LIPID-PEROXIDATION INHIBITS OLEOYL-COA - 1-ACYL-SN-GLYCERO-3-PHOSPHOCHOLINE O-ACYLTRANSFERASE IN RAT CNS AXOLEMMA-ENRICHED FRACTIONS

The effect of phospholipid peroxidation on the acylation of lysoPtdCho (lysophosphatidylcholine) by axolemma-enriched fraction prepared from rat brain stein was investigated. After two types of peroxidative treatments, the in vitro induction of malondialdehyde and conjugated dienes formation in axolemmal membranes correlated to a shift in the ratio of saturated/unsaturated fatty acids. By using an Fe2+ (20 muM)-ascorbate (0.25 mM) peroxidation system, the residual acyltransferase activity was 55% of the initial one. No change in K(m) value for either oleoyl-CoA or lysoPtdCho was found, whereas a loss of 24% in V(max) was observed. After 5 min preincubation with 150 mM t-BuOOH, 70% inactivation of the acylation reaction was observed. A near suppression of enzyme activity was reached with 400 mM. The apparent K(m) for oleoyl-CoA decreased sharply (from 6.6 muM in control preparations to 4.1 muM in t-BuOOH-treated membranes), indicating a 2-fold increase in the enzymatic affinity for this substrate. The apparent K(m) for lysoPtdCho increased markedly (from 1.56 muM in the control preparations to 5.88 muM in t-BuOOH-treated membranes) whereas a decrease of V(max) (from 1.65 to 0.80 nmol/min/mg protein) for the same substrate was observed. Significant enzyme inactivation (loss of 60% of initial activity) was seen when 10 mumol of photooxidized phospholipids were preincubated with axolemmal membranes. Significant dose-dependent enzyme inactivation was brought about by addition of 10-60 mumol of peroxidized PtdEtn/100 mug axolemmal protein. The percent enzyme inhibition by peroxidized PtdCho at equivalent amounts was lower than that by PtdEtn. Linoleic acid hydroperoxides. exogenously added or generated in situ, had a concentration-dependent effect, promoting a marked inhibition of acyltransferase activity. The results suggest a link between the oxygen radical generation and the inhibitory perturbation of the membrane structure in which the enzyme is embedded.