An assay for proteoglycanase (PGase) and it's application to articular chondrocyte cultures.

Abstract

The role of PGase in the degradation of cartilage proteoglycans (PG) is still unclear. Although several assays are available, there still exists some problems in the sensitivity, specificity and simplicity of these procedures. We therefore decided to develop an improved assay for PGase in order to detect small quantities of enzyme activity in the culture medium of articular chondrocytes. As substrates of PGase, we prepared fluorescein isothiocyanate labelled core protein (FITC-PGC) and fluorescein amine labelled hyaluronic acid-proteoglycan subunit complex (FAHA-PGS) from new born calf costal cartilages. PGase activity was estimated from the change of elution profile of Sepharose CL-4B chromatography of fluorescein labelled substrates after incubation with the activated enzyme solution. For measurement of PGase in large numbers of samples, we developed the microultrafiltration method using ultrafiltration membrane (MPS-I kit, Amicon). The concentrated media of articular chondrocyte cultures was used for the investigation of the nature of PGase(s) derived from articular chondrocytes. It was found that APMA, CoCl2, and CaCl2 were needed for activation of the enzyme. Sephadex-G75 gel chromatography of the crude enzyme solution suggests that the molecular weight of PGase from rabbit articular chondrocytes was approximately 5 X 10(4) daltons. From the results of gel chromatography of the digested FITC-PGC and FAHA-PGS it was concluded that one of the sites of attack of the chondrocyte secreted PGase might be near the hyaluronic acid binding region of core protein. The effects of various agents on the production of PGase were also studied. Interleukin-I was found to stimulate PGase production in cultured articular chondrocytes.