Degree Type

Dissertation

Date of Award

1983

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Molecular, Cellular, and Developmental Biology

Abstract

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.

DOI

https://doi.org/10.31274/rtd-180813-3246

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Stephen Gary Carter

Language

en

Proquest ID

AAI8407057

File Format

application/pdf

File Size

149 pages

Included in

Biochemistry Commons

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