Classification of Entamoeba Species by Means of Electrophoretic Properties of Amebal Enzymes

Abstract

Qualitative electrophoretic studies were made on five amebal enzymes from 21 cultures of amebae. The enzymes investigated were glucokinase, EC.2.7.1.1; glucosephosphate isomerase, EC.5.3.1.9; phosphoglucomutase, EC.2.7.5.1; malate dehydrogenase (decarboxylating), EC.1.1.1.40; and unclassified enzymes called NADP diaphorase. A total of 16 different amebal enzyme bands were characterized by direct comparisons on cellulose acetate strips. The amebal enzymes were clearly distinguished from those of the bacterial associate. Ten strains of typical E. histolytica gave a total of eight characterized enzyme bands, six of which were common to all strains. Seven of these strains contained diaphorase d, but not diaphorase e, while in the remaining three strains the reverse was found. Five strains of atypical E. histolytica were distinguished from the 10 typical strains by the absence of a second glucokinase band, by the presence of a different isomerase band, and by the absence of a slowmigrating diaphorase band. The enzyme pattern of one of the atypical strains (Huff) was identical with that of E. moshkowskii, the ameba isolated from sewage. Identical enzyme patterns were found for four cultures of the reptilian parasites, E. invadens and E. terrapinae. Each gave seven characterized enzyme bands only one of which was common to E. histolytica and E. moshkowskii. Recent studies on two glycolytic enzymes revealed similar electrophoretic migration patterns for the enzymes from eight or nine strains of typical, large race Entamoeba histolytica. Reeves et al. (1967) found the same two glucokinase (EC.2.7.1.1) isoenzymes from each of nine strains of the ameba of diverse geographical origin. Montalvo and Reeves (1968) investigated eight of these cultures and found a single glucosephosphate isomerase (EC.5.3.1.9) common to all. These two enzymes were clearly distinguished from the respective enzymes from the atypical strains of E. histolytica (the low temperature strains), and from related species of ameba. The prior studies had disclosed the apparent electrophoretic identity of glucokinase and glucophosphate isomerase, respectively, from the atypical E. histolytica strains and Entamoeba moshkowskii, the free-living ameba found in sewage. This led Montalvo and Received for publication 16 January 1968. * Supported in part by U. S. Public Health Service Grant AI-02951 and in part by the U. S. Army Medical Research and Development Command Contract DA-49-193-MD-2620 under the sponsorship of the Commission on Enteric Infections of the Armed Forces Epidemiological Board. t Public Health Service Research Career Awardee from the National Institute of General Medical Sciences. + Present address: Department of Biochemistry, University of Arizona School of Medicine, Tucson, Arizona. Reeves to echo a suggestion previously made by Goldman and Cannon (1967) that the atypical E. histolytica strains may belong in the species E. moshkowskii. Some further support for this suggestion resulted from the present studies. This report extends electrophoretic studies on amebal enzymes to include three additional enzymes: phosphoglucomutase (EC.2.7.5.1), L-malate: NADP oxidoreductase (decarboxylating) (EC.1.1.1.40), and unclassified enzymes called NADP diaphorase. Additional strains of amebae were introduced into this study, and for these strains observations were made, as well, on glucokinase and isomerase. One of the new enzymes gave evidence of qualitative differences among the strains of typical E. histolytica. To the best of our knowledge this finding provides the first firm biochemical evidence of strain differentiation among this important group of organisms. MATERIALS AND METHODS