Purification of rat intestinal maltase/glucoamylase and its anomalous dissociation either by heat or by low pH.
Maltase-glucoamylase, a microvillous membrane ectoenzyme, was solubilized from rat intestinal mucosa by digestion with papain and subsequently purified to homogeneity with an overall yield of 10--20%. An antibody to the purified enzyme formed a single precipitin line in immunodiffusion experiments with an intestinal homogenate. The enzyme was shown to be an acidic glycoprotein (20% sugar by weight) which contained low amounts of cysteine and no sialic acid. At pH3--6, maltase activity was slowly lost, but the enzyme was re-activated by re-adjustment of the pH to neutrality. However, in the presence of sodium dodecyl sulphate, acid pH values inactivated maltase irreversibly, and at the same time converted the enzyme (mol.wt. 500000 approx.) into five new species with apparent molecular weights ranging from 134000 to 480000 as judged by polyacrylamide-gel electrophoresis. The same five fragments were also formed by boiling the enzyme for brief periods in the presence of sodium dodecyl sulphate or urea either with or without reducing agents. The dissociated species were stable on re-electrophoresis, and amino acid analysis showed them to be very similar to each other and to the original enzyme. The bands migrated anomalously on polyacrylamide gels of different concentration, thereby preventing the assignment of precise molecular weights. It is possible that the five species may represent stable aggregates of a common monomer of the enzyme.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005087:Glucan 1,4-alpha-Glucosidase / Q000302:isolation & purification*; D005959:Glucosidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007413:Intestinal Mucosa / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D051381:Rats; D013696:Temperature; D000520:alpha-Glucosidases / Q000302:isolation & purification*
D000596:Amino Acids; D005959:Glucosidases; D000520:alpha-Glucosidases; D005087:Glucan 1,4-alpha-Glucosidase