Purification and properties of an aminopeptidase from the mid-gut gland of scallop (Patinopecten yessoensis)

Umetsu, Hironori; Arai, Mito; Ota, Toshinori; Kudo, Ryohei; Sugiura, Hitomi; Ishiyama, Hiroyuki; Sasaki, Kazuo

doi:10.1016/j.cbpc.2003.09.008

Cited by 17 publications

(16 citation statements)

References 32 publications

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“…Routinely, aminopeptidase activity was assayed using Leu-MCA as substrate according to the method of Umetsu et al (2003) with some modifications. Ten to 50-folds diluted enzyme (100 ll) was added to 850 ll of 25 mM sodium phosphate buffer (pH 7.0) containing 1 mM DTT and 0.02% NaN 3 (buffer A).…”

Section: Methodsmentioning

confidence: 99%

Identification of an aminopeptidase from the skeletal muscle of grass carp (Ctenopharyngodon idellus)

Zhou

Liu

Sun

et al. 2009

Fish Physiol Biochem

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Aminopeptidases play important roles in turnover of proteins, metabolism of hormones and neurotransmission, cell maturation and immunological regulations. In the present study, an aminopeptidase was purified to homogeneity from the skeletal muscle of grass carp by ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite and Phenyl-Sepharose. The purified enzyme revealed a molecular mass of approximately 105 kDa both on SDS-PAGE and on gel filtration of Superdex 200. The enzymatic activity toward synthetic substrates was optimal at 40°C and pH 7.0-7.5. Metal-chelating agents such as EDTA and EGTA effectively inhibited the enzyme activity while inhibitors to serine, asparatic and cysteine proteinases did not show much effect, suggesting its belonging to metalloproteinase family. A specific aminopeptidase inhibitor bestatin was most effective in suppressing the enzymatic activity and performed in a competitive fashion. The enzymatic activity was slightly enhanced by metal ions of Mg2+ and Mn2+ while inhibited to different extents by Co2+, Cu2+, Zn2+ and Ca2+. Sulfhydryl reagent was necessary to maintain its activity. Purified enzyme demonstrated amidolytic activity most effectively against synthetic aminopeptidase substrate Leu-methylcoumarylamide (MCA) while N-terminal-blocked substrates and myofibrillar proteins were not hydrolyzed. The enzyme purified in the present study was quite possibly a leucine aminopeptidase (LAP) and functions during muscular protein metabolism.

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Section: Methodsmentioning

confidence: 99%

Identification of an aminopeptidase from the skeletal muscle of grass carp (Ctenopharyngodon idellus)

Zhou

Liu

Sun

et al. 2009

Fish Physiol Biochem

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“…16) The results of the action for various peptides and the kinetic study for pNA and MCA substrates showed that scallop aminopeptidase is specific for alanine in the amino-termini of substrates and resembles mammal cytosol alanyl aminopeptidases [4][5][6]8,9) with respect to substrate specificity. Our previous paper 15) demonstrated, however, that the enzyme differs from mammal cytosol alanyl aminopeptidase in molecular weight and sensitivity to puromycin, SH-blocking reagents, and divalent metal ions. Judging by these overall results, the enzyme from the mid-gut gland of the scallop may be classified into a new category different from cytosol alanyl aminopeptidase.…”

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confidence: 91%

“…In the MCA substrates, the values of K m and k cat varied dramatically in the range of 1.12-40.0 M and 0.049-15.1 sec À1 , respectively. The catalytic efficiencies, k cat =K m values for Ala-pNA and Ala-MCA 15) were 921 and 854 s À1 Á mM À1 , respectively. These values were highest in the pNA and MCA substrates.…”

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confidence: 97%

“…These enzymes from mammals are believed to participate in the metabolism of hormones and neurotransmitters. [12][13][14] In a previous paper, 15) we demonstrated that an aminopeptidase, isolated and purified from the mid-gut gland of the scallop (Patinopecten yessoensis), is an enzyme with a MW of 61,000 by SDS-polyacrylamide gel electrophoresis and an isoelectric point of 5.2. The enzyme preferred substrates having Ala or Met as an amino acid residue from the results of relative hydrolysis rates of amino acid-pNAs and MCAs.…”

mentioning

confidence: 99%

“…The aminopeptidase was purified from the mid-gut gland of the scallop and assayed using Ala-pNA as a substrate as in our previous experiment. 15) One katal was the amount of enzyme required to liberate 1 mol p-nitroaniline per second from Ala-pNA at pH 7.0 and 30 C. The fluorometric assay was done as in our previous expriment 15) by the use of amino acid-MCAs as substrates.…”

mentioning

confidence: 99%

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Substrate Specificity of Aminopeptidase from the Mid-gut Gland of the Scallop (Patinopecten yessoensis)

Umetsu

Arai

Ota

et al. 2004

Bioscience, Biotechnology, and Biochemistry

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Application of capillary electrophoresis coupling with electrochemiluminescence detection to estimate activity of leucine aminopeptidas

Ming

Wei

Zhou

et al. 2013

Biomedical Chromatography

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A new method to estimate the leucine aminopeptidase (LAP, EC 3.4.11.1) activity using capillary electrophoresis coupled with electrochemiluminescence (ECL) is described. The liberated proline produced by LAP catalyzing the hydrolysis reaction of leucin-proline was used as an ECL coreagent to enhance Ru(bpy)3 (2+) ECL signals efficiently. The detection limit for proline was 2.88 × 10(-6) m (signal-to-noise ratio 3), which was equal to 9.60 × 10(-8) units of LAP being used to catalyze leucin-proline for 1 min. The Michaelis constant Km (2.07 × 10(-2) mol/L) and the maximum reaction velocity Vmax (1.06 × 10(-5) mol/L/min) of LAP for leucin-proline are reported. The reaction conditions including the concentration of metal ions, incubation temperature and pH were optimized. This method was successfully applied to detect LAP activity in plasma and the results were in good agreement with that obtained by the clinical method.

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Purification and properties of an aminopeptidase from the mid-gut gland of scallop (Patinopecten yessoensis)

Cited by 17 publications

References 32 publications

Identification of an aminopeptidase from the skeletal muscle of grass carp (Ctenopharyngodon idellus)

Identification of an aminopeptidase from the skeletal muscle of grass carp (Ctenopharyngodon idellus)

Substrate Specificity of Aminopeptidase from the Mid-gut Gland of the Scallop (Patinopecten yessoensis)

Application of capillary electrophoresis coupling with electrochemiluminescence detection to estimate activity of leucine aminopeptidas

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