Title page for ETD etd-02072013-040125


Type of Document Master's Thesis
Author Aceti, David John
URN etd-02072013-040125
Title Activation of acetate in acetate-grown Methanosarcina thermophila :purificationm and characterization of acetate kinase
Degree Master of Science
Department Anaerobic Microbiology
Advisory Committee
Advisor Name Title
Ferry, James G. Committee Chair
Dean, Dennis R. Committee Member
White, Robert H. Committee Member
Wilkins, Tracy D. Committee Member
Keywords
  • Methylotrophic microorganisms
Date of Defense 1980-04-05
Availability restricted
Abstract

Extracts of acetate-grown Methanosarcina thermoghila were assayed for the presence of enzymes which might catalyze a proposed activation of acetate as the initial step in the pathway of methanogenesis from acetate by that organism. Acetate kinase and phosphate acetyltransferase activities of 4.9 and 49 μmoles of product/min/mg protein, respectively, were detected. Acetate kinase was purified 102- fold to a specific activity of 656 μmoles ADP formed/min/mg protein and was essentially homogeneous by denaturing gel electrophoresis. The native enzyme (Mr 94,000) was an α2 homodimer with a subunit Mr of 53,000. Activity was optimal between pH 7.0 and 7.4 and was stable to heating at 70°C for 15 min. The apparent Km for acetate was 22 mM (Vmax = 668 μmoles ADP/min/mg protein) and 2.8 mM for ATP (Vmax = 777 pmoles ADP/min/ protein). The enzyme phosphorylated propionate at 602 of the rate with acetate but was unable to use formate. TTP, ITP, UTP, GTP, and CTP replaced ATP as the phosphoryl donor to acetate. One of several divalent cations was required for activity; the maximum rate was obtained with Mn2+.

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