Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2.
Human prohormone convertase PC2 was expressed in Xenopus oocytes and its 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 specificity as the Type-2 endopeptidase, cleaving at the CA-junction (Lys64, Arg65) of human des-31,32-proinsulin to generate insulin; little activity was found toward human des-64,65-proinsulin or proinsulin itself. Recombinant PC2 was maximally active in 5-7 mM Ca2+ (K0.5 = 1.6 mM) whereas the Type-2 endopeptidase was maximally active in 0.5-1 mM Ca2+ (K0.5 = 40 microM). Both enzymes had a pH optimum of 5.0-5.5 but the Type-2 endopeptidase was active over a wider pH range. Two molecular forms of recombinant PC2 (71 kDa and 68 kDa) were found, both had an intact C-terminus but differed by the presence of the propeptide. The endogenous PC2 comprised several overlapping forms (size range 64-68 kDa), approximately two-thirds of which lacked C-terminal immunoreactivity. 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 endogenous PC2 did not account for the different pH and Ca2+ sensitivities shown by the enzymes. A modulating effect of carbohydrate on enzyme activity could not be excluded.
Bailyes
E M
EM
Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, U.K.
Shennan
K I
KI
Usac
E F
EF
Arden
S D
SD
Guest
P C
PC
Docherty
K
K
Hutton
J C
JC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D045706:Proprotein Convertase 2; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D007328:Insulin; D011994:Recombinant Proteins; D010450:Endopeptidases; D013381:Subtilisins; D045706:Proprotein Convertase 2; D002118:Calcium