Lipoprotein Lipase: Some Effects of Activator Proteins

Abstract

This paper considers how apolipoprotein CII from human plasma lipoproteins and T1 and T2 proteins from egg yolk lipoproteins stimulate the activity of lipoprotein lipase. These activator proteins stabilized the enzyme much more effectively than a thousandfold higher concentration of albumin did, indicating a direct interaction with the enzyme. The effects of the activators were seen also at 1 M NaC1. Thus, forces other than electrostatic are implicated. Centrifugation experiments showed that '251-labeled lipase bound equally well to the emulsion droplets in the absence of activator protein as in its presence. This was true even under conditions when the activator caused a severalfold increase in the rate of hydrolysis. Thus, the activator makes enzyme at the interface more effective in hydrolysis. By optimizing the conditions it was possible to obtain almost as high rates of triglyceride hydrolysis in the absence as in the presence of activator. Thus, the main effect of the activator protein is probably not on a rate-limiting chemical step. Under most conditions, the rate of hydrolysis was much below optimal and activator increased it. This was always the case with phosphatidylcholine/triglyceride emulsions, where the activator enhanced hydrolysis of both lipids. Other experiments showed that the activator enhanced triglyceride hydrolysis in the absence of phospholipids and phospholipid hydrolysis in the absence of triglycerides. It is suggested that interaction with activator orientates the enzyme and/or the lipid substrate for effective hydrolysis at the surface of lipoproteins/model substrates.Lipoprotein lipase is a glycoprotein enzyme which hydrolyzes acylglycerols in plasma lipoproteins at the capillary endothelium (review in [l]). Activator proteins, present on the lipoproteins, enhance the enzyme activity [2]. The primary structure of the human activator, apolipoprotein CII, is known [3], and the activity has been localized to its C-terminal part [4,5]. How it promotes hydrolysis is however not known. The action of the lipase involves several steps [6], any of which could be the site of the activator effect; adsorption of the lipase to the lipid-water interface of the insoluble substrate, formation of an enzyme-substrate (Michaelis) complex at the interface, the chemical steps of catalysis, and finally removal of the products from the site of lipolysis. The following paper [7] considers the mechanism of the product inhibition. This paper considers the possible effects of activator proteins on the other three steps. MATERIALS AND METHODSLipoprotein lipase was purified from bovine skim milk by chromatography on heparin-Sepharose as Enzyme. Lipoprotein lipase (EC 3.1.1.34) described [8]. 1251-labeled lipoprotein lipase was prepared by the lactoperoxidase method [9] and then repurified and separated from unreacted iodide by chromatography on heparin-Sepharose. The resulting material had the same specific enzyme activity as the unlabeled preparation, and retained its ability to bind to heparin-Sepharose. Lipa...

Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry

“…Thus, in addition to activation, apoC-II also stabilized LPL. This is in accord with results from previous studies (30). The stabilizing effect of apoC-II is usually not recognized with other substrate systems used for measurements of LPL activity.…”

“…The stabilization of LPL by addition of apoC-II, as seen in the present study, is usually not detected in other substrate systems. Therefore the magnitude of the activation of LPL by apoC-II can easily be overestimated (30). ITC provided a method to carefully investigate possible effects of apoA-V on the catalytic activity of LPL.…”

The Activity of Hepatic Lipase and Lipoprotein Lipase in Glycogen Storage Disease: Evidence for a Circulating Inhibitor of Postheparin Lipolytic Activity

“…Preliminary in vitro studies have shown that increasing oleic acid concentrations result in increasing inhibition of both hepatic and lipoprotein lipase activities in a dose-related manner which could be reversed by the addition of excess albumin (I I). The in vitro inhibition of lipoprotein lipase activity by fatty acids has been reported before (1, 2,26) and it has been suggested that fatty acids may have an important physiological role in the feedback control of lipase activity in vivo. It is unlikely that the increased serum triglyceride concentrations in GSD were inhibitory as postheparin plasma from the patient with type 1 hyperlipoproteinemia did not inhibit normal postherapin plasma activity.…”

Site-directed Mutagenesis of Apolipoprotein CII to Probe the Role of Its Secondary Structure for Activation of Lipoprotein Lipase

“…For measurement of the activation ability of peptide 39 -62 compared with that of peptide 50 -79, three substrates with different properties were used to explore the activation ability of the apoCII fragments. One was the same as that described above for the mutants, whereas the second one was a gum-arabic-stabilized emulsion of 3 H-labeled trioleoylglycerol prepared as described (15). The conditions were otherwise the same for both systems.…”

“…This is the case also for LPL. With substrate emulsions that are less accessible to LPL alone, the activity is more dependent on apoCII, and the -fold activation is, therefore, larger than with simper substrates (15).…”