Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Molecular, Cellular, and Developmental Biology
This dissertation is divided into three parts: (1) Purification and Characterization of a 5'-Nucleotidase from Corn Shoot Microsomes, (2) Characterization of a High Affinity (Ca('2+) + Mg('2+))-ATPase from Corn Shoot Plasma Membranes, and (3) Partial Purification and Characterization of a Ca('2+)-Stimulated ATPase Activity from Corn Shoot Plasma Membranes. In the first part, a 5'-nucleotidase was purified to apparent homogeneity. The native enzyme is a glycoprotein, M(,r)= 49,000. SDS/PAGE indicates the presence of two subunits, M(,r) = 24,500 and 25,500. Relative rates of hydrolysis for substrates are: AMP > GMP > IMP > CMP > UMP >> pNPP. Adenosine is a noncompetitive inhibitor, K(,i) UTP > GTP > CTP >> pNPP. The order of the ability of different divalent cations to stimulate the enzyme is Ca('2+) > Mg ('2+) > Co('2+) > Mn('2+) > Ni('2+) > Zn('2+). The enzyme has a K(,a) value for Ca('2+) of 0.2 (mu)M and shows a dependence on Mg('2+) for the high affinity Ca('2+)-ATPase activity to be functional. Part 3 describes the solubilization and partial purification and characterization of a Ca('2+)-ATPase from corn plasma membranes. The enzyme has an apparent molecular weight of 105,000 by gel filtration and a pH optimum at 6.5. The order of the ability of substrates tested to elicit Ca('2+) stimulation by the enzyme is ATP > UTP > CTP > GTP >> pNPP. The order of different divalent cations to stimulate the enzyme is Ca('2+) > Mg('2+) > Co('2+) > Mn('2+) > Ni('2+) > Zn('2+). The Ca('2+) affinity constant determination indicated the presence of two binding affinities by the enzyme, a high affinity component, K(,a) = 0.06 (mu)M and a low affinity component, K(,a) = 15 (mu)M. The addition of monovalent cations increased the velocity of the reaction while not affecting the affinity constant for Ca('2+) with the enzyme; however, monovalent cations were shown to be required for the low affinity component to be functional.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Stephen Gary Carter
Carter, Stephen Gary, "Characterization of a 5'-nucleotidase and a high affinity Ca2+-stimulated ATPase from microsomes from Zea mays coleoptiles " (1983). Retrospective Theses and Dissertations. 8458.