Identification of beta-galactosidase activity in purified bovine retinal rod outer segments.
We have identified beta-galactosidase activity in purified bovine rod outer segments (ROS), using rho-nitrophenyl-beta-D-galactopyranoside (PNPG) and chlorophenol red-beta-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 beta-galactosidase vs. other hydrolases (N-acetyl-beta-glucosaminidase, alpha- and beta-mannosidase, alpha-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 beta-galactosidase activity increased linearly with time, temperature, and protein concentration, and obeyed Michaelis-Menten kinetics with both substrates. For PNPG, Vmax approximately 88 nmol/h/mg protein and the apparent Km approximately 147 microM. For CPRG, Vmax approximately 33 nmol/h/mg protein and the apparent Km approximately 50 microM. ROS beta-galactosidase activity was affected by carbohydrates and their derivatives: glucose, fucose, sucrose, maltose and N-acetyl-galactosamine were found to stimulate the activity, while D-galactono-gamma-lactone and, to a lesser extent, D-galactose were inhibitory. The enzyme activity also was slightly stimulated by [Cl-] and markedly by dithiothreitol (DTT), while rho-chloro-mercuribenzoic acid (PCMB) and rho-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-beta-D-glucoside was slightly stimulatory.(ABSTRACT TRUNCATED AT 250 WORDS)
Prasad
V V
VV
Anheuser-Busch Eye Institute, Saint Louis University School of Medicine, MO 63104.
Fliesler
S J
SJ
D000818:Animals; D002417:Cattle; D002469:Cell Separation; D002733:Chlorophenols; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D005697:Galactosides; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009598:Nitrophenylgalactosides; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism*
D002733:Chlorophenols; D004791:Enzyme Inhibitors; D005697:Galactosides; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; C074184:chlorophenol red galactopyranoside; D001616:beta-Galactosidase