Fluorometric assay of a calcium-dependent, paired-basic processing endopeptidase present in insulinoma granules.
A novel fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC (RSKR-AMC) was used to characterize Ca(++)-activated proteolytic activity present in purified insulinoma secretory granules. Secretory granules efficiently cleaved this substrate in a time- and protein-dependent manner; the hydrolysis rate was between 2 and 4 pmol/min/ug of protein, with an apparent Km of 55 microM. Greater than 90% of the activity against this substrate was dependent on the presence of Ca++, with half-maximal stimulation obtained at 100 microM Ca++. The pH optimum of enzymatic activity was 5.5-6, and the profile of inhibition by various proteinase inhibitors was similar to that previously described for the type I and II proinsulin processing enzymes. These biochemical characteristics and co-elution of the RSKR-AMC processing activity with the type II endopeptidase activity on anion-exchange chromatography suggest that the new assay selectively detects the Lys-Arg-directed, or type II, proinsulin processing endopeptidase. This fluorogenic assay is more quantitative, sensitive and rapid than methods previously used, and therefore presents a significant improvement for the study of similar Ca(++)-activated processing endopeptidases.
Lindberg
I
I
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Lincoln
B
B
Rhodes
C J
CJ
D000599:Amino Acids, Diamino / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000201:enzymology*; D010450:Endopeptidases / Q000032:analysis* / Q000187:drug effects / Q000302:isolation & purification; D005470:Fluorometry / Q000379:methods*; D007340:Insulinoma / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D051381:Rats
D000599:Amino Acids, Diamino; D002412:Cations; D009842:Oligopeptides; D011480:Protease Inhibitors; D010450:Endopeptidases; D002118:Calcium