Rapid and simple isolation procedure for lipoprotein lipase from human milk.
Lipoprotein lipase (LPL) is an important enzyme in lipid and energy metabolism of all vertebrates. Measurement of its activity in human postheparin plasma has become a standard procedure for diagnosis of Type I hyperlipoproteinemia and other types of hypertriglyceridemias. This paper presents a rapid and simple purification procedure for human lipoprotein lipase and the production of specific polyclonal antibodies. In the isolation procedure, the fat moiety of human milk obtained by centrifugation was delipidated and a buffer-extractable fraction chromatographed sequentially on heparin-Sepharose and phenyl-Sepharose. This three-step procedure provides a high yield of apparently pure LPL with very high specific activity against radiolabeled triacylglycerol substrates. The apparent molecular weight of LPL on SDS-PAGE was 60 kDa. Amino acid analysis and NH2-terminal sequencing proved the identity and the apparent homogeneity of the isolated enzyme. alpha-Lactoferrin and antithrombin III, common contaminants in earlier isolation procedures, were not detectable immunologically. Purified LPL was used to produce in the rabbit a specific polyclonal antiserum that inhibited LPL activity from human postheparin plasma and other tissues. In postheparin plasma from normal individuals, anti-LPL IgG was used in Western blotting to show LPL protein. In preheparin plasma, or in certain patients with Type I hyperlipoproteinemia, no specific signal was detected. The improved purification procedure presented here allows the rapid isolation of human LPL and production of antibodies to the protein, both of which will greatly facilitate future studies of this important enzyme.
Zechner
R
R
Institute of Medical Biochemistry, Karl Franzens University Graz, Austria.
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002417:Cattle; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006801:Humans; D008071:Lipoprotein Lipase / Q000097:blood / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D008895:Milk, Human / Q000201:enzymology*; D008969:Molecular Sequence Data; D011817:Rabbits
D000906:Antibodies; D008071:Lipoprotein Lipase