published_or_final_version / Biochemistry / Master / Master of Philosophy

V-ATPases are enzymes found in all eukaryotic cells. They are organized into a peripheral membrane complex (V1) and an integral membrane complex (V0). VI is responsible for ATP hydrolysis and generates the energy used by Vo to pump protons from the cytosol into the vacuole. Subunit d is a component of Vo possibly located at the interface between V 1 and V. in the V-ATPase complex. We hypothesize that subunit d could be involved in the structural and functional coupling of VI and Vo. This was tested by generating point mutations along the open reading frame of subunit d from yeast. The mutations F94A, H128A, D173A, D217A, D261A, E317A, W325A, E328A and C329A, all in conserved regions of the protein sequence, were characterized by examining their growth phenotype and by assessing their ATPase specific activity, proton transport and V1Vo assembly in purified vacuolar membranes. The mutations E317A, W325A, E328A and C329A had reduced ATPase and proton transport activities. In addition, V1Vo assembly was compromised by the mutation W325A. Our results suggest that residues at the carboxyl-end of subunit d are important for ATPase activity, proton pumping and V1Vo assembly at the membrane. / Department of Chemistry

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1989. / Includes bibliographical references. / Microfiche. / xvii, 206 leaves, bound ill. 29 cm

Thesis (Ph. D.)--Worcester Polytechnic Institute. / Keywords: Zn; Heavy metal; ATPase. Includes bibliographical references (156-180 leaves ).

Thesis--University of Florida. / Description based on print version record Typescript. Vita. Includes bibliographical references (leaves 88-95).

Thesis (Ph. D.)--University of Chicago, Department of Chemistry, December 2002. / Includes bibliographical references. Also available on the Internet.

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

No description available.

The sarcoplasmic reticulum Ca²⁺ -pumping ATPase is the primary system responsible for the removal of calcium from the sarcoplasm during relaxation of skeletal and cardiac muscles. Since the rat heart SR is used frequently in our laboratory to study the Ca²⁺ -transport defects in disease states, the Ca²⁺ - ATPase activity of this system was characterized. Calmodulin (CaM) and cAMP-dependent protein kinase (cAMP-PK) are known to regulate the dog cardiac SR Ca²⁺ -pump. The effects of these regulators on the rat heart SR Ca²⁺ -pump were studied. Studies were also carried out to investigate the effects of Triton X-100 on SR Ca²⁺ -ATPase activity and the regulation of this activity by CaM. The rat heart SR Ca²⁺-ATPase was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Magnesium produced a concentration-dependent increase in the basal ATPase activity without affecting the maximal ATPase activity. This appeared to result in a gradual disappearance of the Ca²⁺ dependency of the ATPase activity. Addition of 100µM CDTA (trans-1,2-diaminocyclo- hexane-N,N,N',N'-tetraacetic acid), in the absence of added magnesium, produced no effect on Ca²⁺ stimulation of ATPase activity. The results appear to indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase. At a constant Mg: ATP ratio, ATP simulated the SR Ca²⁺-ATPase activity in a concentration-dependent manner. Double-reciprocal plots of the data suggest that the true substrate for rat heart SR Ca²⁺-ATPase may be ATP and not Mg.ATP. In the crude SR, CaM did not stimulate total or Ca²⁺-stimulated ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations. CaM also failed to stimulate membrane phosphorylation over a range of Mg²⁺ concentrations. Furthermore, CaM did not produce a significant effect on calcium transport into SR vesicles. The catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating membrane phosphorylation and Ca²⁺ -ATPase activity. Two CaM antagonists, trifluperazine and compound 48/80, did not affect the rat heart SR ATPase activity. The ATPase activity in Triton-washed SR membranes appeared to be increased at low Triton concentrations. This effect was probably due to the removal of non-intrinsic proteins, leaky vesicles or altered membrane fluidity. At higher Triton X-100 concentrations, the ATPase activity was lost, probably due to loss of the phospholipid environment. When SR membranes phosphorylated under conditions similar to those used for the ATPase assay were analysed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) followed by autoradiography, a single phosphorylated protein of 7,500-9,000 dalton was observed. This protein may represent the monomeric form of phospholamban. CaM, however, appeared to have no effect on the phosphorylation of this 7,500-9,000 dalton protein in either untreated or Tritan-washed SR membranes. It is speculated that the rat heart SR contains tightly bound CaM which cannot be removed by treatment with Triton X-100. / Pharmaceutical Sciences, Faculty of / Graduate

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