Activity of isoenzymes A and B of N-acetyl-β-glucosaminidase in renal cancer tissue

In Poland, renal cancer makes up 3-4% of all malignant tumours. Lysosomal glycosidases take part in renal tissue destruction. The exoglycosidases degrade the oligosaccharide chains of glycoconjugates (glycoproteins, proteoglycans and glycolipids). N-acetyl-β-glucosaminidase (E.C.3.2.1.52) (HEX) is an acid lysosomal exoglycosidase which releases N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) residues from the non-reducing ends of oligosaccharide chains of glycoconjugates (glycoproteins, glycolipids, proteoglycans). HEX is a glycoprotein composed of two polypeptide chains a and β. The chains are present in pairs in three possible combinations: isoenzyme A-αβ, isoenzyme B-ββ and isoenzyme S-αα. Isoenzymes A and B demonstrate the highest activity in the tissues; however, the ratio of isoenzymes A/B is different in various tissues. In the natural oligosaccharides, HEX hydrolyzes the b glycoside bond of N-acetylhexosamine with other sugars. The enzyme reacts with artificial substrates such as derivatives of hexosamines with p-nitrophenol and umbelliferone. To determine the activity of isoenzymes A and B in renal tumour and neighbouring macroscopically normal renal tissue in polyacrylamide gel after isoelectrofocusing, we used a-naphtyl-AS- -BI-N-acetyl-b-glucosaminide as a substrate. Electrofocusing is an electrophoretic method of protein separation in polyacrylamide gel which uses the migration of molecules in a pH gradient that is formed after applying an electric field to an ampholyte mixture. The aim of the study was quantitative determination of N-acetyl-β-D hexosaminidase isoenzymes activity by using two methods: colorimetric and electrophoretic and two different substrates.
Materials: Renal specimens harvested from 30 patients during surgery due to renal cancer were used for the study.
Results: In cancerous and normal human renal tissues, isoenzyme A of N-acetyl-b-D hexosaminidase showed statistically higher specific activity, in comparison to isoenzyme B, after determination of both isoenzymes by two different methods.
Conclusion: Determination of HEX isoenzyme activity using the colorimetric method may be considered as a valuable diagnostic marker in renal diseases. The results of HEX isoenzyme activity determination by colorimetric and electrophoretic method are compatible, although for routine determinations the colorimetric method is recommended as the electrofocusing is more technically difficult and more expensive than the colorimetric one.