Purification and characterization of lipoprotein lipase from human postheparin plasma and its comparison with purified bovine milk lipoprotein lipase.
Lipoprotein lipase [EC 3.1.1.34, LpL] was purified from human postheparin plasma (PHP) almost to homogeneity (a 210,000-fold purification) using columns of heparin-Sepharose, hydroxylapatite, and concanavalin A-Sepharose, and its properties were compared with the purified bovine milk LpL. The specific activity of the PHP-LpL was 26 mmol free fatty acids (FFA)/h/mg of protein at 37 degrees C; close to that of bovine milk LpL (35 mmol FFA/h/mg). For both enzyme preparations, the pH optimum (about 8.7) and the inhibition by sodium chloride were almost the same. The apparent Michaelis constants were also similar; 2.5 mM for human PHP-LpL and 2.1 mM for bovine milk LpL. The apparent molecular weight of the purified human PHP-LpL was 58,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, slightly larger than that of the bovine milk LpL (56,000). Although the amino acid composition of the two LpL preparations had only slight differences, antibody raised against bovine milk LpL cross-reacted very weakly with purified human PHP-LpL. With 1% bovine serum albumin, bovine milk LpL was highly stable, but the human PHP-LpL was unstable; it lost 60% of its activity within 60 min at 0 degrees C. In the absence of apolipoprotein C-II (apo C-II), the activity of human PHP-LpL was very weak. However, human PHP-LpL was activated by apo C-II more strongly than bovine milk LpL; the fold activation of human PHP-LpL by apo C-II was 7-8 times that of bovine milk LpL. The apparent Km value of human PHP-LpL for apo C-II (1.00 +/- 0.58 microM) was larger than that of bovine milk LpL (0.15 +/- 0.03 microM).
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D005260:Female; D006493:Heparin; D006801:Humans; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000097:blood / Q000302:isolation & purification*; D008892:Milk / Q000201:enzymology*; D008970:Molecular Weight; D010949:Plasma
D000596:Amino Acids; D005230:Fatty Acids, Nonesterified; D006493:Heparin; D008071:Lipoprotein Lipase