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.