A new fluorometric method for measuring the action of C apolipoproteins on milk lipoprotein lipase.
Monolayer vesicles containing pyrene-labelled nonanoyltriglyceride (1-2 ditetradecyl 3-pyrene nonanoyl glyceride) were used as a substrate to measure bovine milk lipoprotein lipase activity. The activation of lipoprotein lipase by synthetic fragments of apolipoprotein C II and apo C III was measured. Fragments 30-78 and 43-78 had actions similar to that of the entire apo C II. Fragments 50-78 and 55-78 were 50% active, fragment 60-78 was 10% active and fragment 66-78 was inactive. Thus the activating capacity depended on the length of the carboxyterminal fragment. Replacing tyrosine 62 in apo C II by glycine removed all lipoprotein lipase activating capacity, while making Tyr 62 less accessible for binding to lipids and enzyme decreased apo C II activating capacity. Apo C III1 inhibited both basal lipoprotein lipase activity (no apo C II) and lipoprotein lipase activated by apo C II. Apo C III, fragment A (1-40) which did not bind lipids, had no inhibitory effect, while fragment B(41-79) had the same effect as whole apo C III,. Apo AI, AII and C I also inhibited lipoprotein lipase. The fluorometric assay is easy to perform, and suitable for metabolic studies such as fatty-acid exchanges between lipoproteins, as it produces no alteration in the reaction products. It also avoids the use of a radio-labelled substrate.
Lambert
D A
DA
I.N.S.E.RM. U. 308, Faculté de Médecine, Vandoeuvre, France.
Catapano
A L
AL
Smith
L C
LC
Sparrow
J T
JT
Gotto
A M
AM
D000818:Animals; D053304:Apolipoprotein C-II; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D005470:Fluorometry / Q000379:methods*; D006868:Hydrolysis / Q000187:drug effects; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008894:Milk Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D011721:Pyrenes; D014280:Triglycerides / Q000378:metabolism
D053304:Apolipoprotein C-II; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D008894:Milk Proteins; D010455:Peptides; D011721:Pyrenes; D014280:Triglycerides; D008071:Lipoprotein Lipase