Structural equivalents of latency for lysosome hydrolases.
1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: beta-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl beta-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of beta-glucuronidase and beta-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the beta-glycerophosphate concentration. The fractional free activity of beta-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free beta-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with beta-glycerophosphate or p-nitrophenyl beta-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of "intact" lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl beta-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane.
D000118:Acetylglucosaminidase; D000135:Acid Phosphatase; D000818:Animals; D001665:Binding Sites; D004072:Digitonin / Q000494:pharmacology; D005696:Galactosidases; D005966:Glucuronidase; D005994:Glycerophosphates / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D008566:Membranes / Q000201:enzymology; D009598:Nitrophenylgalactosides / Q000378:metabolism; D009995:Osmosis; D010042:Ouabain / Q000494:pharmacology; D010539:Permeability; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats
D005994:Glycerophosphates; D009598:Nitrophenylgalactosides; D011092:Polyethylene Glycols; D010042:Ouabain; D006867:Hydrolases; D000135:Acid Phosphatase; D005696:Galactosidases; D005966:Glucuronidase; D000118:Acetylglucosaminidase; D004072:Digitonin