Expression and sorting of rat plasma kallikrein in POMC-producing AtT-20 cells.
A vaccinia virus (VV) vector was used to express rat plasma kallikrein (rPK) in the constitutively secreting cells, BSC-40, and in the endocrine regulated cells, AtT-20. Using a specific rPK antibody and a fluorogenic substrate, Phe-Phe-Arg-AMC, we demonstrated that in both cell lines VV infections resulted in the synthesis of an immunoreactive enzyme predominantly present as a zymogen which can be activated with trypsin. Stimulation of VV:rPK-infected AtT-20 cells with either 5mM 8-bromo-cAMP or 56 mM KCl resulted in a different pattern of rPK and ACTH secretion, strongly suggesting that rPK follows the constitutive secretory pathway. Finally, the 10% rPK activity found within AtT-20 cell extracts had no effect on pro-opiomelanocortin (POMC) processing either intracellularly or extracellularly. The above data show that the biosynthetic machinery of both cell lines analyzed does not allow the efficient activation of plasma prekallikrein. Finally, despite the PK's demonstrated ability to cleave various hormone precursors in vitro at pairs of basic residues, in vivo, we did not obtain evidence that this hepatic enzyme can also act as an intracellular pro-protein processing enzyme.
Sawyer
N
N
J.A. DeSève Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Quebec, Canada.
Rondeau
N
N
Chrétien
M
M
Seidah
N G
NG
D000324:Adrenocorticotropic Hormone / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D007610:Kallikreins / Q000235:genetics / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000235:genetics
D000906:Antibodies; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D007610:Kallikreins