Hydrolysis of guinea pig nascent very low density lipoproteins catalyzed by lipoprotein lipase: activation by hjman apolipoprotein C-II.

The kinetics of bovine milk lipoprotein lipase (LPL) were studied in order to determine the reaction mechanism of this enzyme. Reaction velocities were determined at varying concentrations of emulsified trioleoylglycerol (TG) and different fixed concentrations of apolipoprotein C-II (C-II) or at varying C-II concentrations and different fixed concentrations of TG. Neither the apparent Km(TG) nor the apparent Km(C-II) was affected by varying the concentrations of C-II or TG, respectively. However, C-II increased the apparent Vmax for the enzyme about 20-fold. The following kinetic parameters were calculated from Lineweaver-Burk plots: Km(C-II) = 2.5 X 10(-8) M and Km (TG) = 2.5 X 10(-3) M. The dissociation constant (KS) of the enzyme-TG binary complex was determined from Scatchard plots to be 7.6 X 10(-8) M. Heparin was found to be a competitive dead-end inhibitor against both TG and C-II. Tricapryloylglycerol represented a competitive inhibitor against TG but a noncompetitive inhibitor against C-II. C-II was shown to interact with dansylated bovine milk LPL, increasing its fluorescent emission by inducing a conformational change in the enzyme. Based on these studies, it was concluded that the LPL-catalyzed reaction follows a random, bireactant, rapid-equilibrium mechanism and the role of C-II in the activation process involves an increase in the catalytic rate constant (Kp) resulting from conformational changes of LPL induced by C-II.

Section: Discussioncontrasting

confidence: 99%

Kinetics of bovine milk lipoprotein lipase and the mechanism of enzyme activation by apolipoprotein C-II

Posner¹,

Wang²,

McConathy³

1983

“…The differences in lipid binding constants of LpL and apoC-II suggest that LpL will preferentially bind to a PC interface in the presence of apoC-II. Several lines of evidence support the hypothesis that apoC-II does not increase the binding of LpL to lipid surfaces, including both kinetic (Fitzharris et al, 1981;Shirai et al, 1983;Shinomiya et al, 1983Shinomiya et al, , 1984 and equilibrium (Jackson et al, 1980;Shirai et al, 1981b;Posner, 1983) studies. Furthermore, the association constant for LpL and a dansylated fragment of apoC-II is increased 40-fold on addition of lipid vesicles to a value (Kd = 0.05 µ ; Voyta et al, 1983) similar to the K¿ for LpL and C14-ether-PC vesicles.…”

Section: Discussionmentioning

confidence: 81%

Interaction of lipoprotein lipase and apolipoprotein C-II with sonicated vesicles of 1,2-ditetradecylphosphatidylcholine: comparison of binding constants

McLean¹,

Jackson²

1985

The interaction of lipoprotein lipase (LpL) and its activator protein, apolipoprotein C-II (apoC-II), with a nonhydrolyzable phosphatidylcholine, 1,2-ditetradecyl-rac-glycero-3-phosphocholine (C14-ether-PC), was studied by fluorescence spectroscopy. A complex of 320 molecules of C14-ether-PC per LpL was isolated by density gradient ultracentrifugation in KBr. The intrinsic tryptophan fluorescence emission spectrum of LpL was shifted from 336 nm in the absence of lipid to 330 nm in the LpL-lipid complex; the shift was associated with a 40% increase in fluorescence intensity. Addition of C14-ether-PC vesicles to apoC-II caused a 2.5-fold increase in intrinsic tryptophan fluorescence and a shift in emission maximum from 340 to 317 nm. LpL and apoC-II/C14-ether-PC stoichiometries and binding constants were determined by measuring the increase in the intrinsic tryptophan fluorescence as a function of lipid and protein concentrations; for LpL the rate and magnitude of the fluorescence increases were relatively independent of temperature in the range 4-37 degrees C. A stoichiometry of 270 PC per LpL for the LpL-lipid complex compares favorably with the value obtained in the isolated complex. The dissociation constant (Kd) of the complex is 4.3 X 10(-8) M. For apoC-II, the stoichiometry of the complex is 18 PC per apoprotein, and the Kd is 3.0 X 10(-6) M. These data suggest that LpL binds more strongly than apoC-II to phosphatidylcholine interfaces.

“…Even though PNPB and trioleoylglycerol hydrolyses appear to be catalyzed by the same LpL active site, the effect of apoC-II on the two reactions is different. It is well-known that apoC-II is an activator of the LpL-catalyzed hydrolysis of various water-insoluble lipid substrates (Fitzharris et al, 1981;Matsuoka et al, 1981;Krauss et al, 1973;Fielding, 1973; to Figures 2 and 4. (B) Replot of the intercepts of the Lineweaver-Burk plots of part A.…”

Section: Resultsmentioning

confidence: 99%

“…Such could occur if the conformation change that occurs when apoC-II and the enzyme interact produces an active site that is catalytically more potent for hydrolysis of lipid substrates (a Fmax effect) and/or has a higher affinity for lipid substrate monomer (a Km effect). Interestingly, the latter effect may apply to stimulation by apoC-II of LpLcatalyzed hydrolysis of emulsified trioleoylglycerol described by Schrecker & Greten (1979) and of triacylglycerols in VLDL described by Fitzharris et al (1981) and Matsuoka et al (1981), all of which are manifested by a large decrease in Km with a small or no effect on Fmax. Fielding (1973), on the other hand, showed that apo VLDL (which contains apoC-II) both increases Fmax and decreases Km of LpL-catalyzed hydrolysis of lecithin-emulsified triacylglycerols and diacylglycerols.…”

Section: Discussionmentioning

confidence: 99%

Lipoprotein lipase-catalyzed hydrolysis of water-soluble p-nitrophenyl esters. Inhibition by apolipoprotein C-II

Quinn¹,

Shirai²,

Jackson³

et al. 1982

Bovine milk lipoprotein lipase (LpL) catalyzes the hydrolysis of the water-soluble esters p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB). The same protein and same active site are involved in hydrolysis of water-soluble p-nitrophenyl esters and emulsified trioleoylglycerol since (a) trioleoylglycerol hydrolysis and PNPB hydrolysis activities coelute from the heparin-Sepharose affinity column used to purify LpL and (b) LpL-catalyzed hydrolyses of trioleoylglycerol and PNPB are inhibited to equal extents by phenylmethanesulfonyl fluoride. The effect of apolipoprotein C-II (apoC-II) on the LpL-catalyzed hydrolysis of PNPA and PNPB has been determined. ApoC-II inhibits hydrolysis of both esters, with a maximum extent of inhibition of 70-90%. Inhibition of the LpL-catalyzed hydrolysis of PNPB is specific for apoC-II, since apolipoproteins A-I, C-I, and C-III-2 have little effect on this reaction, and is partial noncompetitive in form. KI values for apoC-II inhibition of the LpL-catalyzed hydrolysis of PNPA and PNPB are in the range 0.26-0.83 microM. The effect of apoC-II on the temperature dependences of LpL-catalyzed hydrolysis of both esters and on NaCl inhibition of LpL-catalyzed PNPB hydrolysis is consistent with a change in rate-determining step with LpL and apoC-II interact. These results indicate not only that there is an interaction between apoC-II and LpL in aqueous solution in the absence of a lipid interface but also that this interaction conformationally modulates the active site of the enzyme.