Purification and characterization of phospholipase A2 from rat stomach.
Phospholipase A2, which is localized in the mucosal part of the corpus of rat stomach (Hirohara et al. (1987) Biochim. Biophys. Acta 919, 231-238), was purified 990-fold from the supernatant of a tissue homogenate by heat treatment at acidic pH, ammonium sulfate fractionation, ion-exchange chromatography, gel-filtration and reverse-phase high-performance liquid chromatography (reverse-phase HPLC). The purified enzyme gave a single protein band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with a molecular mass of approx. 17 kDa. The enzyme had a pH optimum of 8.0 and hydrolyzed the 2-arachidonoyl residue of phosphatidylcholine preferentially to the 2-oleoyl residue, the Vmax and Km values for the two being 227 and 29 mumol/min per mg protein and 0.037 and 0.019 mM, respectively. The activity was calcium-dependent and was markedly increased by SDS and dimethyl sulfoxide (DMSO). The enzyme showed typical product inhibition. Free unsaturated fatty acids (oleic, arachidonic and docosahexaenoic acids), which are supposedly the main enzymatic products in vivo, inhibited the activity. Arachidonic acid caused noncompetitive inhibition and its concentration for its maximal inhibition (50% inhibition) was 5 x 10(-5) M. Lysophosphatidylcholine, free saturated fatty acids (palmitic and stearic acids) and arachidonic acid metabolites (leukotrienes and prostaglandins) had no effect on the activity.
Yasuda
T
T
Department of Medical Chemistry, Kansai Medical School, Osaka, Japan.
Hirohara
J
J
Okumura
T
T
Saito
K
K
D000645:Ammonium Sulfate; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008297:Male; D008970:Molecular Weight; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D051381:Rats; D011919:Rats, Inbred Strains; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013270:Stomach / Q000201:enzymology*
D005231:Fatty Acids, Unsaturated; D012967:Sodium Dodecyl Sulfate; D010741:Phospholipases A; D054467:Phospholipases A2; D000645:Ammonium Sulfate; D002118:Calcium; D004121:Dimethyl Sulfoxide