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Lipoprotein lipase-catalyzed hydrolysis of p-nitrophenyl butyrate. Interfacial activation by phospholipid vesicles.

K. Shirai,Richard,Jackson

Published 1982 in Journal of Biological Chemistry

ABSTRACT

The mechanism of action of bovine milk lipoprotein lipase (LpL) was studied with a water-soluble substrate of p-nitrophenylbutyrate (PNPB). The calculated maximal velocity ( Vmm) and Michaelis constant (Km) values at 37 "C were 2.0 pmol of p-nitrophenol released/min/ mg of LpL and 0.52 mM, respectively. The addition of phospholipid vesicles enhanced the rate of PNPB hydrolysis by LpL. In the presence of dipalmitoyl phosphatidylcholine (DPPC) vesicles at 37 "C, the V,, and K,,, values were 8.8 pmol ofp-nitrophenol released/min/ mg of LpL and 0.55 mM, respectively, indicating that the enhancement of LpL activity was due to an increase in the Vma. The phospholipid-induced enhancement of LpL activity for PNPB was not correlated to the increase in the concentration of PNPB associated with the phospholipid vesicles. The effects of phospholipid vesicles on LpL activity for PNPB were influenced by the phase transition temperature (T,) of the lipid. At 17 "C, dimyristoyl phosphatidylcholine (DMPC) (Tc, 24 "C) and DPPC (TC, 41 "C) both caused an 800% increase in LpL activity. At 33 "C, the increase in activity by DMPC and DPPC were 190% and 800%, respectively. At 42 "C, neither DMPC nor DPPC affected enzyme activity. Diether DMPC (Tc, 27 "C) and sphingomyelin (Tc, 37 "C), two lipids which are not substrates for LpL, also caused an 800% increase in the activity of the enzyme for PNPB at 17 "C. In the presence of both DMPC vesicles and DPPC vesicles, the temperature dependence of LpL activity for PNPB was nearly identical with that of DPPC vesicles. When the enzymic reaction was first performed at 26 "C in the presence of DMPC vesicles and then DPPC vesicles were added, PNPB hydrolysis by LpL was enhanced. These results support the hypothesis that LpL interacts with lipid interfaces to increase the catalytic activity toward a water-soluble substrate. Furthermore, for interfacial activation, LpL prefers the lipid interface in the gel phase to that in the liquid-crystalline phase. A decrease in the enhancing effect of lipids in the liquid-crystalline phase on LpL activity toward PNPB suggests that interfacial activation is less effective and/or that the phospholipid is the preferred substrate for LpL.

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