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Epitopes of the Human Erythrocyte Ca²⁺-Mg²⁺ ATPase Pump in Human Osteoblast-Like Cell Plasma Membranes^*

JAMES L. BORKE, ERIK F. ERIKSEN, JUNZABURO MINAMI, PHILIP KEETING, KENNETH G. MANN, JOHN T. PENNISTON, B. LAWRENCE RIGGS and RAJIV KUMAR

Department of Medicine, Divisions of Nephrology and Endocrinology (J.L.B., E.F.E., P.K., B.L.R., R.K.), and the Department of Biochemistry and Molecular Biology (J.M., J.T.P., R.K.), Mayo Clinic and Foundation Rochester, Minnesota 55905
The Department of Biochemistry, University of Vermont (K.G.M.) Burlington, Vermont 05405

Address requests for reprints to: R. Kumar, Division of Nephrology, Mayo Clinic, Rochester, Minnesota 55905.

Human osteoblast-like cells were examined for the presence of the Ca²⁺-Mg²⁺ ATPase pump. The osteoblastlike cells had characteristic features of the osteoblast phenotype, including the presence of osteonectin, bone GLA protein, and type I collagen. The cells were able to mineralize matrix, their production of cAMP increased in response to PTH, and their alkaline phosphatase activity increased in response to 1,25-dihydroxyvitamin D₃. Immunocytochemical staining of the osteoblast-like cells with a monoclonal antibody against human red cell Ca²⁺-Mg²⁺ ATPase demonstrated the presence of an epitope of the Ca²⁺-Mg²⁺ ATPase in these cells; staining of paraffin-embedded osteoblast-like cell sections demonstrated anti-Ca²⁺-Mg²⁺ ATPase staining only in cell plasma membranes. Western blot analysis of osteoblast-like cell homogenates showed that the monoclonal antibody to human erythrocyte Ca²⁺-Mg²⁺ ATPase bound to a major band at 140,000 mol wt, similar to the mol wt of known plasma membrane Ca²⁺-Mg²⁺ ATPases. The presence in the osteoblast-like cells of a Ca²⁺-Mg²⁺ ATPase similar to the human red cell calcium pump suggests that this enzyme may play a role in osteoblast intracellular calcium homeostasis.

^* This work supported by NIH Grants HD-9140, GM-28835, DK-25409, and AG-04875 and the Mellon Foundation.

Supported by NIH Training Grant AM-07013.

Received April 15, 1988.