Purification of rat intestinal maltase/glucoamylase and its anomalous dissociation either by heat or by low pH

Abstract

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.

Amphiphilic Pig Intestinal Microvillus Maltase/Glucoamylase

“…The specific activity obtained was smaller (20-35 Ujmg) than reported earlier for both papain and detergent-solubilized forms of rat maltase/glucoamylase [5,11], but of the same size as reported for papain-solubilized rabbit maltase/glucoamylase [7].…”

“…Rat intestinal maltase/glucoamylase in both papain-solubilized and Triton-solubilized forms has been reported to have a molecular weight of 500000 [5,11], with subunits having molecular weights of 245000 and 134000. Analytical ultracentrifugation studies [S, 301 performed on the papain-solubilized forms of human and rabbit maltase/glucoamylase have revealed molecular weights of 21 0000-200000, which fit our data on the papain maltase/glucoamylase.…”

Studies on the size and shape of rabbit intestinal glucoamylase-maltase complex

“…Unlike other amylases, it can catalyse the hydrolysis of the 1,6-a-glycosidic linkage. This enzyme complex has been purified to varying degrees from different animal species (Seetharam et al, 1970;Schlegal-Haueter et al, 1972;Kelly & Alpers, 1973) and to homogeneity from rat (Flanagan & Forstner, 1978) and rabbit * To whom reprint requests should be addressed. (Sivakami & Radhakrishnan, 1973).…”

Purification and properties of neutral maltase from human granulocytes

“…In addition to a major band of Mr 500000, which corresponds to the neutral maltase peak of 550000 observed on gel filtration, this procedure reveals a minor fraction of an Mr-260 000 active protein. This minor band may be attributed to a partial dissociation of a polymeric or aggregated protein under the electrophoresis conditions (Flanagan & Forstner, 1978;Sorensen et al, 1982;Lee & Forstner, 1984;Noren et al, 1986). Further studies on the quaternary structure of human granulocytic neutral maltase are in progress.…”