Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2
| Autor: | Kevin Docherty, Kathleen I.J. Shennan, John C. Hutton, E. F. Usac, E M Bailyes, Paul C. Guest, Susan D. Arden |
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| Rok vydání: | 1995 |
| Předmět: |
endocrine system
Xenopus Prohormone convertase Proprotein convertase 2 Endogeny Biology Biochemistry Catalysis Substrate Specificity law.invention law Endopeptidases Animals Humans Insulin Subtilisins Cloning Molecular Protein precursor Molecular Biology Proinsulin chemistry.chemical_classification Cell Biology Hydrogen-Ion Concentration Molecular biology Recombinant Proteins Endopeptidase Rats Proprotein Convertase 2 Enzyme chemistry Recombinant DNA Calcium Research Article |
| Zdroj: | Biochemical Journal. 309:587-594 |
| ISSN: | 1470-8728 0264-6021 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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