the tac promoter gene of the E. coli plasmids. The products expressed were then purified on Sephadex G-50 and HPLC DEAE-5PW columns. The substitutions in cystatin A were confirmed by the amino acid compositions, N-terminal amino acid sequences and elution positions on ion-exchange chromatography of the products. The Ki values of these products for the cysteine proteinases, papain and cathepsins B, H and L, were determined in comparison with those of wild type recombinant cystatin A. Results showed that the cystatin A mutants had similar inhibitory activities to those of wild type recombinant cystatin A. Namely replacement of amino acids in the QVVAG sequence of cystatin A did not significantly affect the inhibitory activities on these proteinases. The results suggest that the QVVAG region is less important than the N-terminal region of cystatin for inhibitory activities on cysteine proteinases.

Amino Acid Sequence↗
PubMed1989

Gene structure of rat cathepsin H.

The gene structure of rat cathepsin H was determined. It comprises at least 12 exons of various lengths (32-433 bp) spanning in total more than 17.5 kbp. The gene structure does not correspond well to the functional unit of the proteinase. The region around the active site Cys residue, the most conserved region among cysteine proteinases, is split by an intron. This is a common characteristic among the gene structures of cysteine proteinases.

Amino Acid Sequence↗
PubMed1989

Involvement of tryptase-related cellular protease(s) in human immunodeficiency virus type 1 infection.

Trypstatin, a new cellular Kunitz-type protease inhibitor purified from rat mast cells, inhibited syncytium formation in human immunodeficiency virus type 1 (HIV-1)-infected CCRF-CEM and uninfected Molt-4 clone 8 at a concentration of 1 microM. Anti-rat tongue mast cell tryptase antibodies reacted with Molt-4 clone 8 cells, as determined by Western blot and by immunofluorescence. In addition, the antibody inhibited syncytium formation. These findings along with homologous sequences with trypstatin and a neutralizing epitope of gp120 of HIV-1 suggest that a tryptase-like cellular enzyme(s) is involved in HIV-1 infection.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Diacylglycerols enhance the anti-tumor effect of glucocorticoid on L5178Y lymphoblasts in vivo.

1,2-Dioleoyl-rac-glycerol, a potent activator of protein kinase C, was found to enhance the growth-inhibitory effect of triamcinolone acetonide on L5178Y lymphoblasts in adrenalectomized, male DBA/2 mice. On the other hand, in mice without adrenalectomy, it markedly inhibited tumor growth without increasing the plasma level of corticosterone or adrenocorticotropic hormone or reducing the body weight. These results suggest that diacylglycerol enhances the action of endogenous glucocorticoid to a sufficient level to inhibit the growth of lymphoblasts. Of various diacylglycerols with different carbon chain lengths tested, 1,2-dioleoyl-rac-glycerol was the most potent growth inhibitor and was maximally effective at a dose of above 30 micrograms/100 g body weight. This finding suggests that diacylglycerols may be useful for enhancing the antitumor effect of a low dose of glucocorticoid or endogenous glucocorticoid on lymphoblasts without any significant side effect.

Adrenalectomy↗
PubMed1989

An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart.

To examine localization of cathepsin B, a representative lysosomal cysteine protease, in atrial myoendocrine cells of the rat heart, immunohistochemistry at the light and electron microscopic level was applied to the atrial tissue, using a monospecific antibody for rat liver cathepsin B. In serial semi-thin sections, immunoreactivity for cathepsin B and atrial natriuretic peptides (ANP) was detected in the para-nuclear region of atrial myoendocrine cells. Several large granules and many fine granules in the region of the cells were positively stained by the cathepsin B antibody. Gold particles indicating cathepsin B antigenicity labeled secretory granules in the cells, which were also labeled by those indicating ANP, using thin sections of the Lowicryl K4M-embedded material. Moreover, some granules labeled densely by immunogold particles for cathepsin B seemed to be lysosomes. By double immunostaining using thin sections of the Epon-embedded material, gold particles indicating cathepsin B and ANP antigenicities were co-localized in secretory granules of the cells. By enzyme assay, activity of cathepsin B was three times higher in atrial tissue than ventricular tissue. The results suggest that co-localization of cathepsin B and ANP in secretory granules is compatible with the possibility that cathepsin B participates in the maturation process of ANP.

Animals↗
PubMed1989

Cysteine proteinase-inhibitory activity of ras gene product is not affected by mutations at GTPase-activating protein-binding sites.

We have investigated the proteinase-inhibitory activity against cathepsin L of some c-Ha-ras gene products (p21s) with point mutations at position 12, 33 or 35, which affect the interaction with GTPase-activating protein (GAP). All of the full-length p21s examined showed similar inhibitory activities irrespective of the point mutation and the transforming activity. These results suggest that mutations at GAP-interacting sites have no effect on the proteinase-inhibitory activity of p21s and that the proteinase-inhibitory activity alone is not sufficient for the transformation caused by p21s.

Amino Acid Sequence↗
PubMed1989

Potentiation of cysteine proteinase-inhibitor activity of full-length Ha-ras oncogene products by denaturation-renaturation.

We have investigated protease-inhibitory activity against cathepsin L of human and murine Ha-ras oncogene products (p21s) produced by Escherichia coli. The inhibitory activity of full-length p21s was much weaker than that of truncated p21s, however, the inhibitory activity was potentiated after denaturation with 8 M urea and 2 M NaCl followed by renaturation. These suggest that p21 can be folded into multiple three-dimensional conformations which have different protease-inhibitory activities.

Animals↗
PubMed1989

Mechanisms and regulation of lysosomal proteolysis.

We have determined the sequence of cDNAs encoding the precursors of rat cathepsins H and L. In order to understand the mechanism and regulation of expression and processing of these genes we have also determined the genomic restriction maps and the genomic structures in the extension sequences of the 5'-regions. We have found that the intracellular locations of cathepsins B, H and L in various cells and organs are different. Lysosomes are shown to have different amounts of cathepsins B, H and L and this heterogeneity may reflect their different functions in autophagy and heterophagy. The intracellular role of cystatins, endogenous inhibitors of cathepsins, in suppressing intralysosomal proteolysis has been clarified. Electron microscopic studies of islet cells of the pancreas using double gold particle immunostaining for insulin and cystatin beta, indicate that cystatin beta is located in the insulin secretory granules. We propose the following hypothesis for the mode of action of cystatin beta: cystatin beta is secreted from its primary location in the secretory granules and is then taken up by target cells which direct it into lysosomes for the regulation of intralysosomal cathepsin activities. The inhibitory activity of cystatin beta is controlled by formation of a mixed-disulfate with glutathione and cysteine at position 3: the free form is active and the "glutathionated" form is inactive. The changes in glutathione balance (oxidized/reduced) in cells may regulate the inhibitory activities of cystatin beta. Further, the regulation of cathepsins under various nutritional and hormonal conditions, as well as the abnormal expressions of cathepsins in various pathological conditions, are discussed.

Amino Acid Sequence↗
PubMed1988

Kunitz-type protease inhibitor found in rat mast cells. Purification, properties, and amino acid sequence.

A low molecular weight serine protease inhibitor, named trypstatin, was purified from rat peritoneal mast cells. It is a single polypeptide with 61 amino acid residues and an Mr of 6610. Trypstatin markedly inhibits blood coagulation factor Xa (Ki = 1.2 x 10(-10) M) and tryptase (Ki = 3.6 x 10(-10) M) from rat mast cells, which have activities that convert prothrombin to thrombin. It also inhibits porcine pancreatic trypsin (Ki = 1.4 x 10(-8) M) and chymase (Ki = 2.4 x 10(-8) M) from rat mast cells, but not papain, alpha-thrombin, or porcine pancreatic elastase. Trypstatin forms a complex in a molar ratio of 1:1 with trypsin and one subunit of tryptase. The complete amino acid sequence of this inhibitor was determined and compared with those of Kunitz-type inhibitors. Trypstatin has a high degree of sequence homology with human and bovine inter-alpha-trypsin inhibitors, A4(751) Alzheimer's disease amyloid protein precursor, and basic pancreatic trypsin inhibitor. However, unlike other known Kunitz-type protease inhibitors, it inhibits factor Xa most strongly.

Amino Acid Sequence↗
PubMed1988

Total synthesis of the cystatin alpha gene and its expression in E. coli.

A gene encoding cystatin alpha has been chemically synthesized, cloned and expressed in E. coli. The gene of 318 base pairs was assembled by enzymatic ligation of 19 oligonucleotides and cloned into a pBR322-derived expression plasmid down stream of the tac promoter. The expression product of the synthetic gene has been purified by Sephadex G-50 column chromatography and shown to have the same properties as those of the authentic protein isolated from rat epidermis.

Amino Acid Sequence↗
PubMed1988

Amino acid sequence of rat liver cathepsin L.

The complete amino acid sequences of the heavy and light chains of rat liver cathepsin L (EC 3.4.22.15) were determined at the protein level. The heavy and light chains consisted of 175 and 44 amino acid residues, respectively, and their Mr values without glycosyl groups calculated from these sequences were 18941 and 5056, respectively. The amino acid sequence was also determined from the N-terminal sequences of the heavy and light chains, and the sequences of cleavage fragments of the heavy chain with lysylendopeptidase and cyanogen bromide. The fragments were aligned by comparison with the amino acid sequence deduced from the sequence of cDNA of rat preprocathepsin L. The sequence of rat liver cathepsin L determined at the protein level was identical with that deduced from the cDNA sequence except that in the heavy chain, residues 176-177 (Asp-Ser) were not present at the C-terminus and alanine was replaced by proline at residue 125. Asn-108 in the heavy chain is modified with carbohydrate.

Amino Acid Sequence↗
PubMed1988

Comparative studies of the sugar chains of aminopeptidase N and dipeptidylpeptidase IV purified from rat kidney brush-border membrane.

Asparagine-linked sugar chains of rat kidney aminopeptidase N and dipeptidylpeptidase IV were investigated comparatively. Oligosaccharides released from the two enzymes by hydrazinolysis were fractionated by paper electrophoresis, serial chromatographies on columns of immobilized Aleuria aurantia lectin, concanavalin A, phytohemagglutinin E4, and Datura stramonium agglutinin, and Bio-Gel P-4 (less than 400 mesh) column chromatography. Structures of oligosaccharides in each fraction were assumed by their effective molecular sizes and behaviors on the four lectin columns and then confirmed by sequential exoglycosidase digestion and methylation analysis. The sugar chains of aminopeptidase N and dipeptidylpeptidase IV are almost the same as those of rat kidney gamma-glutamyltranspeptidase reported previously [Yamashita, K., Hitoi, A., Matsuda, Y., Tsuji, A., Katunuma, N., & Kobata, A. (1983) J. Biol. Chem. 258, 1098-1107]. They are a mixture of 5 high mannose type sugar chains and 32 (for aminopeptidase N) or 26 (for dipeptidylpeptidase IV) mono-, bi-, tri-, and tetraantennary complex type sugar chains. The unique feature of the complex-type sugar chains of both enzymes is that they all contain the bisecting N-acetylglucosamine residue and are incompletely galactosylated in their outer-chain moieties.

Aminopeptidases↗
PubMed1988

Degradation of epidermal growth factor receptors by cathepsin L-like protease: inhibition of the degradation by c-Ha-ras gene products.

Extract of NIH3T3 mouse fibroblasts contains a protease which can cleave epidermal growth factor receptor (EGF receptor). This protease was tentatively named cathepsin X and purified to near homogeneity. The characteristics of cathepsin X were similar to those of cathepsin L and the proteolytic activity of cathepsin X was inhibited by c-Ha-ras gene products.

Animals↗