β-Glucosidase (phlorizin hydrolase) activity of the lactase fraction isolated from the small intestinal mucosa of infant rats, and the relationship between β-glucosidases and β-galactosidases

Sephadex G-200 chromatography reveals the presence of two β-glucosidase (phlorizin hydrolase) peaks in the supernatant obtained after papain treatment of mucosal homogenate from the jejunum of infant rats, the major one being identical with the neutral β-galactosidase (lactase) peak in position, shape, and pH optimum (pH 5–6). The other one, also with a nearly neutral pH optimum, emerges shortly after the acid (lysosomal) β-galactosidase, which has pH optimum 3.5. In the 105 000 × g supernatant of the mucosal homogenate, not previously treated with papain, the soluble (lysosomal) acid β-galactosidase, which has no phlorizin hydrolase (β-glucosidase) activity, predominates. Kinetic data (mutual inhibition between β-glucosides and β-galactosides, inhibition by Tris and aldonolactones) show common properties of the β-glucosidase and β-galactosidase active site(s) in the chromatographic fraction of the neutral β-galactosidase (lactase). However, with the artifical substrates (β-glucoside and β-galactoside) the mixed type of mutual inhibition was obtained, which cannot unequivocally exclude, at present, the existence of more than a single binding site for the two substrates in the lactase fraction. The acid β-galactosidase is more specific to the glycone part of the substrate molecule (C-4 configuration) than the neutral β-galactosidase, as the former enzyme does not effectively split cellobiose and hetero-β-glucosides (phlorizin and p-nitrophenyl-β-glucoside) and is not inhibited by phlorizin and gluconolactone. On the other hand, the neutral β-galactosidase (lactase) fraction, which is released from the mucosal membrane by the proteolytic action of papain similarly as the other phlorizin hydrolase (hetero-β-glucosidase), splits β-glucosides as well as β-galactosides and the lactase activity is competitively inhibited by phlorizin. Phloretin β-galactoside is hydrolyzed by the two β-galactosidases and exhibits a competitive inhibitory effect on both enzymes. Unlike the lactase fraction, the second phlorizin hydrolase (hetero-β-glucosidase) does not apparently split cellobiose and is inhibited by o.r mM p-chloromercuribenzoate.