ENZYMATIC CHARACTERIZATION OF IMMUNOPURIFIED PROHORMONE CONVERTASE .2. POTENT INHIBITION BY A 7B2 PEPTIDE FRAGMENT

Prohormone convertases (PCs) are thought to mediate the controlled pro teolysis of prohormones and neuropeptide precursors. While recombinant PC1 and furin are currently available, thus far it has not been possi ble to produce recombinant PC2. We have used conditioned medium obtain ed from the mouse insulinoma cell Line beta TC3 to generate a working preparation of enzymatically active PC2 through immunopurification. Im munopurified PC2 cleaved the fluorogenic substrate Cbz-Aro-Ser-Lys-Arg -AMC in a time- and calcium-dependent manner. It was half-maximally st imulated at 75 mu M Ca2+, had an optimum pH of 5, and exhibited PCMS a nd EDTA sensitivity similar to that reported for furin and PC1. The ti ght-binding inhibitor 27 kDa 7B2 was used to calculate the K-d for thi s inhibitor and the active enzyme concentration. The K-d was 7.3 +/- 1 .7 nM, and the turnover rate of PC2 was 5.2 molecules substrate per en zyme molecule per minute. The specific activity was 4.9 nmol/mu g/h (a ssuming a molecular mass for PC2 of 64 kDa). The enzyme preparation wa s able to cleave recombinant proenkephalin at at least four of the exp ected paired basic sites in the absence, but not in the presence, of 2 7 kDa 7B2. Since 21 kDa 7B2 is functionally inactive as a proteinase i nhibitor, we examined the inhibitory activity of the carboxy-terminal portion of 27 kDa 7B2 (7B2 CT-peptide). Synthetic peptides were used t o demonstrate that the 7B2 CT-peptide (a) represents a potent inhibito r of PC2 (K-i = 57 nM), (b) can block the conversion of proPC2 to PC2, and (c) can block the PC2-mediated conversion of proenkephalin to sma ller peptide fragments. This peptide thus may represent a useful tool in the study of prohormone conversion.