Richard L. Jackson

Hydrolysis of human plasma high density lipoprotein2-phospholipids and triglycerides by hepatic lipase

Summary To determine the physiological role of hepatic lipase in lipoprotein metabolism, human plasma very low (VLDL), low (LDL) or high (HDL 2 and HDL 3 ) density lipoproteins were labeled with [ 14 C] triolein or [ 14 C] dipalmitoyl phosphatidylcholine and the rates of lipid hydrolysis were determined using human heparin-releasable hepatic lipase. Compared to LDL and HDL 3 , HDL 2 phospholipids were the preferred substrate for hepatic lipase; 17% of HDL 2 phospholipids were hydrolyzed. [ 14 C] Triolein-labeled HDL 2 were also hydrolyzed by hepatic lipase. However, purified bovine milk lipoprotein lipase had negligible activity on HDL 2 -triglycerides. After incubation of HDL 2 with hepatic lipase, there was a decrease in the size of the particle as demonstrated by gel filtration and an increase in the density as shown by zonal ultracentrifugation. These results suggest that hepatic lipase may play a role in the interconversion of HDL subfractions.

Binding of a high reactive heparin to human apolipoprotein E: Identification of two heparin-binding domains

Ligand-blotting and dot-blotting procedures were used to investigate the binding of [125I]-heparin to apolipoprotein E, its thrombin fragments E22 (residues 1-191) and E12 (residues 192-299), and to nine apolipoprotein E synthetic fragments. E22 and E12 bound [125I] heparin indicating multiple heparin-binding domains. Synthetic peptides of apoE corresponding to residues 129-169, 139-169, and 144-169, but not 148-169, bound [125I] heparin suggesting that residues 144-147 (Leu-Arg-Lys-Arg) in E22 are important for binding. Peptide 202-243 and 211-243 but not 219-243 bound [125I] heparin suggesting that residues 211-218 (Gly-Glu-Arg-Leu-Arg-Ala-Arg-Met) comprise a portion of the E12 heparin-binding domain.

Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase.

The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4 -yl) amino]-caproyl] phosphatidylcholine (C6-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A2 (PA2) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C6-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca++ was required for PA2 catalysis. Identification of the products of hydrolysis showed cleavage at the sn-1 and sn-2 positions for LpL and PA2, respectively. For PA2, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme.

Comparison of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins and trioleoylglycerol/phosphatidylcholine-stabilized particles as substrates for lipoprotein lipase

The effect of apolipoproteins C-II and C-III on the lipoprotein lipase-catalyzed hydrolysis of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins and particles of trioleoylglycerol stabilized with a phosphatidylcholine monolayer was investigated. For both triacylglycerol-rich lipoproteins and artificial lipid particles, maximal lipoprotein lipase activity occurred at a constant apolipoprotein C-II/phospholipid mol ratio of 2.0 X 10(-4) and was independent of particle size, indicating that the amount of apolipoprotein C-II bound to the surface of the substrate is important for enzyme activation. The effect of apolipoprotein C-II on lipoprotein lipase activity with apolipoprotein C-II-deficient lipoproteins as substrate was to decrease the apparent Michaelis constant (Kmapp) from 7.1 to 1.0 mM with minor changes on the apparent maximal velocity (Vmax) (22.2 mmol free fatty acid released/h per mg enzyme). In contrast, apolipoprotein C-II increased the apparent Vmax from 2.4 to 20.0 mmol free fatty acid/h per mg enzyme for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol/phospholipid particles with little change in Kmapp (1.0 mM). Addition of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins or high-density lipoproteins to trioleoylglycerol/phospholipid particles in the presence of apolipoprotein C-II inhibited lipoprotein lipase activity. Lipoprotein lipase activity was also inhibited by the addition of a large excess of lipid-free apolipoprotein C-III to the artificial particles. The decrease in lipoprotein lipase activity correlated with the amount of bound apolipoprotein C-II. We suggest that the reported discrepancies on the effect of apolipoproteins C-II and C-III on lipoprotein lipase catalysis is related to differences in substrates and to the amount of added apolipoproteins.

Visualization of heparin-binding proteins by ligand blotting with 125I-heparin

A ligand-blotting procedure which allows detection of heparin-binding proteins is described. Crude commercial heparin was fractionated by chromatography on a column of human plasma low-density lipoproteins immobilized to Sepharose CL-4B. Chromatography yielded an unbound and a bound fraction of heparin, designated URH and HRH, respectively. The HRH fraction was reacted with the N-hydroxysuccinimidyl ester of 3-(p-hydroxyphenyl)propionic acid and then labeled with 125I. Proteins were separated by 3-20% pore-gradient gel electrophoresis, transferred to nitrocellulose, and then assayed for their ability to bind 125I-labeled HRH. Human plasma apolipoproteins B-100, B-48, and E of chylomicrons, very low-density lipoproteins, and low-density lipoproteins bound the 125I-labeled HRH; the radiolabeled heparin did not bind to serum albumin, ferritin, catalase, and lactate dehydrogenase. The ligand-blotting procedure should facilitate the purification of heparin-binding domains from these proteins and, moreover, may be applicable to the investigation of heparin-protein interactions in general.

Effect of calcium-channel-blocking drugs on lysosomal function in human skin fibroblasts

Recent reports suggest that certain Ca2+-channel-blocking drugs reduce the severity of atherosclerosis in cholesterol-fed animals. To determine whether the suppression of atherogenesis is related to altered lipoprotein metabolism, we have assessed the effects of these drugs on the catabolism of plasma low density lipoproteins (LDL) by human skin fibroblasts. The Ca2+-channel-blocking drugs verapamil and diltiazem inhibit the lysosomal degradation of LDL by these cells; degradation of epidermal growth factor was also inhibited by the same drugs, suggesting a general effect of these drugs on lysosomal function. In contrast, nifedipine did not affect the degradation of LDL or epidermal growth factor. None of the drugs affected phospholipid or protein synthesis. Entry of LDL into the lysosomes also was not affected. [3H]Diltiazem, which inhibited LDL degradation, accumulated in the lysosome-rich fraction, whereas [3H]nimodipine, a drug structurally and functionally similar to nifedipine, did not accumulate. We suggest that the inhibitory effect of some of the Ca2+-channel-blocking drugs on lysosomal function is due to their basic nature, causing them to accumulate in lysosomes, thereby increasing intralysosomal pH.

Complete exchange of phospholipids between microsomes and plasma lipoproteins mediated by liver phospholipid-exchange proteins

It is known that cholesterol and phospholipids are in dynamic equilibrium and spontaneously exchange both in vitro and in vivo between individual classes of plasma lipoproteins [l-4] . Spontaneous transfer of phospholipids has also been shown between lipoproteins and erythrocytes [5],mitochondria [6] ,liver microsomes [7] , liver slices [8] and cells in culture [9]. In these examples of phospholipid exchange it is presumed that transfer occurs upon formation of a collision complex. Evidence for a protein-facilitated transfer of lipoprotein phospholipids in serum is controversial [7,10]. On the other hand, eukaryotic cells contain proteins designated as phospholipid exchange proteins (PLEP) that do facilitate the transfer of phospholipids from one membrane to another [ 1 I-131 . A purified PLEP from beef liver shows a highly specific affinity for phosphatidylcholine (PC-PLEP [ 141). Two proteins have been isolated from bovine cerebral cortex which stimulate both the transfer of phosphatidylinositol (PI) and PC (PI-PLEP [IS] ). Recently, exchange proteins have been isolated from rat liver [ 161 and hepatoma [ 171 which lack specificity in the transfer of phospholipid. The purpose of the present study was to determine whether and if so, to what extent, purified PCand PI-exchange proteins catalyze the transfer of phospholipids between rat liver microsomes and the individual classes of lipoproteins. It will be shown that the protein-mediated transfer of labeled PC and PI from microsomes to very low density lipoproteins (VLDL), LDL and HDL was 5-lo-fold higher than

Effect of Ca2+ blocking agents on the metabolism of low density lipoproteins in human skin fibroblasts

Abstract The effect of Ca 2+ entry blockers (prenylamine, fendiline, verapamil, diltiazem and nifedipine) and calmodulin inhibitors (trifluoperazine and dibucaine) on the biosynthesis of cholesterol and on the metabolism of low density lipoproteins (LDL) was studied in human skin fibroblasts in culture. Addition of prenylamine, fendiline, trifluoperazine or dibucaine (5–10 μM) to the cells incubated with lipoprotein-deficient serum decreased the LDL-mediated inhibition of the incorporation of [1- 14 C] acetate into sterols by greater than 75%. LDL-mediated inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase was also decreased by these drugs. Verapamil and diltiazem were also effective at 50–100 μM concentrations. The drugs did not prevent the inhibition of sterol synthesis by 25-hydroxycholesterol, suggesting that they affect LDL metabolism and not HMG-CoA reductase itself. The drugs decreased the cellular degradation of LDL and enhanced the accumulation of LDL in the cells.

INTERACTION OF PLASMA APOLIPOPROTEINS WITH LIPID MONOLAYERS

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidy[14C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipids by apolipoprotein C-III. The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface. Although apolipoprotein A-I and arginine-rich protein gave surface pressure increases, phospholipid was only slightly removed fromthe interface by the addition of liposomes. Based on these findings, we conclude that the apolipoproteins C interact specifically with phosphatidylcholine at the interface. This interaction is important as it relates to the transfer of the apolipoproteins C and phospholipids from very low density lipoproteins to other plasma lipoproteins. The addition of human plasma high density lipoproteins or very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of phosphatidyl[14C]choline from the interface 3--4 fold. Low density lipoproteins did not affect the rate of decrease. During lipolysis of very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of with the lipid monolayer. Lipolysis experiments were performed in a monolayer trough containing a surface film of egg phosphatidyl[14C]choline and a subphase of very low density lipoproteins and bovine serum albumin. Lipolysis was initiated by the addition of purified milk lipoprotein lipase to the subphase. As a result of lipolysis, there was a decrease in surface radioactivity of phosphatidylcholine. The pre-addition of high density lipoproteins decreased the rate of decrease in surface radioactivity...

Effect of a sustained release formulation of diltiazem on the development of atherosclerosis in cholesterol-fed rabbits

Cholesterol-fed rabbits were used to test potential anti-atherosclerotic effects of diltiazem, a calcium antagonist of the benzothiazepine type. Two groups of 7 rabbits each were fed standard laboratory chow supplemented with 1% cholesterol. One group received a 60 mg sustained release diltiazem capsule twice a day and the other group received a placebo capsule twice a day. A third group of control animals were fed an unmodified basal diet under conditions exactly the same as the experimental groups. All groups were studied over a period of 16 weeks. The cholesterol-fed animals showed a marked increase in plasma total cholesterol which was not significantly different for the diltiazem and placebo groups. Plasma calcium levels, blood pressure, and heart rate were also unchanged from the control animals. In the diltiazem-treated animals, 47.5 +/- 10.5% of the aortae showed atherosclerotic lesions; the value for the placebo group was 43.1 +/- 8.1%. Similar results were obtained for the coronary arteries. These results show that diltiazem treatment in the doses employed in this study had no effect on the reduction of atherosclerosis in this animal model.