Effects of Cations, Phospholipases, and Neuraminidase on Calcium Binding to "Gas-Dissected " Membranes from Cultured Cardiac

SUMMARY. Sarcolemmal membranes prepared by 'gas dissection ' from monolayers of cultured neonatal rat heart cells were studied with respect to their ability to bind calcium. Lanthanum displacement of calcium was 168 ± 7 nmol/mg sarcolammel protein. This represents 3.21 mmol Ca/kg dry weight original cells on the basis of the measured membrane protein:dry cell weight ratio of 19.1 g/kg. Lanthanum-displaceable calcium from whole cells was essentially equal (3.32 mmol/kg dry weight), which indicates that all calcium displaceable from whole cells by lanthanum is localized to sarcolemmal sites. The potency of a series of divalent cations for calcium displace-ment from the sarcolemma was according to similarity of their crystal radii to that of calcium (cadmium> manganese> magnesium). This order was the same for the cations ' ability to displace calcium from whole cells and for their ability to uncouple excitation from contraction in neonatal papillary muscle. The membranes were treated with four enzymes: phospholipase A ^ phospho-lipase C, phopholipase D, and neuraminidase. Phospholipase A2 and phospholipase D produced significantly increased calcium-binding. The increased binding secondary to phospholipase A2 treatment was eliminated by an albumin wash which was indicative of binding to the fatty acid product of hydrolysis. The increase after phospholipase D treatment can be attributed to a