Partial purification and kinetic characterization of the microsomal phospholipase A2 from thermally acclimated rainbow trout (Salmo gairdneri).
Phospholipase A2 (PLA2) was extracted from liver microsomal membranes of both 5 and 20 degrees C-acclimated rainbow trout (Salmo gairdneri), using the non-ionic detergent, Triton X-100. Further purification was achieved by precipitation with 35-65% ammonium sulfate followed by gel filtration chromatography in the presence of 0.1% Triton X-100 on Sephadex G-200. These procedures resulted in a 30-fold purification and the removal of all traces of phospholipid from the enzyme of both warm- and cold-acclimated trout. Column elution profiles were similar for both acclimation groups, yielding a molecular weight estimate for the trout liver enzyme of 73,000. Comparisons of activity levels and kinetic parameters of PLA2 from warm- and cold-acclimated fish indicated that compensation for temperature at non-saturating substrate concentrations was an attribute of both the particulate (microsomal) enzyme and the lipid-free protein. Cold acclimation resulted in higher activity below Vmax due primarily to decreased apparent Km values. These adaptations to temperature could not be attributed to the interaction of the enzyme with the membrane lipids, but were due to qualitative changes in the enzyme that resulted from acclimation. Other adaptive qualities of PLA2, such as reduced Km in response to acute decreases in temperature in warm-acclimated fish, were only apparent in particulate preparations, and thus were a function of the protein-lipid complex. These data suggest that an acclimation-induced increase in the activity of PLA2 may result in the activation of a deacylation-reacylation cycle at cold temperatures.
D000064:Acclimatization; D000818:Animals; D003902:Detergents; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D017830:Octoxynol; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D011092:Polyethylene Glycols; D013696:Temperature; D014337:Trout
D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2