Identification and isolation of the phosphorylated intermediate of the calcium pump in rat intestinal basolateral membranes.
Transport of Ca2+ by the ATP-dependent Ca2+ pump has been demonstrated previously in rat intestinal basolateral-membrane vesicles. To identify the Ca2+-pump protein, duodenal basolateral membranes were phosphorylated with [gamma-32P]ATP in the presence of Ca2+ and La3+, under conditions conducive for maximal formation of the phosphorylated intermediate of the Ca2+ pump. Four major phosphoprotein bands were seen on autoradiograms of acidic SDS/polyacrylamide gels; the properties of a phosphoprotein (pp) at 130 kDa (pp130) were consistent with those expected for the plasma-membrane Ca2+ pump. This phosphoprotein was markedly enhanced by La3+, exhibited the characteristics of an acyl-phosphate bond, was preferentially phosphorylated from ATP and inhibited by micromolar concentrations of vanadate. Another phosphoprotein of 115 kDa possibly represented the endoplasmic reticulum Ca2+ pump or a fragment of pp130. Other phosphoproteins of 75 and 95 kDa were predominantly expressions of alkaline phosphatase. Formation of pp130 was highest in duodenal basolateral-membrane preparations when compared with those of jejunum and ileum or other subcellular fractions. A similar correlation between Ca2+-pump activity and pp130 formation was not found in membranes from villus-tip and crypt cells or in vitamin D-deficient animals. pp130 was isolated as a single phosphoprotein by calmodulin-affinity chromatography. We conclude that pp130 represents the phosphorylated intermediate of the rat intestinal basolateral-membrane Ca2+ pump, which can be separated from other phosphoproteins using its properties as a calmodulin-binding protein.
Wajsman
R
R
Division of Gastroenterology and Nutrition, State University of New York, Buffalo General Hospital 14203.
Walters
J R
JR
Weiser
M M
MM
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity
D008565:Membrane Proteins; D010750:Phosphoproteins; D002118:Calcium