Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. I. Characterization of a specific antibody and relationships between the intracellular and secreted pools of the enzyme.
Polyclonal antibodies have been raised in rabbits against homogeneous lipoprotein lipase (LPL) purified from the media of adipose 3T3-F442A cells. The antibody is able to inhibit the apolipoprotein C-II-dependent activity of LPL, to immunoprecipitate LPL under nondenaturating conditions from media and cellular extracts. A dot-blot immunoassay of secreted LPL is also described (range 0.1-0.7 milliunits). The secretion potential mu, taken as the ratio of total releasable activity or antigen to initial cellular activity or antigen, was determined. This was shown in cells treated with heparin and cycloheximide to be equal to 1 for LPL antigen but significantly greater than 1 for LPL activity assayed under standard conditions. No LPL was actually degraded within the cells. A dramatic enhancement of the intracellular activity was induced by a mere dilution of detergent-treated cell lysates with no change in LPL antigen. The total intracellular activity reached a plateau at a value which now became identical to that obtained in the medium of cells exposed to heparin and cycloheximide. The existence of an inhibitor of LPL activity has been excluded as well as that of an increase in the catalytic activity of LPL during its secretion, before or after exposure to heparin. Our results indicate a systematic underestimation of LPL intracellular activity and suggest that LPL is present within intracellular cisternae in a cryptic state. This potential activity can be fully unmasked in vitro. In agreement with other data (Vannier, C., and Ailhaud, G., (1989) J. Biol. Chem. 264, 13206-13216), our results appear to exclude the existence of a reservoir of catalytically inactive LPL molecules within adipose cells.
Vannier
C
C
Centre de Biochimie, Centre National de la Recherche Scientifique (UPR 7300), Nice, France.
Deslex
S
S
Pradines-Figuères
A
A
Ailhaud
G
G
D000273:Adipose Tissue / Q000201:enzymology* / Q000378:metabolism; D000818:Animals; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D002417:Cattle; D002460:Cell Line; D003593:Cytoplasm / Q000201:enzymology* / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D015151:Immunoblotting; D008071:Lipoprotein Lipase / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D011233:Precipitin Tests
D000941:Antigens; D006493:Heparin; D008071:Lipoprotein Lipase