Rat intestinal maltase--glucoamylase. Purification of the detergent-solubilized enzyme in the presence of protease inhibitors: properties and identification of a protease-sensitive subunit.
Failure to develop clear-cut, distinguishing characteristics for hydrophobic and hydrophilic forms of maltase-glucoamylase led us to attempt the purification of the detergent-extracted enzyme in the continuous presence of protease inhibitors (phenylmethylsulfonyl fluoride and N-ethylmaleimide). The enzyme was purified by molecular exclusion, anion-exchange, and affinity column chromatography to a final specific maltase activity of 80 U/mg protein, comparable to previously solubilized enzymes. Both detergent (d-maltase) and proteolytically (p-maltase) solubilized enzymes had identical Km's for maltose and similar glycogenase activity. d-Maltase was clearly amphipathic. Whereas 95% of p-maltase was eluted with aqueous buffer from an octyl-Sepharose CL-4B column, the elution of d-maltase required solutions containing Triton X-100 and ethylene glycol. On density gradient centrifugation and sodium dodecyl sulfate (SDS)--polyacrylamide gels, p-maltase migrated as one high molecular weight species of 500,000. In contrast d-maltase migrated heterogeneously and the smallest maltase-active forms delineated by these two techniques, as well as by high pressure liquid chromatography, had molecular weights which ranged from 120,000 to 15,0000. Both p- and d-maltase were dissociated by heat in SDS, forming five prominent species as we have previously described. In contrast to p-maltase, in which the smallest species, band 1, equalled 36.7% of the total mass, band 1 of d-maltase accounted for 66.5%. Band 1 was separable when smaller amounts of enzyme were applied to slab gels and stained with silver, into two proteins of 130,000 and 145,000 daltons. The 145,000 dalton protein was absent in p-maltase and was replaced by a faint band of 140,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D005087:Glucan 1,4-alpha-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism; D005959:Glucosidases / Q000302:isolation & purification*; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010206:Papain; D051381:Rats; D013379:Substrate Specificity; D014325:Tromethamine / Q000494:pharmacology; D000520:alpha-Glucosidases / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D046911:Macromolecular Substances; D014325:Tromethamine; D005959:Glucosidases; D000520:alpha-Glucosidases; D005087:Glucan 1,4-alpha-Glucosidase; D010206:Papain