Trypanosoma lewisi: Alterations in membrane function in the rat

EXPERIMENTAL

PARASITOLOGY

48,

Trypanosomo

(1979)

lewisi:

WAYNE Department

15-26

of Zoology,

Alterations in Membrane in the Rat

Function

P. SCHRAW AND GERALD L. VAUGHAN 1 University

(Accepted

of Tennessee,

for publication

Knoxville,

14 November

Tennessee 37916,

U.S.A.

1978)

SCHRAW, W. P., AND VAUGHAN, G. L. 1979. Trypanosoma lewisi: Alterations in membrane function in the rat. Experimental Parasitology 48, 15-26. DEAE-celhdosepurified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (nondividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K’ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V,,, 4.00 * 1.02 nmoles/ 1 X 108 cells/min) with respect to 7-day celb (V,,,,, 1.83 * 0.62 nmoles 1 X 10’ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K, = 0.30 2 0.02 mM) than in ‘I-day cells (K, = 0.59 -1- 0.11 mM). When leucine and K’ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V,,,) of both substrates were observed for 4-day cells; K, values for leucine and K’ were not altered by the stage of infection. For leucine, the V,,, and K, for 4-day cells were 2.40 * 0.50 nmoles/l X 10” cells/30 set and 78 k 7 pM, respectively; corresponding values in 7-day cells were 1.06 2 0.02 nmoles/l X 10” cells/30 set and 66 * 11 PM. For K’, the I’,,, an d K, for 4-day cells were 15.97 * 0.38 nmoles/l X 108 cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 f 1.82 nmoles/l X 10’ cells/min and 1.05 mM. The observed increlse in the rate of K’ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K’ efflux was noted when 4- and ‘I-day cells were compared (t: of K’ leak for 4- and 7-day cells were 68.1 ? 9.3 and 67.9 2 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5’-nucleotidase, was observed when 4- and 7-day cells were compared. INDEX DESCRIPTORS: Trypanosomu lewisi; Hemoflagellate; Protozoa, parasitic; Memchromatography; Glucose; branes; Transport; Rat; Ablastin; Metabolism; DEAE-cellulose Leucine: Potassium.

given the name ablastin (Taliaferro 1932) and has been shown to be a 6 S protein ( D’Alesandro 1959). Numerous reports have dealt with metabolic alterations in T. lewisi which appear concurrently with the production of ablastin. Moulder (1948a) demonstrated decreased glucose oxidation and increased O2 consumption in growth-

INTRODUCTION

Early work by Taliaferro (1932) has shown that rodents infected with Typanosoma lewisi produce an antibody which inhibits cell division of the parasite without affecting its viability. This antibody was 1 To whom reprint requests should be addressed. 15

0014-4894/79/040015-12$02.00/O Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

16

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AND

inhibited cells, and attributed this difference to an ablastic effect. Taliaferro and Pizzi (1960) found decreased [“Slmethionine and [‘*C ] adenine incorporation in inhibited organisms in uivo; they concluded that ablastin inhibited protein and nucleic acid synthesis. D’Alesandro and Sherman (1964) and D’Alesandro (1964) found reduced activity of several glycolytic and phosphogluconate pathway enzymes in antibody-inhibited cells. These reductions were attributed to indirect effects of the antibody on cellular metabolism. This conclusion was based on the observed nonpenetration of antibody into intact cells ( D’Alesandro 1970). Patton ( 1970, 197%) demonstrated enzymatically the existence of ouabain-sensitive, Na+, K+, Mg’ -dependent ATPase activity in T. lewisi. The activity of this enzyme was depressed by IgG-enriched immune serum. Strickler and Patton ( 1975) implicated cyclic 3,‘5’-AMP as a factor in ablastic inhibition of cell division by demonstrating increases in the intracellular concentration of nucleotide in inhibited cells. Several investigators have suggested that ablastin exerts its influence at the plasma membrane ( D’Alesandro 1970; Bawden 1975 and Patton 1975) by influencing transport, membrane-bound enzyme activity or other structural components. While numerous studies have been concerned with metabolite transport in trypanosomes (Sanchez and Read 1969; Southworth and Read 1969, 1970; Manjra and Dusanic 1972, 1973; Ruff and Read 1974a, b; Sanchez 1974; Patton 1975; Goldberg et al. 1976; and Jackson and Fisher 1977), only that of Patton (1975) has dealt with comparative aspects of transport during normal infections. Patton ( 1975) cited evidence that glucose transport in T. lewisi was depressed by ablastic serum. A decrease in the V,,,,, for glucose transport was observed; the affinity of the carrier, however, was unchanged.

VAUGHAN

This investigation compares membrane function (glucose, leucine, and potassium transport; S-nucleotidase activity) in dividing and ablastin-inhibited T. lewisi. The aim of this investigation was to determine whether the glucose carrier is the unique locus of ablastin action or if glucose transport is only one of several membrane-bound activities influenced by the antibody. MATERIALS

AND

METHODS

Rloodstream forms of the trypanosome, Typanosoma lewisi, were maintained in 250 to 400-g male Sprague Dawley albino rats (Charles River Breeders, Wilmington, Mass.) by weekly intraperitoneal injections of 2 to 4 X 10’ cells in 5 ml of 145 mM NaCl. The strain of T. lewisi used was provided by Dr. J. L. Shaw, Department University of Kansas, of Microbiology, and was originally isolated by Dr. W. H. Taliaferro. On either the fourth (dividing) or the seventh day (nondividing trypanosomes) after infection (the day of injection was considered Day 0), rats were bled by cardiac puncture into 2 ml of 37 C, 0.2 M phosphate-buffered saline, pH 8.0, (Lanham 1968; Lanham and Godfrey 1970) per 8 ml blood. The phosphatebuffered saline (PBS) contained 150 units of sodium heparin. Penthrane (Abbott Labs, Chicago, Ill.) was used to anesthetize rats. Blood was centrifuged at 15OOg for 10 min at 0 to 2 C and the plasma removed. The buffy coat, containing trypanosomes, was resuspended in 4 ml icecold PBS, pH 8.0. For amino acid- and K+-transport experiments, the PBS used in the isolation and washing of trypanosomes contained 10 mit4 glucose. For glucosetransport and S-nucleotidase experiments, the PBS was glucose free. Cell suspensions were centrifuged at 1500g for 2 min at 0 to 2 C, and the upper layer containing trypanosomes, platelets, and white blood cells was removed from the erythrocytes

Trttpanosoma

kwisi:

below. This process was repeated a total of three times. Pooled cells were resuspended in 20 ml ice-cold PBS, pH 8.0, centrifuged 8 min at 15OOg (0 to 2 C), and the supernatant removed. The pellet was resuspended in 1 to 2 ml ice-cold PBS, pH 8.0, and was applied to a 23 x loo-mm DEAE-cellulose column (DE-52, Whatman) containing 20 ml packed resin (Lanham, 1968). Each column was used once. Ice-cold PBS, pH 8.0, was applied to the column, and 50 ml of effluent containing the trypanosomes collected. The effluent was monitored microscopically to determine the efficiency of the purification. No contamination with host cells was observed. Purified organisms were centrifuged at 15OOg for 8 min (0 to 2 C) and the supernatant removed. Transport. Tris-buffered saline was used in the handling and preparation of trypanosomes for transport experiments. The Tris buffer contained 25 mM Tris, 120 mM NaCl, 4.8 mM KCI, 2.6 mM CaC12, and 1.2 mM MgS04 (total cation concentration approximately 130 mM, Moulder referred to as 194813)) and is hereafter TBS 7.4. For glucose-transport experiments, the TBS 7.4 was used as described, while experiments involving leucine transport utilized TBS 7.4 containing 10 mM glucose. Finally, K+-free TBS 7.4 containing 10 mM glucose was used for K+-transport experiments. Column-purified cells were resuspended in 20 ml of the appropriate ice-cold TBS 7.4 and centrifuged at 15OOg for 8 min at 0 to 2 C. The pellet was resuspended in 20 to 25 ml of the appropriate ice-cold TBS 7.4. For K+ experiments, the buffer contained in addition 0.2% bovine serum albumin (BSA, Sigma Fraction V), which was required to minimize cell lysis during the K+-efflux experiments to be described. Radioactive D- [ 3H] glucose (sp. act. 15.1 Ci/mmol), L- [ 3H]leucine (sp. act. 60 Ci/ mmol), and the potassium analog, ““Rb (sp. act. 4.5 Ci/mg) were obtained from

MEMBGANE

PUNCTION

17

New England Nuclear Corporation, Boston, Mass. All other chemicals used were of the highest purity obtainable commercially. Purity of [3H] glucose was monitored by thin-layer chromatography in 30: 35: 5 chloroform, glacial acetic acid, and Hz0 on Gelman ITLC-SA plates. For an experiment, 0.6 ml of trypanosome suspension in the appropriate TBS 7.4 was preincubated 4 min at 37 C. An equal volume of 37 C buffer containing the desired Hor 86Rb-labeled substrate was added, the mixture vortexed, and incubations carried out in a 37 C water bath with shaking for the desired times. Nonradioactive glucose, leucine, or potassium was added to the appropriate label so that after dilution the following specific activities were obtained: 0.5 to 1.0 @/~mole (glucose), 1.0 &i/ pmole (leucine), and 0.1 to 0.2 &i/qole (K’) . Final concentrations of trypanosomes determined by hemocytometer count were between 3 and 5 x lo7 cells/ml. Glucose and leucine incubations were terminated by injecting 1.0 ml of cell-label suspension into 5 ml of ice-cold TBS 7.4 suspended over 25-mm Gelman fiberglass filters of 0.45-pm pore size (Fisher Scie-rtific Co.). After vacuum filtration (Vaughan and Cook 1972), filters were washed twice with 10 ml ice-cold TBS 7.4. Care was taken not to let filters dry during the wash procedure as this significantly influenced the results. After the final wash, filters were dried at 110 C for 30 min and were placed in vials containing 5 ml of 4 g Omnifluor (New England Nuclear) per liter toluene. Vials were counted in a Beckman LS 230 liquid scintillation counter at 40% efficiency. Potassum incubation (using s”Rb as the K+ tracer) was terminated by injecting 1.0 ml cell-label suspension into 5 ml icecold 20 mM K+ TBS 7.4 suspended over Gelman filters. After filtration, filters were washed twice with 10 ml of the same buffer, and were placed in vials containing 10 ml distilled HZ0 (Vaughan and Cook

1s

SCHRAW

AND

1972). The amount of “‘;Rb in the cells was determined by Cerenkov counting on a Beckman LS 230 counter (40% efficiency). Nonspecific absorption of radioactivity to cells and filters was measured by performing incubations at 0 C (Manjra and Dusanic 1972). The nonspecific absorption at 0 C was subtracted from the total radioactivity measured after incubations at 37 C. The difference of these two values was considered the specific uptake associated with cells. Potassium e@~x. Column-purified cells were resuspended in 20 ml ice-cold K-free TBS 7.4 containing 10 mA4 glucose and centrifuged at 1500g for 8 min at 0 to 2 C. The pellet was resuspended in 10 ml of Kc-free 0.2% BSA TBS 7.4 containing 10 mM glucose. The cell density was 1 x 10R cells/ml. Cells were preincubated 5 min at 37 C, and 10 ml of 37 C 8”RB-KC1 added. After dilution, a specific activity of 0.2 &i/pmole and a final K+ concentration of 5 mM were obtained. Following a 5-min incubation period with label, cell-free radiolabel was removed (Goldfine et al. 1972) following centrifugation at 15006 for 5 min (0 to 2 C). The pellet was resuspended in 20 ml ice-cold 5 mM K+ TBS 7.4 containing 10 mM glucose. After a final centrifugation at 1500g for 5 min (0 to 2 C ) , cells were resuspended in 20 ml of 37 C 0.17~ BSA TBS 7.4 containing 5.0 mM K+ and 10 ml\4 glucose. At the desired times, l-ml aliquots were injected into 5 ml of ice-cold 20 mM K+ TBS 7.4 layered over Gelman filters, filtered, washed, and counted as described for K+ transport. Potassium influx: azide and ouabain inhibition. Column-purified, washed (see K+ efflux) 4-day cells were resuspended in K+free 0.2% BSA TBS 7.4 containing 10 mM glucose. The cell density was 1.25 to 1.5 x lo* cells/ml. Ice-cold cell suspension (0.6 ml) was added to 0.6 ml ice-cold K-free TBS 7.4 with or without 10 mii4 sodium azide. Four samples were used per treat-

VAUGHAN

merit. Cells were preincubated for 30 min at 37 C. At this time, 0.5 &i of *‘;RB (100 ~1) in K-free TBS 7.4 was added, and incubations carried out for an additional 5 min. One-milliliter aliquots were taken, filtered, washed, and counted for radioactivity as described for K+ transport. Using the same cell preparations described for azide, the effect of ouabain (Sigma) on K+ influx was investigated. To 0.6 ml of cells at 37 C was added 0.6 ml 37 C K+-free TBS 7.4 with or without 10 mM ouabain. Four samples were used for each treatment. Cells were preincubated 1 hr at 37 C with shaking, and 0.5 &i ““Rb (100 ~1) was added to each cell suspension. Incubations were terminated 5 min later, and LO-ml aliquots were taken, filtered, washed, and counted for radioactivity as described for K+ transport. Assay of S-nucleotidase activity. A modification of the method of Gentry and Olsson (1975) was used in which 5’-nucleotidase activity (5’-ribonucleotide phosphohydrolase, EC 3.1.3.5) is the difference between total phosphatase activity and activity in the presence of a,p-methylene adenosine 5’-diphopshate (P-L Biochemicals, Milwaukee, Wis.), a specific inhibitor of 5’-nucleotidase. Cells purified by ion-exchange chromatography (Lanham 1968) in glucose-free PBS, pH 8.0, were resuspended in 20 ml ice-cold Tris maleate, pH 7.1, and were centrifuged at 1500g for 10 min (0 to 2 C). This process was repeated twice. The Tris buffer was 50 mM Tris, 3 mM MgC12, and 120 mM titrated to pH 7.1 (25 C) with maleic acid. Cells were resuspended in icecold Tris maleate MgClz (TMM 7.1; 50 mM Tris, 3 mM MgCla) at a ratio of 1 ml buffer per 100 ~1 packed cells. Cells were disrupted by two freeze-thaw cycles in an ethanol-CObath. Lysates were centrifuged at 1500g for 10 min (0 to 2 C), and the supernatant discarded. The pellet was resuspended twice in 20 ml of icecold TMM 7.1 and centrifuged as above.

Trypanosomu

lewisi:

The pellet was resuspended in TMM 7.1 at a ratio of 1.0 ml buffer/100 ,IJ packed trypanosome ghost. Protein was determined by the method of Lowry et aE. (1951) using BSA (Sigma) as the standard. A 100-~1 aliquot of crude membrane prepared as described contained approximately 100 pg protein. Assays were performed in duplicate at 37 C in a total volume of 0.5 ml using 85 to 100 pg protein/tube. Total phosphatase activity was measured in TMM 7.1 containing 100 pM S-AMP and 2.0 &i/ pmole 5’- [3H]AM,P (New England Nuclear, sp. act. 13.5 Ci/mmol). Inhibitorinsensitive phosphatase activity was measured in TMM 7.1 containing 100 PM 5’-AMP, 2.0 &i/pmole 5’-[ 3H]AMP and 50 PM a,/+methylene ADP. All samples contained 0.1% final concentration Triton X-100 which was added to the incubation buffer to eliminate variations caused by vesicle formation. Reactions were initiated by addition of trypanosome ghost to tubes preincubated 3 min at 37 C. Incubations were terminated by addition of 0.5 ml each of 0.15 M ZnSo4 and 0.15 Ba(OH)2 with

MEMBRANE

FUNCTION

19

vortexing. Tubes were centrifuged 15 min at 1500g at 20 C to pellet precipitated protein and unreacted S-AMP from the product ( [3H]adenosine) in the supernatant. One-half milliliter of the clear supernatant and 0.5 ml Hz0 were added to 10 ml scintillation fluid (8.5 g Omnifluor/liter toluene to which was added 500 ml Triton X-100), and counted in a Beckman LS 230 counter ( 35% efficiency ) . Determination of cell viability. Hemocytometer counts were made to estimate T. Zewisi viabihty after each experiment. Cell suspensions were diluted with 0 C isotonic 0.2% BSA TBS 7.4, and the proportion of viable cells determined. Nonviable trypanosomes were identified by the absence of motility and by a decrease in refractive index. RESULTS

DEAE-cellulose-purified Trypanosoma lewisi remained viable (95 to 99% ) and infective to the rat under all conditions described. Cell suspensionswere devoid of other blood components when examined microscopically. Glucose Transport

I

2

3

MlNUTES

FIG. 1. Initial entry of D-glucose into dividing and nondividing Trypanosoma lewisi at an extracellular glucose concentration of 0.5 mM. Incubations were performed at 37 C for the times indicated and the specific uptake (nmole glucose/ liter x IO” cells) was determined for dividing (0) and nondividing ( l ) cells, Each point is the mean of duplicate determinations.

Initial entry rates of glucose into dividing (4-day) and nondividing (?-day) trypanosomes were measured at an extracellular glucose concentration of 0.5 mM (Fig. 1). Uptake was linear for 1 min in both cell populations. The initial velocity of glucose influx was markedly greater in 4-day cells. The rate of glucose entry into 4- and 7-day organisms was measured at various extracellular substrate concentrations (Fig. 2). One-minute incubation times were chosen to eliminate, as much as possible, the effects of metabolism (Salter and Cook 1976) and membrane transconcentration (Goldfine et al. 1972; Whitesell et al. 1972; Whitesell et al. 1977; and Heaton and Gelehrter 1977). Influx was dependent

20

SCHRAW

AND

VAUGHAN

-

GLUCOSE

“T

(mM)

2. Glucose influx into dividing and nondividing Trypanosoma lewisi as a function of the extracellular glucose concentration. One-minute incubations were performed at 37 C and the specific uptake (nmole glucose/liter x 10’ cells/min) was determined for dividing ( 0) and nondividing ( l ) cells at each glucose concentration tested. Each point is the mean of triplicate determinations.

GSE

FIG.

on the extracellular glucose concentration with entry following typical MichaelisMenten kinetics. Glucose entered by carrier-mediated processes;no diffusional component of entry was observed. Doublereciprocal plots (Lineweaver and Burk 1934) of the saturable component (O.l2.0 mM) of glucose influx (Fig. 3) revealed the following changes in kinetic parameters. At 4 days postinfection, the V,,,,, and K, for initial glucose entry were 4.00 * 1.02 nmole/liter x lo8 cells/min and 0.30 t 0.02 mM, respectively. At 7 days, corresponding values were 1.83 t 0.62 nmole/liter X IO8 cells/min and 0.59 * 0.11 mM, respectively. Values are the mean -t the standard deviation (SD) of triplicate determinations performed on different days. Leucine Transport Figure 4 illustrates the initial entry of L-leucine into 4- and 7-day cells. At an extracellular leucine concentration of 20 PM, entry was linear for 30 sec. Negligible

(mM )

3. Double-reciprocal plots of the saturable component of glucose influx into dividing and nondividing Trypanosoma lewisi. l/Glucose influx is the reciprocal of velocity (nmole/liter X 10” cells/min) and l/Glucose is the reciprocal of the extracellular glucose concentration (m&f) for dividing ( 0) and nondividing ( l ) cells. Lines were drawn by least-squares regression. FIG.

incorporation of 3H into TCA-insoluble material occurred during the 2-min entry period studied. The initial velocity of leutine entry into 7-day cells was approximately 50% of that observed in 4-day cells.

MINUTES

FIG. 4. Initial entry of L-leucine into dividing and nondividing Trypanosoma lewisi at an extracellular leucine concentration of 20 pM. Incuba. tions were performed at 37 C for the times indicated and the specific uptake (nmole leucine/liter X 10’ cells) was determined for dividing ( 0) and nondividing ( l ) cells. Each point is the mean of duplicate determinations.

Typanosoma

kwisi:

Initial entry rates of leucine into 4- and 7-day cells were measured as a function of the extracellular leucine concentration (Fig. 5). Influx was concentration dependent displaying both saturable and nonsaturable components. Figure 6 illustrates the method used to interpret measured influx values depicted in Fig. 5 for the 7-day cells. Information relative to influx in 4-day trypanosomes was treated in a similar manner. Below 0.05 mM extracellular leucine, entry increased linearly with increasing amino acid concentration in the medium. Above this value, a second linear relationship was established between measured influx and the extracellular concentration of leucine. This bimodal dependence of entry on the extracellular leucine concentration was resolved into two components (Vaughan and Cook 1972 and Goldfine et al. 1972), a saturable and a diffusional component. The diffu-

MEMBRANE

21

FUNCTION

LEUCINE

hhll

FIG. 6. Leucine influx into nondividing TryZewisi as a function of the extracellular 1eucine concentration. Measured leucine influx ( 0) has been resolved into a saturable ( l ) and a diffusional ( A ) component (see text). The linear portion of the measured influx curve was derived by least-squares regression of the values obtained between 0.05 and 0.5 mM leucine. Each point is the mean of triplicate determinations. panosoma

sional component (Fig. 6) was calculated from the product of the slope of the measured influx and the leucine concentration at each substrate concentration tested. These calculated values were subtracted from the measured influx to obtain values for the saturable component. Measured leucine influx (V) was related to the extracellular leucine concentration (S) by the equation

V v = 2Ky; 0.1

0.2 LEIJCINE

0.3

0.4

0.5

(mM)

FIG. 5. Leucine influx into dividing Trypanosomu Zewisi as a function of the extracellular leutine concentration. Incubations (30 set) were performed at 37 C and the specific uptake (nmole leucine/liter X 10’ cells/30 set) was determined for dividing ( 0 ) and nondividing (l ) cells. Values are the mean -C SD of triplicate determinations.

s

+sLs,

where V,,,, is the maximal leucine influx described by the saturable component, K,, is the leucine concentration at V = V,,,/2 for the saturable component, and SL is the slope of the diffusional component. When s<< Km, this equation reduces the form V = I’,,,, + SLS illustrating that V,, can be determined by extrapolating the line defining measured influx at high substrate concentrations to the ordinate.

23

SCHRAW

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VAUGHAN 60-

MINUTES

-0 I

LEUCINE

(mM)

FIG. 7. Double-reciprocal plots of the saturable component of leucine influx into dividing and nondividing Trypanosoma lewisi. IjLeucine inllux is the reciprocal of velocity (nmole/liter X 10’ cells/30 set) and l/leucine is the reciprocal of the extracellular leucine concentration (mM) for dividing ( 0) and nondividing ( l ) cells. Lines were drawn by least-squares regression.

Double-reciprocal plots (Fig. 7) of the saturable component of leucine influx for 4- (0.01 to 0.02 mM) and ‘I-day (0.01 to 0.1 mM) cells provided the following kinetic parameters, At 4 days postinfection, the V,,,,, and K,, for initial entry were 2.40 f 0.50 nmole/liter x lo8 cells/30 set and 78 * 7 PM, respectively. At 7 days, the corresponding values were 1.06 I 0.02 x 10s cells/30 set and 66 inmole/liter 11 PM, respectively. All values cited are the mean + SD of triplicate determinations performed on different days.

FIG. 8. Initial entry of potassium into dividing and nondividing Trypanosoma Zewisi at an extracellular K+ concentration of 5 mM. “Rubidium was used as the K’ tracer. Incubations were performed at 37 C for the times indicated, and the specific uptake (nmole K/liter X 10’ cells) was determined for dividing ( 0) and nondividing ( l ) cells. Each point is the mean of duplicate determinations.

for 1 min in both cell populations, and K entered 4-day cells at a greater rate than 7-day cells. 60 I

1 0

Po+assium Transport Using ““Rb as a potassium tracer, initial entry rates of K+ into 4- and 7-day cells were measured at an extracellular K+ concentration of 5 mM (Fig. 8). *“Rb was used instead of 42K because of its longer half-life and its similarity to K+ in other systems (Vaughan and Cook 1972; Banerjee and Bosmann 1976). Influx was linear

20

40

60

MINUTES

FIG. 9. Release of “Rb (K’) from dividing and nondividing Trypanosoma lewisi. Experiments were performed as described under Potassium efflux. Application of the first sample to the filtration apparatus occurred at 0 min. Points represent the mean of the amount of K+ remaining ( nmole K+/liter X 10’ cells) in dividing ( 0 ) and nondividing ( o ) cells at the time of sampling for four determinations. Lines were drawn by least-squares regression.

Trypanosoma

lewisi:

25 1

I

2

5

IO

K+hM)

FIG. 10. Initial entry of K’ into dividing and nondividing Trypanosoma lewisi as a function of the extracellular K’ concentration. Specific uptake (nmole K/liter X 10’ cells/min) was determined for dividing (0) and nondividing ( l ) cells after I-min incubations at 37 C. The V,,, for duplicate determinations was obtained by extrapolation of the linear component (5 to 10 mA4) of measured influx to the ordinate (dashed lines). K, is the intersection of the dotted lines with the abscissa.

Potassium-8GRb-loaded cells were allowed to leak into 5.0 mM K’, and radioactivity remaining in the cells determined as a function of time (Fig. 9). Potassium e&x was linear in both cell populations over the period studied. Extrapolation of the line defining measured efllux to the abscissa enabled estimation of the turnover time for K+ in both cell populations. From this value, calculated half-times for leak were derived for 4-day (68.1 * 9.3 min ) and 7-day (67.9 -+ 15.2 min) cells (mean -t SD of four determinations performed on different days ) . The similarity of these values indicates that ablastin does not influence the rate of cation efflux in T. lewisi. Initial influx of K’ into 4- and 7-day trypanosomes was measured as a function of the extracellular potassium concentra-

MEMBRANE

FUNCTION

23

tion (Fig. 10). Over the concentration range studied, entry was dependent on the K+ concentration of the medium. The curve defining measured K+ was resolved into saturable and nonsaturable components, and was analyzed as previously described for leucine. The V,,,,, for K+ entry was determined by extrapolation of the linear component of measured influx (dashed lines ) to the ordinate. V,,l,, values of 15.97 -t 0.38 and 4.76 -t 1.82 nmole/ liter x 10s cells/min (mean * SD of duplicate determinations performed on different days) were obtained for 4- and 7-day cells, respectively. The K, was the substrate concentration where V = V,,,/2 (Fig. 10, dotted lines). Values of 1.20 and 1.05 mM were obtained for 4- and 7-day cells, respectively. K+ entry was apparently sigmoidal at low substrate concentrations (0.2 to 1.0 mM). Potassium influx was inhibited by azide but not by ouabain. Azide (5 mM, 30 min) inhibited 30% of the total K+ flux into 4-day cells (4601* 258 and 6575 * 728 cpm/liter x lOa cells/5 min *“Rb pulse in treated and control cells, respectively). Ouabain (5 mM, 60 min) had no effect on K+ influx (6732 * 165 and 6934 +- 184 cpm/liter x lOa cells/5 min 86Rb pulse in treated and control cells). All values reported are the mean F SD of triplicate determinations performed on different days. Decreased K+ influx in azide did not appear to be caused by cell injury; 90 to 95% cell viability was estimated for cells with and without azide. In order to determine if all membranebound activity is depressed in 7-day cell populations, the activity of 5’-nucleotidase was measured in 4- and 7-day cells. This enzyme has been shown to be localized predominantly, if not exclusively, in the plasma membrane (Avruch and Wallach 1971; Glastris and Pfeiffer 1974), and has been used as a plasma membrane marker. Using the radioassay of Gentry and Olsson ( 1975)) 5’-nucleotidase activity was found

24

SCHRAW AND VAUGHAN

Y

: 5

IO

15

4 20

MINUTES

FIG. 11. 5’-Nucleotidase activity in dividing and nondividing Trypanosoma lewisi as a function of time. 5’-Nucleotidase activity (Fmole adenosine formed/mg protein) for dividing (A) and nondividing ( V ) cells was obtained by subtracting inhibitor-insensitive phosphatase activity from total phosphatase activity. Other symbols are: total phosphatase activity in dividing (0) and nondividing ( l ) cells; inhibitor-insensitive phosphatase activity in dividing ( X) and nondividing ( X .) cells. Each point is the mean of duplicate determinations.

to be linear for 10 min in both 4- and 7-day parasites (Fig. 11). No significant difference in activity was found when these populations were compared.

transport for glucose (Fig. 3), leucine (Fig. 7), and potassium (Fig. 10). In contrast to the observed effects on carriermediated activities, cessation of growth did not influence passive membrane permeability to potassium (Fig. 9) or the activity of membrane-bound 5’-nucleotidase (Fig. 11). These results indicate that not all membrane parameters are equally susceptible to modification by ablastin. Unlike Patton ( 1975), were have observed alterations in both the K,,, and V,,, for glucose transport (Fig. 3). The altered K,,, for glucose (without concomitant changes in K,, for leucine and potassium) suggests a membrane perturbation adjacent to or involving the glucose carrier. Because of the importance of glucose to the trypanosome (Moulder 1948b; Ryley 1951; Bowman et al. 1972; Clarkson and Brohn 1976; Jackson and Fisher 1977) we propose that alteration of glucose transport (coupled with subsequent changes in glucose metabolism) may be one of the initial events associated with the onset of ablastic inhibtion. To test this hypothesis, we are investigating the influence of glucose transport and metabolism on other Trypanosoma leu;isi functions known to be influenced by ablastin.

DISCUSSION

Numerous investigations (see Bawden 1975 for review) have attributed growth inhibition of the rat trypanosome, Trypanosoma lewisi, to host production of an antibody ( ablastin). Several authors (D’Alesandro 1970; Bawden 1975; and Patton 1975) have suggested that ablastin interacts with structural or functional components of the parasite plasma membrane. We have examined membrane function in T. lewisi isolated at 4 (dividing) and 7 (nondividing cells) days postinfection after removal of contaminating host cells on ionexchange columns (Lanham 1968). Under these circumstances, growth inhibition of the parasite was shown to be associated with a decline in the specific activity of

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