Demonstration of the phosphorylated intermediates of the Ca 2+-transport ATPase in a microsomal fraction and in a [formula omitted] purified from smooth muscle by means of calmodulin affinity chromatography

Abstract

Ca 2+-dependent hydroxylamine-sensitive phosphorylated proteins can be demonstrated in a microsomal fraction of porcine antrum (stomach) smooth muscle and in a Ca 2+-transport ATPase (( Ca 2+ + Mg 2+)- ATPase) purified from this tissue by means of a calmodulin affinity technique. These phosphoproteins represent the phosphorylated intermediates of the ( Ca 2+ + Mg 2+)- ATPases . In the ( Ca 2+ + Mg 2+)- ATPase purified from smooth muscle the phosphorylated intermediate has an M r of 130 000 corresponding to the value found for erythrocyte ( Ca 2+ + Mg 2+)- ATPase . In the smooth muscle microsomal fraction this 130 kDa phosphoprotein can also be seen, although its intensity is usually very low compared to a corresponding phosphorylation at M r 100 000. Including La 3+ together with Ca 2+ during phosphorylation of the microsomes increased selectively the steady state-level of the 130 kDa phosphoprotein over the value of the 100 kDa one. The 100 kDa Ca 2+-dependent phophoprotein could either indicate the presence of a ( Ca 2+ + Mg 2+)- ATPase of the same type of sarcoplasmic reticulum of skeletal muscle, or it could represent a proteolytic product of the 130 kDa phosphoprotein.