In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity.
Previous studies have established that human hepatocellular carcinoma cells (Hep G2) secrete into serum-free medium the pro form of apolipoprotein A-I (proapo-A-I) suggesting that its conversion to mature apo-A-I occurs after secretion. In order to assess the mode and site of proapo-A-I to apo-A-I conversion, we incubated the medium from [3H]proline-labeled Hep G2 cells with either human plasma, serum, lymph, or fractions thereof obtained by density gradient ultracentrifugation. The conversion was monitored by two-dimensional gel electrophoresis and by Edman degradation. Human plasma, serum, or mesenteric lymph all induced proapo-A-I to apo-A-I conversion; this was time dependent, unaffected by the serine protease inhibitor phenylmethylsulfonyl fluoride and inhibited by EDTA. Purified radiolabeled proapo-A-I bound to lymph chylomicrons and plasma high density lipoproteins. The converting enzyme was associated with both of these particles. Activity was also found in the d greater than 1.21-g/ml fraction and may have been derived from high density lipoprotein after displacement by high salts and/or ultracentrifugal force. We conclude that the conversion of proapo-A-I to apo-A-I occurs extracellularly and is probably effected by a metallo-enzyme which may act at the amphiphilic surface of either chylomicrons or high density lipoproteins.
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I; D001053:Apolipoproteins / Q000235:genetics* / Q000302:isolation & purification; D001054:Apolipoproteins A; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood; D053440:Lipoproteins, HDL3; D008113:Liver Neoplasms / Q000378:metabolism; D008196:Lymph / Q000378:metabolism; D008666:Metalloendopeptidases; D011498:Protein Precursors; D011499:Protein Processing, Post-Translational
D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D001054:Apolipoproteins A; D008075:Lipoproteins, HDL; D053440:Lipoproteins, HDL3; D011498:Protein Precursors; D010450:Endopeptidases; D008666:Metalloendopeptidases