Rabbit myocardial cytosolic lysophospholipase. Purification, characterization, and competitive inhibition by L-palmitoyl carnitine.
Rabbit myocardial lysophospholipase was purified 27,000-fold to near homogeneity by ammonium sulfate precipitation, DEAE-Sephacel, gel filtration, chromatofocusing, and hydroxylapatite chromatography. Chromatofocusing demonstrated two activity peaks, each with a molecular mass of 23,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both activity peaks had similar kinetic parameters (Vmax = 7 mumols/mg/min, Km = 9-11 microM) and similar pH profiles (7.5 optimum). Each activity peak was competitively inhibited by L-palmitoylcarnitine with similar inhibitory constants (KI = 10-11 microM). In addition, palmitic acid competitively inhibited myocardial lysophospholipase (KI = 37 microM). A rapid loss of lysophospholipase activity resulted from heating at 37 degrees C in the absence of substrate (t1/2 = 3 min). This thermal denaturation was attenuated similarly by either lysophosphatidylcholine (15 microM) or L-palmitoylcarnitine (15 microM). Thus, L-palmitoylcarnitine complexes with purified myocardial lysophospholipase and competitively inhibits a major pathway of lysophosphatidylcholine catabolism, thereby potentially contributing to accumulation of lysophosphatides in ischemic myocardium and ventricular dysrhythmia.
D000818:Animals; D002331:Carnitine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D008245:Lysophospholipase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D009206:Myocardium / Q000201:enzymology*; D010172:Palmitoylcarnitine / Q000494:pharmacology*; D010740:Phospholipases / Q000302:isolation & purification*; D011817:Rabbits; D013379:Substrate Specificity
D010172:Palmitoylcarnitine; C037735:linoleoylcarnitine; D010740:Phospholipases; D008245:Lysophospholipase; D002331:Carnitine