EXPERIMENTAL

PARASITOLOGY

41,

370-384

Entamoeba RAM~N Departments

(1977)

histolytica:

Membrane

Fractions

SERRANO,~ JANE E. DEAS, AND LIONEL

G. WARREN 2

of Biochemistry, and Tropical Medicine and Medical Parasitology, Louisiana State University Medical Center, New Orleans, Louisiana 70112, U.S.A. (Accepted

for publication

24 May 1976)

SERRANO, R., DEAS, J. E., AND WARREN, L. G. 1977. Entamoeba hi.sto&ica. Experimental Parasitology 41, 370384. A cell fractionation study was made of Entamoeba histolytica, a parasitic amoeba which lacks many typical subcellular organelles. Two membrane fractions were isolated from amoeba1 homogenates. Fraction V was pelleted at 5 XlO”g min and purified by density centrifugation at the interphase between sucrose layers of densities 1.13 and 1.23. It consists of homogeneous empty vesicles of 1.2 pm. Fraction M, isolated by centrifugation of the 5 X 1O’g min supernatant at 3 X IO’g min, is mainly composed of small vesicles and cistemae (0.1-0.5 pm), glycogen, and electron-dense bodies. Fraction V contains the plasma membrane since sugar transport with properties similar to that of intact cells was demonstrated, and 5’-nucleotidase (EC 3.1.3.5) was exclusively in fraction V. The presence of digestive vacuoles is suggested by the localization of acid phosphatase (EC 3.1.3.2) in this fraction. Both enzymes, distributed identically during differential and density centrifugation, and the acid phosphatase could not be solubilized by osmotic shock and/or repeated freezing and thawing. Fraction V and M contain, respectively, 0.57 and 0.54 mg of phospholipid/mg of protein, 0.04 and 0.11 mg of RNA/‘mg of protein, and 0.39 and 0.22 mole of cholesterol/mole of phospholipid. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed both specific and common polypeptides between both fractions. These data show that in E. histolytica there are two functional types of membranes sharing some similarities of composition. Fraction V would be common to the plasma membrane and to the digestive vacuoles (structures that are probably in continuous interchange through endocytosis and ameboid movement), and would contain hydrolases which, in typical cells, are in the lumen of the lysosomes. Fraction M would form cytoplasmic structures of the smooth ER type. INDEX DESCRIPTORS: Entamoeba histolytica Schaudinn, 1903; Amoeba; Membranes; Cell fractionation; Phagosomes; Enzymes; Transport; Chemical composition; Axenic cultures; Electron microscopy; Electrophoresis; Glucokinase (EC 2.71.12); Phosphoglucose isomerase (EC 5.3.1.9); Amylase (EC 3.2.1.1-2); ATPase (EC 3.6.1.3); 5’-Nucleotidase (EC 3.1.3.5); Acid phosphatase (EC 3.1.3.2). INTRODUCTION The ultrastructure of the parasitic amoeba Entamoeba histolytica has been examined 1 Present address: Institute de Enzimologia de1 C.S.I.C., Facultad de Medicina de la Universidad Autbnoma, Madrid, 34 Spain. 2 Address reprint requests to Dr. L. G. Warren, Department of Tropical Medicine and Medical Parasitology, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, La. 70112, U.S.A.

recently in several laboratories, (ElHashimi and Pittman 1970; Griffin and Juniper 1971; Rosenbaum ‘and Wittner 1970) and some striking differences are observed when this amoeba is compared with other animal cells. The most abundant structures in the cytoplasm are: (1) vacuoles of diameter 0.2 to 2 pm; (2) glycogen rosettes; (3) smaI1 cisternae of smooth endoplasmic reticulum (ER); (4)

370 zii%iP’

1977 by Academic Press, Inc. t3 09 rcpnuIllction in 8nY form -ed.

ISSN

0014-4894

Entarrweba helical arrays of ribosomes; ,and (5) the so-called “electron-dense bodies.” Although mitochondria, lysosomes, Golgi vesicles, and rough ER are lacking in E. hktolytica, these organelles are present in free-living amoebae (Bowers and Korn 1968; Daniels 1973). The large vacuoles are known to be involved in phagocytic processes (ElHashimi and Pittman 1970; Griffin and Juniper 1971; Rosenbaum and Wittner 1970), and the helical polyribosomes appear to be the site of protein synthesis as described for several animal cells in culture (Weiss and Grover 1968). The physiology of the cytoplasmic structures of E. histoZytica remains mainly speculative because a subcellular fractionation of this amoeba has never been done. This organism is an obligate anaerobe obtaining energy via a glycolytic pathway which differs from the classical EmbdenMeyerhoff pathway in several steps (Reeves 1972). Ethanol, acetate, carbon dioxide, and hydrogen are the end products, but the last steps of the fermentation and the mechanism of hydrogen evolution remain unknown. In another anaerobic protozoan, Tritrichomonas foetus, a new kind of organelle seems to be involved in these processes (Lindmark and Miiller 1973). Similar structures can be expected in E. histolytica. Although the factors responsible for the pathogenic properties of E. histolytica remain essentially unknown (Brandt and Perez Tamayo 1970)) recently two different experimental approaches have suggested that the properties of the surface membrane of the amoeba are involved in the pathogenic mechanism (Eaton, Meerovitch, and Costerton 1970; Martinez-Palomo, Gonzalez-Robles, and de la Torre 1973). We have made a cell fractionation study of E. histolytica as a preliminary step toward our understanding of the biology of this organism. In the following results we describe the isolation of two membrane fractions from amoeba1 homogenates, and their salient biochemical characteristics.

histolytica

371 MATERIALS

Cultivation

AND METHODS

of Amoebae

Entamoeba histolytica, strain 200: NIH, was grown in axenic culture as described by Diamond ( 1968), with added penicillin (700 units/ml). Amoebae were harvested after 72-90-hr cultivation, and sedimented by gentle centrifugation. Tbe pelleted amoebae were washed at room temperature with a salt solution containing 100 mM NaCl, 20 mM K2HP04, 0.5 mM Mg&, and 0.1 mM Ca( NOa)2, adjusted to pH 7.0 with HCl. Packed cell volume in the above solution was determined in a graduated 12-ml centrifuge tube following centrifugation for 3 min at 7OOg,,, (International clinical centrifuge). The disruption of the amoebae and subsequent subcellular fractionation are described under Results. Enzyme Assays All enzyme activities were assayed at 37°C. The activity was recorded during the linear portion of the activity curve. The amount of enzyme employed was within the range where activity was proportional to enzyme concentration. One unit of enzyme activity corresponds to the amount of enzyme that gives rise to 1 rmole of productlmin under the specified conditions of assay. Glucokinase (EC 2.71.12) and phosphoglucose isomerase (EC 5.3.1.9) were assayed as described by Reeves, Montalvo, and Sillero (1967). Amylase (EC 3.2.1.1-2) was assayed in a final volume of 0.1 ml containing 40 mM imidazole-HCl, pH 7.0, OSOJOglycogen (Sigma, Type II), and 2-40 mU of enzyme. At 0 and 20 min the reaction was halted by the addition of 0.1 ml of dinitrosalicylic acid reagent (Bemfeld 1955). The activity was expressed as reducing glucose equivalents. When assaying membrane fractions for the above three enzymes, Triton X-100 (Emulsion Engineering Inc., Elk Grove Village, Ill., U.S.A.) was added to a final

372

SEXRANO,

DEAS

concentration of O.l%,, to eliminate permeability barriers and to decrease turbidity. Mg-stimulated ATPase (EC 3.6.1.3) was assayed in a final volume of 0.2 ml, containing 40 mM Tris-HCl, pH 7.5, 5 mM MgC&, 2.5 mM ATP (Sigma), and l-10 mU of enzyme. S-Nucleotidase (EC 3.1.3.5) was similarly assayed but ATP and MgCls were omitted and the assay mixture contained 2.5 mM AMP (Boehringer) and 0.05% sodium deoxycholate. In both assays the reaction was stopped at 0 and 20 min by the addition of 0.8 ml of Fiske and Subbarow reagent (Leloir and Cardini 1957). Ascorbic acid, at lo/O final concentration, was added after centrifugation of the precipitate. Acid phosphatase (EC 3.1.3.2) was assayed in a final volume of 1.0 ml containing 2 mM p-nitrophenyl phosphate ( Nutritional Biochemical Corp. ), 40 mM sodium acetate buffer, pH 5.0, 5 mM MgC&, 0.1% Triton X-100, and l-30 mU of enzyme. The reaction was stopped at 0 and 2 min with 1 drop of 40% KOH, and the color was read at 400 nm. The extinction coefficient of p-nitrophenol under these conditions is 19000 M-l cm-l. Chemical

Analysis

Protein was determined by the method of Lowry, Rosebrough, Farr, and Randall (1951) using bovine serum albumin (Nutritional Biochemical Corporation) as standard. Prior precipitation of amoeba1 protein with 5% trichloroacetic acid did not alter values obtained subsequently by the Lowry procedure. Ribonucleic acid (RNA) was determined with or&o1 reagent following removal of acid soluble compounds, lipids, and proteins (Schneider 1957). To minimize errors due to insoluble glycogen the differences in absorbancy at 660 and 580 nm were used (Ashwell 1957). Phospholipids were extracted with chloroform-methanol, 2: 1, (Radin 1969) and washed with perchloric acid (Dittmer and Wells 1969). The inorganic phosphate was

AND

WARREN

then determined by the method of King (Lindberg and Ernster 1956). It was assumed that 1 mg of amoeba1 phospholipid contained 1 pmole of phosphate (Sawyer, Bischoff, Guidry, and Reeves 1967). Polysaccharide was determined as follows: Up to 50 ~1 of amoeba1 homogenate fractions were precipitated with 10 ml of 95% ethanol for 5 min. The suspension was filtered through a glass fiber filter (Schleicher & Schuell, Inc., 21 mm) and washed with 10 ml of 95% ethanol. The filters containing the precipitated polysaccharide were placed in 2 ml of distilled water and mildly agitated for 5 min. The glass filters were removed, and the liquid assayed for polysaccharide by the phenolsulfuric acid method (Dubois, Gilles, Hamilton, Rebers, and Smith 1956). Results are expressed as milligrams of glucose equivalents assuming a molecular weight of 162 for the glucose units in the polysaccharide. Cholesterol was assayed by the Liebermann-Burchard reaction (Stadtman 1957). Polyacrylamide

Gel Electrophoresis

Essentially the methods of Fairbanks, Steck, and Wallach (1971) were followed with concentrations of acrylamide (Eastman) and sodium dodecyl sulfate (Sigma) of 5.670 and 1.070, respectively, Proteins were stained with Coomassie blue (Mann Research Labs). Gel densitometry was done using a Gilford spectrophotometer and model 2410 linear transport accessory. Scanning was made through a O.l-mm slit at 550 nm. The sources of the molecular weight markers were as follows: ovalbumin, bovine serum albumin, trypsin, and ctyochrome c from Nutritional Biochemical Corp.; phosphorylase a and myoglobin (type II) from Sigma. Electron

microscopy

Pellets from subcellular fractions, equivalent to 0.1 ml of cells, were resuspended in 30-50 v01 of cold 3% glutaraldehyde in

Entumoeba 0.1. M cacodylatcHC1 (pH 7.3) buffer for 3 hr. The glutaraldehyde-fixed fractions were centrifuged ,at 7OOOg/min, washed three times with cold 0.25 M cacodylateHCl (pH 7.3) buffer, and stored overnight in this buffer at 4 C. The fractions were then washed once more with 0.25 M cacodylate buffer, postfixed in 1% osmium tetroxide-cacodylate buffer ( pH 7.3)) rinsed in deionized water, and dehydrated through a graded series of ethanols to propylene oxide. The embedding was in Spurr low viscosity embedding medium (Polysciences, Inc.). Sections were cut with a diamond knife on a Reichert OMU-2 ultramicrotome, mounted on naked 300mesh copper grids, stained with many1 acetate and lead citrate, and viewed in an Hitachi HU-11-A electron microscope at 50 kV. Uptake

of Glucose by Isoluted

Membranes

Isolated membranes were incubated for uptake studies at a concentration of 6-7 mg of protein/ml at a temperature of 25 C, in a sucrose solution containing 10 mM Tris-HCl, pH 7.5, and the final volume was 50-100 ~1. After a lo-min preincubation, the uptake was initiated by addition of [ U-14C]glucose ( ICN, Cleveland, Ohio, U.S.A.) in a final concentration of l-2 mM and a specific activity of about 4000 cpm/nmole. Samples of 5 or 10 ~1 were taken at appropriate time intervals, and mixed with 5-7 ml of ice-cold 0.25 M sucrose. They were filtered immediately, through Millipore filters (type HA, 0.45pm pore diameter), and the filters were dried under an infrared lamp. The filtration procedure took about 20 sec. The dried filters were transferred to a liquid scintillation vial, and radioactivity was measured as previously described (Serrano and Reeves 1974). Two types of controls were run in each experiment: In one the complete incubation mixture was kept at 0 C, and in the other the membranes were omitted,

histolyticu

373 &SULTS

Isolation

of Membrane

Fractions

All the operations were made at 04 C. Sucrose solutions were buffered with 10 mM Tris-HCl, pH 7.5, and contained 0.5 mM dithiothreitol ( Sigma). An anoptral phase microscope (Reichert, Vienna, Austria) was used to monitor the appearance of the homogenate at each step of fractionation. Centrifugations were made in glass tubes in a Sorvall Model RC2 centrifuge, rotor SS34, except when indicated, The g values correspond to maximum radii, Packed cells (1 to 2 ml) were resuspended in 50 mM sucrose to a concentration of 9 to 14% by volume. At this osmolarity the cells swell, but after 20 min extensive breakage is not yet observed. Homogenization is then accomplished by three to five strokes with the tight-fitting pestle of a Dounce homogenizer (Kontes Glass Co., Vineland, N.J., 15-ml capacity). This procedure ruptured more than 99% of the cells. The preswelling of the cells facilitates the rapid rupture in the Dounce homogenizer. Immediately after homogenization, sufficient 2 M sucrose was added to produce a final concentration of 0.25 M. The isotonically adjusted preparations were designated “whole homogenate,” and fractionated as described below and summarized in Fig. 1. An initial centrifugation was made for 90 set at 700g (International clinical centrifuge) . The supernatant was centrifuged again under the same conditions, to separate completely the debris. About 1% of the total protein, and of the total acid phosphatase activity, appeared in these pellets. Phase microscopic observation of the debris revealed the presence of large pieces of cells and of nuclei. The second supernatant was centrifuged for 10 min at 5000g and the resulting supernatant was centrifuged again under the same conditions. The final supernatant was centrifuged for 110 mm at 27,000g to yield “fraction M” (pellet) and ‘soluble phase” (supernatant ).

374

SERRANO,

DEAS

AND

WARREN

Osmotic swelling I” 50mM Sucrose

Resuspend in 0.25M Sucrose IO5 g x min

Supernatant

discard

I-

Resuspend in l.SM Sucrose Overlaid l.OM Sucrose

3 x IO6 q x min

FIG. 1. Scheme of the procedure

for the isolation of membrane

fractions from Entumoeba

histolytica.

The pellets of the SO,OOOg/min centrifugations, which consist primarily of vesicles but contain some nuclei, were washed in 10 ml of 0.25 M sucrose and centrifuged for 10 min at 10,OOOg.The supernatant of this washing was turbid indicating some loss of material. The washed 50,00Og/min pellet was resuspended in 4 ml of 1.8 M sucrose (density 1.23). A layer of 4 ml of 1.0 M sucrose (density 1.13) was overlaid and the preparation centrifuged for 110 min at 27,000g. Two visible fractions appeared in the tubes: (1) a pellet (“heavy particles”) and (2) an interphase (“fraction V”). The pellet was composed of nuclei and some large vesicles. The interphase contained only vesicles. The interphase material was diluted to a sucrose concentration of about 0%0.3M and centrifuged for 10 min at 10,OOOg.This supernatant was

perfectly clear indicating no extensive loss of material at this washing step. All the pelleted fractions were resuspended in 0.25 M sucrose to a volume ranging from 20 to 100% of the original packed cell volume used for the fractionation. Processing for electrophoretic analysis and for electron microscopy was made immediately after preparation of the fractions (about 4 hr following rupture of the cells). For chemical analysis, enzymatic assaysand transport studies frozen fractions, used within 48 hr, gave identical results as observed with freshly prepared fractions. Electron M~CTOSCO~ZJ of Membrane Fractions Two main membrane fractions were obtained by the above fractionation procedure: (1) fraction V, pelleted at relatively

Entamoeba low speed and purified through a sucrose gradient; and (2) fraction M, which required more prolonged centrifugation to be pelleted, and was not submitted to further purification. Freshly prepared fractions V and M were examined for ultrastructure. Fraction V (Fig. 2) consisted of closed empty vesicles 1 to 2 pm in diameter. In some cases small vesicles appear trapped inside the big ones. In general, the preparation is very homogeneous. In contrast, fraction M (Fig. 3) is heterogeneous: Vesicles 0.1 to 0.5 pm in diameter are abundant, but elongated cisternae, glycogen rosettes, electron-dense bodies, round bodies, and amorphous aggregates are also present. Properties and Distribution Marker Enzymes

of the

The total and specific activities observed in “whole homogenates” for the subcellular enzyme markers are shown in Table I. The phosphatase activity of amoeba1 homoge-

histolyticu

375

nates, as measured with p-nitrophenyl phosphate as substrate, was strictly dependent upon an acidic pH. The activities observed at pH 7.5, Tris-HCI buffer) and at pH 9.5 (triethanolamine buffer) were, respectively, 0.6 and less than 0.1% of the activity obtained with the standard assay at pH 5.0 (acetate buffer). Thus, the amoebae contain little alkaline phosphatase (EC 3.1.3.1). Th e acid phosphatase activity of whole homogenates is increased by about 100% by the addition of either MgCl, (5 mM) or Triton X-100 (O.l%), both effects being additive. The addition of Triton X-100 (0.1%) has no effect on the activity of glucokinase, phosphoglucoisomerase, and amylase. The amylase activity was relatively insensitive to pH, i.e., at pH 5.0 (acetate buffer) the activity was 70% that observed at pH 7.0 (imidazole buffer). The hydrolysis of glycogen by amoeba1 homogenates yielded primarily oligosaccharides as evidenced by the fact that free glucose enzymatically determined (with hexokinase and glucose 6-phosphate

FIG. 2. Electron micrograph of Entamoebahktolytka membrane fraction V. Typical vesicles showing structural homogeneity of the fraction, Bar represents 1 JAIIL

376

-NO,

DEAS

AND

WARREN

FIG. 3. Electron micrograph of Entamoebahktolytica membrane fraction M. Preparation is heterogeneous containing small vesicles (V) up to 1 pm in diameter, dense bodies (B), round bodies (RB), amorphous material (A), cisternae (C), and glycogen (G). Note that small vesicles constitute most of the preparation with other structures scattered between the vesicles. Bar represents 1 Frn.

dehydrogenase) was less than 7% of the reducing sugar value. The ATPase activity of amoeba1 homogenates was not affected by 0.050/o sodium deoxycholate, 50 mM NaCl, 25 mM KCl, and/or 0.25 mM ouabain. MgClz (5 mM) stimulates about twofold. Ca2+ is also an activator, and resulted in a five-fold stimulation. The activation by both divalent cations was not additive. The 5’-nucleotidase activity was not affected by the presence of MgClz (5 mM), but 0.05% sodium deoxycholate increased the activity by about 40%. The relative activities of the marker enzymes in the subcellular fractions are shown in Table I. About one-third of the total activity of 5’-nucleotidase and acid phosphatase is recovered in “fraction V” with a specific activity nine times greater than the whole homogenate. No other fraction has com-

parable total or specific activity. This is a strong indication that both enzymes, acid phosphatase and S-nucleotidase, are located almost exclusively in the membrane structures isolated in fraction V. Conversely, both fractions V and M contained substantial amounts of Mg ATPase, and in both fractions the specific activity was greater than in the whole homogenate. Thus, Mg-ATPase is a particle bound enzyme with a wide distribution among the subcellular structures of E. histolytica. The very low activity of acid phosphatase, 5’nucleotidase, and Mg ATPase in the soluble phase indicates all these enzymes are virtually localized in particulate fractions. The amylase activity was distributed in all the fractions. The major amount (66% of total) was in the soluble phase, but the highest specific activity was in the “heavy particles” (5.3 times greater than the whole homogenate). Glucokinase and phospho-

Entamoeba

histolytica

TABLE Act)ivity

of Marker

Fraction*

Whole

Acid

homogenate

TA SA

17.X 0.27’

Washed large particles pellet

TA 8.~ EF

8.0 1.3 5.6

Fraction

TA sa EF

0.1 2.4 x.9

TA SA EF

0.7 0.7 2.8

1’

Euxymes

i 1.3 f0.02

i 0.8 It 0.2

I

in Subcellular

Fractions

5’-Nucleotidase

phosphstase

jai

1.50 0.02

(2) (2)

0.8 0.13 ti.5

(4) (5)

0.5 0.18 9.0

(2) (2)

Fraction

hl

TA SA EF

1.8 0.3 1.1

f f

0.2 0.03

(4) (4)

Soluble

phase

TA S.-i EF

0.2 0.03 0.1

f f

0.1F 0.003

(3) (5)

f i

377

from

Entumorba

n1g ATPase

0.3 0.005

Amylase

(4) (4)

3.20 0.04

(2) (2)

1.0 0.1 3.5

Ik 0.05 f 0.04

(4) (4)

0.0 0.2 5.0

4 0.00 f 0.03

(4) (3)

2.7 0.0 4.5

0.1 0.1 5.0

* f

0.02 0.03

(3) (3)

0.1 0.1 2.5

f

(3)

0.1 0.03 1.3

f 0.03 * 0.01

(4) (4)

0.7 0.1 2.5

f f

0.14 0.02

n