Identification of β-galactosidase activity in purified bovine retinal rod outer segments

We have identified β-galactosidase activity in purified bovine rod outer segments (ROS), using -nitrophenyl-β-D-galactopyrano-side (PNPG) and chlorophenol red-β-D-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 β-galactosidase vs. other hydrolases (N-acetyl-β-glucosaminidase, α- and β-mannosidase, α-fucosidase, 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 β-galactosidase activity increased linearly with time, temperature, and protein concentration, and obeyed Michaelis - Menten kinetics with both substrates. For PNPG, Vmax 88 nmol/h/mg protein and the apparent Km 147 μM. For CPRG, Vmax 33 nmol/h/mg protein and the apparent Km 50 μM. ROS β-galactosidase activity was affected by carbohydrates and their derivatives: glucose, fucose, sucrose, maltose and N-acetylgalactosamine were found to stimulate the activity, while D-galactono-γ-lactone and, to a lesser extent, D-galactose were inhibitory. The enzyme activity also was slightly stimulated by [CI−] and markedly by dithiothreitol (DTT), while -chloromercuribenzoic acid (PCMB) and -hydroxymercuribenzoic 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-β-D-glucoside was slightly stimulatory. The potential roles of ROS β-galactosidase in late, post-translation processing of rhodopsin (and/or other galactosylated glycoproteins) and in ROS disc membrane morphogenesis are discussed.