Kinetic analysis and substrate specificity of a lysophospholipase from the macrophage-like cell line P388D1.
The kinetics of the lysophospholipase purified from the P388D1 macrophage-like cell line (Zhang and Dennis (1988) J. Biol Chem. 263, 9965-9972) have been explored. Three different lysophospholipids were used in these studies: 1-hexadecanoyllysophosphatidylcholine, 1-tetradecanoyllysophosphatidylcholine, and 1-hexadecanoyllysophosphatidylglycerol. Since all of the substrate dependence data for these substrates fit a Hill model, the enzyme's activity appears to be cooperative requiring at least two lipid molecules for full enzymatic activity. The enzyme did not show a preference for any of these substrates since their kcat ranged from 1.2 to 1.5 mumol min-1 mg-1 and their half-maximal activities ([S]0.5) were achieved at substrate concentrations between 15 and 33 microM. Enzymatic activity also appeared to be independent of the aggregation state of the substrate. No dramatic changes in rate could be associated with substrate aggregation at the critical micelle concentration. This is in marked contrast to some phospholipases A2 that exhibit dramatic activations when their substrates aggregate. In very dilute solutions, less than 0.5 micrograms/ml protein, the lysophospholipase loses activity irreversibly within minutes. This effect of low protein concentrations can be overcome by maintaining the enzyme in the presence of greater than 10 microM lysophospholipid. This inactivation can affect kinetic studies, since the [S]0.5s for this enzyme are usually in this range. We have found that increasing the protein concentration with a 'non-specific protein', e.g., cytochrome c, can protect the enzyme without affecting activity and, thus, allow valid kinetic data to be obtained over the full substrate concentration range.
Stafford
R E
RE
Department of Chemistry, University of California, San Diego, La Jolla 92093-0601.
Zhang
Y Y
YY
Deems
R A
RA
Dennis
E A
EA
D000818:Animals; D002460:Cell Line; D004795:Enzyme Stability; D007700:Kinetics; D008245:Lysophospholipase / Q000378:metabolism*; D008264:Macrophages / Q000201:enzymology*; D013379:Substrate Specificity
D008245:Lysophospholipase