Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide.
Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D005450:Fluorescamine / Q000494:pharmacology*; D008239:Lysine; D009132:Muscles / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013141:Spiro Compounds / Q000494:pharmacology*
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D013141:Spiro Compounds; D005450:Fluorescamine; D000252:Calcium-Transporting ATPases; C017251:1,2-oleoylphosphatidylcholine; D008239:Lysine; D002118:Calcium