PURIFICATION AND ENZYMATIC CHARACTERIZATION OF RECOMBINANT PROHORMONECONVERTASE-2 - STABILIZATION OF ACTIVITY BY 21 KDA 7B2

Although previous efforts to produce significant quantities of purifie d prohormone convertase 2 from either recombinant or natural sources h ave been unsuccessful, our recent finding that the neuroendocrine poly peptide 7B2 is necessary for the biosynthesis of enzymatically active prohormone convertase 2 (PC2) has enabled us to obtain active recombin ant enzyme from the conditioned medium of PC2-producing CHO cells supe rtransfected. with cDNA coding for 21 kDa 7B2, The recombinant enzyme was purified to apparent homogeneity, with a 40% recovery, in milligra m quantities, Two protein bands of M(r)s 71 and 75 kDa were observed a fter SDS-PAGE followed by either Coomassie staining or Western blottin g with PC2 antiserum. Spontaneous conversion of the 71- and 75-kDa spe cies to the 66-kDa form occurred during incubation at pH 5.0; the degr ee of conversion correlated with a dramatic increase in activity. K(m) s of 124 and 131 mu M and K(cat)s of 0.49 and 0.81 s(-1) were obtained far the substrates Cbz-Arg-Ser-Lys-Arg-AMC and Pyr-Arg-Thr-Lys-Arg-AM C, respectively. The pH optimum was 5.0, and the enzyme was inhibited by h7B2(155-185') p-CMS, and EDTA but not by other inhibitors tested, Interestingly, 21 kDa 7B2 was observed to copurify with the enzyme in a molar ratio of about 1:100 (7B2:PC2); Prior addition of recombinant 21 kDa 7B2 to activated 66 kDa PC2 provided significant protection aga inst thermal denaturation. When coassociated 7B2 was mostly removed fr om activated PC2 through gel filtration, subsequent addition of recomb inant 7B2 exerted a significant stabilizing effect on enzyme activity, Millimolar Ca2+ and pHs between 5 and 6 were required to observe this effect. Since these conditions resemble those thought to occur within secretory granules, and since 21 kDa 7B2 represents a stored secretor y granule protein, our data suggest a physiological role for 21 kDa 7B 2 in the stabilization of PC2 activity. (C) 1996 Academic Press, Inc.