The Experts below are selected from a list of 135 Experts worldwide ranked by ideXlab platform

Katsuji Nishi - One of the best experts on this subject based on the ideXlab platform.

  • Puromycin-sensitive alanyl aminopeptidase from human Liver Cytosol: purification and characterization
    Forensic Science International, 2000
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Ikuko Ushiyama, Akiyoshi Nishimura, Iwao Ohkubo, Katsuji Nishi
    Abstract:

    Abstract A Cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS–PAGE in the presence of β-ME. These findings suggest that the enzyme exists as a monomeric form in human Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys–Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K cat / K m values of AAP-S at the optimal pH was Arg->Arg–Arg->Met->Leu->Lys->Phe->Lys–Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1,10-phenanthroline, DFP, PCMBS, Zn 2+ , Cd 2+ , Co 2+ , Cu 2+ , Hg 2+ and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat Liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human Liver Cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of Liver cells and renal tubules, and was ubiquitously localized in various human tissues.

  • Puromycin-sensitive alanyl aminopeptidase from human Liver Cytosol: purification and characterization.
    Forensic science international, 2000
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Ikuko Ushiyama, Akiyoshi Nishimura, Iwao Ohkubo, Katsuji Nishi
    Abstract:

    A Cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat Liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human Liver Cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of Liver cells and renal tubules, and was ubiquitously localized in various human tissues.

  • dipeptidyl peptidase iii from rat Liver Cytosol purification molecular cloning and immunohistochemical localization
    Biological Chemistry, 1999
    Co-Authors: Iwao Ohkubo, Yoshio Yamamoto, Yao Hua Li, Toshinaga Maeda, Takuya Yamane, Peige Du, Katsuji Nishi
    Abstract:

    : Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat Liver Cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS-PAGE in the absence or presence of beta-mercaptoethanol. These findings suggest that the enzyme exists in a monomeric form in rat Liver Cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Gly-Arg-MCA in the pH range of 7.5 to 9.5. The Km, k(cat) and k(cat)/Km values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 62.1 s(-1) x nM(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 36.2 s(-1) x nM(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS and NEM. These findings suggest that DPP III is an exo-type peptidase with characteristics of a metallo- and serine peptidase. For further information on the molecular structure, we screened a rat Liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG antibodies, determined the cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRDIII-11, is composed of 2640 bp and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. The DPP III antigen was detected in significant amounts in the Cytosol of various rat tissues by immunohistochemical examination.

  • isolation and characterization of an alanyl aminopeptidase from rat Liver Cytosol as a puromycin sensitive enkephalin degrading aminopeptidase
    Biological Chemistry, 1998
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Iwao Ohkubo, Kai Huang, Katsuji Nishi
    Abstract:

    : Alanyl aminopeptidase (AAP-S) was purified to homogeneity from rat Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 100,000 on Sephacryl S-200 HR and to be 90,000 on SDS-PAGE in the presence of beta-mercaptoethanol. These findings suggested that the enzyme exists as a monomeric form in rat Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Tyr- and Met-MCAs, and moderately hydrolyzed Arg-, Lys-, Leu-, Phe- and Lys-Ala-MCAs at pHs ranging from 7.5to 8.0. The enzyme also hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order for k(cat)/Km values of AAP-S at the optimal pH (pH 7.5) was Lys->Met->Arg->Ala->Leu->Phe->Tyr->Lys-Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, PCMBS, Zn2+, Cd2+, Co2+, Cu2+ and Hg2+, and puromycin. The amino acid sequence of the first 43 residues of the enzyme was determined as Pro1-Glu-Lys-Arg-Pro5-Phe-Glu-Arg-Leu-Pro10-Thr-Glu-Val-Ser-Pro 15-Ile-Asn-Tyr-Ser-Leu20-(Cys)-Leu-Lys-Pro-Asp25-Leu-Leu- Asp-Phe-Thr30-Phe-Glu-Gly-Lys-Leu35-Glu-Ala-Ala-Ala-Gln40 -Val-Arg-Gln-. This N-terminal amino acid sequence is almost identical with those of puromycin-sensitive enkephalin-degrading aminopeptidases in rat and human brains, and the mouse neuroblastoma cell line Neuro2A. These findings suggest that the AAP-S from rat Liver Cytosol is a puromycin-sensitive aminopeptidase. Furthermore, with immunohistochemistry the enzyme was strongly stained in the Cytosol of the rat Liver cells.

Yoshio Yamamoto - One of the best experts on this subject based on the ideXlab platform.

  • Puromycin-sensitive alanyl aminopeptidase from human Liver Cytosol: purification and characterization
    Forensic Science International, 2000
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Ikuko Ushiyama, Akiyoshi Nishimura, Iwao Ohkubo, Katsuji Nishi
    Abstract:

    Abstract A Cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS–PAGE in the presence of β-ME. These findings suggest that the enzyme exists as a monomeric form in human Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys–Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K cat / K m values of AAP-S at the optimal pH was Arg->Arg–Arg->Met->Leu->Lys->Phe->Lys–Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1,10-phenanthroline, DFP, PCMBS, Zn 2+ , Cd 2+ , Co 2+ , Cu 2+ , Hg 2+ and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat Liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human Liver Cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of Liver cells and renal tubules, and was ubiquitously localized in various human tissues.

  • Puromycin-sensitive alanyl aminopeptidase from human Liver Cytosol: purification and characterization.
    Forensic science international, 2000
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Ikuko Ushiyama, Akiyoshi Nishimura, Iwao Ohkubo, Katsuji Nishi
    Abstract:

    A Cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat Liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human Liver Cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of Liver cells and renal tubules, and was ubiquitously localized in various human tissues.

  • dipeptidyl peptidase iii from rat Liver Cytosol purification molecular cloning and immunohistochemical localization
    Biological Chemistry, 1999
    Co-Authors: Iwao Ohkubo, Yoshio Yamamoto, Yao Hua Li, Toshinaga Maeda, Takuya Yamane, Peige Du, Katsuji Nishi
    Abstract:

    : Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat Liver Cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS-PAGE in the absence or presence of beta-mercaptoethanol. These findings suggest that the enzyme exists in a monomeric form in rat Liver Cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Gly-Arg-MCA in the pH range of 7.5 to 9.5. The Km, k(cat) and k(cat)/Km values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 62.1 s(-1) x nM(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 36.2 s(-1) x nM(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS and NEM. These findings suggest that DPP III is an exo-type peptidase with characteristics of a metallo- and serine peptidase. For further information on the molecular structure, we screened a rat Liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG antibodies, determined the cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRDIII-11, is composed of 2640 bp and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. The DPP III antigen was detected in significant amounts in the Cytosol of various rat tissues by immunohistochemical examination.

  • isolation and characterization of an alanyl aminopeptidase from rat Liver Cytosol as a puromycin sensitive enkephalin degrading aminopeptidase
    Biological Chemistry, 1998
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Iwao Ohkubo, Kai Huang, Katsuji Nishi
    Abstract:

    : Alanyl aminopeptidase (AAP-S) was purified to homogeneity from rat Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 100,000 on Sephacryl S-200 HR and to be 90,000 on SDS-PAGE in the presence of beta-mercaptoethanol. These findings suggested that the enzyme exists as a monomeric form in rat Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Tyr- and Met-MCAs, and moderately hydrolyzed Arg-, Lys-, Leu-, Phe- and Lys-Ala-MCAs at pHs ranging from 7.5to 8.0. The enzyme also hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order for k(cat)/Km values of AAP-S at the optimal pH (pH 7.5) was Lys->Met->Arg->Ala->Leu->Phe->Tyr->Lys-Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, PCMBS, Zn2+, Cd2+, Co2+, Cu2+ and Hg2+, and puromycin. The amino acid sequence of the first 43 residues of the enzyme was determined as Pro1-Glu-Lys-Arg-Pro5-Phe-Glu-Arg-Leu-Pro10-Thr-Glu-Val-Ser-Pro 15-Ile-Asn-Tyr-Ser-Leu20-(Cys)-Leu-Lys-Pro-Asp25-Leu-Leu- Asp-Phe-Thr30-Phe-Glu-Gly-Lys-Leu35-Glu-Ala-Ala-Ala-Gln40 -Val-Arg-Gln-. This N-terminal amino acid sequence is almost identical with those of puromycin-sensitive enkephalin-degrading aminopeptidases in rat and human brains, and the mouse neuroblastoma cell line Neuro2A. These findings suggest that the AAP-S from rat Liver Cytosol is a puromycin-sensitive aminopeptidase. Furthermore, with immunohistochemistry the enzyme was strongly stained in the Cytosol of the rat Liver cells.

Iwao Ohkubo - One of the best experts on this subject based on the ideXlab platform.

  • Puromycin-sensitive alanyl aminopeptidase from human Liver Cytosol: purification and characterization
    Forensic Science International, 2000
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Ikuko Ushiyama, Akiyoshi Nishimura, Iwao Ohkubo, Katsuji Nishi
    Abstract:

    Abstract A Cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS–PAGE in the presence of β-ME. These findings suggest that the enzyme exists as a monomeric form in human Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys–Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K cat / K m values of AAP-S at the optimal pH was Arg->Arg–Arg->Met->Leu->Lys->Phe->Lys–Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1,10-phenanthroline, DFP, PCMBS, Zn 2+ , Cd 2+ , Co 2+ , Cu 2+ , Hg 2+ and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat Liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human Liver Cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of Liver cells and renal tubules, and was ubiquitously localized in various human tissues.

  • Puromycin-sensitive alanyl aminopeptidase from human Liver Cytosol: purification and characterization.
    Forensic science international, 2000
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Ikuko Ushiyama, Akiyoshi Nishimura, Iwao Ohkubo, Katsuji Nishi
    Abstract:

    A Cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat Liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human Liver Cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of Liver cells and renal tubules, and was ubiquitously localized in various human tissues.

  • dipeptidyl peptidase iii from rat Liver Cytosol purification molecular cloning and immunohistochemical localization
    Biological Chemistry, 1999
    Co-Authors: Iwao Ohkubo, Yoshio Yamamoto, Yao Hua Li, Toshinaga Maeda, Takuya Yamane, Peige Du, Katsuji Nishi
    Abstract:

    : Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat Liver Cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS-PAGE in the absence or presence of beta-mercaptoethanol. These findings suggest that the enzyme exists in a monomeric form in rat Liver Cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Gly-Arg-MCA in the pH range of 7.5 to 9.5. The Km, k(cat) and k(cat)/Km values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 62.1 s(-1) x nM(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 36.2 s(-1) x nM(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS and NEM. These findings suggest that DPP III is an exo-type peptidase with characteristics of a metallo- and serine peptidase. For further information on the molecular structure, we screened a rat Liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG antibodies, determined the cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRDIII-11, is composed of 2640 bp and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. The DPP III antigen was detected in significant amounts in the Cytosol of various rat tissues by immunohistochemical examination.

  • isolation and characterization of an alanyl aminopeptidase from rat Liver Cytosol as a puromycin sensitive enkephalin degrading aminopeptidase
    Biological Chemistry, 1998
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Iwao Ohkubo, Kai Huang, Katsuji Nishi
    Abstract:

    : Alanyl aminopeptidase (AAP-S) was purified to homogeneity from rat Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 100,000 on Sephacryl S-200 HR and to be 90,000 on SDS-PAGE in the presence of beta-mercaptoethanol. These findings suggested that the enzyme exists as a monomeric form in rat Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Tyr- and Met-MCAs, and moderately hydrolyzed Arg-, Lys-, Leu-, Phe- and Lys-Ala-MCAs at pHs ranging from 7.5to 8.0. The enzyme also hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order for k(cat)/Km values of AAP-S at the optimal pH (pH 7.5) was Lys->Met->Arg->Ala->Leu->Phe->Tyr->Lys-Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, PCMBS, Zn2+, Cd2+, Co2+, Cu2+ and Hg2+, and puromycin. The amino acid sequence of the first 43 residues of the enzyme was determined as Pro1-Glu-Lys-Arg-Pro5-Phe-Glu-Arg-Leu-Pro10-Thr-Glu-Val-Ser-Pro 15-Ile-Asn-Tyr-Ser-Leu20-(Cys)-Leu-Lys-Pro-Asp25-Leu-Leu- Asp-Phe-Thr30-Phe-Glu-Gly-Lys-Leu35-Glu-Ala-Ala-Ala-Gln40 -Val-Arg-Gln-. This N-terminal amino acid sequence is almost identical with those of puromycin-sensitive enkephalin-degrading aminopeptidases in rat and human brains, and the mouse neuroblastoma cell line Neuro2A. These findings suggest that the AAP-S from rat Liver Cytosol is a puromycin-sensitive aminopeptidase. Furthermore, with immunohistochemistry the enzyme was strongly stained in the Cytosol of the rat Liver cells.

Yao Hua Li - One of the best experts on this subject based on the ideXlab platform.

  • Puromycin-sensitive alanyl aminopeptidase from human Liver Cytosol: purification and characterization
    Forensic Science International, 2000
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Ikuko Ushiyama, Akiyoshi Nishimura, Iwao Ohkubo, Katsuji Nishi
    Abstract:

    Abstract A Cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS–PAGE in the presence of β-ME. These findings suggest that the enzyme exists as a monomeric form in human Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys–Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K cat / K m values of AAP-S at the optimal pH was Arg->Arg–Arg->Met->Leu->Lys->Phe->Lys–Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1,10-phenanthroline, DFP, PCMBS, Zn 2+ , Cd 2+ , Co 2+ , Cu 2+ , Hg 2+ and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat Liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human Liver Cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of Liver cells and renal tubules, and was ubiquitously localized in various human tissues.

  • Puromycin-sensitive alanyl aminopeptidase from human Liver Cytosol: purification and characterization.
    Forensic science international, 2000
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Ikuko Ushiyama, Akiyoshi Nishimura, Iwao Ohkubo, Katsuji Nishi
    Abstract:

    A Cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat Liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human Liver Cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of Liver cells and renal tubules, and was ubiquitously localized in various human tissues.

  • dipeptidyl peptidase iii from rat Liver Cytosol purification molecular cloning and immunohistochemical localization
    Biological Chemistry, 1999
    Co-Authors: Iwao Ohkubo, Yoshio Yamamoto, Yao Hua Li, Toshinaga Maeda, Takuya Yamane, Peige Du, Katsuji Nishi
    Abstract:

    : Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat Liver Cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS-PAGE in the absence or presence of beta-mercaptoethanol. These findings suggest that the enzyme exists in a monomeric form in rat Liver Cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Gly-Arg-MCA in the pH range of 7.5 to 9.5. The Km, k(cat) and k(cat)/Km values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 62.1 s(-1) x nM(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 36.2 s(-1) x nM(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS and NEM. These findings suggest that DPP III is an exo-type peptidase with characteristics of a metallo- and serine peptidase. For further information on the molecular structure, we screened a rat Liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG antibodies, determined the cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRDIII-11, is composed of 2640 bp and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. The DPP III antigen was detected in significant amounts in the Cytosol of various rat tissues by immunohistochemical examination.

  • isolation and characterization of an alanyl aminopeptidase from rat Liver Cytosol as a puromycin sensitive enkephalin degrading aminopeptidase
    Biological Chemistry, 1998
    Co-Authors: Yoshio Yamamoto, Yao Hua Li, Iwao Ohkubo, Kai Huang, Katsuji Nishi
    Abstract:

    : Alanyl aminopeptidase (AAP-S) was purified to homogeneity from rat Liver Cytosol. The molecular weight of the purified enzyme was calculated to be approximately 100,000 on Sephacryl S-200 HR and to be 90,000 on SDS-PAGE in the presence of beta-mercaptoethanol. These findings suggested that the enzyme exists as a monomeric form in rat Liver Cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Tyr- and Met-MCAs, and moderately hydrolyzed Arg-, Lys-, Leu-, Phe- and Lys-Ala-MCAs at pHs ranging from 7.5to 8.0. The enzyme also hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order for k(cat)/Km values of AAP-S at the optimal pH (pH 7.5) was Lys->Met->Arg->Ala->Leu->Phe->Tyr->Lys-Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, PCMBS, Zn2+, Cd2+, Co2+, Cu2+ and Hg2+, and puromycin. The amino acid sequence of the first 43 residues of the enzyme was determined as Pro1-Glu-Lys-Arg-Pro5-Phe-Glu-Arg-Leu-Pro10-Thr-Glu-Val-Ser-Pro 15-Ile-Asn-Tyr-Ser-Leu20-(Cys)-Leu-Lys-Pro-Asp25-Leu-Leu- Asp-Phe-Thr30-Phe-Glu-Gly-Lys-Leu35-Glu-Ala-Ala-Ala-Gln40 -Val-Arg-Gln-. This N-terminal amino acid sequence is almost identical with those of puromycin-sensitive enkephalin-degrading aminopeptidases in rat and human brains, and the mouse neuroblastoma cell line Neuro2A. These findings suggest that the AAP-S from rat Liver Cytosol is a puromycin-sensitive aminopeptidase. Furthermore, with immunohistochemistry the enzyme was strongly stained in the Cytosol of the rat Liver cells.

Masayoshi Yamaguchi - One of the best experts on this subject based on the ideXlab platform.

  • regucalcin increases superoxide dismutase activity in rat Liver Cytosol
    Biological & Pharmaceutical Bulletin, 2004
    Co-Authors: Yuko Fukaya, Masayoshi Yamaguchi
    Abstract:

    The effect of regucalcin, a regulatory protein in the intracellular signaling process, on superoxide dismutase (SOD) activity in the Cytosol of rat Liver was investigated. The presence of zinc sulfate (10−6 or 10−5 m) or cupric sulfate (10−6 m) in the enzyme reaction mixture caused a significant increase in SOD activity, indicating that Cu/Zn-SOD may be present in the Liver Cytosol. SOD activity was significantly increased by the addition of regucalcin (0.1, 0.25, or 0.5 μM) to the reaction mixture. The presence of dithiothreitol (DTT; 0.1, 0.5, or 1.0 mM), a protective reagent for the sulfhydryl group, caused a significant decrease in SOD activity. The effect of regucalcin (0.25 μM) in increasing SOD activity was not seen in the presence of DTT (1.0 mM). Meanwhile, SOD activity was significantly raised by the addition of N-ethylmaleimide (NEM; 0.5 or 1.0 mM), a modifying reagent for the sulfhydryl reagent. Regucalcin (0.25 μM) caused a significant increase in SOD activity in the presence of NEM (1.0 mM). The effect of regucalcin in increasing SOD activity may not involve the sulfhydryl group of SOD. This study demonstrates that regucalcin has an activatory effect on SOD in the Liver Cytosol of rats.

  • Suppressive role of endogenous regucalcin in the enhancement of nitric oxide synthase activity in Liver Cytosol of normal and regucalcin transgenic rats.
    Journal of Cellular Biochemistry, 2003
    Co-Authors: Masayoshi Yamaguchi, Hiroko Takahashi, Yoshinori Tsurusaki
    Abstract:

    The suppressive role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the enhancement of nitric oxide (NO) synthase activity in the Liver Cytosol of rats was investigated. The enzyme activity was measured in a reaction mixture containing either vehicle or calcium chloride (1–20 μM) in the absence or presence of regucalcin (0.1, 0.25, or 0.5 μM). NO synthase activity was significantly increased by the addition of calcium (5–20 μM). This increase was completely abolished in the presence of trifluoperazine (TFP; 10–50 μM), an antagonist of Ca2+/calmodulin. The addition of regucalcin (0.1–0.5 μM) caused a significant fall in the calcium-increased enzyme activity. The effect of regucalcin (0.25 μM) in decreasing NO synthase activity was seen in the presence of ethylene glycol bis-(2-aminoethylether) N,N,N′,N′-tetraacetic acid (EGTA, 1 mM) or TFP (20 μM), indicating that regucalcin acts independent on Ca2+/calmodulin. NO synthase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10–50 ng/ml) in the reaction mixture. The effect of the antibody (50 ng/ml) or calcium (10 μM) in elevating NO synthase activity in the Liver Cytosol of normal rats was not seen in the Liver Cytosol obtained from regucalcin transgenic rats. Moreover, the increase in NO synthase activity in the Liver Cytosol of normal rats induced by a single intraperitoneal administration of calcium (5.0 mg/100 g body weight) was significantly enhanced in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. The administration of calcium caused a significant increase in regucalcin level in the Liver Cytosol of normal rats. The present study demonstrated that endogenous regucalcin plays a suppressive role in the enhancement of NO synthase activity in the Liver Cytosol of rats. J. Cell. Biochem. 88: 1226–1234, 2003. © 2003 Wiley-Liss, Inc.

  • Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat Liver Cytosol.
    Molecular and Cellular Biochemistry, 1992
    Co-Authors: Masayoshi Yamaguchi
    Abstract:

    The effect of regucalcin, isolated from rat Liver Cytosol, on neutral proteolytic activity in the hepatic Cytosol was investigated. The Ca2+-requiring proteinase required 5–10 µM Ca2+ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25–2.0 µM) in the absence or presence of Ca2+ (5.0 µM) added. The effect of regucalcin, however, was the greater in the absence of Ca2+ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca2+ (5.0 µM). In the absence of Ca2+, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 µg/ml), and heavy metals (25 µM cadmium or 25 µM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat Liver Cytosol, and that regucalcin may activate Ca2+-independent neutral cysteinyl-proteinase.

  • hepatic calcium binding protein regucalcin decreases ca2 calmodulin dependent protein kinase activity in rat Liver Cytosol
    Chemical & Pharmaceutical Bulletin, 1990
    Co-Authors: Seiichi Mori, Masayoshi Yamaguchi
    Abstract:

    The effect of regucalcin, a calcium-binding protein isolated from rat Liver Cytosol, on Cytosolic Ca2+/calmodulin-dependent protein kinase activity was investigated. The increase in Cytosolic Ca2+/calmodulin-dependent protein kinase activity with passage of incubation time was clearly prevented by the presence of regucalcin (1.0 μM). An appreciable effect of regucalcin was seen at 0.5 μM. The Cytosolic Ca2+/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca2+ (0.25-1.0 mM). This increase was clearly blocked by the presence of regucalcin (1.0 μM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2.5-15 μg). In the presence of regucalcin (1.0 μM), trifluoperazin (50 μM), an antagonist of calmodulin, significantly decreased the Cytosolic Ca2+/calmodulin-dependent protein kinase activity. These results suggest that regucalcin can regulate the Ca2+-calmodulin effect in Liver Cytosol.