Effects of cations, phospholipases, and neuraminidase on calcium binding to "gas-dissected" membranes from cultured cardiac cells.
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 displacement from the sarcolemma was according to similarity of their crystal radii to that of calcium (cadmium greater than manganese greater than 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 A2, phospholipase 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 an increase in phosphatidate, with attendant increase in net anionic charge on the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D066298:In Vitro Techniques; D007473:Ion Channels / Q000378:metabolism; D007811:Lanthanum / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D009439:Neuraminidase / Q000494:pharmacology*; D010740:Phospholipases / Q000494:pharmacology*; D051381:Rats; D012508:Sarcolemma / Q000378:metabolism*
D002413:Cations, Divalent; D007473:Ion Channels; D007811:Lanthanum; D010740:Phospholipases; D009439:Neuraminidase; D002118:Calcium