title: Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2.
abstract: 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.
authors.affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, U.K.
authors.forename: E M
authors.initials: EM
authors.lastname: Bailyes
authors.forename: K I
authors.initials: KI
authors.lastname: Shennan
authors.forename: E F
authors.initials: EF
authors.lastname: Usac
authors.forename: S D
authors.initials: SD
authors.lastname: Arden
authors.forename: P C
authors.initials: PC
authors.lastname: Guest
authors.forename: K
authors.initials: K
authors.lastname: Docherty
authors.forename: J C
authors.initials: JC
authors.lastname: Hutton
mesh_terms: 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
chemical_list: D007328:Insulin; D011994:Recombinant Proteins; D010450:Endopeptidases; D013381:Subtilisins; D045706:Proprotein Convertase 2; D002118:Calcium