The multiple forms and kinetic properties of the N-acetyl-β-d-hexosaminidases from colonic tumours and mucosa of rats treated with 1,2-dimethylhydrazine

Abstract

The separation and purification of the N-acetyl-beta-D-hexosaminidase activities from tumours induced by 1,2-dimethylhydrazine in the rat colon and from colonic mucosa of tumour-bearing animals are reported. Mucosa contained N-acetylhexosaminidases A and B, as well as a third form whose properties with regard to electrophoretic mobility and thermostability lay between those of A and B. Tumours contained only N-acetylhexosaminidase A and B activities. Each form possessed both N-acetylglucosaminidase (EC 3.2.1.30) and N-acetylgalactosaminidase (EC 3.2.1.53) activities, which could not be separated by a variety of techniques. The alteration of the ratio of the two specific activities in each form during purification, together with differences in the kinetic inhibition constants and behaviour during inactivation by various reagents or a temperature of 50 degrees C, supported the belief that each form contains the two enzyme activities, glucosaminidase and galactosaminidase, at separate active sites. This model is in contrast with that reported for these activities from a number of other sources. A variety of treatments reported to cause the conversion of form A into a form resembling B failed to produce such an effect on the rat colonic hexosaminidases.

Alteration in glycosidases from well-differentiated colorectal adenocarcinoma of rat.

“…The specific activity determined was 3.58 ,u mol / hr / mg of protein. The value was much higher than that (0.0111umol/hr/mg of protein) of rat colonic mucosa of a different strain reported by Mian et al (1979), but was comparable with that (1.95 ,u mol/hr/mg of protein) of human colonic mucosa (Tsao et al 1979). These data suggested that the enzyme solubilization was achieved to an extent which is comparable with or much higher than that reported previously.…”

“…On the other hand, no significant differences in this proportion were shown in rat colonic adenocarcinoma and human prostatic carcinoma (Mian et al 1979;Whitehurst et al 1982). In the present study, two components of /3-N-acetylglucosaminidase corresponding chromatographically to the A and B isozymes were termed the A and B forms, respectively, according to the designation by Mian et al (1979).…”

“…Ih and Table 2). In the order of elution from the column, they were identified as the B, intermediate, and A forms according to the designation by Mian et al (1979). In contrast, the tumor contained the intermediate form representing only 7% of the total activity ( Fig.…”

“…The differences in glycosidase levels and their isozyme patterns have also been frequently reported for various tumors (Brattain et al 1979; Mian et al 1979: Okochi et al 1979: Varani et al 1979; Dobrossy et al 1981), and Bosmann and Hall (1974) have suggested the possibility that the lysosomal enzymes may modify the cell surface components.…”

“…The activities of glycosidases were measured with 2.4 mM 4MU-N-acetyl-/3-glucosaminide at pH 4.5, 1.2 mM 4MU-/3-galactoside at pH 4.5, 2.4 mM 4MU-/3-glucuronide at pH 4.5, 1.2 mM 4MU-a-L-fucoside at pH 5.5, 0.5 mM 4MU-a-galactoside at pH 4.5 and 1.2 mM 4MU-/3-glucoside at pH 6.0 for respective enzymes. The activity for p-nitrophenyl-N-acetyl-/3-D-glucosaminide of /3-N-acetylglucosaminidase was measured by the method of Mian et al (1979). The activities of /3-N-acetylglucosaminidase, /3-galactosidase, /3-glucuronidase and a-L-fucosidase in the fractions obtained by DEAE-cellulose column chromatography were separately determined with 1.2 mM of the relevant substrates.…”