J. Biol. Chem., Vol. 261, Issue 24, 10990-10995, Aug, 1986
Efficiency of reconstitution of the membrane-associated sn-glycerol 3- phosphate acyltransferase of Escherichia coli
MA Scheideler and RM Bell
Membrane-associated enzymes are often solubilized with detergents,
purified, and then reconstituted with phospholipid cofactors to regain
function. Insofar as most purification and reconstitution procedures are
not quantitative, the final reconstituted preparations could reflect a
population of molecules ranging from fully functional to completely
inactive. Quantitative studies on the efficiency of reconstitution of the
Triton X-100-solubilized sn-glycerol 3-phosphate (glycerol-P)
acyltransferase of Escherichia coli cytoplasmic membrane were undertaken at
each step of purification. Physical recovery of the 83,000 Mr polypeptide
was quantitated in polyacrylamide gels using membranes from cells labeled
with [3H]leucine. The 83,000 Mr polypeptide in such gels was demonstrated
to consist exclusively of the glycerol-P acyltransferase peptide by V8
peptide mapping. Comparison between physical recovery of 83,000 Mr
polypeptide and reconstituted activity allowed the efficiency of
reconstitution to be determined. Unexpectedly, disproportionalities
occurred during the purification. However, the final purification of
reconstituted enzyme activity matched that of the 83,000 Mr polypeptide.
This method also allowed measurement of the specific activities of the
glycerol-P acyltransferase in membranes from a wild type E. coli strain and
from plasmid-containing strains which express the plsB gene product to
different extents. The physical amounts of the 83,000 Mr polypeptide and
glycerol-P acyltransferase activity measured in membranes were not strictly
proportional. In strains where the amount of 83,000 Mr polypeptide was
enhanced, a larger proportion of latent activity was observed following
solubilization and reconstitution. The results establish the suitability of
the reconstituted preparations of glycerol- P acyltransferase for detailed
kinetic analysis and permit inferences pertaining to regulation.
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