The plasma membrane system in cancer cells is a site of many well-documented biological and biochemical changes. These include cell-cell interaction, response to growth factors, and enhanced transport of ions and nutrients, which may be driven by ATP. Relatively high Mg?2+?-ATPase activity that was insensitive to ouabain was observed in the purified plasma membranes of several human tumor xenografts in the athymic mice. One of our research objectives is to determine if the ATPase is implicated in the transport processes across the cell membrane. We have since discovered that the Mg?2+?-ATPase activity, i.e., Mg?2+?-dependent ATP hydrolysis, is contributed by several enzymes. Four ATP hydrolyzing activities can be demonstrated in the plasma membranes of human oat cell carcinoma. These are a low affinity Ca?2+?-ATPase; a Mg?2+?-ATPase; a monovalent ion-stimulated, Mg?2+?-dependent ATPase; and an ATP diphosphohydrolase. The low affinity Ca?2+?-ATPase, a 30 kilodalton protein, has been partially purified from oat cell carcinoma, which is especially abundant in this enzyme. The functions of these ATPases are not yet known, although it is certain that physiological functions are in part dictated by the sidedness of the ATPases. Both ecto-Mg?2+?-ATPase and ectoCa?2+?-ATPase are detected in cultured human tumor cells. In one hepatoma cell line, which appears to be an autocrine system, we found EGF, cholera toxin, and hydrocortisone induced the ecto-ATPase 5-to-10-fold. Our future research plan includes purification of these ATPases, their characterization and preparation of antibodies, and determination of their membrane orientation. Experimental evidence for the implication of these ATPases in transport will be provided by reconstitution techniques. (A)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA027117-05
Application #
3167534
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1981-03-01
Project End
1987-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
5
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Shi, X J; Knowles, A F (1994) Prevalence of the mercurial-sensitive EctoATPase in human small cell lung carcinoma: characterization and partial purification. Arch Biochem Biophys 315:177-84
Murray, S L; Knowles, A F (1990) Butyrate induces an ectoMg2(+)-ATPase activity in Li-7A human hepatoma cells. J Cell Physiol 144:26-35
Knowles, A F (1990) Synergistic modulation of ectoCa2(+)-ATPase activity of hepatoma (Li-7A) cells by epidermal growth factor and cyclic AMP. Arch Biochem Biophys 283:114-9
Knowles, A F (1988) Inhibition of growth and induction of enzyme activities in a clonal human hepatoma cell line (Li-7A): comparison of the effects of epidermal growth factor and an anti-epidermal growth factor receptor antibody. J Cell Physiol 134:109-16
Knowles, A F (1988) Differential expression of ectoMg2+-ATPase and ectoCa2+-ATPase activities in human hepatoma cells. Arch Biochem Biophys 263:264-71
Imes, S S; Kaplan, N O; Knowles, A F (1987) Plasma membrane-associated phosphatase activities hydrolyzing [32P]phosphotyrosyl histones and [32P]phosphatidylinositol phosphate. Anal Biochem 161:316-22
Knowles, A F (1986) Endogenous phosphorylation of proteins and phosphatidylinositol in the plasma membranes of a human astrocytoma. Arch Biochem Biophys 249:76-87
Knowles, A F; Lawrence, C M (1985) Enzymatic synthesis and hydrolysis of [32P]phosphatidylinositol phosphate. Biochem Biophys Res Commun 129:220-5
Knowles, A F; Salas-Prato, M; Villela, J (1985) Epidermal growth factor inhibits growth while increasing the expression of an ecto-Ca2+-ATPase of a human hepatoma cell line. Biochem Biophys Res Commun 126:8-14