Production, purification, and characterization of recombinant prohormone convertase 5 from baculovirus-infected insect cells.
The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual pro-hormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a K(m) in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms.
Cain
Brian M
BM
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA. bcain@opal.tufts.edu
Vishnuvardhan
Daesety
D
Wang
Wenge
W
Foulon
Thierry
T
Cadel
Sandrine
S
Cohen
Paul
P
Beinfeld
Margery C
MC
D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D051379:Mice; D045702:Proprotein Convertase 5; D011480:Protease Inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D018411:Spodoptera / Q000821:virology
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D045702:Proprotein Convertase 5; D012697:Serine Endopeptidases; C473805:propeptide convertase 5, mouse