Nucleotide-induced alteration of rat liver microsome calcium pump activity

Abstract

Rat liver microsomes sequester calcium by an energy dependent process that may be a nonmuscle cell analog of the sarcoplasmic reticulum Ca 2+ pump of skeletal muscle (Moore, L., Chen, T.S., Knapp, H.R., Jr. and Landon, E.J. (1975) J. Biol. Chem. 250, 4562–4568). Homogenization of rat liver in the presence of ATP (5 mM) results in a 2-fold increase of the specific activity of the microsome Ca 2+ pump. The effect of ATP is concentration dependent and is detected at ATP levels as low as 0.1 mM. ATP will produce this effect if added before homogenization, after homogenization or after any of the centrifugation steps of microsome isolation. Homogenization of rat liver in the presence of ADP and AMP also increases specific activity of the microsome Ca 2+ pump, but to a lesser extent than ATP. Other nucleoside triphosphates have been tested and are in general less effective than ATP in increasing microsome Ca 2+ pump activity. The phosphate group of nucleotides appears to be important to this effect in that adenosine does not affect Ca 2+ pump activity, while sodium pyrophosphate will increase pump activity but to a smaller extent than ATP. The presence of nucleotides or pyrophosphate during microsome isolation results in the release of a small amount of protein material from microsomes. These proteins can be detected in 105 000 × g supernatant by SDS-polyacrylamide gel electrophoresis. Three bands of molecular weight 46 000, 42 000 and 36 000 comprise the majority of protein material released from microsomes. The ability of nucleotides to release one of the these proteins, the 42 000 molecular weight band, from microsomes correlates with the ability of the nucleotide to increase microsome Ca 2+ pump activity. Preliminary evidence indicates that the protein released from ATP-treated microsomes is able to suppress the stimulated calcium uptake measured in ATP-treated microsomes. It is possible that this protein functions to regulate Ca 2+ pump activity in the endoplasmic reticulum of liver.