Identification of βgalactosidase activity in purified bovine retinal rod outer segments

We have identified βgalactosidase activity in purified bovine rod outer segments (ROS), using ρnitrophenyl-βD-galactopyrano-side (PNPG) and chlorophenol red-βD-galactopyranoside (CPRG) as substrates. This glycosylhydrolase activity did not appear to represent contamination from other retinal subcellular fractions, based upon the relative specific activities of βgalactosidase vs. other hydrolases (N-acetyl-βglucosaminidase, α and βmannosidase, αfucosidase, and acid phosphatase) in bovine retina and ROS homogenates. Using PNPG as a substrate, two pH optima were observed (at 3.5 and 5.5), while the hydrolysis of CPRG exhibited a single, broad pH optimum centered at 5.5. In contrast, hydrolysis of PNPG and CPRG by retinal homogenates exhibited single pH optima, at 3.5 and 5.5., respectively. ROS βgalactosidase activity increased linearly with time, temperature, and protein concentration, and obeyed Michaelis - Menten kinetics with both substrates. For PNPG, V_max ≈88 nmol/h/mg protein and the apparent K_m ≈147 μM. For CPRG, V_max ≈33 nmol/h/mg protein and the apparent K_m ≈50 μM. ROS βgalactosidase activity was affected by carbohydrates and their derivatives: glucose, fucose, sucrose, maltose and N-acetylgalactosamine were found to stimulate the activity, while D-galactono-γlactone and, to a lesser extent, D-galactose were inhibitory. The enzyme activity also was slightly stimulated by [CI^-] and markedly by dithiothreitol (DTT), while ρchloromercuribenzoic acid (PCMB) and ρhydroxymercuribenzoic acid (PHMB) inactivated the enzyme. In addition, the enzymatic activity was also found to be differentially sensitive to various anionic and nonionic detergents. However, n-octyl-βD-glucoside was slightly stimulatory. The potential roles of ROS βgalactosidase in late, post-translation processing of rhodopsin (and/or other galactosylated glycoproteins) and in ROS disc membrane morphogenesis are discussed.