A procedure for the isolation of malic enzyme from muscle tissue of the roundworm Ascaris suum is described. The fractionation method yields relatively large quantities of the enzyme,with a specific activity of fifteen moles of malate converted to pyruvate and carbon dioxide per min per mg at 25ยบ. Homogeneity was established with analytical ultracentrifugation, zone electrophoresis, isoelectric focusing, and rechromatography. The molecular weight of the enzyme was 250,000, and it is dissociated under several conditions into four identical monomers of 64,000 daltons. The enzyme exists as a single electrophoretic form and prefers manganous and NAD over other cations and NADP. Ammonium sulfate competes with manganous for the active site and titration with DTNB yields eight thiol groups per mole. Titration of the first four thiol groups is accompanied by a complete loss in enzyme activity. Equilibrium dialysis, product inhibition, and initial velocity studies suggest a rapid-equilibrium random sequential mechanism for the Ascaris suum malic enzyme. The presence of 1.3 binding sites per subunits was determined for L-ma late. Antisera prepared against A. suum malic enzyme reacted to a small extent with the NAD malic enzymes from two free-living nematodes, Panarellus redivivus and Turbatrix aceti. A correlation coefficient of 0.911 was obtained upon comparing the amino acid composition of A. suum and E. coli malic enzymes. Some sequence homology is predicted between these malic enzymes. The physiological interpretation favors the binding of malate initially, with the subsequent addition of NAD to the enzyme.
| Identifer | oai:union.ndltd.org:unt.edu/info:ark/67531/metadc504259 |
| Date | 12 1900 |
| Creators | Fodge, Douglas W. |
| Contributors | Harris, Ben G., Gracy, Robert W., Booth, John A., Schlueter, Edgar A., Redden, David R. |
| Publisher | North Texas State University |
| Source Sets | University of North Texas |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Format | vi, 148 leaves: ill., Text |
| Rights | Public, Fodge, Douglas W., Copyright, Copyright is held by the author, unless otherwise noted. All rights reserved. |
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