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Electrophoretic characterization of amebal glucose phosphate isomerases
EXPERIAIENTAL
PARASITOLOGY
22, 129-136
( 1968)
Electrophoretic
Characterization
Glucose Francisco Department
Phosphate Montalvo
of Biochemistry, New (Submitted
of Amebal
Isomerases”
and
Richard
E. Reeves’
Louisiana State University Orleans, Louisiana 70112
for publication,
26 September
School of Medicine, 1967)
MOXTALVO, F., AND REEVES, R. E. 1967. Electrophoretic characterization of amebal glucose phosphate isomerase. Experimental Parasitology 22, 129-136. An electrophoretic method for the characterization of glucose phosphate isomerase has been developed. This method was applied to the enzyme from 18 different cultures of amebae. Five strains of atypical Entamoeba histolytica and two strains of E. moshkowskii yielded a fast-migratin, 0 isomerase. Two strains of E. invadens and one strain of E. terrapinae gave a slow-migrating isomerase. Eight strains of typical, large race E. histolytica gave an enzyme which migrated intermediately between these fast- and slow-migrating enzymes. All of the amebal enzymes were electrophoretically different from crystalline yeast glucose phosphate isomerase, and frcm the isomerase extracted from Bacteroides symbiosrrs, the associate organism. The electrophoretic behavior of glucose phosphate isomerase on cellulose acetate strips is proposed as a convenient means of classifying ameba cultures and of distinguishing the typical strains of E. histolytica from atypical strains and related species. The culture relationships based on isomerase were identical with those previously reported based on the enzyme glucokinase. The biochemical properties of the amebal enzymes were similar to each other and resembled those reported for glucose phosphate isomerase from other sources.
Entamoeba lzistolytica and closely related species are rich in soluble glucose phosphate isomerase (n-glucose B-phosphate ketolisomerase E. C. 5.3.1.9). This is the enzyme which reversibly catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate. The activity of 1 This investigation was supported in part by U.S. Public Health grant AI-02952, and in part by the United States Army Research and DevelContract DA-49-193-MDopment Command, 2620, under the sponsorship of the Commission on Enteric Infections of the Armed Forces Epidemiological Board. Some of the data were taken from a thesis submitted by Francisco Montalvo to Louisiana State University (1967) in partial fulfillment of the requirements for the degree Master of Science. ? Public Health Service Research Career Awardee from the National Institute of Medical Sciences.
this soluble and stable enzyme is sufficiently great that it may be directly assayed by spectrophotometric methods in crude dialyzed amebal extracts. During these investigations, a method was developed for the localization of glucose phosphate isomerase activity on cellulose acetate strips following electrophoresis. This report concerns the application of this method to the study of comparative enzymatic relationships among the various strains and species of amebae. Reeves et al. (1967) have recently reported a similar study of many of these cultures based upon the properties of their glucokinases. MATERIALS AND METHODS Organisms. The strains of typical E. histolytica studied and the date and location 129
130
MONTALVO
of isolation follows: DKB strain, England, 1924; NRS strain, England (from a MacaGUS sp. monkey), 1929; 200 strain, Washington, D.C., 1947; K-9 strain, Korea, 1951; the F-22, BH, JH, and JS strains, New Orleans, 1947, 1955, 1956, and 1959, respectively. All ,strains except NRS had been obtained from naturally infected, diseased humans. The five strains of atypical E. histolytica (Laredo, Huff, AG, 403, and JA) had been isolated from infected humans showing no symptoms, or minimal symptoms of disease. The isolation of these five strains has been reviewed by Richards et al. (1966). The two strains of E. invadens (IP and PZ) and the E. terrapinae strain had been isolated from reptiles. The two investigated cultures of E. moshkowskii were those designated FIC and CA-2. Cultivation of amebae. Tube cultures of amebae were maintained in a medium containing Trypticase, glucose, horse serum, thiomalate, penicillin, and cells of a penicillin-sensitive anaerobic bacteria designated Bacteroides symbiosus (Reeves et al., 1957). Inoculae taken from the tube cultures were used to seed Petri-dish cultures. These were grown in an anaerobic incubator, harvested, and lyophilized under the conditions described by Reeves and Ward ( 1965). The typical E. histolytica organisms and the Laredo strain were grown at 36°C the others at 2832°C. The trophozoites were active, and free from cysts. The lyophilized amebae, sealed in glass under vacuum, were stored at -20°C until used. Materials. Glucose 6-phosphate dehydrogenase and crystalline yeast glucose phosphate isomerase were from Boehringer, Mannheim; NADP3 was obtained from Sigma Chemical Company, St. Louis, 3 Abbreviations used: NADP, nicotinamide adenine dinucleotide phosphate; F-6-?, D-fructose 6phosphate; G-SD, n-glucose ‘6-phosphate; EDTA, ethylenediamine tetraacetic acid; Tris, trihydroxymethylaminomethane.
AND
REEVES
Missouri; p-nitroblue tetrazolium chloride, from Nutritional Biochemicals Corporation, Cleveland, Ohio; and phenazine methosulfate, from Aldrich Chemical Company, Inc., Milwaukee, Wisconsin. Studies on glucose phosphate isomerase working in the reverse direction (F-6-P -+ G-6-P) require fructose 6-phosphate which is relatively free from contamination by glucose 6-phosphate. Many commercial samples of fructose 6-phosphate are unsatisfactory for this purpose (Schwartz and Bodansky, 1965). Most of our reported kinetic studies employed a lot of fructose 6-phosphate obtained from Sigma which contained less than 0.75% of the glucose phosphate impurity, however, the substrate at this purity has not been regularly available from this source. The following simple purification procedure yielded F-6-P free from detectable amounts of interfering substrates; one gm of Boehringer barium fructose g-phosphate (lot No. 6195531) was dissolved in 40 ml of water, and stirred at room temperature with 0.5 gm Darko-G60 carbon, filtered, rinsed, and made up to a volume of 50 ml. This solution, pH 7, contained 1.5 mmoles of F-6-P and .05 mmoles G-6-P, by enzymatic assay. To the solution was added, with shaking, 0.1 ml bromine and it was allowed to stand in the dark at room temperature for 1 hour. The excess bromine was then removed by a current of air. The solution was then free of G-6-P but it contained 6-phosphogluconate equivalent to the original G-6-P content. After passage through a Dowex50 (Na salt) column it was suitable for the electrophoretic experiments. Further purification from the 6-phosphogluconate was achieved by passing the barium-free effluent through a column containing 1 gm of Dowex-1 (acetate form, 0.72 mE/gm), collecting the e&rent in j-ml fractions. Each fraction was assayed enzymatically for F-6-P, G-6-P, and 6-phosphogluconate. The earlier F-6-P-containing
AMEBAL
ISOMERASES
fraction yielded 0.3 mmoles of F-6-P which was free from the two contaminants. This method of purification of the substrate appears to offer advantages over that employed by Kahana et al. (1960) and Salas et al. ( 1965). The horizontal paper electrophoresis apparatus and the cellulose acetate strips, designated Sepraphore III, were obtained from Gelman Instrument Company, Ann Arbor, Michigan. Biogel P-300 was obtained from Calibiochem, Los Angeles, Calif. DEAE Sephadex was obtained from Pharmacia Fine Chemicals, Inc., Pescataway, N. J. Amebal extracts. The lyophilized amebae, representing known volumes and numbers of centrifugally packed cells, were suspended in mM EDTA, pH 7, and mixed for 30 minutes at 0°C. The extracts of typical E. histmolytica, E. terrapinae, and E. invadens were made to represent 50 million cells/ml while the atypical E. histolytica and E. moshkowskii were made up I- or 200 million/ml. The cellular debris was removed by centrifugation at 30,000 g for 10 minutes. The supernatant fluid was employed, before and after dialysis, in the electrophoretic experiments. Dialysis was against 100 volumes of mM EDTA, pH 7, in the cold, overnight. The dialyzed extracts were used for enzyme assays. Fresh amebae were suspended in the EDTA solution in a plastic tube which was floated in ice water in the chamber of a g-kc Raytheon magnetostriction apparatus. The full power was applied for 3 minutes after which the sonically disrupted suspensions were centrifuged and dialyzed as described above. The crude or purified enzyme was stable toward dialysis, storage at refrigeration temperatures, and to freezing and thawing. Enzyme assay. Glucose phosphate isomerase activity was assayed by a spectrophotometric method measuring the rate of
131
formation of glucose 6-phosphate from fructose g-phosphate. The isomerase was linked with an excess of glucose 6-phosphate dehydrogenase and the rate of reduction of NADP was observed at 340 mp. A Beckman DU spectrophotometer with the Gilford model-220 attachments was used. The standard assay solution contained 1 mM fructose 6-phosphate, 0.3 mM triphosphopyridine nucleotide, 4 pg/ ml glucose B-phosphate dehydrogenase, and 50 mM Tris buffer, pH 8.0. It was preincubated for 5 minutes to get rid of the glucose B-phosphate contaminant. Amebal enzyme was then added and the change in optical density was recorded for 3 minutes. The molar absorbancy of reduced NADP was taken as 6.27 X 10” at 340 rnp. A unit of activity is defined as that producing 1 pmole of glucose 6-phosphate/ minute at 25°C under the standard assay conditions noted above. Specific activity is reported as units of enzyme activity per milligram protein. Electrophoresis. The cellulose acetate strips were soaked for 10 minutes or more in a buffer containing 4.88 gm/liter sodium barbital, and 3.24 gm/liter sodium acetate, adjusted to pH 8.0 with HCl. Excess liquid was removed on paper towels. The strips were then stretched over the electrophoretic bridge and streaked with 5-10 ~1 of amebal extract, Electrophoresis was for 1 or 2 hours at 12V/cm at room temperature. At the end of the run, the strips were developed for glucose phosphate isomerase activity. The developing solution contained 50 mM Tris-HCI pH 8.0; 0.3 mM NADP; 1mM fructose 6-phosphate; 0.4 mg/ml p-nitroblue tetrazolium chloride; 25 pg/ml glucose 6-phosphate dehydrogenase and 20 pg/ml phenazine methosulfate. The isomerase bands appeared within five minutes at room temperature and about fifteen minutes was required to develop their maximal color.
132
MONTALVO AND REEVES
Omission of the phenazine methosulfate resulted in the formation of spurious bands. RESULTS
Enzyme In typical Activity
preparation experiments
and purification. listed in Table I
TABLE I of Glucose Phosphate Isomerases from Amebal Trophozoites
Source Typical E. histolytica DKB DKB DKB DKB Atypical E. histolytrca Laredo Laredo Laredo Huff E. moshkowslcii E. invackns
Fresh (F) or lpophiliaed (L)
C/ml packed cells
U/mg protein in amebal extract
F F L L
29.5 20.0 14.0 12.5
.78 -.33 .29
L L L F F L
21.7 11.9 29.0 27.0 34.0 12.0
.43 .32 .82 1.00 1.25 .44
and lyophilized cells fresh trophozites yielded 1234 units of soluble isomerase activity per milliliter of packed cells. Amebal glucose phosphate isomerase was separated from other enzymes of the glycolytic pathway and extensively purified in the following manner: An extract of fresh DKB trophozoites was placed on a 3 cm2 x 32-cm column of Biogel P-300 which had previously been conditioned with 0.02 M potassium phosphate, pH 6.4, containing 0.001 M EDTA. The column was eluted with the same buffer and the fractions containing isomerase were combined. The recovery of activity was quantitative. The enzyme had been purified 7-fold based on its protein content. It still contained significant amounts of glucokinase and phosphoglucose mutase activity. The product was concentrated to 5 ml by vacuum dialysis against the phosphate buffer, and applied to a 3 cm2 X 14-cm
column of DEAE Sephadex previously conditioned with, 0.02 M potassium phosphate, pH 7, containing mM EDTA. This column was eluted with the pH 7 buffer and a leading peak of isomerase emerged, followed by the glucokinase. The tubes containing the first half of the isomerase peak were of the highest specific activity. They were combined to yield six units of isomerase. The overall purification of the enzyme amounted to 91-fold. This enzyme solution was free from glucokinase and phosphoglucose mutase. Its electrophoretic and kinetic properties were similar to those of the crude enzyme. Electrophoretic results. The electrophoretic mobilities of the isomerase from the DKB and 200 strains are compared in Fig. IA. Comparisons such as this were made by placing the DKB amebal extract along the starting line on one half of a cellulose acetate strip and extract from a second strain or species of ameba on the other half. Results of this work indicated that the isomerases from the DKB, 200, K-9, F-22, JH, JS, NRS, and BH strains all had identical electrophoretic mobilities. However, the extracts from the atypical E. histolytica strains all showed isomerases migrating much faster than did that of the DKB strain. For the fast-migrating isomerases, that of the Laredo strain was chosen as standard and comparisons were made against the Laredo enzyme. Results of this study showed that electrophoretically identical fast-migrating isomerases were obtained from the Laredo, Huff, JA, AG, and 403 strains, and from each ‘of the two cultures of E. moshkowskii. Figure 1B illustrates the results obtained with enzyme from three of the atypical E. histolytica strains and one strain of E. moshkowskii. For comparison, enzyme from the DKB strain (typical E. histolytica) was included in this experiment. Electrophoretic comparison between en-
AMEBAL
133
ISOMERASES
DKB
E mod. 403 AG Laredo
FIG. 1. Electrophoretic comparisons of glucose phosphate isomerases from various sources. A. A typical result illustrating the electrophoretic identity of isomerase from two strains of typical E. kistolytica (strains DKB and 200). B. A typical result illustrating the electrcphoretic identity of isomerase from three strains of atypical E. histolytica (strains Laredo, AC, and 403) with the enzyme from E. moshkouzkii (strain FIC). For comparison the enzyme from a typical strain of E. histolytica (DKB) was included in this experiment. C. Half of this strip was streaked with a mixture prepared from enzymes E. inoadens (PZ strain), E. histolytica (DKB and Laredo strains) and yeast isomerase. The other half of the strip was spotted with the enzymes, singly. The results show that a mixture of isomerases can be resolved by electrophoresis. D. Migration of enzyme from the Laredo strain of ameba is compared with that extracted from Bacteriodes symbiosus, the associate organism in the ameba cultures. Print A was made from a color-film positive, prints B, C, and D from black and white negatives.
zymes from the DKB strain and E. invadens revealed that both cultures of the latter organism contained an isomerase which migrated more slowly than did the enzyme of the DKB strain. The isomerase of E. tewapinae was indistinguishable from that of E. invadens by this technique. Figure 1C shows, on a single electropherogram, the results obtained with slow-, intermediate-, and fast-migrating amebal isomerases. For comparison this experiment also included a sample of crystalline yeast isomerase. To the top of this figure all four isomerases were mixed and placed together on the starting line; below, each was spotted individually along the starting line. Inspection of the figure reveals that the isomerases of the typical and atypical E. histoZytica strains (DKB and Laredo, respectively) are widely separated by electrophoresis, that yeast isomerase migrates between these two, and that the isomerase from E. invadens is the slowest migrating of these enzymes. Kineiic studies. Reported in Table II are
some results of biochemical kinetic studies on the amebal isomerases. Some published results on isomerases from other sources have also been included in the table. That the inhibition of amebal isomerase by 6phosphogluconate is competitive with substrate was evidenced by Lineweaver-Burk plots of data from all investigated amebal isomerases. Bacterial isomerase. Fresh B. symbiosus cells, 1.8 gm, were suspended in 16.2 ml of .02 A4 KPO, buffer (pH 6.4) containing mM EDTA and subjected to sonic disintegration in the Raytheon apparatus. This solution was placed directly in the sample cup and the treatment was continued, with periods. The cooling, for two Sminute solution was then centrifuged for 30 minutes at 27,000 g to remove unbroken cells and cell debris. The solution contained 304 units of glucose phosphate isomerase by the standard assay and 195 mg of protein. Ten ml of the centrifuged bacterial extract was fractionated with ammonium sulfate. Protein fractions insoluble at 50, 65,
134
MONTALVO
AND
REEVES
helminths, bacteria, and amebae. The biochemical kinetic properties of the enzyme from all investigated sources appear Inhibition by to be quite similar. The amebal isomerases 6-phosphoKm (F-6-P), gluconate were electrophoretically distinct from those Source mM Ki, mM of yeast or B. symbioses, the associate organism. Results from this worke The electrophoretic mobilities of the B. hzstolytica amebal glucose phosphate isomerases fell DKB strain .08 ,013 ,066 into three groups. The five atypical E. Laredo .12 ,015 histolytica strains and the two E. naoshHuff 19 ,025 kowskii strains had a fast-migrating enE. invadens :09 ,015 E. zyme; all eight strains of typical E. moshkowskii .09 ,028 histolytica yielded an intermediately miYeast .14 ,023 Selected results from the literature grating enzyme; and the two strains of Yea& .15 ,015 E. invadens and the E. terrapinae yielded Rabbit muscled 10 a slow-migrating enzyme. The classificaRabbit muscle and .03 ,005 tion of amebal cultures based upon the bra& properties of amebal isomerases was idenS. mansonid .lO Human erythrocytese .03 ,005 tical to that previously proposed by Reeves Bovine mammary .07 et al. (1967) based on studies of amebal gland’ glucokinases. There is a striking uniformity in the electrophoretic properties of the a Km’s and Ki’s were determined from Lineweaver-Burk plots of data from four appropriate enzyme from typical E. lzistolytica of substrate concentrations. The conditions used were diverse geographical origin, from England those of the st,andard assay (see Materials and to Korea, and having dates of isolation Methods) exceptf for substrate and inhibitor conranging from 1924 to 1959. centrations. Crude, dialyzed amebal enzyme soluThere is a growing realization of the tions were employed except in one noted instance. 6 This value was obtained with column purified marked differences between the two enzyme and purified F-6-P. groups of organisms herein designated as c Salas et al. (1965). typical and atypical E. histolytica. Entner d Buediny and MacKinnon (1955). and Most (1965) applied the terms “rege Kahana et al. (1960). ular” or “true” to the former. The term f Hines and Wolfe (1963). strains” has been “room-temperature widely applied to the latter since, unlike and 80% saturation were collected, redisthey multiply at or the typical strains, solved in buffer, and dialyzed. The fracbelow room temperature. Entner et al. tion representing 65-80% saturation con( 1962), Entner and Most ( 1965), and tained 12.5 mg protein and 125 units of enzyme activity. Upon electrophoresis this Albach et al. (1966) have reported differences in drug sensitivities between these bacterial enzyme was found to migrate groups of organisms. Richards et al. (1966) considerably faster than the enzyme from have summarized the earlier findings on the Laredo strain of amebae (see Fig. 1D). the two groups and have suggested that DISCUSSION only the former may be associated with severe human disease. Albach and Shaffer Glucosephosphate isomerase is probably (1965) have noted differences in free an ubiquitous enzyme. It has been obamino acid content, and Goldman et al. served in many mammalian tissues, yeasts, Kinetic
Properties
TABLE II n.f Glu,cose Phosphate Isomerases
AMEBAL
ISOMEFiASES
135
CLG medium. Journal of Protozoology 12, ( 1960) and Goldman and Cannon (1967) 659-665. have noted immunological differences beALBACH, R. A,, SHAFFER, J. G., AND WATSON, tween the typical and atypical strains. Our R. H. 1966. A comparison of in vitro drug present studies and those of Reeves et al. sensitivities of strains of Entamoeba which ( 1967) emphasize qualitative enzymatic grow at 37°C and at room temperature. The American Journal of Tropical Medicine differences between the two groups. and Hygiene 15, 855-859. Entner and Most (1965) have suggested BUEDING, T., AND MACKINNON, J. A. 1955. that differentiation of species is required Studies of the phosphoglucose isomerase of between the “true” or typical strains and Schistosoma mansoni. The Journal of Biologatypical or “room-temperature” strains of ical Chemistry 215, 507-513. E. histolytica. It appears to us that prior ENTNER, N., AND MOST, H. 1965. Genetics of Entumoeba: characterization of two new to the assignment of a new species desigparasitic strains which grow at room tempernation serious consideration should be ature (and at 37’C). Journal of Protozoolaccorded to the possibility that the atypical ogy 12, 10-13. E. histolytica organisms belong to the ENTNER, N., EVANS, L. A., AND GONZALES, C. species E. moshkowskii. This suggestion 1962. Genetics of Entamoeba histolytica. has also been made by Goldman and CanDifferences in drug sensitivity between Laredo and other strains of Entamoeba hisnon (1967) based on their immunological tolytica. Journal of Protozoology 9, 466-469. findings. A significant aspect of our present GOLDMAN, M., AND CANNON, R. T. 1967. Antifindings is the apparent identity of the genie analysis of Entamoeba histolytica by isomerase from the atypical E. lzistolytica means of fluorescent antibody. V. Comparito that from E. moshkowskii. son of 15 strains of Entamoeba with information on their pathogenicity to guinea pigs. Bueding and MacKinnon (1955) reThe American Journal of Tropical Medicine sorted to immunological procedures to and Hygiene 16, 245-254. distinguish between the isomerases of GOLDMAN, M., CARVER, R. K., AND GLEASON, Schistosoma and rabbit muscle. The imN. N. 1960. Antigenic analysis of Entamoeba munological technique failed to distinguish histolytica by means of fluorescent antibody. between the enzymes of three schistosoma II. E. .histoZytica and E. hartmanni. Experimental Parasitology 10, 366-388. species: S. mansoni, S. japonicum, and S. HINES, M. C., AND WOLFE, R. G. 1963. Phoshematobium. It might be fruitful to apply phoglucose isomerase. II. Influence of pH on the electrophoretic technique to such probkinetic parameters. Biochemistry 2, 770-775. lems of isomerase characterization. ACKNOWLEDGMENT We are indebted to Dr. James G. Shaffer for cultures of K9, 200, and F22 strains; to Dr. Quentin M. Geiman for the DKB strain; to Dr. William Balamuth for the two strains of E. inwadens, and E. terrupinae; to Dr. Frank Connell for the Laredo strain; to Dr. Morris Goldman for the JA, AG, 403, and Huff strains and for E. moshkowskii, strain FIC; and to Professor Armando Ruiz G. for a culture of I?. moshkowskii, strain CA-2. REFERENCES ALBACH, R. A., AND SHAFFER, J. G. 1965. Free amino acid analyses of 4 strains of Entamoeba histolytica and E. invadens in the
KAHANA, S. E., LOWRY, 0. H., SCHULZ, D. W., PASSONNEAU, J. V., AND CRAWFORD, E. J. 1960. The kinetics of phosphoglucoisomerase. The Journal of Biological Chemistry 235, 2178-2184. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin Phenol reagent. Journal of Biological Chemistry 193, 265-275. REEVES, R. E., AND WARD, A. B. 1965. Large lot cultivation of Entamoeba histolytica. Journal of Parasitology 51, 321-324. REEVES, R. E., MELENEY, H. E., AND FRYE, W. W. 1957. A modified Shaffer-Frye technique for the cultivation of Entamoeba hi.stoZytica and some observations of its carbohydrate requirements. American Journal of Hygiene 48, 124-130.
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MONTALVO
REEVES, R. E., MONTALVO, F., AND SILLERO, A. 1967. Glucokinase from Entamoeba histolytica and related organisms. Biochemistry ~6, 1752-1760. RICHARDS, C. S., GOLDMAN, M., AND CANNON, L. T. 1966. Cultivation of Entamoeba histolytica and Entamoeba histolytica-like strains at reduced temperature and behavior of the amebae in diluted media. American Journal of Tropical Medicine and Hygiene 15, 648-655.
AND
REEVES
SALAS, M., VINUELA, E., AND SOLS, A. 1965. Spontaneous and enzymatically catalyzed anomerization of glucose 6-phosphate and anomeric specificity of related enzymes. The Chemistry 246, 561Journal of Biological 568. SCHWARTZ, M. K., AND BODANSKY, 0. 1965. Use of commercially available preparations of fructose B-phosphate in the determination of phosphohexose isomerase activity. Analytical Bioc.hemistvy 11, 48-53.
PARASITOLOGY
22, 129-136
( 1968)
Electrophoretic
Characterization
Glucose Francisco Department
Phosphate Montalvo
of Biochemistry, New (Submitted
of Amebal
Isomerases”
and
Richard
E. Reeves’
Louisiana State University Orleans, Louisiana 70112
for publication,
26 September
School of Medicine, 1967)
MOXTALVO, F., AND REEVES, R. E. 1967. Electrophoretic characterization of amebal glucose phosphate isomerase. Experimental Parasitology 22, 129-136. An electrophoretic method for the characterization of glucose phosphate isomerase has been developed. This method was applied to the enzyme from 18 different cultures of amebae. Five strains of atypical Entamoeba histolytica and two strains of E. moshkowskii yielded a fast-migratin, 0 isomerase. Two strains of E. invadens and one strain of E. terrapinae gave a slow-migrating isomerase. Eight strains of typical, large race E. histolytica gave an enzyme which migrated intermediately between these fast- and slow-migrating enzymes. All of the amebal enzymes were electrophoretically different from crystalline yeast glucose phosphate isomerase, and frcm the isomerase extracted from Bacteroides symbiosrrs, the associate organism. The electrophoretic behavior of glucose phosphate isomerase on cellulose acetate strips is proposed as a convenient means of classifying ameba cultures and of distinguishing the typical strains of E. histolytica from atypical strains and related species. The culture relationships based on isomerase were identical with those previously reported based on the enzyme glucokinase. The biochemical properties of the amebal enzymes were similar to each other and resembled those reported for glucose phosphate isomerase from other sources.
Entamoeba lzistolytica and closely related species are rich in soluble glucose phosphate isomerase (n-glucose B-phosphate ketolisomerase E. C. 5.3.1.9). This is the enzyme which reversibly catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate. The activity of 1 This investigation was supported in part by U.S. Public Health grant AI-02952, and in part by the United States Army Research and DevelContract DA-49-193-MDopment Command, 2620, under the sponsorship of the Commission on Enteric Infections of the Armed Forces Epidemiological Board. Some of the data were taken from a thesis submitted by Francisco Montalvo to Louisiana State University (1967) in partial fulfillment of the requirements for the degree Master of Science. ? Public Health Service Research Career Awardee from the National Institute of Medical Sciences.
this soluble and stable enzyme is sufficiently great that it may be directly assayed by spectrophotometric methods in crude dialyzed amebal extracts. During these investigations, a method was developed for the localization of glucose phosphate isomerase activity on cellulose acetate strips following electrophoresis. This report concerns the application of this method to the study of comparative enzymatic relationships among the various strains and species of amebae. Reeves et al. (1967) have recently reported a similar study of many of these cultures based upon the properties of their glucokinases. MATERIALS AND METHODS Organisms. The strains of typical E. histolytica studied and the date and location 129
130
MONTALVO
of isolation follows: DKB strain, England, 1924; NRS strain, England (from a MacaGUS sp. monkey), 1929; 200 strain, Washington, D.C., 1947; K-9 strain, Korea, 1951; the F-22, BH, JH, and JS strains, New Orleans, 1947, 1955, 1956, and 1959, respectively. All ,strains except NRS had been obtained from naturally infected, diseased humans. The five strains of atypical E. histolytica (Laredo, Huff, AG, 403, and JA) had been isolated from infected humans showing no symptoms, or minimal symptoms of disease. The isolation of these five strains has been reviewed by Richards et al. (1966). The two strains of E. invadens (IP and PZ) and the E. terrapinae strain had been isolated from reptiles. The two investigated cultures of E. moshkowskii were those designated FIC and CA-2. Cultivation of amebae. Tube cultures of amebae were maintained in a medium containing Trypticase, glucose, horse serum, thiomalate, penicillin, and cells of a penicillin-sensitive anaerobic bacteria designated Bacteroides symbiosus (Reeves et al., 1957). Inoculae taken from the tube cultures were used to seed Petri-dish cultures. These were grown in an anaerobic incubator, harvested, and lyophilized under the conditions described by Reeves and Ward ( 1965). The typical E. histolytica organisms and the Laredo strain were grown at 36°C the others at 2832°C. The trophozoites were active, and free from cysts. The lyophilized amebae, sealed in glass under vacuum, were stored at -20°C until used. Materials. Glucose 6-phosphate dehydrogenase and crystalline yeast glucose phosphate isomerase were from Boehringer, Mannheim; NADP3 was obtained from Sigma Chemical Company, St. Louis, 3 Abbreviations used: NADP, nicotinamide adenine dinucleotide phosphate; F-6-?, D-fructose 6phosphate; G-SD, n-glucose ‘6-phosphate; EDTA, ethylenediamine tetraacetic acid; Tris, trihydroxymethylaminomethane.
AND
REEVES
Missouri; p-nitroblue tetrazolium chloride, from Nutritional Biochemicals Corporation, Cleveland, Ohio; and phenazine methosulfate, from Aldrich Chemical Company, Inc., Milwaukee, Wisconsin. Studies on glucose phosphate isomerase working in the reverse direction (F-6-P -+ G-6-P) require fructose 6-phosphate which is relatively free from contamination by glucose 6-phosphate. Many commercial samples of fructose 6-phosphate are unsatisfactory for this purpose (Schwartz and Bodansky, 1965). Most of our reported kinetic studies employed a lot of fructose 6-phosphate obtained from Sigma which contained less than 0.75% of the glucose phosphate impurity, however, the substrate at this purity has not been regularly available from this source. The following simple purification procedure yielded F-6-P free from detectable amounts of interfering substrates; one gm of Boehringer barium fructose g-phosphate (lot No. 6195531) was dissolved in 40 ml of water, and stirred at room temperature with 0.5 gm Darko-G60 carbon, filtered, rinsed, and made up to a volume of 50 ml. This solution, pH 7, contained 1.5 mmoles of F-6-P and .05 mmoles G-6-P, by enzymatic assay. To the solution was added, with shaking, 0.1 ml bromine and it was allowed to stand in the dark at room temperature for 1 hour. The excess bromine was then removed by a current of air. The solution was then free of G-6-P but it contained 6-phosphogluconate equivalent to the original G-6-P content. After passage through a Dowex50 (Na salt) column it was suitable for the electrophoretic experiments. Further purification from the 6-phosphogluconate was achieved by passing the barium-free effluent through a column containing 1 gm of Dowex-1 (acetate form, 0.72 mE/gm), collecting the e&rent in j-ml fractions. Each fraction was assayed enzymatically for F-6-P, G-6-P, and 6-phosphogluconate. The earlier F-6-P-containing
AMEBAL
ISOMERASES
fraction yielded 0.3 mmoles of F-6-P which was free from the two contaminants. This method of purification of the substrate appears to offer advantages over that employed by Kahana et al. (1960) and Salas et al. ( 1965). The horizontal paper electrophoresis apparatus and the cellulose acetate strips, designated Sepraphore III, were obtained from Gelman Instrument Company, Ann Arbor, Michigan. Biogel P-300 was obtained from Calibiochem, Los Angeles, Calif. DEAE Sephadex was obtained from Pharmacia Fine Chemicals, Inc., Pescataway, N. J. Amebal extracts. The lyophilized amebae, representing known volumes and numbers of centrifugally packed cells, were suspended in mM EDTA, pH 7, and mixed for 30 minutes at 0°C. The extracts of typical E. histmolytica, E. terrapinae, and E. invadens were made to represent 50 million cells/ml while the atypical E. histolytica and E. moshkowskii were made up I- or 200 million/ml. The cellular debris was removed by centrifugation at 30,000 g for 10 minutes. The supernatant fluid was employed, before and after dialysis, in the electrophoretic experiments. Dialysis was against 100 volumes of mM EDTA, pH 7, in the cold, overnight. The dialyzed extracts were used for enzyme assays. Fresh amebae were suspended in the EDTA solution in a plastic tube which was floated in ice water in the chamber of a g-kc Raytheon magnetostriction apparatus. The full power was applied for 3 minutes after which the sonically disrupted suspensions were centrifuged and dialyzed as described above. The crude or purified enzyme was stable toward dialysis, storage at refrigeration temperatures, and to freezing and thawing. Enzyme assay. Glucose phosphate isomerase activity was assayed by a spectrophotometric method measuring the rate of
131
formation of glucose 6-phosphate from fructose g-phosphate. The isomerase was linked with an excess of glucose 6-phosphate dehydrogenase and the rate of reduction of NADP was observed at 340 mp. A Beckman DU spectrophotometer with the Gilford model-220 attachments was used. The standard assay solution contained 1 mM fructose 6-phosphate, 0.3 mM triphosphopyridine nucleotide, 4 pg/ ml glucose B-phosphate dehydrogenase, and 50 mM Tris buffer, pH 8.0. It was preincubated for 5 minutes to get rid of the glucose B-phosphate contaminant. Amebal enzyme was then added and the change in optical density was recorded for 3 minutes. The molar absorbancy of reduced NADP was taken as 6.27 X 10” at 340 rnp. A unit of activity is defined as that producing 1 pmole of glucose 6-phosphate/ minute at 25°C under the standard assay conditions noted above. Specific activity is reported as units of enzyme activity per milligram protein. Electrophoresis. The cellulose acetate strips were soaked for 10 minutes or more in a buffer containing 4.88 gm/liter sodium barbital, and 3.24 gm/liter sodium acetate, adjusted to pH 8.0 with HCl. Excess liquid was removed on paper towels. The strips were then stretched over the electrophoretic bridge and streaked with 5-10 ~1 of amebal extract, Electrophoresis was for 1 or 2 hours at 12V/cm at room temperature. At the end of the run, the strips were developed for glucose phosphate isomerase activity. The developing solution contained 50 mM Tris-HCI pH 8.0; 0.3 mM NADP; 1mM fructose 6-phosphate; 0.4 mg/ml p-nitroblue tetrazolium chloride; 25 pg/ml glucose 6-phosphate dehydrogenase and 20 pg/ml phenazine methosulfate. The isomerase bands appeared within five minutes at room temperature and about fifteen minutes was required to develop their maximal color.
132
MONTALVO AND REEVES
Omission of the phenazine methosulfate resulted in the formation of spurious bands. RESULTS
Enzyme In typical Activity
preparation experiments
and purification. listed in Table I
TABLE I of Glucose Phosphate Isomerases from Amebal Trophozoites
Source Typical E. histolytica DKB DKB DKB DKB Atypical E. histolytrca Laredo Laredo Laredo Huff E. moshkowslcii E. invackns
Fresh (F) or lpophiliaed (L)
C/ml packed cells
U/mg protein in amebal extract
F F L L
29.5 20.0 14.0 12.5
.78 -.33 .29
L L L F F L
21.7 11.9 29.0 27.0 34.0 12.0
.43 .32 .82 1.00 1.25 .44
and lyophilized cells fresh trophozites yielded 1234 units of soluble isomerase activity per milliliter of packed cells. Amebal glucose phosphate isomerase was separated from other enzymes of the glycolytic pathway and extensively purified in the following manner: An extract of fresh DKB trophozoites was placed on a 3 cm2 x 32-cm column of Biogel P-300 which had previously been conditioned with 0.02 M potassium phosphate, pH 6.4, containing 0.001 M EDTA. The column was eluted with the same buffer and the fractions containing isomerase were combined. The recovery of activity was quantitative. The enzyme had been purified 7-fold based on its protein content. It still contained significant amounts of glucokinase and phosphoglucose mutase activity. The product was concentrated to 5 ml by vacuum dialysis against the phosphate buffer, and applied to a 3 cm2 X 14-cm
column of DEAE Sephadex previously conditioned with, 0.02 M potassium phosphate, pH 7, containing mM EDTA. This column was eluted with the pH 7 buffer and a leading peak of isomerase emerged, followed by the glucokinase. The tubes containing the first half of the isomerase peak were of the highest specific activity. They were combined to yield six units of isomerase. The overall purification of the enzyme amounted to 91-fold. This enzyme solution was free from glucokinase and phosphoglucose mutase. Its electrophoretic and kinetic properties were similar to those of the crude enzyme. Electrophoretic results. The electrophoretic mobilities of the isomerase from the DKB and 200 strains are compared in Fig. IA. Comparisons such as this were made by placing the DKB amebal extract along the starting line on one half of a cellulose acetate strip and extract from a second strain or species of ameba on the other half. Results of this work indicated that the isomerases from the DKB, 200, K-9, F-22, JH, JS, NRS, and BH strains all had identical electrophoretic mobilities. However, the extracts from the atypical E. histolytica strains all showed isomerases migrating much faster than did that of the DKB strain. For the fast-migrating isomerases, that of the Laredo strain was chosen as standard and comparisons were made against the Laredo enzyme. Results of this study showed that electrophoretically identical fast-migrating isomerases were obtained from the Laredo, Huff, JA, AG, and 403 strains, and from each ‘of the two cultures of E. moshkowskii. Figure 1B illustrates the results obtained with enzyme from three of the atypical E. histolytica strains and one strain of E. moshkowskii. For comparison, enzyme from the DKB strain (typical E. histolytica) was included in this experiment. Electrophoretic comparison between en-
AMEBAL
133
ISOMERASES
DKB
E mod. 403 AG Laredo
FIG. 1. Electrophoretic comparisons of glucose phosphate isomerases from various sources. A. A typical result illustrating the electrophoretic identity of isomerase from two strains of typical E. kistolytica (strains DKB and 200). B. A typical result illustrating the electrcphoretic identity of isomerase from three strains of atypical E. histolytica (strains Laredo, AC, and 403) with the enzyme from E. moshkouzkii (strain FIC). For comparison the enzyme from a typical strain of E. histolytica (DKB) was included in this experiment. C. Half of this strip was streaked with a mixture prepared from enzymes E. inoadens (PZ strain), E. histolytica (DKB and Laredo strains) and yeast isomerase. The other half of the strip was spotted with the enzymes, singly. The results show that a mixture of isomerases can be resolved by electrophoresis. D. Migration of enzyme from the Laredo strain of ameba is compared with that extracted from Bacteriodes symbiosus, the associate organism in the ameba cultures. Print A was made from a color-film positive, prints B, C, and D from black and white negatives.
zymes from the DKB strain and E. invadens revealed that both cultures of the latter organism contained an isomerase which migrated more slowly than did the enzyme of the DKB strain. The isomerase of E. tewapinae was indistinguishable from that of E. invadens by this technique. Figure 1C shows, on a single electropherogram, the results obtained with slow-, intermediate-, and fast-migrating amebal isomerases. For comparison this experiment also included a sample of crystalline yeast isomerase. To the top of this figure all four isomerases were mixed and placed together on the starting line; below, each was spotted individually along the starting line. Inspection of the figure reveals that the isomerases of the typical and atypical E. histoZytica strains (DKB and Laredo, respectively) are widely separated by electrophoresis, that yeast isomerase migrates between these two, and that the isomerase from E. invadens is the slowest migrating of these enzymes. Kineiic studies. Reported in Table II are
some results of biochemical kinetic studies on the amebal isomerases. Some published results on isomerases from other sources have also been included in the table. That the inhibition of amebal isomerase by 6phosphogluconate is competitive with substrate was evidenced by Lineweaver-Burk plots of data from all investigated amebal isomerases. Bacterial isomerase. Fresh B. symbiosus cells, 1.8 gm, were suspended in 16.2 ml of .02 A4 KPO, buffer (pH 6.4) containing mM EDTA and subjected to sonic disintegration in the Raytheon apparatus. This solution was placed directly in the sample cup and the treatment was continued, with periods. The cooling, for two Sminute solution was then centrifuged for 30 minutes at 27,000 g to remove unbroken cells and cell debris. The solution contained 304 units of glucose phosphate isomerase by the standard assay and 195 mg of protein. Ten ml of the centrifuged bacterial extract was fractionated with ammonium sulfate. Protein fractions insoluble at 50, 65,
134
MONTALVO
AND
REEVES
helminths, bacteria, and amebae. The biochemical kinetic properties of the enzyme from all investigated sources appear Inhibition by to be quite similar. The amebal isomerases 6-phosphoKm (F-6-P), gluconate were electrophoretically distinct from those Source mM Ki, mM of yeast or B. symbioses, the associate organism. Results from this worke The electrophoretic mobilities of the B. hzstolytica amebal glucose phosphate isomerases fell DKB strain .08 ,013 ,066 into three groups. The five atypical E. Laredo .12 ,015 histolytica strains and the two E. naoshHuff 19 ,025 kowskii strains had a fast-migrating enE. invadens :09 ,015 E. zyme; all eight strains of typical E. moshkowskii .09 ,028 histolytica yielded an intermediately miYeast .14 ,023 Selected results from the literature grating enzyme; and the two strains of Yea& .15 ,015 E. invadens and the E. terrapinae yielded Rabbit muscled 10 a slow-migrating enzyme. The classificaRabbit muscle and .03 ,005 tion of amebal cultures based upon the bra& properties of amebal isomerases was idenS. mansonid .lO Human erythrocytese .03 ,005 tical to that previously proposed by Reeves Bovine mammary .07 et al. (1967) based on studies of amebal gland’ glucokinases. There is a striking uniformity in the electrophoretic properties of the a Km’s and Ki’s were determined from Lineweaver-Burk plots of data from four appropriate enzyme from typical E. lzistolytica of substrate concentrations. The conditions used were diverse geographical origin, from England those of the st,andard assay (see Materials and to Korea, and having dates of isolation Methods) exceptf for substrate and inhibitor conranging from 1924 to 1959. centrations. Crude, dialyzed amebal enzyme soluThere is a growing realization of the tions were employed except in one noted instance. 6 This value was obtained with column purified marked differences between the two enzyme and purified F-6-P. groups of organisms herein designated as c Salas et al. (1965). typical and atypical E. histolytica. Entner d Buediny and MacKinnon (1955). and Most (1965) applied the terms “rege Kahana et al. (1960). ular” or “true” to the former. The term f Hines and Wolfe (1963). strains” has been “room-temperature widely applied to the latter since, unlike and 80% saturation were collected, redisthey multiply at or the typical strains, solved in buffer, and dialyzed. The fracbelow room temperature. Entner et al. tion representing 65-80% saturation con( 1962), Entner and Most ( 1965), and tained 12.5 mg protein and 125 units of enzyme activity. Upon electrophoresis this Albach et al. (1966) have reported differences in drug sensitivities between these bacterial enzyme was found to migrate groups of organisms. Richards et al. (1966) considerably faster than the enzyme from have summarized the earlier findings on the Laredo strain of amebae (see Fig. 1D). the two groups and have suggested that DISCUSSION only the former may be associated with severe human disease. Albach and Shaffer Glucosephosphate isomerase is probably (1965) have noted differences in free an ubiquitous enzyme. It has been obamino acid content, and Goldman et al. served in many mammalian tissues, yeasts, Kinetic
Properties
TABLE II n.f Glu,cose Phosphate Isomerases
AMEBAL
ISOMEFiASES
135
CLG medium. Journal of Protozoology 12, ( 1960) and Goldman and Cannon (1967) 659-665. have noted immunological differences beALBACH, R. A,, SHAFFER, J. G., AND WATSON, tween the typical and atypical strains. Our R. H. 1966. A comparison of in vitro drug present studies and those of Reeves et al. sensitivities of strains of Entamoeba which ( 1967) emphasize qualitative enzymatic grow at 37°C and at room temperature. The American Journal of Tropical Medicine differences between the two groups. and Hygiene 15, 855-859. Entner and Most (1965) have suggested BUEDING, T., AND MACKINNON, J. A. 1955. that differentiation of species is required Studies of the phosphoglucose isomerase of between the “true” or typical strains and Schistosoma mansoni. The Journal of Biologatypical or “room-temperature” strains of ical Chemistry 215, 507-513. E. histolytica. It appears to us that prior ENTNER, N., AND MOST, H. 1965. Genetics of Entumoeba: characterization of two new to the assignment of a new species desigparasitic strains which grow at room tempernation serious consideration should be ature (and at 37’C). Journal of Protozoolaccorded to the possibility that the atypical ogy 12, 10-13. E. histolytica organisms belong to the ENTNER, N., EVANS, L. A., AND GONZALES, C. species E. moshkowskii. This suggestion 1962. Genetics of Entamoeba histolytica. has also been made by Goldman and CanDifferences in drug sensitivity between Laredo and other strains of Entamoeba hisnon (1967) based on their immunological tolytica. Journal of Protozoology 9, 466-469. findings. A significant aspect of our present GOLDMAN, M., AND CANNON, R. T. 1967. Antifindings is the apparent identity of the genie analysis of Entamoeba histolytica by isomerase from the atypical E. lzistolytica means of fluorescent antibody. V. Comparito that from E. moshkowskii. son of 15 strains of Entamoeba with information on their pathogenicity to guinea pigs. Bueding and MacKinnon (1955) reThe American Journal of Tropical Medicine sorted to immunological procedures to and Hygiene 16, 245-254. distinguish between the isomerases of GOLDMAN, M., CARVER, R. K., AND GLEASON, Schistosoma and rabbit muscle. The imN. N. 1960. Antigenic analysis of Entamoeba munological technique failed to distinguish histolytica by means of fluorescent antibody. between the enzymes of three schistosoma II. E. .histoZytica and E. hartmanni. Experimental Parasitology 10, 366-388. species: S. mansoni, S. japonicum, and S. HINES, M. C., AND WOLFE, R. G. 1963. Phoshematobium. It might be fruitful to apply phoglucose isomerase. II. Influence of pH on the electrophoretic technique to such probkinetic parameters. Biochemistry 2, 770-775. lems of isomerase characterization. ACKNOWLEDGMENT We are indebted to Dr. James G. Shaffer for cultures of K9, 200, and F22 strains; to Dr. Quentin M. Geiman for the DKB strain; to Dr. William Balamuth for the two strains of E. inwadens, and E. terrupinae; to Dr. Frank Connell for the Laredo strain; to Dr. Morris Goldman for the JA, AG, 403, and Huff strains and for E. moshkowskii, strain FIC; and to Professor Armando Ruiz G. for a culture of I?. moshkowskii, strain CA-2. REFERENCES ALBACH, R. A., AND SHAFFER, J. G. 1965. Free amino acid analyses of 4 strains of Entamoeba histolytica and E. invadens in the
KAHANA, S. E., LOWRY, 0. H., SCHULZ, D. W., PASSONNEAU, J. V., AND CRAWFORD, E. J. 1960. The kinetics of phosphoglucoisomerase. The Journal of Biological Chemistry 235, 2178-2184. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin Phenol reagent. Journal of Biological Chemistry 193, 265-275. REEVES, R. E., AND WARD, A. B. 1965. Large lot cultivation of Entamoeba histolytica. Journal of Parasitology 51, 321-324. REEVES, R. E., MELENEY, H. E., AND FRYE, W. W. 1957. A modified Shaffer-Frye technique for the cultivation of Entamoeba hi.stoZytica and some observations of its carbohydrate requirements. American Journal of Hygiene 48, 124-130.
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REEVES, R. E., MONTALVO, F., AND SILLERO, A. 1967. Glucokinase from Entamoeba histolytica and related organisms. Biochemistry ~6, 1752-1760. RICHARDS, C. S., GOLDMAN, M., AND CANNON, L. T. 1966. Cultivation of Entamoeba histolytica and Entamoeba histolytica-like strains at reduced temperature and behavior of the amebae in diluted media. American Journal of Tropical Medicine and Hygiene 15, 648-655.
AND
REEVES
SALAS, M., VINUELA, E., AND SOLS, A. 1965. Spontaneous and enzymatically catalyzed anomerization of glucose 6-phosphate and anomeric specificity of related enzymes. The Chemistry 246, 561Journal of Biological 568. SCHWARTZ, M. K., AND BODANSKY, 0. 1965. Use of commercially available preparations of fructose B-phosphate in the determination of phosphohexose isomerase activity. Analytical Bioc.hemistvy 11, 48-53.