Preparation and properties of immobilized lipoprotein lipase

doi:10.1016/0005-2760(80)90211-8

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism

Volume 620, Issue 2, 7 November 1980, Pages 308-316

Biochimica et Biophy...

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Abstract

Purified bovine milk lipoprotein lipase has been covalently attached to CH-Sepharose with water-soluble carbodiimide. The immobilized enzyme retained enzymic activity and was stimulated 7-fold by the addition of human apolipo-protein C-II. Both [³H] heparin and ¹²⁵I-labeled apolipoprotein C-II bound to the immobilized enzyme; unlabeled heparin and apolipoprotein C-II competed for binding of their respective labeled compounds. Apolipoprotein C-II did not compete for binding of [³H] heparin and vice versa. Human apolipoprotein C-III did not bind to the immobilized enzyme nor did it compete for apolipoprotein C-II binding. We conclude from these studies that both apolipoprotein C-II and heparin interact with immobilized lipoprotein lipase and that they have different binding sites.

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To clarify the role of defective lipoprotein lipase (LPL) in hypertriglyceridemia, the LPL masses and LPL activities in post-heparin plasma (PHP) were studied in severe hypertriglyceridemias. The developed sandwich enzyme immunoassay for the LPL was sensitive from 0.5 to 20 ng/ml of LPL in human PHP. The plasma LP mass increased by heparin injection (30 USP units/kg) and was found to positively correlate with LPL activity. The mean LPL activity from PHP of normal controls was $2,960 ± 1,057 nmol/ml/h$ . The mean LPL masses from human pre- and 15-min post-heparin plasma from normal subjects were $25 ± 5 ng/ml$ and $224 ± 60 ng/ml$ , respectively. Thus the specific activity of LPL from PHP of normal controls was calculated to be 13.3 μmol FFA released/h/μg LPL. Among hypertriglyceridemic patients with over 1,000 mg/dl of serum triglyceride, the incidence of patients with LPL masses less than −2 standard deviations (S.D.) of those of average normal control subjects was found to be 27%. Seventy percent of patients showed specific activities within + 2 S.D. of those of average control LPL, and 30% showed significantly low specific activities less than −2 S.D. despite the fact that LPL masses were not less than −2 S.D. of the average normal controls. These results suggest that the evaluation of LPL masses in PHP would be useful for finding functionally defective LPL in patients with hypertriglyceridemia, and that up to 30% severe hypertriglyceridemias may have functionally defective LPL.
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Effect of heated skim milks on purified milk lipoprotein lipase activity against intralipid was correlated (.75) with their ability to affect spontaneous lipolysis in whole milks. Purified hydrophobic fraction of proteose-peptone that contained principally component 3 inhibited purified lipoprotein lipase activity. In contrast, fractions poor in proteose-peptone component 3 activated lipoprotein lipase activity. Study of interactions between activating and inhibiting effects suggested that the net effect of the milk proteose-peptone fraction on lipoprotein lipase activity resulted mainly from its inhibitory component, rather than from its activating components.
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^☆: The lipoprotein lipase referred to in this paper is EC 3.1.1.3. The recommended (I.U.B.) name of this enzyme is triacylglycerol lipase.

^∗: Present address: The Second Department of Internal Medicine, Chiba University School of Medicine, Chiba 280, Japan.

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Preparation and properties of immobilized lipoprotein lipase☆

Abstract

J. Lipid Res.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Clin. Chim. Acta

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Lipid Res.

J. Biol. Chem.

J. Biol. Chem.

J. Lipid Res.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

FEBS Lett.

FEBS Lett.

Annu. Rev. Biochem.