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The rates of reaction of human lipoprotein lipase (EC 3.1.1.34) with triacylglycerol and partial acylglycerol substrates have been compared as a function of the concentration of lipase cofactor protein (apolipoprotein C-II). The data indicate that the dissociation constant for monoacylglycerol is approximately three orders of magnitude greater than for diacylglycerols, indicating that only when the concentrations of higher acylglycerols become vanishingly small will significant monoacylglycerol hydrolysis (from 1-monoacylglycerol generated by isomerization of the 2-substituted primary product) be mediated by the lipase. This is in spite of the fact that maximal reaction velocities with each of the potential substrates are similar. A 'lipolytic cycle' is proposed to explain binding and dissociation of substrates with cofactor-lipase complex during catabolism of triacylglycerols.

The activity of lipoprotein lipase isolated from rat postheparin plasma has been determined with synthetic lipids, in the presence and absence of apoprotein of the natural substrate very low density lipoprotein, as a function of medium ion-pair concentration of a number of different inorganic salts. The several kinetic effects of lipoprotein protein on lipase activity were specifically and quantitatively reversed in the presence of molar sodium chloride or solutions of equivalent effective ion concentrations of other salts. Salt-mediated inhibition was fully reversible by silution and was independent of substrate concentration. Inhibition was a function of the identity of the salt anion within a Hofmeister (lyotropic) series: I- greater than SCN- greater than NO3- greater than Cl- greater than F-, and, in these terms, was not significantly different for a series of inorganic chlorides (Li+, Na+, K+, Cs+). The effects of salts on the natural lipoprotein substrates, chylomicrons, and very low density lipoproteins were similar to those obtained with a synthetic lipid-protein substrate complex. These findings are discussed in the light of recent ideas on the activation of lipoprotein lipase.

The chemical and biochemical properties of cholesterol-enriched and cholesterol-poor chylomicrons from rat lymph have been compared. The enriched particles, prepared from cholesterol-containing lipid dispersions, passed into the duodenum, had four to ten times the cholesteryl ester content of the control chylomicrons but had the same content of total "core" (cholesteryl ester + triglyceride) lipid. Both chylomicron species had the same protein composition, the same phospholipid composition, and the same composition of triglyceride fatty acids. The rate of hydrolysis of chylomicron triglyceride for enriched and control particles was determined using both soluble and membrane-supported lipoprotein lipase (LPL) species from heart and adipose tissues. The lipase that was functional in the isolated perfused heart showed no significant difference in initial catabolic rate with cholesterol-enriched and control chylomicrons. The same result was obtained with this isolated LPL species in vitro. The lipase that was functional in isolated perfused epididymal adipose tissue showed a slightly lower catabolic rate with cholesterol-enriched particles (84% of that obtained with control chylomicrons). The same result was obtained with isolated adipose tissue LPL. It is concluded that cholesteryl ester content of chylomicrons under these conditions neither affects their protein composition nor has a major effect on their rate of reaction with lipoprotein lipase.

Kinetic analysis of activation of lipoprotein lipase by apo C-2 indicates that the lipase co-protein increase the rate of lipolysis by adsorption to enzyme at the lipid interface, with formation of a 1:1 molar complex, whose dissociation constant in the presence of triglyceride substrate is about 3 X 10(-13) moles cm-3. Activation by apo C-2, like that by the entire lipoprotein apoprotein moiety (Fielding and Fielding, 1976) is reversible by inorganic salts and the dependence of activation on medium ion-pair activity supports the concept that such inhibition is mediated through a single specific anion-binding site of the lipase co-protein.

The catalytic rate of membrane-supported lipoprotein lipase has been determined for chylomicron and very low density lipoprotein substrates during the formation of triglyceride-depleted ("remnant") particles. Both lipoprotein species and their generated remnant products were competitive substrates for lipase activity. Remnant formation from each species was associated with decreasing kc but an unchanged apparent Km. This finding was confirmed from the rate of plot of total triglyceride catabolism by lipase at low substrate concentrations. When compared with the major very low density lipoprotein fraction (Sf 100-400), a fraction isolated from plasma with a lower flotation rate (Sf 40-100) had a lipid composition and decreased kc compatible with this representing a physiological remnant particle.

A lipoprotein lipase species (mol wt 69 250) has been isolated from rat postheparin plasma, which differs from the low-molecular-weight species previously characterized in its amino acid composition and hexosamine content, and in its lower affinity for triglyceride-rich lipoprotein substrates. However, both enzymes are activated by the same coprotein (C-terminal glutamic acid, apo-C-2) from human very low density lipoprotein and have a similar specificity for lipid esters. Neither purified enzyme is activated by heparin. Both are inhibited by molar sodium chloride. Both enzyme species can be recovered from the same plasma samples. The possible relationship of these proteins to the different functional lipoprotein lipase activities of muscle and adipose tissues is discussed.

Chylomicrons isolated form rat intestinal lymph were incubated with plasma. Protein transfer to chylomicrons, reaction rate with purified lipoprotein lipase, and content of lipase cofactor were determined. While the overall protein content of chylomicrons was increased 3--4-fold, and the content of lipase cofactor increase 4-fold, reaction velocity of the activated particles with lipoprotein lipase was increased only 1.3-fold. Maximal rate of hydrolysis was achieved in the presence of much smaller quantities of activator than the lipoprotein particles were capable of binding, and chylomicrons were fully activated for triclyceride hydrolysis in the presence of only 10% plasma for triglyceride concentrations of up to 3 mg/ml. Cofactor protein was not rate-limiting for hydrolysis of triglyceride from chylomicrons. These results are discussed in the light of recent concepts of the regulation of lipoprotein lipase activity.

The activity of lipoprotein lipase in the vascular bed represents the major pathway by which triglyceride fatty acid is cleared from the plasma and made available to the epripheral tissues. Recent studies on the properties of the enzyme, both solubilized and membrane-bound have provided new information on the regulation of its activity with the major triglyceride-rich lipoprotein substrates. A key role for this enzyme in the regulation of plasma triglyceride levels is indicated from studies of lipase levels in human adipose tissue and in blood plasma obtained after injection of heparin.

Rat plasma contains monoglyceride hydrolase activities against both 1(3)- and 2-monoglycerides. These activities are present as lipoprotein complexes recovered by either density flotation or by agarose gel chromatography with plasma high density lipoprotein. However neither activity is complexed with the major apoproteins (apo-A-I, apo-E) of this lipoprotein class. 2-Monoglyceride hydrolase (but not 1(3)-monoglyceride hydrolase) activity associates with the triglyceride-rich lipoprotein class. The two activities are also noncompetitive with respect to substrate, and differ in pH- and cofactor-dependence and sensitivity to inhibition by diethyl p-nitrophenyl phosphate. Rat platelets also contain both 1(3)- and 2-monoglyceride hydrolase activities. These differ in reactivity with antiesterase and after solubilization and electrophoretic migration with each other and with the corresponding plasma activities. Studies with the isolated perfused rat heart suggest that a major role in the catabolism of 2-monoglyceride generated from lipoprotein lipase activity at the coronary bed is played by the plasma 2-monoglyceride hydrolase activity.

The kinetic constants for membrane-supported lipoprotein lipase have been determined for the enzyme active in lipoprotein triglyceride catabolism in perfused heart and adipose tissues, using a nonrecirculating system. Heart endothelial lipoprotein lipase reacted as a single population of high-affinity substrate binding sites (Km' 0.07 mM triglyceride). Km' (apparent Michaelis constant for the supported enzyme species) was independent of flow rate and the enzyme was rapidly released by heparin, suggestive of a superficial membrane binding site. Lipoprotein lipase active in perfused adipose tissue had significantly different kinetic properties, including a low substrate affinity (Km' 0.70 mM triglyceride), diffusion dependence of Km' at low flow rates, and slow release of enzyme by heparin. Adipose tissue may contain a small proportion of high affinity sites. While only a small proportion of total heart tissue lipoprotein lipase was directly active in triglyceride hydrolysis, this study suggests that the major part of lipoprotein lipase in adipose tissue may be involved in the hydrolysis of circulating lipoprotein triglyceride.