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Published on: January 7, 2013
Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2
E M Bailyes1, K I Shennan, E F Usac
1Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, U.K.
The Biochemical Journal
|July 15, 1995
Summary
This study compares human prohormone convertase PC2 (proprotein convertase 2) expressed in Xenopus oocytes with rat insulin secretory granule endopeptidase. Recombinant PC2 shows similar substrate specificity but different calcium sensitivities, suggesting post-translational modifications influence enzyme activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Endocrinology
Background:
- Prohormone convertase PC2 (proprotein convertase 2) is a key enzyme in hormone processing.
- Understanding PC2's properties is crucial for deciphering insulin biosynthesis and secretion.
- Previous studies identified a Type-2 endopeptidase in rat insulin secretory granules as PC2.
Purpose of the Study:
- To compare the biochemical properties of recombinant human prohormone convertase PC2 (PC2) with the endogenous Type-2 endopeptidase from rat insulin secretory granules.
- To investigate the substrate specificity, pH, and calcium (Ca2+) dependency of both enzyme forms.
- To analyze the molecular forms and post-translational modifications of recombinant and endogenous PC2.
Main Methods:
- Expression of human prohormone convertase PC2 in Xenopus oocytes.
- Enzyme activity assays using modified proinsulin substrates.
- Determination of substrate specificity, pH optimum, and Ca2+ kinetics.
- Analysis of molecular forms using SDS-PAGE and Western blotting with C-terminal specific antibodies.
- Assessment of N-glycosylation patterns.
Main Results:
- Recombinant PC2 exhibited identical substrate specificity to the rat Type-2 endopeptidase, efficiently cleaving human des-31,32-proinsulin to insulin.
- Maximal activity for recombinant PC2 occurred at 5-7 mM Ca2+, significantly higher than the 0.5-1 mM Ca2+ for the Type-2 endopeptidase.
- Both enzymes showed a pH optimum of 5.0-5.5, but the endogenous enzyme had broader pH activity.
- Recombinant PC2 existed as 71 kDa and 68 kDa forms, while endogenous PC2 comprised 64-68 kDa forms, with differences potentially due to N-glycosylation and proteolytic processing.
- Discrepancies in Ca2+ and pH sensitivity were not fully explained by observed post-translational modifications, suggesting a potential role for carbohydrate modulation.
Conclusions:
- Recombinant human PC2 expressed in Xenopus oocytes mirrors the substrate specificity of the endogenous rat enzyme.
- Significant differences in Ca2+ and pH sensitivity exist between recombinant and endogenous PC2, potentially influenced by post-translational modifications like N-glycosylation.
- Further investigation is needed to elucidate the precise mechanisms modulating PC2 activity, including the role of carbohydrate moieties.
