ATP-dependent regulation of rabbit myocardial cytosolic calcium-independent phospholipase A2.
We report here that rabbit myocardial cytosolic calcium-independent phospholipase A2 exists as a high molecular weight complex comprised of catalytic and regulatory polypeptides whose activity and stability are influenced by specific interactions with ATP. Multiple lines of evidence document the functional significance of interactions between the catalytic complex and ATP including: 1) adenine nucleotide triphosphates attenuate the rate of thermal denaturation of native cytosolic phospholipase A2; 2) ATP augments the initial rate of phospholipid hydrolysis in a manner independent of the concentration, interfacial properties, and physical state of aggregated substrate; 3) adenine nucleotide triphosphates attenuate the reactivity of an essential thiol residue to covalent modification by 5,5'-dithiobis(2-nitrobenzoic acid); and 4) the catalytic complex specifically and reversibly binds to ATP affinity matrices, although the purified 40-kDa catalytic subunit neither binds to ATP affinity matrices nor is subject to ATP-dependent activation and stabilization. The catalytic and regulatory elements were functionally resolved by differential thermal inactivation and the ATP-regulatable phospholipase A2 catalytic complex was reassembled by reconstitution of highly purified catalytic and partially purified regulatory proteins. Thus, alterations in ATP concentration influence the activity and longevity of the myocardial cytosolic calcium-independent phospholipase A2 catalytic complex, thereby potentially modulating the release of lipidic second messengers and facilitating adaptive alterations in membrane physical properties.
Hazen
S L
SL
Molecular and Cellular Cardiovascular Biochemistry Section, Washington University School of Medicine, St. Louis, Missouri 63110.
Gross
R W
RW
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002853:Chromatography, Liquid; D004789:Enzyme Activation; D006358:Hot Temperature; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology* / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000302:isolation & purification; D011489:Protein Denaturation; D011817:Rabbits
D010743:Phospholipids; D000255:Adenosine Triphosphate; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium