Abstract
Human lipoprotein lipase and hepatic triglyceride lipase were purified to homogeneity from post-heparin plasma. These enzymes were purified 250,000- and 100,000-fold with yields of 27 +/- 15 and 19 +/- 6%, respectively. Molecular weight determination by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents yielded Mr of 60,500 +/- 1,800 and 65,200 +/- 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. These lipase preparations were shown to be free of detectable antithrombin by measuring its activity and by probing of Western blots of lipases with a monospecific antibody against antithrombin. In additions, probing of Western blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. Four stable hybridoma-producing distinct monoclonal antibodies (mAb) to hepatic triglyceride lipase were isolated. The specificity of one mAb, HL3-5, was established by its ability to immunoprecipitate hepatic triglyceride lipase catalytic activity. Interaction of HL3-5 with this lipase did not inhibit catalytic activity. The three other mAb interacted with hepatic triglyceride lipase only after denaturation of the enzyme with detergents. The relatedness of these two enzymes was examined by comparing under the same conditions the thermal inactivation, the sensitivity to sulfhydryl and reducing agents, amino acid composition, and the mobility of peptide fragments generated by cyanogen bromide cleavage. The results of these studies strongly support the view that the two enzymes are different proteins. Immunological studies confirm this conclusion. Four mAb to hepatic triglyceride lipase did not interact with lipoprotein lipase in Western blots, enzyme-linked immunosorbent assay, and immunoprecipitation experiments. These immunological studies demonstrate that several epitopes of the hepatic triglyceride lipase protein moiety are not present in the lipoprotein lipase molecule.
Highlights
These lipase preparations were shown to be free of detectable antithrombinby measuring its activity and endothelial cells [3]
There are more than 20 plasma proteins which have been reported[31]to bind tightly to heparin-Sepharose 4B, a matrix commonly employed as a single purification step for the purification of plasma lipases
Antithrombin presentin plasmaa t a concentration of 0.2 mg/ ml has been a common contaminant in purified plasma lipase preaprations.Ostlund-Lindqvist and Boberg [11] were the firstauthorsto call attentiontothecontamination with antithrombin of plasma lipases purified by a single step on heparin-Sepharose columns
Summary
100 ml of plasma, 67 ml of 0.65 M NaC1, 30% glycerol, 5 mM phosphate, p H 7.0, were added.Theantiproteolyticagent, Trasylol (FBA Pharmaceuticals)w, as included toa final concentration of 100 units/ml This diluted plawsmasa loaded on two heparin-Sepharose 4B columns (X5 15 cm) in parallel at a combined rate of 200 ml/h. Eight-five per cent of elutes a t 0.75 M NaC1, lipolytic activity (lipoprotein lipase) in the antithrombin was recovered in the column washing with the second is sensitive to1M NaCl and elutesa t 1.5 M NaCl the NaCl gradient, and no protein could be detected in the (data not shown). The mean overall recovery for the purification of hepatic triglyceride lipase was 19 f 6%
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