Asparagine And Glutamine Metabolism In A Euryhaline Chlamydomonas Species
Date of Award
Doctor of Philosophy (Ph.D.)
A euryhaline Chlamydomonas species that could utilize NO(,3)('-), NH(,4)('+) or L-asparagine equally well as a nitrogen source for photoautotrophic growth was isolated from a subtropical beach. Whole cells converted ('14)C-(U)-L-asparagine to ('14)C-aspartate with little intracellular accumulation of radioactivity, apparently by the action of an L-asparaginase located at the cell surface. L-asparaginase activity was detected in whole cells and cell-free extracts but not in culture filtrates. Cells grown under nitrogen-limitation (0.1 mM NO(,3)('-), NH(,4), or L-asparagine) possessed four to six times the activity of cells grown at high concentrations (1.0-5.0 mM) of these three nitrogen sources. Regulation of L-asparaginase activity was apparently through the repression of synthesis only, and derepression in the absence of combined nitrogen was a photosynthetic, energy-dependent process. Cellular levels of biosynthetic glutamine synthetase were regulated in a similar fashion to L-asparaginase, although cells grown under nitrogen limitation only possessed 1.5-2.0 times the activity of cells grown at high concentrations of combined nitrogen. Glutamate dehydrogenase was apparently regulated by a different mechanism, greatest activity occurring when cells were grown at high concentrations of asparagine or ammonium but not nitrate. L-asparaginase was purified to near homogeneity (over 600 fold) and the partially-purified enzyme possessed no glutaminase activity. The reaction end products aspartate and ammonium did not inhibit enzyme activity. The enzyme possessed optimum activity at pH 7.8 and at a temperature of 46(DEGREES). The molecular weight of the enzyme was 275,000 as determined by gel filtration and the Km for L-asparagine was 1.1-1.3 x 10('-4) M. The partially-purified enzyme (78 IU mg('-1) protein) possessed little antitumor activity against the 6C3HED lymphosaroma in C3H mice.
Paul, John Heiks Iii., "Asparagine And Glutamine Metabolism In A Euryhaline Chlamydomonas Species" (1980). Dissertations from ProQuest. 1180.