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Related Experiment Videos

Basic arginine esterase from human seminal plasma: purification and some properties.

T Kobayashi1, J Y Park, Y Matsuda

  • 1First Department of Biochemistry, Meiji College of Pharmacy, Tokyo, Japan.

Archives of Andrology
|November 1, 1991
PubMed
Summary

A novel human seminal plasma amidase was purified and characterized. This enzyme exhibits distinct properties differentiating it from other known seminal proteases.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protease research

Background:

  • Human seminal plasma contains various enzymes, including proteases, that play roles in reproduction.
  • Characterization of these enzymes is crucial for understanding their functions and potential clinical applications.
  • Previous studies have identified several proteases in semen, but a comprehensive understanding of all amidolytic activities remains incomplete.

Purpose of the Study:

  • To purify and characterize a basic arginine esterase (amidase) from human seminal plasma.
  • To investigate the substrate specificity and inhibition profile of the purified enzyme.
  • To differentiate the enzyme's characteristics from other known human seminal proteases like tissue kallikrein, acrosin, and seminin.

Main Methods:

  • Purification of the enzyme from human seminal plasma using CM-cellulose chromatography.

Related Experiment Videos

  • Determination of specific activity using N-alpha-tosyl-L-arginine methyl ester (Tos-Arg-Me) as a substrate.
  • Analysis of molecular weight via SDS-PAGE.
  • Evaluation of inhibition by various protease inhibitors (aprotinin, SBTI, leupeptin, antipain, alpha 1-antitrypsin, OTI) and EDTA.
  • Testing hydrolysis of synthetic basic amino acid derivatives and specific p-nitroanilide substrates (Tos-Gly-Pro-Arg-pNA, Boc-Leu-Pro-Arg-pNA).

    • Main Results:

    • A basic arginine esterase was purified approximately 230-fold, exhibiting a specific activity of 3.2 mumol Tos-Arg-Me esterolysis per A280.
    • The purified enzyme appeared as a single band on SDS-PAGE with an estimated molecular weight of 3.4-4.1 x 10(4).
    • Enzyme activity was significantly suppressed by aprotinin, soybean trypsin inhibitor (SBTI), leupeptin, and antipain, but weakly affected by alpha 1-antitrypsin, ovomucoid trypsin inhibitor (OTI), EDTA, and chymostatin.
    • The enzyme efficiently hydrolyzed synthetic basic amino acid derivatives, with Tos-Gly-Pro-Arg-pNA and Boc-Leu-Pro-Arg-pNA identified as optimal substrates.
    • The enzymatic properties clearly distinguished this enzyme from tissue kallikrein, acrosin, and seminin.

    Conclusions:

    • A novel amidase with characteristics distinct from known human seminal proteases has been isolated and purified.
    • The enzyme's inhibition profile suggests a serine protease mechanism, with a preference for basic amino acid residues.
    • This purified amidase represents a unique enzymatic entity within human seminal plasma, warranting further investigation into its physiological role.