Cleavage of CCK 33 by recombinant PC2 in vitro.
The specificity and kinetic properties of CCK 33 cleavage by recombinant prohormone convertase 2 (PC2) was investigated using a combination of Sephadex G-50 chromatography, HLPC and RIA methods. It is shown that CCK 33 can be cleaved effectively by PC2 to form CCK 8, the reaction of which has a K(m) of 104.8 microM. No CCK 22 or other products were detected and the reaction is completely inhibited by the PC2 inhibitor, p-CMS (p-chloromercuriphenylsulfonic acid), suggesting that the cleavage is PC2 specific. The time course of this reaction shows an initial lag phase followed by a rapid increase in velocity consistent with a previously reported spontaneous transformation from a 71 kDa relatively inactive form into a more active form of 62 kDa (1). PC2 did not cleave recombinant pro CCK or CCK 1-21. These results demonstrate that PC2, an enzyme usually associated with dibasic cleavages, can cleave easily at single basic residues.
Wang
W
W
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Beinfeld
M C
MC
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002766:Cholecystokinin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D045706:Proprotein Convertase 2; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012844:Sincalide / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; D002731:4-Chloromercuribenzenesulfonate; C060249:procholecystokinin; D002766:Cholecystokinin; C044926:cholecystokinin 22 C-terminal fragment; D013381:Subtilisins; D045706:Proprotein Convertase 2; D012844:Sincalide