DIFFERENCES BETWEEN THE CATALYTIC PROPERTIES OF RECOMBINANT HUMAN PC2AND ENDOGENOUS RAT PC2

Human prohormone convertase PC2 was expressed in Xenopus oocytes and i ts properties were compared with those of the Type-2 endopeptidase of rat insulin secretory granules, previously identified as PC2 [Bennett, Bailyes, Nielson, Guest, Rutherford, Arden and Hutton (1992) J. Biol. Chem. 267, 15229-15236], Recombinant PC2 had the same substrate speci ficity as the Type-2 endopeptidase, cleaving at the CA-junction (Lys(6 4), Arg(65)) Of human des-31,32-proinsulin to generate insulin; little activity was found toward human des-64,65-proinsulin or proinsulin it self. Recombinant PC2 was maximally active in 5-7 mM Ca2+ (K-0.5 = 1.6 mM) whereas the Type-2 endopeptidase was maximally active in 0.5-1 mM Ca2+ (K-0.5 = 40 mu M). Both enzymes had a pH optimum of 5.0-5.5 but the Type-2 endopeptidase was active over a wider pH range. Two molecul ar forms of recombinant PC2 (71 kDa and 68 kDa) were found, both had a n intact c-terminus but differed by the presence of the propeptide. Th e endogenous PC2 comprised several overlapping forms (size range 64-68 kDa), approximately two-thirds of which lacked C-terminal immunoreact ivity. Part of the size difference between recombinant and endogenous PC2 was attributable to differences in N-glycosylation. The different post-translational proteolytic modifications of recombinant and endoge nous PC2 did not account for the different pH and Ca2+ sensitivities s hown by the enzymes. A modulating effect of carbohydrate on enzyme act ivity could not be excluded.