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Shin-ichi Ishii - One of the best experts on this subject based on the ideXlab platform.

  • primary structure of streptomyces griseus Metalloendopeptidase ii
    Bioscience Biotechnology and Biochemistry, 1998
    Co-Authors: Shuichi Kojima, Takashi Kumazaki, Shin-ichi Ishii, Kinichiro Miura
    Abstract:

    Streptomyces griseus Metalloendopeptidase II (SGMPII) is a unique protease, since it shows anomalous susceptibility to the proteinaceous “serine protease inhibitors” produced by Streptomyces, such as Streptomyces subtilisin inhibitor (SSI) and its homologous proteins. In this study, we analyzed the amino acid sequence of SGMPII by analyzing various peptide fragments produced enzymatically. The sequence of SGMPII, which is composed of 334 amino acids, showed no extensive similarity to SSI-insensitive metalloproteases produced by other species of Streptomyces, except for the amino acid residues essential for catalysis and zinc binding. However, SGMPII is 35-41% similar to thermolysin and its related metalloproteases, which are not inhibited by SSI, and the residues presumed to be critical for catalysis and zinc-binding are well conserved in SGMPII. Glu137 in a “His-Glu-Xaa-Xaa-His” motif of SGMPII was identified as the residue modified by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2, an active-site-directed irreversibl...

  • Application of bimane-peptide substrates to spectrofluorometric assays of Metalloendopeptidases.
    Journal of Biochemistry, 1991
    Co-Authors: Kenta Kajiwara, Takashi Kumazaki, Eisuke Sato, Yuichi Kanaoka, Shin-ichi Ishii
    Abstract:

    : A spectrofluorometric method for sensitive determination of Metalloendopeptidase activity has been developed by using a bimane-peptide containing a tryptophan residue, i.e. 1,7-dioxo-2,5,6-trimethyl-1H,7H-pyrazolo[1,2-alpha]pyrazol-3-yl-methyl- thiomethylcarbonyl-phenylalanyl-tryptophanyl-leucine (Bim-SCH2CO-Phe-Trp-Leu-OH). Such an "intramolecularly quenched" substrate was originally designed for a sensitive assay of angiotensin I converting enzyme (ACE) [Sato, E. et al. (1989) Chem. Pharm. Bull. 37, 145-147]. All the typical Metalloendopeptidases tested, such as thermolysin, Pseudomonas aeruginosa (Ps.) elastase, Streptomyces griseus Metalloendopeptidases I and II (SGMPI and SGMPII), and alkinonase A, a Metalloendopeptidase from Streptomyces violaceorectus, cleaved this substrate strictly at a Phe-Trp bond, leading to a marked increase in fluorescence. Kinetic parameters of the enzymatic hydrolyses of five kinds of analogous bimane substrates were compared to examine how the nature of neighboring amino acid residues on either side of the cleavable bond affects the catalytic efficiency of each of the Metalloendopeptidases. Bim-SCH2CO-Phe-Trp-Leu-OH was most efficiently hydrolyzed by all of these enzymes. The use of this substrate made it possible to determine minute amounts of Metalloendopeptidases, especially those originating from Streptomycetes (for example, as little as 10 fmol of SGMPII).

  • Purification and characterization of Streptomyces griseus Metalloendopeptidases I and II.
    Journal of Biochemistry, 1991
    Co-Authors: Hideaki Tsuyuki, Kenta Kajiwara, Takashi Kumazaki, Akira Fujita, Shin-ichi Ishii
    Abstract:

    : Two Metalloendopeptidases, designated as Streptomyces griseus Metalloendopeptidases I and II (SGMPI and SGMPII), were isolated from a commercial Pronase P by a method including affinity chromatography on carbobenzoxy-L-alaninyl-triethylenetetraminyl-Sepharose (Z-Ala-T-Sepharose). The two enzymes differed from each other in behavior on ion-exchange chromatography but showed the same amino-terminal sequence at least up to the 20th residue. Their molecular weights were both estimated to be 37,000 by SDS-polyacrylamide gel electrophoresis. Elemental and amino acid composition analyses indicated that both of them contained about 1 g atom of zinc and one cystine residue per mol of protein. Cleavage specificities of the two enzymes toward synthetic peptide-substrates were very similar to those observed with thermolysin. EDTA, o-phenanthroline, and phosphoramidon strongly inhibited these enzymes, while typical serine-protease inhibitors and cysteine-protease inhibitors had no effect. The findings clearly indicate that SGMPI and SGMPII can be classified into the family of zinc-endopeptidases. It was unexpectedly found, however, that these Metalloendopeptidases were strongly inhibited by protein serine-protease inhibitors produced by Streptomycetes, such as Streptomyces subtilisin inhibitor (SSI), alkaline protease inhibitor-2c' (API-2c'), and plasminostreptin (PS).

  • interactions of streptomyces serine protease inhibitors with streptomyces griseus Metalloendopeptidase ii
    Journal of Biochemistry, 1991
    Co-Authors: Kenta Kajiwara, Takashi Kumazaki, Hideaki Tsuyuki, Akira Fujita, Shin-ichi Ishii
    Abstract:

    : Streptomyces griseus Metalloendopeptidase II (SGMPII) was shown to form tight complexes with several Streptomyces protein inhibitors which had been believed to be specific to serine proteases, such as Streptomyces subtilisin inhibitor (SSI), plasminostreptin (PS), and alkaline protease inhibitor-2c' (API-2c'), as well as with Streptomyces metalloprotease inhibitor (SMPI). The dissociation constants of complexes between SGMPII and these inhibitors were successfully determined by using a novel fluorogenic bimane-peptide substrate. The values ranged from nM to pM. The results of studies by gel chromatographic and enzymatic analyses indicated that SGMPII is liberated from the complex with SSI by the addition of subtilisin BPN'. SGMPII and subtilisin BPN' proved, therefore, to interact with SSI in a competitive manner, despite the difference in the chemical nature of their active sites.

James L. Roberts - One of the best experts on this subject based on the ideXlab platform.

  • secretion of Metalloendopeptidase 24 15 ec 3 4 24 15
    DNA and Cell Biology, 1999
    Co-Authors: Emer S Ferro, Marc J Glucksman, John W Tullai, James L. Roberts
    Abstract:

    The Metalloendopeptidase EP24.15 (EC3.4.24.15) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly. Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment. We utilized a model system of neuroendocrine secretion, the AtT20 cell. According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested ...

  • Structural and functional studies of the Metalloendopeptidase (EC 3.4.24.15) involved in degrading gonadotropin releasing hormone
    Biophysical Journal, 1992
    Co-Authors: Marc J Glucksman, Marian Orlowski, James L. Roberts
    Abstract:

    Converging biophysical and molecular biological techniques including genetic and computer aided engineering are being utilized in the study of zinc-Metalloendopeptidase E.C. 3.4.24.15 (EP 24.15), a peptidase involved in the degradation of Gonadotropin releasing hormone (GnRH), the master regulatory decapeptide involved in reproduction. This 71 kDa enzyme rapidly cleaves the Tyr-Gly6 bond in GnRH, the rate limiting reaction in hormone inactivation. EP 24.15 has been identified and isolated (1, 2) and the full length rat cDNA has recently been sequenced, and used to direct the expression of the functional 645 amino acid protein (3). The sequence of EP 24.15 shows no sequence identity with any known Metalloendopeptidases beyond the commonly shared active site motif, -H-E-x-x-H-, found in this family of enzymes. EP 24.15 is a predominantly cytosolic enzyme that is stable, not glycosylated, and can be modeled with other globular proteins. Hydrophobic Cluster Analysis, (HCA) (4), is a heuristic algorithm ascertaining structural domains by detecting patterns of secondary structure and sequence homology in a two-dimensional analysis. This method has been used to determine if thermolysin may be used in modeling EP 24.15, and extending this analysis to enkephalinase and angiotensin converting enzyme two other members of the zinc-Metalloendopeptidase family. Information gained from this study would be a step towards modeling substrate and inhibitor interactions to elucidate the conformation of this enzyme, as a probe for the function of this enzyme in vivo, and as a target for pharmacological intervention.

  • molecular cloning and primary structure of rat testes Metalloendopeptidase ec 3 4 24 15
    Biochemistry, 1990
    Co-Authors: Adrian R Pierotti, Marc J Glucksman, Marian Orlowski, Ke Wen Dong, James L. Roberts
    Abstract:

    : The complete amino acid sequence of rat testes Metalloendopeptidase (EC 3.4.24.15) was deduced from the nucleotide sequence of a cDNA clone isolated by screening a rat testes library with a polyclonal antibody raised against a homogeneous preparation of the rat testes enzyme. The correctness of the sequence was verified by N-terminal amino acid sequence analysis of the isolated enzyme and by partial amino acid sequence analysis of three tryptic peptides located near the N-terminus, the middle, and C-terminus of the native protein. The enzyme is composed of 645 amino acids with a molecular weight of 72,985. This value is close to that of the purified rat testes and brain enzyme as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. The enzyme contains the putative active-site sequence -H-E-F-G-H- that is homologous to the sequence in the active site of thermolysin and several other related bacterial enzymes, as well as to active-site sequences of several mammalian zinc metallopeptidases. No amino acid sequence homology, beyond this active site, was found with thermolysin, a bacterial zinc Metalloendopeptidase, nor with several mammalian zinc metallopeptidases. Northern blot hybridization analyses showed the presence of mRNA encoding the enzyme in rat testes, but not in other rat tissues in spite of the finding that enzyme activity is widely distributed in all tissues and that relatively high activities are present in rat brain and pituitary.

Adrian R Pierotti - One of the best experts on this subject based on the ideXlab platform.

  • n arginine dibasic convertase a Metalloendopeptidase as a prototype of a class of processing enzymes
    Proceedings of the National Academy of Sciences of the United States of America, 1994
    Co-Authors: Adrian R Pierotti, Annik Prat, Valerie Chesneau, Florence Gaudoux, Annemarie Leseney, Thierry Foulon, Paul Cohen
    Abstract:

    Abstract N-Arg dibasic convertase is a Metalloendopeptidase from rat brain cortex and testis that cleaves peptide substrates on the N terminus of Arg residues in dibasic stretches. By using both an oligonucleotide and antibodies to screen a rat testis cDNA library, a full-length cDNA was isolated. The sequence contains an open reading frame of 1161 codons corresponding to a protein of 133 kDa that exhibits 35% and 48% similarity with Escherichia coli protease III (pitrilysin, EC 3.4.99.44) and rat or human insulinase (EC 3.4.99.45), respectively. Moreover, the presence of the HXXEH amino acid signature (XX = FL) clearly classifies N-Arg dibasic convertase as a member of the pitrilysin family of zinc-Metalloendopeptidases. In addition, a Cys residue that may be responsible for the thiol sensitivity of the insulinase and N-Arg dibasic convertase was proposed. The protein sequence contains a distinctive additional feature consisting of a stretch of 71 acidic amino acids. We hypothesize that this Metalloendopeptidase may be a member of a distinct class of processing enzymes.

  • molecular cloning and primary structure of rat testes Metalloendopeptidase ec 3 4 24 15
    Biochemistry, 1990
    Co-Authors: Adrian R Pierotti, Marc J Glucksman, Marian Orlowski, Ke Wen Dong, James L. Roberts
    Abstract:

    : The complete amino acid sequence of rat testes Metalloendopeptidase (EC 3.4.24.15) was deduced from the nucleotide sequence of a cDNA clone isolated by screening a rat testes library with a polyclonal antibody raised against a homogeneous preparation of the rat testes enzyme. The correctness of the sequence was verified by N-terminal amino acid sequence analysis of the isolated enzyme and by partial amino acid sequence analysis of three tryptic peptides located near the N-terminus, the middle, and C-terminus of the native protein. The enzyme is composed of 645 amino acids with a molecular weight of 72,985. This value is close to that of the purified rat testes and brain enzyme as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. The enzyme contains the putative active-site sequence -H-E-F-G-H- that is homologous to the sequence in the active site of thermolysin and several other related bacterial enzymes, as well as to active-site sequences of several mammalian zinc metallopeptidases. No amino acid sequence homology, beyond this active site, was found with thermolysin, a bacterial zinc Metalloendopeptidase, nor with several mammalian zinc metallopeptidases. Northern blot hybridization analyses showed the presence of mRNA encoding the enzyme in rat testes, but not in other rat tissues in spite of the finding that enzyme activity is widely distributed in all tissues and that relatively high activities are present in rat brain and pituitary.

Marc J Glucksman - One of the best experts on this subject based on the ideXlab platform.

  • The role of neuropeptide processing enzymes in endocrine (prostate) cancer: EC 3.4.24.15 (EP24.15).
    Protein and Peptide Letters, 2004
    Co-Authors: Todd A. Swanson, Amanda Pabon, Keith D. Philibert, Mina Wang, Michael P. Myers, Marc J Glucksman
    Abstract:

    The zinc Metalloendopeptidase EC3.4.24.15 [EP24.15, thimet oligopeptidase], a neuropeptide processing enzyme, is central to the formation and degradation of many bioactive peptides in the neural proteome, and is highly expressed in normal prostate. EP24.15 actions are increased in androgen-dependent prostate cancer compared to androgen-independent; augmented by androgen treatment, and inhibited by clinical GnRH analogs. The “neural” prostate includes: neuropeptides, cognate receptors and processing enzymes regulating signaling of peptide-mediated neural inputs.

  • secretion of Metalloendopeptidase 24 15 ec 3 4 24 15
    DNA and Cell Biology, 1999
    Co-Authors: Emer S Ferro, Marc J Glucksman, John W Tullai, James L. Roberts
    Abstract:

    The Metalloendopeptidase EP24.15 (EC3.4.24.15) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly. Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment. We utilized a model system of neuroendocrine secretion, the AtT20 cell. According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested ...

  • Structural and functional studies of the Metalloendopeptidase (EC 3.4.24.15) involved in degrading gonadotropin releasing hormone
    Biophysical Journal, 1992
    Co-Authors: Marc J Glucksman, Marian Orlowski, James L. Roberts
    Abstract:

    Converging biophysical and molecular biological techniques including genetic and computer aided engineering are being utilized in the study of zinc-Metalloendopeptidase E.C. 3.4.24.15 (EP 24.15), a peptidase involved in the degradation of Gonadotropin releasing hormone (GnRH), the master regulatory decapeptide involved in reproduction. This 71 kDa enzyme rapidly cleaves the Tyr-Gly6 bond in GnRH, the rate limiting reaction in hormone inactivation. EP 24.15 has been identified and isolated (1, 2) and the full length rat cDNA has recently been sequenced, and used to direct the expression of the functional 645 amino acid protein (3). The sequence of EP 24.15 shows no sequence identity with any known Metalloendopeptidases beyond the commonly shared active site motif, -H-E-x-x-H-, found in this family of enzymes. EP 24.15 is a predominantly cytosolic enzyme that is stable, not glycosylated, and can be modeled with other globular proteins. Hydrophobic Cluster Analysis, (HCA) (4), is a heuristic algorithm ascertaining structural domains by detecting patterns of secondary structure and sequence homology in a two-dimensional analysis. This method has been used to determine if thermolysin may be used in modeling EP 24.15, and extending this analysis to enkephalinase and angiotensin converting enzyme two other members of the zinc-Metalloendopeptidase family. Information gained from this study would be a step towards modeling substrate and inhibitor interactions to elucidate the conformation of this enzyme, as a probe for the function of this enzyme in vivo, and as a target for pharmacological intervention.

  • molecular cloning and primary structure of rat testes Metalloendopeptidase ec 3 4 24 15
    Biochemistry, 1990
    Co-Authors: Adrian R Pierotti, Marc J Glucksman, Marian Orlowski, Ke Wen Dong, James L. Roberts
    Abstract:

    : The complete amino acid sequence of rat testes Metalloendopeptidase (EC 3.4.24.15) was deduced from the nucleotide sequence of a cDNA clone isolated by screening a rat testes library with a polyclonal antibody raised against a homogeneous preparation of the rat testes enzyme. The correctness of the sequence was verified by N-terminal amino acid sequence analysis of the isolated enzyme and by partial amino acid sequence analysis of three tryptic peptides located near the N-terminus, the middle, and C-terminus of the native protein. The enzyme is composed of 645 amino acids with a molecular weight of 72,985. This value is close to that of the purified rat testes and brain enzyme as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. The enzyme contains the putative active-site sequence -H-E-F-G-H- that is homologous to the sequence in the active site of thermolysin and several other related bacterial enzymes, as well as to active-site sequences of several mammalian zinc metallopeptidases. No amino acid sequence homology, beyond this active site, was found with thermolysin, a bacterial zinc Metalloendopeptidase, nor with several mammalian zinc metallopeptidases. Northern blot hybridization analyses showed the presence of mRNA encoding the enzyme in rat testes, but not in other rat tissues in spite of the finding that enzyme activity is widely distributed in all tissues and that relatively high activities are present in rat brain and pituitary.

Judith S. Bond - One of the best experts on this subject based on the ideXlab platform.

  • activation mechanism of meprins members of the astacin Metalloendopeptidase family
    Journal of Biological Chemistry, 1997
    Co-Authors: Gary D Johnson, Judith S. Bond
    Abstract:

    : Meprins are mammalian zinc Metalloendopeptidases with protease domains structurally related to astacin, the prototype of the "astacin family" of metalloproteases. Mature, active astacins are produced by proteolytic removal of an activation peptide to generate a new NH2-terminal residue. Structural studies indicate that the NH2-terminal ammonium group inserts into a water-filled cavity adjacent to the active site to form a salt bridge with a Glu residue that is conserved in all astacins. A similar interaction is known to play a crucial role in the activation of trypsin, resulting in the hypothesis that this salt bridge is required for the activation of astacin-like proteases. In this study, we have used the mouse meprin alpha subunit as a model to test this hypothesis of zymogen activation of the astacins. Mutants were generated to vary the NH2-terminal residue of the mature meprin alpha subunit (Asn78) and its putative salt bridge partner (Glu178). In addition, mutants creating NH2-terminal extensions and truncations were expressed in human embryonic kidney 293 cells. The recombinant proteins were activated by limited protease digestion and assayed for enzymatic activity and thermal stability. Point mutations of Asn78 resulted in enzymes with activity comparable to the wild-type enzyme, indicating that the structure of this side chain is not essential for activity. NH2-terminal extension mutants of meprin alpha retained partial activity, with greater decreases against peptide relative to protein substrates. A mutant with a deletion of Asn78 to disrupt salt bridge formation with Glu178 had full activity, indicating that the putative salt bridge with Glu178 is not essential for enzyme activity. However, all changes in meprin alpha subunit NH2-terminal structure were found to decrease the thermal stability of the enzyme. These observations and additional data indicate that the zymogen activation mechanism of meprin and other astacins differs from that of the trypsin family of enzymes, and has some features in common with matrixins. It is proposed that prosequence removal of astacins allows the formation of hydrogen bonds involving the two NH2-terminal residues that are critical for enzyme structure.

  • Membrane Metalloendopeptidases in Immune Function and Disease
    Advances in Experimental Medicine and Biology, 1997
    Co-Authors: Judith S. Bond, Weiping Jiang
    Abstract:

    The enzymes that compose the ‘Metallopeptidases’ are a diverse group1. Forty-seven distinct evolutionary families of metallopeptidases have been identified in the last eight years; more than for any other protease classes, i.e., the serine/threonine, cysteine, or aspartic classes of proteases (see the Peptidase World Wide Web sites:http://www.qmw.ac.uk/~ugca000/iupac/enzyme/ and htpp://prolysis.phys.univ-tours.fr/Prolysis). In 1987, the primary amino acid sequence of very few metallopeptidases and of only one mammalian Metalloendopeptidase (human fibroblast collagenase) were known, and the 3-dimensional structures of very few metallopeptidases (thermolysin, carboxypeptidase A and B) were solved2. 3. Now hundreds of sequences of members of this Class are known, and many x-ray structures have been determined to high resolution (see, for example, reference 4). Thus our information about this class is expanding rapidly.

  • expression of subunits of the Metalloendopeptidase meprin in renal cortex in experimental hydronephrosis
    American Journal of Physiology-renal Physiology, 1996
    Co-Authors: Sharon D Ricardo, Judith S. Bond, Gary D Johnson, John Kaspar, Jonathan R Diamond
    Abstract:

    Meprin A is a Metalloendopeptidase in the proximal tubular epithelium of rodents that is capable of hydrolyzing a great variety of peptides and proteins. The aim of the present investigation was to...

  • The astacin family of Metalloendopeptidases
    Protein Science, 1995
    Co-Authors: Judith S. Bond, Robert J. Beynon
    Abstract:

    The astacin family of Metalloendopeptidases was recognized as a novel family of proteases in the 1990s. The crayfish enzyme astacin was the first characterized and is one of the smallest members of the family. More than 20 members of the family have now been identified. They have been detected in species ranging from hydra to humans, in mature and in developmental systems. Proposed functions of these proteases include activation of growth factors, degradation of polypeptides, and processing of extracellular proteins. Astacin family proteases are synthesized with NH2-terminal signal and proenzyme sequences, and many (such as meprins, BMP-1, tolloid) contain multiple domains COOH-terminal to the protease domain. They are either secreted from cells or are plasma membrane-associated enzymes. They have some distinguishing features in addition to the signature sequence in the protease domain: HEXXHXXGFXHEXXRXDR. They have a unique type of zinc binding, with pentacoordination, and a protease domain tertiary structure that contains common attributes with serralysins, matrix Metalloendopeptidases, and snake venom proteases; they cleave peptide bonds in polypeptides such as insulin B chain and bradykinin and in proteins such as casein and gelatin; and they have arylamidase activity. Meprins are unique proteases in the astacin family, and indeed in the animal kingdom, in their oligomeric structure; they are dimers of disulfide-linked dimers and are highly glycosylated, type I integral membrane proteins that have many attributes of receptors or integrins with adhesion, epidermal growth factor-like, and transmembrane domains. The alpha and beta subunits are differentially expressed and processed to yield latent and active proteases as well as membrane-associated and secreted forms. Meprins represent excellent models of hetero- and homo-oligomeric enzymes that are regulated at the transcriptional and posttranslational levels.

  • Families of Metalloendopeptidases and their relationships.
    FEBS Letters, 1992
    Co-Authors: Weiping Jiang, Judith S. Bond
    Abstract:

    Crystal structures available for four Metalloendopeptidases have revealed zinc ligands for these enzymes. New sequence information has made it possible to compare the primary structures of the zinc-binding site in Metalloendopeptidases. A scheme based on the zinc-binding site is proposed to classify Metalloendopeptidases into five distinct families: thermolysin, astacin, serratia, matrixin, and snake venom metalloproteinases. Two histidines and one glutamate are zinc-ligands in the thermolysin family. Three histidines and one tyrosine are zinc ligands in other four families, which are further distinguished by the identity of the residue following the third histidine and by the environment surrounding the tyrosine.