Soluble neutral and acid maltases in the suckling-rat intestine. The effect of cortisol and development

Abstract

The 100000g supernatants from 13-day-old suckling-rat intestinal homogenates contained 43.5% of the total intestinal maltase activity, compared with 7.1% in weaned adult rats aged 40 days. The soluble maltase activity was separated on Sepharose 4B into two quantitatively equal fractions at pH6.0, one containing a maltase with a neutral pH optimum and the other a maltase with an acid pH optimum. The neutral maltase was shown to be a maltase-glucoamylase identical with membrane-bound maltase-glucoamylase in molecular weight, heat-sensitivity, substrate specificity, K(m) for maltose and K(i) for Tris. The soluble enzyme was induced by cortisol, but the ratio of the soluble to bound enzyme fell during induction. Solubility of the neutral maltase was not accounted for by the action of endogenous proteinases under the preparative conditions used. It is postulated that the soluble neutral maltase is a membrane-dissociated form of the bound enzyme and that the relationship between these two forms is modulated by cortisol. The acid maltase generally resembled acid maltase of liver, muscle and kidney. It was shown to be a maltase-glucoamylase with optimal activity at pH3.0, and molecular weight of 136000 by density-gradient centrifugation. At pH3.0 its K(m) for maltose was 1.5mm. It was inhibited by turanose (K(i)=7.5mm) and Tris (K(i)=5.5mm) but not by p-chloromercuribenzoate or EDTA. Some 55% of its activity was destroyed by heating at 50 degrees C for 10min. The acid maltase closely resembled beta-glucuronidase and acid beta-galactosidase in its distribution in the intestine, response to tissue homogenization in various media, and decrease in activity with cortisol treatment and weaning, indicating that it was a typical lysosomal enzyme concentrated in the ileum.

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

“…Density gradient centrifugation in both sucrose and mannitol gradients by the method of Martin & Ames (1961) showed that the enzyme virtually co-sedimented with f-galactosidase (mol.wt. 540000; 16.6S), a result which confirms earlier studies with the impure enzyme (Galand & Forstner, 1974a Dissociation of the enzyme Whereas it is well recognized that maltase activity persists in the presence of SDS (Maestracci et al, 1973(Maestracci et al, , 1975Critchley et al, 1975), our preliminary experiments showed poor recovery of activity in enzyme samples which had been treated with SDS at low pH. Several pH effects are shown in Fig.…”

“…2). Interestingly, the antibody also reacted with a naturally soluble 'heavy' maltase, which Galand & Forstner (1974a) showed was present in young rats. No precipitate was formed with sucrase/isomaltase (Fig.…”

“…This was carried out by the method of Martin & Ames (1961) as described previously (Galand & Forstner, 1974a) by using 5-20% (w/v) mannitol or sucrose gradients, except that the buffer used was 0.1 M-Tris/HCI, pH 7.4.…”

“…Published values of the molecular weight by a number of techniques have varied from approx. 500000 (Forstner, 1971;Maestracci et al, 1973Maestracci et al, , 1975Galand & Forstner, 1974a) to about half that value (Kelly & Alpers, 1973). It seems probable that a molecule of such size must consist of more than a single polypeptide chain, and the present study was undertaken to investigate this possibility.…”

Intestinal Glycosidase Activities in one Adult and two Suckling Echidnas: Absence of a Neutral Lactase (b-D-Galactosidase).

“…That of the sucklings, however, had a pH optimum of 4· 5 and was predominantly soluble. It appears, therefore, that in the echidna, as in the tammar wallaby , the intestinal maltase activity of the sucklings is due mainly to a lysosomal acid maltase similar to that described in suckling rats (Galand and Forstner 1974).…”