Search

1. 1. Optimal assay conditions were determined for a triacylglycerol lipase purified from human post-heparin plasma. The characteristics of this enzyme were compared to those of crude post-heparin plasma and adipose tissue lipoprotein lipase activities. 2. 2. Assay in solutions of high ionic strength (i.e. l M NaCl) resulted in activation of the purified enzyme. This activation was shown to be associated with a marked reduction in apparent molecular weight by gel filtration experiments. 3. 3. A pH optimum of 9.0 was observed for the purified triacylglycerol lipase. Evidence is presented for the existence of a factor in pre-heparin plasma which can cause a shift in the pH optimum to 7.6. 4. 4. The characteristics of the crude post-heparin plasma triacylglycerol lipase activity are compatible with the presence of both this enzyme and a lipase with the properties of adipose tissue lipoprotein lipase.

Valine apolipoprotein, glutamic acid apolipoprotein, and alanine apolipoprotein (apoLP-Ala) (designated by their COOH-terminal amino acids) are three small apolipoproteins which constitute more than half of the protein in human plasma very low density lipoprotein. These proteins were isolated free of lipid by techniques previously described. As determined by sedimentation equilibrium analysis and estimated from amino acid analyses, the approximate molecular weights of the apolipoproteins were: valine apolipoprotein, 7,000; glutamic acid apolipoprotein, 10,000; and apoLP-Ala, 10,000. Circular dichroism and optical rotatory dispersion spectra for valine apolipoprotein were consistent with a high content of α helix, while the spectra obtained with apoLP-Ala suggested its structure to be mainly of random coil. Previously reported polymorphism of apoLP-Ala was found to be attributed to differences in content of sialic acid.

The protein components of human plasma very low density lipoproteins (Sf g 20) were studied following partial and total delipidation. After the neutral lipids were extracted with heptane, the resulting phospholipid-protein complexes contained at least one immunochemical reactant different from the major apoproteins of high density and low density lipoproteins. Purification required total delipidation with ethanol-ether, gel filtration, and diethylaminoethyl cellulose chromatography. Two proteins were then isolated that differed from the proteins of high density or low density lipoproteins, and their purity was established by immunochemical analysis and polyacrylamide gel electrophoresis. One of these had γ mobility, NH2-terminal threonine, COOH-terminal valine, and no tyrosine, histidine, cysteine, or cystine. The second, α2-migrating protein had NH2-terminal serine, COOH-terminal alanine, and no isoleucine, cysteine, or cystine. These two proteins constituted approximately half of the total protein in very low density lipoprotein.

Summary The effects of two apolipoproteins isolated from human very low density lipoproteins (apoLp-Glu and apoLp-Ala) on lipoprotein lipase (LPL) activity have been studied. ApoLp-Glu markedly stimulated LPL as reported by others. ApoLp-Ala isolated by techniques previously described also activated LPL at low levels. However, with further purification by hydroxylapatite chromatography all activation by apoLp-Ala was eliminated with the removal of a small contaminant immunochemically identical to apoLp-Glu. ApoLp-Ala consistently inhibits LPL when present at levels above 2% of the substrate (w/w). This inhibition was not overcome by addition of phospholipid, apoLp-Glu, or more enzyme.

The lipase activities of post-heparin plasma have been shown to hydrolyze many classes of lipids, including: a) triglycerides (TG), diglycerides (DG) and monoglycerides (MG) (Shore and Shore, 1960; Greten et al., 1969; Fielding, 1972; Greten, 1972), b) phospholipids (Vogel and Zieve, 1964; Vogel and Bierman, 1967) and c) long-chain fatty acyl coenzyme A derivatives (Jansen and Hulsman, 1973). Attempts to physically separate enzymes specific for the hydrolysis of each of these lipid classes have been unsuccessful. Recent studies have shown that at least two triglyceride lipase activities exist in post-heparin plasma (LaRosa et al., 1972; Greten et al., 1972; Fielding, 1970). They have been characterized after partial purification from swine post-heparin plasma (Ehnholm et al., 1972). One of these was similar in its characteristics to the lipoprotein lipase of adipose tissue (Bensadouw et al., unpublished) and the other differed from lipoprotein lipase in its lack of activation by plasma cofactor and in being stimulated rather than inhibited by exposure to solutions high in ionic strength. This hydrolase was also active against phospholipids. The techniques used successfully in the separation of swine triglyceride lipase have now been applied to the study of human post-heparin plasma.

Lipoprotein lipase was purified from acetone powders of pig adipose tissue. Extraction of acetone powders with 1.2 m NaCl in 0.005 m sodium-barbital buffer, pH 7.4, or heparin (200 units per ml) in distilled water, was 6 times as effective as extraction with 0.025 m NH4OH-NH4Cl buffer, pH 8.6, the commonly used extractant for lipoprotein lipase. At pH values below 7.5, over 85% of the activity extracted into 1.2 m NaCl could be recovered after 4 hours. The partially purified enzyme at later stages was stabilized by the inclusion of 20% glycerol in the buffers. Most of the purification was accomplished by affinity chromatography on Sepharose 4B columns containing covalently bound heparin. At this step, the preparation was purifed 600-fold. This purified enzyme binds reversibly to columns containing concanavalin A covalently bound to Sepharose. Lipolytic activity was eluted from concanavalin A-Sepharose column with 0.2 m α-methyl-d-mannoside, 1.0 m NaCl and 0.005 m sodium-barbital, pH 7.0. At this stage, the enzyme was purified 2100-fold. Isoelectric focusing yielded a single major peak of activity with an isoelectric point of 4.0. Minimum molecular weight determination by gel filtration in buffers containing 1.0 m NaCl and by disc gel electrophoresis in sodium dodecyl sulfate yielded values of 62,000 and 60,000, respectively. The crude enzyme, and that eluted from heparin-Sepharose columns, did not show stimulation by heparin, whereas that obtained after isoelectric focusing exhibited a 60 to 100% stimulation at 22 µg of heparin per ml. Activation by dialyzed serum was dependent on the stage of purification. The crude enzyme showed a 20-fold stimulation by serum but showed some activity in its absence; that purified by isoelectric focusing exhibited a complete dependence on the presence of serum for hydrolysis of triolein emulsions stabilized with gum arabic. Of the three very low density lipoprotein apoproteins studied, only apoLp-Glu could substitute for serum as an activator. In the presence of serum in the assay system, apoLp-Ser was as potent an inhibitor of lipoprotein lipase as apoLp-Ala.

An apparently new form of abetalipoproteinemia, homozygous hypobetalipoproteinemia, was studied in progeny of a mating of two parents each heterozygous for familial hypobetalipoproteinemia, which was characterized by three-generation vertical transmission on both maternal and paternal sides of the family. The two children, with an apparent homozygous form of hypobetalipoproteinemia, had, in addition to abetalipoproteinemia, acanthocytosis, intestinal etpithelial and hepatic steatosis and steatorrhea. No low desity lipoprotein (LDL) was detected by immunodiffusion with antisera to LDL or apoLDL. The defects in apolipoproteins in these two children were the same as those reported in children with abetalipoproteinemia inherited as an autosomal recessive trait. The genetic defect of hypobetalipoproteinemia, when homozygous, can lead to all of the known clinical and biochemical features of abetalipoproteinemia.

This presentation will describe the use of diet, Clofibrate, and one other drug on the treatment of different kinds of familial hyperlipidemia. The latter disorders were classified as different types of hyperlipoproteinemia (1). The use of such grouping of patients has many distinct advantages. Probably these are greatest in pharmacologic or dietary experiments and the extension of the results to practical management of affected patients. This has been demonstrated in the systematic trial of Clofibrate (CPIB, Atromid-S®) on each of the five major types of familial hyperlipoproteinemia. The companion effects of drug and diet have also been carefully evaluated. A fuller description of each type of hyperlipoproteinemia is presented elsewhere in this symposium (2).