Release of the prohormone convertase PC1 from AtT-20 cells.
AtT-20 cells are known to synthesize two molecular weight forms of the prohormone converting enzyme PC1 with molecular masses of 87 and 66 kDa. In this study we have analyzed basal and stimulated secretion of these proteins. Western blot results show that basal secretion medium of cultured AtT-20 cells contained low concentrations of both the 87 and 66 kDa forms of PC1 with the former protein predominant. During the stimulation period with CRF, cAMP and cAMP + BaCl2, increased release of both proteins was observed, but the 66 kDa protein predominated. Secretion medium obtained from stimulated and unstimulated cells was enzymatically active against the Cbz-Arg-Ser-Lys-Arg-AMC fluorogenic substrate as well as against 35S-proenkephalin. This activity was Ca+2 dependent and was inhibited by the chelating agent EDTA. The activity was insensitive to acid and thiol proteinase inhibitors as well as to N-alpha-p-tosyl-L-Lys-chloromethyl ketone; it was slightly sensitive to phenylmethyl sulfonyl fluoride and was strongly inhibited by D-Tyr-Ala-Lys-Arg-chloromethyl ketone. This inhibitor profile exhibits strong similarities to furin and kexin. After partial purification of medium by gel filtration chromatography, a portion of the enzymatic activity and immunoreactivity for both 87 kDa and 66 kDa proteins eluted with an apparent molecular weight of 400 kDa (suggesting aggregation); however the highest activity appeared in the elution position of the 66 kDa monomer. When the 87 kDa protein was removed from the medium by means of an affinity column containing an antibody against the carboxyl terminal portion of PC1, the column flow-through, which included the 66 kDa protein, still remained enzymatically active. These data support the notion that the 66 kDa protein, which is the most concentrated PC1 product stored in AtT-20 cells and is released during stimulation, is enzymatically active.
Vindrola
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Lindberg
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D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D017609:Barium Compounds / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010911:Pituitary Neoplasms; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D017609:Barium Compounds; D002712:Chlorides; D007527:Isoenzymes; D009842:Oligopeptides; D011480:Protease Inhibitors; C024986:barium chloride; D003346:Corticotropin-Releasing Hormone; D004492:Edetic Acid; D000242:Cyclic AMP; D043484:Proprotein Convertases; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D016282:Aspartic Acid Endopeptidases; D002118:Calcium