Characterization of beta-glucuronidase in the retinal pigment epithelium.

PURPOSE

To study the biochemical and molecular characteristics of the lysosomal enzyme beta-glucuronidase (GUSB) in the retinal pigment epithelium (RPE) and other tissues of different species.

METHODS

Freshly isolated and cultured cells were harvested, and GUSB activity was measured fluorimetrically in cell homogenates or tissue culture media using the synthetic substrate 4-methyl-umbelliferyl beta-D-glucuronide (4-MUG). The temperature and pH optima, and thermal stability of GUSB in the RPE and fibroblasts were established. Distribution of glycosaminoglycans (GAGs), the natural substrates of GUSB, in the RPE and fibroblast cell layer and media was examined by cellulose acetate electrophoresis following 72 h of metabolic labeling with Na2(35)SO4. Total or poly A(+)-RNA isolated from cells or tissues of different species were examined in Northern blots to identify GUSB mRNA transcripts.

RESULTS

Among all the species, the activity of GUSB and its mRNA level was found to be consistently high in RPE cells. In RPE cultures, the activity was detected in the cell layer and the media, and the activity decreased in both compartments with serial passage. While the temperature and pH optima for the enzyme activity was similar across the species, the thermal stability was remarkably different. The GAG profiles in RPE cells were different from fibroblasts. Supplementation of the cultured cells with selected GAGs moderately increased the GUSB activity. A GUSB transcript was detected in all the tissues examined. In man, mouse, dog, and cat the size of the transcript was 2.4 kb, while the rat GUSB transcript was 2.7 kb.

CONCLUSIONS

The ubiquitous distribution of GUSB was evident from the biochemical and molecular studies. Presence of a high level of GUSB activity in the RPE makes it an ideal model for studies of this enzyme both in normal as well as in diseases resulting in GUSB deficiency.