The enzymic activity of transient sodium dodecyl sulphate-protein complexes of rat intestinal maltase-glucoamylase formed during dissociation [proceedings].

Maltase-glucoamylase (a-D-ghcoside glucohydrolase, EC 3.2.1.20) is a glycoprotein ectoenzyme of the intestinal brush-border membrane. It appears to be the largest component of this membrane (Galand & Forstner, 19746; Maestracci et al., 1975) and its mol.wt. has been estimated by a number of methods to be 4 x 105-5x lo5 (Forstner, 1971 ; Galand & Forstner, 1974~ ; Maestracci et af., 1975). Despite this great size, its subunit composition, if any, is unknown. The enzyme releases glucose from maltose (maltase) and starch (glucoamylase) and also hydrolyses artificial a-glucosides such as p-nitrophenyl a-D-glucoside. However, the maltase activity of the rat enzyme is about 50 times higher than either the glucoamylase or a-glucosidase activity. The activity of the pure enzyme persists remarkably in solutions of sodium dodecyl sulphate at temperatures below 45°C (pH7.4), but is quickly inactivated by boiling (Flanagan & Forstner, 1976). Polyacrylamide-gel electrophoresis showed that loss of enzyme activity was accompanied by complete conversion of the enzyme into five major bands. These changes were also accomplished without heat, by exposing the enzyme to pH values below 5.2 in the presence of sodium dodecyl sulphate (Fig. Id). Both methods of dissociation gave identical gel patterns. The mobilities of bands 1-5 (Fig. Id) were 0.55, 0.37, 0.26, 0.20 and 0.16 respectively. Minor bands with mobilities of 0.28, 0.51 and 0.62 were occasionally seen. The distribution of protein as judged by densitometry was as follows: band 1,36%; band 2,24%; band 3,13%; band 4,19%; band 5 , 8 % . Each of the bands also stained faintly for carbohydrate with the periodic acidlschiff stain. When maltase-glucoamylase was incubated at temperatures between 45°C and 87°C and pH7.4 or at 25°C and pH5.2-6.4 in the presence of 1 % (wlv) sodium dodecyl sulphate, the enzyme was only partially inactivated. This was accompanied by gradual dissociation of the enzyme. Initially some transient protein bands appeared which did not correspond to any of the final species. These bands showed enzyme activity. Progressively these bands increased in intensity, then faded and were replaced by the final dissociated species, which were enzymically inactive. This can be seen in Fig. 1. The first protein bands to appear on the gels during pH-induced dissociation were those with mobilities 0.24, 0.31 and 0.40. All of these displayed enzyme activity (Fig. la) and did not correspond to any of the final associated species. As the dissociation process continued more intermediate protein bands were seen, particularly a strong transitory band with a mobility of 0.22 and iiffuse band at 0.38-0.40, and both of these were enzymically active (Fig. 16). In the final stages, the only activity remaining was associated with the transitory band of mobility 0.40. On lowering the pH to 5, even this was destroyed, and the fully dissociated protein pattern was obtained (Fig. Id). Preliminary data appear to indicate that dissociation of maltase-glucoamylase does not result in dissociation of the maltase activity of the enzyme from its p-nitrophenyl a-Dglucosidase activity or from its starch-hydrolysing activity.