Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.
Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an  isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd
D000135:Acid Phosphatase / Q000032:analysis; D000596:Amino Acids; D000818:Animals; D005966:Glucuronidase / Q000032:analysis; D006038:Glyoxylates; D006596:Hexosaminidases / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000032:analysis*; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D009439:Neuraminidase; D009578:Nitrobenzenes; D010072:Oxamic Acid / Q000031:analogs & derivatives; D011487:Protein Conformation; D051381:Rats; D013429:Sulfatases / Q000032:analysis
D000596:Amino Acids; D006038:Glyoxylates; D009578:Nitrobenzenes; C009783:4-nitrophenyloxamic acid; D006867:Hydrolases; D000135:Acid Phosphatase; D013429:Sulfatases; D006596:Hexosaminidases; D009439:Neuraminidase; D005966:Glucuronidase; D010072:Oxamic Acid