MOLECULAR ANALYSIS OF 2 HEXOKINASE ISOENZYMES FROM ENTAMOEBA-HISTOLYTICA

The zymodemes, electrophoretic patterns of hexokinase, phosphoglucomut ase and glucose phosphate isomerase isoenzymes, have been widely used to determine the pathogenicity of Entamoeba histolytica isolates. Alth ough pathogenic and nonpathogenic forms of E. histolytica differ clear ly in sequences of many homologous genes, a conversion between pathoge nic and nonpathogenic zymodemes has been reported by several laborator ies. To approach the question what might be the basis for the observed conversion, we examined the molecular biology of the hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzymes in pathogenic E. histolytica. We isolated two different cDNAs pHXK1 and pHXK2 coding f or polypeptides with significant sequence similarity to hexokinases an d deduced molecular masses of 49.8 kDa and 49.4 kDa. The two hexokinas e sequences differed by 11% on the amino acid and by 8% on the nucleot ide level. Expression of the cDNAs in Escherichia coli as nonfusion pr oteins gave two polypeptides with hexokinase activity. The recombinant Hxk1 and Hxk2 polypeptides comigrated with the more basic and more ac idic isoforms of pathogenic amoebae in starch gel electrophoresis, as well as in low and high resolution isoelectric focussing gels. This id entified the observed hexokinase isoenzymes of pathogenic E. histolyti ca as the products of two genes, hxk1 and hxk2.