A neutral proteinase of monkey liver microsomes solubilization, partial purification, and properties

Kazuhiro Sogawa, Kenji Takahashi

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    8 Citations (Scopus)

    Abstract

    Conditions for the solubilization of membrane-bound neutral proteinase associated with monkey liver microsomes were investigated. Among the reagents tested, deoxycholate, cholate, and some nonionic detergents, including Triton X-100, with hydrophilic-lipophilic balance values of around 13, were effective. The solubilization profile indicated that the enzyme is bound to the microsomal membranes by strong hydrophobic interaction.The enzyme was partially purified from monkey liver microsomal fraction, previously washed with 1 m KC1 and 0.05% sodium dodecyl sulfate, by Triton X-100 extraction, followed by chromatography on columns of hydroxylapatite and Sepharose CL-6B. The apparent molecular weight of the enzyme was estimated to be about 88,000 from the elution position on Sepharose CL-6B column chromatography in the presence of 0.5% sodium cholate. It was optimally active at pH 8.0 with heat-denatured casein as a substrate. It was strongly inhibited by diisopropyl phosphorofluoridate and phenylmethanesulfonyl fluoride, indicating that the enzyme is a serine proteinase. EDTA, EGTA, and chymostatin also inhibited the enzyme strongly. Among urea-denatured protein substrates tested, calf thymus histone was hydrolyzed most rapidly, followed by casein, hemoglobin, and bovine serum albumin, whereas practically no hydrolysis occurred with denatured ovalbumin, fibrinogen, and α-globulin as substrates.

    Original languageEnglish
    Pages (from-to)1313-1322
    Number of pages10
    JournalJournal of biochemistry
    Volume86
    Issue number5
    DOIs
    Publication statusPublished - 1979 Nov

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology

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