The ablastin phenomenon: Inhibition of membrane function

EXPERIMENTAL PARASITOLOGY 38, 357-369 (1975)

The Ablastin

Phenomenon:

Inhibition

of Membrane

Function

CURTIS L. PATTON Department

of Microbiology,

New Haven,

Yale University School of Medicine, Connecticut 06510

(Accepted for publication April 17, 1975) PATTON, C. L. 1975. The ablastin phenomenon: Inhibition of membrane function. Experimental Parasitology 38, 357-369. Autoradiography of Trypanosoma lewisi labeled in vioo with ‘H-thymidine

( sHTdR) shows that the shortest doubling time for labeled organisms is 8 hr in intact and immunosuppressed rats. The parasite doubling time increases progressively after the fourth day of infection to 12 hr in immunosuppressed rats and to 24 hr or more in intact rats. The number of days following infection during which the trypanosomes reproduce is prolonged in immunosuppressed rats. In vitro studies of ablastin using ‘HTdR-labeled trypanosomes confirmed that cell reproduction halts in the presence of ablastin, but resumes when the parasites are removed from the antibody. Several lines of evidence have been obtained, indicating that the primary effects of ablastin may be on membrane function. Thus, the saturable component for glucose transport in reproducing and ablastin inhibited trypanosomes has an average K, value of 2.8 x 10m4M, but the average V,,, values for glucose transport are reduced from 3.15 nmole/min/l.25 X 10’ reproducing parasites to an average of 1.8 nmole/min/l.25 X 10’ nonreproducing forms. Glucose transport is competitively inhibited by 2-deoxyo-glucose (2DOG). The exit and counterflow of “‘C-2DOG from previously loaded trypanosomes is restricted in the presence of antiserum. Metabolism; Membranes; Immunity; INDEX DESCFUPTORS: Trypanosoma kwisi; Ablastin.

Trypanosomes are unique protozoa in that they live in the blood plasma of vertebrate hosts as a part of their life cycle. Not only do these parasites swim in the materials they require for their nutrition, but they also bathe in antibodies against them soon after they parasitize the bloodstream. Some of these antibodies are trypanocidal; others affect the physiology of the parasites more subtly (Moulder 1948; Masseyeff and Gambert 1963; Pizzi and Taliaferro 1960; Taliaferro and Pizzi 1960; Patton 1970). In trypanosomiasis a broad This paper is eighth in a series developed from a workshop from Ablastin held at the Rockefeller University, New York, N.Y. on June 21-22, 1973.

assemblage of antibodies is serologically demonstrable, but they often bear no relationship to the degree of functional immunity. Largely because of the immune responses it stimulates in the rat during parasitemia, Trypanosoma lewisi has held the interest of parasitologists for much of the present century. The most remarkable aspect of infections with T. bwisi is the development of antibody, called ablastin (Taliaferro 1932), that inhibits the reproduction of the parasites without otherwise damaging them. The course of T. lewisi infections is described by an exponential rise in para357

Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

CURTIS L. PATTON

sitemia during the first week (Figs. 1, 2). After this time, the rate of increase in parasitemia diminishes in intact rats (Fig. 2B) due to a complex of host defenses and heralds a precipitous drop in parasitemia. The first crisis is brought about by trypanocidal antibody (Coventry 1930; Taliaferro 1932). The point of inflection and plateau of the parasitemia curve in intact rats clearly precedes this crisis and corresponds with the development of ablastin (Taliaferro 1924, 1929, 1932). Ablastin and the antibody associated with the crisis

have been described as 7 S proteins ( D’Alesandro 1959). Though the departure from the exponential course of infection described by hemocytometer counts may be due to inhibition of reproduction of trypanosomes (Fig. 2B), it also reflects destruction of trypanosomes by phagocytes (Patton 1972a) and sequestration (Ormerod 1963; Greenblatt and Tyroler 1971) . and mensural criteria Morphological ( Taliaferro 1932)) Yj-amino acids and Wadenine incorporation in Vito (Taliaferro

FIG. 1 (A-D). Photographs showing incorporation of “H-TdR by Trypanosoma Zewisi as indicated by the black dots representing autoradiographs of the nucleus (N) and kinetoplast (K). Fig. 1A is a photograph of a blood smear from an intact rat infected with 7’. Zewisi for 3 days. Eight hours prior to making the smear, 500 UC of 3H-TdR (Sp AC = 3.5 mC/mmole) was injected into the animal. The labeled trypanosome left of center shows a cluster of black dots covering a single nucleus and kinetoplast. The labeled trypanosome right of center in the process of dividing shows a pattern of dots distributed over a pair of nuclei and a pair of kinetoplasts. A photograph of a bloodsmear from the same rat 16 hr after the rat had been injected with “H-TdR is shown in B. The photographs shown in C and D are of dividing trypanosomes pulsed for 2 hr during axenic culture in the presence of 3 UC of “H-TdR ( Sp AC = 3.5 mC/mmole) as an addition in 3 ml of medium. Trypanosomes shown in C were transferred after the pulse to ablastic medium for 22 hr. Trypanosomes shown ill D were placed in ablastic mediunl after the pulse and kept there for 12 hr before lacing transferred to normal medhml for an adclitional 12 hr.

INHIBITION

OF

MEMBRANE

FUNCTION

al---J0

Hours

after

‘H-TdR

Pu I se

I6 Hours after

32 3H-Tdf?

46 Pulse

0

FIG. 2 (A and B). Parasitemia (---) and mean grain counts (-) over 100 labeled trypanosomes following injection of 500 UC “H-TdR (Sp AC = 3.5 mC/mmole) into two intact rats and two rats treated with dexamethasone. Hemocytometer counts indicate that the doubling time for T. lewisi in rats infected for 3 days (A) is 8.5 hours in dexamethasone treated (- -0--) and intact rats --•--). Mean grain counts over labeled trypanosomes decreased by half after 8 hours over trypanosomes from rats given dexamethasone (-0-) and intact rats (-•-). When another set of rats was injected with “H-TdR 5 days after infection with T. Zewisi (B), the mean grain count over labeled organisms was reduced by half after 12 hr in dexamethasone treated rats (-0-) and 24 hr in intact rats (-•-). The increase in parasitemia in dexamethasone treated (--O-) and intact rats (--a--) ceased to be logarithmic at this time.

and Pizzi 1960; Pizzi and Taliaferro 1960), “H-thymidine incorporation in viva (Patton and Clark 1965) and in vitro (Patton 1972b) have been used to measure cell synthesis and reproduction in T. leu;isi. These studies support the conclusions that ablastin halts synthetic activity and arrests cell division. The parasites remaining in the peripheral circulation after the first crisis, therefore, live there with no apparent reproduction. A second crisis effected by 17 S (D’Alesandro 1959) try-

panocidal antibody (Coventry 1930; Taliaferro 1932) terminates the infection. Trypanosomes surviving the first crisis differ antigenically from reproducing forms (Entner and Gonzales 1966; Entner 1968; D’Alesandro 1966; Lincicome and Watkins 1965) and are resistant to the early try(Taliaferro 1932; panocidal antibody Coventry 1936). Thus, it appears that T. lewisi is capable of developing antigenic variants like the relapsing African trypanosomes. However, unlike the relapsing try-

360

CURTIS

panosomes, it expresses only two or three antigenic types (Entner and Gonzales 1966; Entner 1968). It is well known that during the course of infections with the brucei-subgroup, the trypanosomes have the confounding ability to change antigenitally (Frank 1905). Each relapse population of trypanosomes is of a different antigenic character from the population preceding it (Inoke et al. 1952; Soltys 1957; Cunningham and Vickerman 1962). As many as 22 antigenic variants of T. brucei were recognized by Ritz (1916), and they appear to develop in orderly succession (Gray, 1962, 1965). A single antigenie variant is maintained until the first appearance of antibody, and thereafter new variants appear every 3 days until the host dies (Gray, 1965). There is cause to speculate that ablastin limits antigenic variation in T. kwisi by halting cell synthesis. The changes that occur in morphology as dividing forms become adult forms are not collateral with antigenic changes. Before the first crisis, the coefficient of variation in total length drops. The trypanosomes appear uniform and have the morphology of adult forms. Most of these organisms are destroyed at the time of the first crisis, which indicates that these parasites have division form antigens. Immunodiffusion studies of division form antigens and adult antigens (Entner and Gonzales 1966; Entner 1968) show that transformation into morphologic adults precedes the corresponding antigenie transformation. Only the trypanosomes that complete the antigenic alteration before the first crisis escape destruction. If adult forms of T. Zewisi are used to initiate infections, they revert back to original antigenic forms when they begin to reproduce. D’Alesandro (1970) postulated that T. lewisi has evolved after a long association with ablastin to a point where it is now limited to expressing only the antigens which distinguish adults from reproducing forms and it has lost any

L.

PATTON

ability it may once have had for more extensive variation. One most important feature of the infection is that both reproducing and adult forms are susceptible to ablastin, which suggests that ablastinogen is a common antigen. Taliaferro (1941) proposed that formation of antibodies which check the reproduction of the parasites plus trypanocidal antibodies is the immunologic basis of nonpathogenic infections. Nonpathogenic trypanosome infections are made virulent by immunosuppressive procedures, and under such conditions T. lewisi reproduces until the host dies (Patton and Clark 1968; Sherman and Ruble 1967). In pathogenic trypanosomiasis, periodic crises occur due to trypanocidal antibodies. However, persistent reproduction of surviving trypanosomes results in repopulation of the bloodstream by antigenic variants, terminating in the death of the host. A fundamental difference, then, between pathogenic and nonpathogenic infections is continuous reproduction of trypanosomes in pathogenic infections. Pathogenic species of trypanosomes produce benign infections in wild ungulates. Though some antelopes die after experimental infections (Carmichael 1934; Ashcroft et al. 1959), those which do survive produce trypanolytic and respiration inhibiting antibodies (Desowitz 1960). The actual immunological basis for controlled infections in some African wild game animals is not known. Van Saceghem (1923) assumed it was associated with an immunity that inhibited the reproduction of the parasites, but the evidence for this is wanting. There are predictable metabolic differences between reproducing and inhibited trypanosomes. In general, in ablastininhibited trypanosomes, enzymes outside the Kreb’s cycle (D’Alesandro and Sherman 1964; D’Alesandro 1966) and glucose utilization are reduced, more oxygen is used per molecule of glucose consumed

INHIBITION

OF

MEMBRANE

(Moulder 1948), protein synthesis is severely restrained, and nucleic acid synthesis is halted (Pizzi and Taliaferro 1960; Taliaferro and Pizzi 1960). The following studies provide additional evidence that ablastin arrests cell division, and new evidence that the primary effect of ablastin is on membrane function. Assimilation of 3H-thymidine followed by autoradiography was used to study reproduction of T. Zewisi (Fig. 1). Generation times or doubling times approximated from grain counts over nuclei and kinetoplasts are predicated on the assimilation of 3H-thymidine as an indication of DNA synthesis. The time required to halve the mean grain count over trypanosomes labeled and removed from 3H-TdR approximates the doubling time and is independent of exponential growth, destruction, or sequestration of parasites. 3H-TdR, when injected into rats, is rapidly distributed throughout the body by the circulatory system and either promptly removed by synthesizing cells or is eliminated (Grenlich et al. 1961; Mossier and Lebland 1960). Only the labeled population of trypanosomes was considered in this study. Trypanosomes labeled on the third day of the infection in intact rats and in rats treated with dexamethasone have a doubling time of about 8.5 hr based on hemocytometer counts, and 8 hr based on the amount of time necessary to reduce the mean grain count by half over labeled trypanosomes (Fig. 2A). Organisms labeled on the 5th day of the infection in dexamethasone-treated rats required 12 hr to reduce the mean grain count by half (Fig. 2B). Whereas, in intact rats the mean grain count halving time was 24 hr (Fig. 2B). The three-fold increase in time to half the mean grain count, or the threefold increase in doubling time, if attributed entirely to ablastin, indicates approximately 75% efficient suppression of the ablastic response in dexamethasone treated rats. The period during which the trypanosomes

FUNCTION

Xl

reproduce in intact rats is abbreviated and in immunosuppressed rats is prolonged (Patton and Clark 1968; Sherman and Ruble 1967). This interpretation and the data supporting it are in direct contradiction with explanations that presume the ablastic response is not a restriction of parasite division but is rather a selective elimination or sequestration of dividing forms (Greenblatt and Tyroler 1971; Ormerod 1963). Ablastin is distinguished in a fundamental way in that it is not removed from immune serum by adsorption with living trypanosomes ( Taliaferro 1932). Though this property is cause for controversy over the true antibody nature of ablastin, it is also a useful property for separating it from the more conventional antibodies that develop during infection. Except for an exceptionally low avidity, ablastin has the usual properties of antibodies (D’Alesandro 1970). D’Alesandro (1962) demonstrated ablastin activity in vitro and clearly showed that the antireproducing activity in immune serum is titratable. The medium he described was used in the following studies of ablastic serum. Reproducing trypanosomes taken from dexamethasone-treated rats 5 days postinfection were pulsed for 2 hr in medium containing 3H-TdR. After the pulse they were centrifuged, washed in fresh medium, and then transferred to medium containing ablastic serum or normal serum. Autoradiography showed that 95% of the organisms were labeled. During 22 hr in culture medium containing adsorbed ablastic serum, the trypanosomes retained 88% of the label. In medium containing normal serum treated as if to adsorb trypanocidal antibodies, the mean grain count was halved over trypanosomes that had been in culture for 18 hr, and after 24 hr in culture the mean grain count was reduced by 66%. The average number of grains was reduced by half over trypanosomes grown for 8.5 hr in medium

3G2

CURTIS

containing untreated normal strum, and after 24 hr the count was reduced by 70% (Fig. 3, left). Th ese data show that cell synthesis ceases in axenically cultured T. ZeuGi in the presence of ablastic serum. Cell synthesis was halted in trypanosomes removed at various intervals during 24 hr from medium containing normal serum to medium containing (Fig. 3, right) ablastic serum. In these experiments the trypanosomes transferred earliest from normal medium to ablastic medium retained a greater percentage of the 3H label. Conversely, trypanosomes removed from medium containing ablastic serum to medium containing normal serum were released from inhibition. The earlier trypanosomes were transferred, the longer they had to incorporate unlabeled TdR and thus reduce the mean grain count per cell (see Fig. lC, D). These results are consistent with Taliaferro’s (1963) suggestion that ablastin is extremely nonavid. The easy dissociation of ablastin and trypanosomes results in their release from inhibition, Nonavid antibodies dissociate rapidly and combine slowly (Jerne 1951; Talmage 1957). The intermolecular forces which hold together antibody and antigen are no different from protein-protein interactions which happen between any two unrelated macromolecules (Kabat 1968). In the case of ablastin, the measure of the strength of binding between the antibody and antigen has its only basis in the interpretation one gives to the biological activity of ablastin. Though ablastin clearly interrupts the and though reproduction of T. let&i, there is a considerable body of knowledge available concerning the metabolism of ablastin-inhibited trypanosomes, the primary effect of ablastin has been a subject of speculation, The general changes associated with ablastin-inhibited trypanosomes appear to be indirectly controlled. It has been suggested that ablastin may act at the cell surface and thereby directly affect some process in membrane transport ( D’Alesandro 1970). There are several

L.

PATTON

. After 5

1

J

2hr

o Ablastic

medijm

A Treated normal medium

1

p N;ryal,me,diu,m,

0

8 Hours

I6 24 in Culture

q

n

i

Ablastic

to Ablaslic

0 Normal

to

Normal

l Ablostic 1

,

0

, 6

Hours

NormGjl

to Abtostic ,

,

I2 before

to Normal , , , I8 Transfer

, 24

3. The effect of adsorbed ablastic serum the mean grain count halving time of “H-TdR labeled dividing trypanosomes in axenic culture. Trypanosomes were pulsed 2 hr in 3 ml of medium to which 3 UC of ‘H-TdR (Sp AC = 3.5 mC/mmole) were an addition. Trypanosomes were removed to fresh medium (left) containing adsorbed ablastic serum (-•-), normal serum treated as if to adsorb trypanocidal antibodies (-A--) and normal serum (-0-). The decrease in grain count was followed during 24 hr in culture. The graph on the right shows the effect of adsorbed ablastic serum on the mean train count halving time of “H-TdR labeled trypanosomes before and after the organisms were transferred to medium containing normal serum. After the pulse, trypanosomes were placed in fresh medium containing ablastic serum and periodically transferred from ablastic medium to fresh medium containing either normal serum (-•-) or ablastic serum (-•-), or they were pulsed and then placed in medium containing normal serum and then transferred periodically to fresh medium containing ablastic serum (-¤--) or normal serum (-0-). The standard errors represent the mean grain counts over labeled trypanosomes on four slides. The mean grain counts per slide were calculated by averaging the number of grains over 50 trypanosomes. On the left, grain counts were made at the time points indicated; on the right, all counts were made after a total time of 24 hr in citro. FIG.

on

INHIBITION

OF

MEMBRANE

lines of evidcncc that would support this idea. Proteins are the only molecules with the degree of specificity required for discriminating between possible substrates. Studies have shown in other biological systems that a protein component required for transport of specific solutes is present in the membrane ( Anraku 1967, 1968a, 196813, 1968c; Nossal and Heppel 1966; Pardee 1966, 1968). No transport sites or structures have been isolated and characterized from trypanosomes, but if they do exist they are likely to be antigenic. Antibodies directed against such functional membrane components might impose constraints on architecture and physiological events associated with membrane transport. The details of isolation and safekeeping of trypanosomes for transport studies as well as the technique and methodology of separating cells from radioactive exposing solutions have been described elsewhere (Patton and Balber, in press). Transport experiments were carried out by adding I%-labeled sugar to suspensions of washed trypanosomes and then, at intervals of 10 to 15 set, separating cell-associated radiolabel from cell-free radiolabel. This separation was effected by centrifuging the organisms through a discontinuous gradient into a layer of perchloric acid. The gradients were composed by layering 250 ~1 of cell suspension [Hank’s balanced salt solution (HBSSA) containing 50 mg ‘j% (w/v) fraction V bovine serum albumin] on top of 50 ~1 of silicone (General Electric Versilube F-50; sp gr = 1.05; viscosity = 70 centistokes) which had itself been layered upon 100 ~1 of 1.5 N HClOd (PCA) in a 500 pl capacity plastic microfuge tube (Beckman). After the tubes were fitted into one of four microfuges (Beckman Model 152), samples were promptly spun for 1 min at 7000g. When trypanosomes are spun through the silicone layer into the PCA layer, the cell-free radiolabel is retained in the aqueous layer above the silicone; radiolabel in the acid soluble portion of the cells is released in the PCA layer

FUNCTION

3G3

below the silicone; and the material precipitated by the acid is pelleted. Time points represent the number of seconds elapsed between the addition of l”C-substrate and the beginning of centrifugation. As shown in Fig. 4A, rates of uptake may be linear for as long as 2 min. Initial rates of glucose entry are concentration dependent, as if the parasites have a limited number of independent adsorption sites. The net rate of glucose uptake increases as the external substrate concentration is increased and approaches a maximum value (Fig. 4A). These kinetic data indicate that there is some saturable component of the transfer process (Fig. 5). This component behaves according to Michaelis-Menten kinetics (Km = 2.8 x lo-‘M). Km values over a series of 10 experiments range between 2.5 and 3.4 x 10m4 M with an average value of 3 x 10e4 M. Glucose transport is competitively inhibited by 2-deoxy-D-glucose (2 DOG) ( Fig. 4 ( A-D) ). Lineweaver-Burke plots show the characteristic straight lines of differing slopes intersecting at a common X lo7 intercept (0.32 nmole/min/l.25 cells)-’ in Fig. 5. The intercept is the inverse of the maximal rate of transfer (Vmax), and its value is a function of the capacity of the carrier system and the adsorbed molecules which dissociate in the forward direction. It depends on the number of carrier sites per unit area of membrane, the number of substrate molecules which can be adsorbed on each site, and the number of carrier-containing regions of membrane per cell (Barnett, et al. 1968; Blasberg 1968; Joanny et al. 1969; Oxender and Christensen 1963). The Vmax for glucose uptake is not altered in the presence of 2 DOG. However, the apparent Km values for glucose transport in the presence of 2 DOG are greater than the Km for glucose uptake in the absence of 2 DOG by the amount of increase in the intercept of the l/S axis (Fig. 5). The percentage of inhibition is thus a function of the ratio of concentrations of 2 DOG and not a

CURTIS L. PATTON

3G4 14

14C - Glucose

C-Glucose

5.01

n*

2 4.0 -I

14C - Glucose ond 0.5mM 2 DOG

of Z-DOG

0

I.OmM

v 0.5rnM

B

7

1

“C-Glucose ond I.OmM 2 DOG

!

* =

D

z 3.0

“0 X

\

8

A 0.25mM o no addition

r

/

I//

1

2-:

Addllions

and

2 DOG

0.25mM

2.0

50

100

seconds

0

-2

0

2

4

I /‘4C-Glucose

6

8

IO

12

14

[mM]

FIG. 5. Lineweaver Burke plots of initial rates of glucose uptake by Trypanosoma let&i and inhibition of glucose uptake by 2 DOG. Data from Fig. 4 (A-D) were used to generate the lines.

0 0

-4

50

100

seconds

FIG. 4. The uptake of 14C-glucose (Sp AC = 250 uC/mmole) by dividing Trypanosoma ZeuZsi in HBSSA at 37 C with no additions (A: -0-)), in the presence of 0.25 mM 2 DOG (B: -A-), in the presence of 0.5 mM 2 DOG (C: -V-) and in the presence of 1 mM 2 DOG (D: -n-). The external concentrations of glucose were 0.078125 mM (l), 0.15625 mM (2), 0.3125 mM (3), 0.625 mM (4), 1.25 mM (5) and 2.5 mM (6).

function of the absolute concentration. The Km value for the uptake of 2 DOG (Patton and Balber, in press) is approximately equal to the Ki value on glucose uptake

(1.9 x 1O-4 M). Glucose uptake as a function of the substrate concentration revealed comparable Km values for reproand ablastin-inhibited trypanoducing somes. Thus, it appears that the transport system in both the inhibited and reproducing trypanosomes exhibits the same affinity for the substrate. However, a comparison of Vmax values for the two forms reveals that the maximum velocity for glucose uptake is reduced from an average of 3.15 nmole/min/l.25 x lo7 reproducing trypanosomes to an average of 1.8 nmole/ min/1.25 x lo7 ablastin-transformed adult trypanosomes (Patton and Balber, in press). Trypanosomes were loaded with 2-deoxy14C-glucose and the outward flow of “C into fresh solutions containing 1 mM glucose or glycerol was measured (Fig. 6). Glucose stimulated the flow of 2-deoxy14C-glucose as though the two sugars share some component of the transport system

INHIBITION

Intracellular in the

14C-2 presence 0 Glucose 0

DOG “C-2DDG

Glycerol

T

0

Uptake

IV

n

‘s E4

6

2

z Oo 50

100

sec.

50

100 seconds

“C-2DOG presence of

A Glucose Glucose Ablastic

v

E 6

150

365

FUNCITON

Intracellular .in the

of

Extracellular “C-2DOG in the presence of l Glucose . Glycerol

01

OF MEMBRANE

“C-200G

and Serum

Extracellular “C-2DOG in the presence of A Glucose v Glucose and Ablast ic Serum

50

100

Uptake

*) ‘0 ‘9 6 ; 6 a4 -2 0 K

0

so 100 sec.

150

seconds

FIG. 6. Trypanosoma lewisi (dividing forms) was loaded with ‘C-2 DOG (Sp AC = 62.35 mC/mmole) in the presence of 1 mM glycerol as an energy substrate (inserts) contained or ablastic serum (-A-). After loading the cells, in 40% desalted normal ( -•-) external “C-2 DOG was removed by centrifugation and the loaded trypanosomes were resuspended in 40% normal serum (left) containing l/mM glycerol (-_O-) or l/mM glucose ( -_o-). On the right loaded trypanosomes were resumended in 40% ablastic glucose serum containing l/mM glucose (-V-) OI 40% normal serum Containing l/mM (--a-_).

such as a carrier molecule in the membrane inward and outward cycling between states (Parclee 1968; Stein 1967). To test whether ablastin affected this process, sera from normal rats and rats that had been infected with T. leurisi for 11 days (ablastic sera) were adsorbed with trypanosomes (D’AIesandro 1962) and desalted on a G-25 Sephadex column to remove serum glucose. Outward flow studies were carried out in solutions containing 405% desalted sera. Although glucose as an addition stimulated the outward 0ow of 2-deoxyW-glucose in all instances, the exit of 14C was clearly restricted when the trypano-

somes were in solutions containing ablastic serum (Fig. 6). Uptake of W-thymidine in reproducing and ablastin-inhibited trypanosomes revealed that 14C-TdR uptake is restricted in inhibited organisms (Patton, 1970). The specific activities of the TclR in exposing solutions due to the presence of serum were unknown, nor were the sera adsorbed. However, since the uptake study was made in the same serum from which the parasites were isolated, it is unlikely that the results were due to a trypanociclal effect. In any interpretation of these data, one

36G

CtJRTIS

has to take iirto accoaut that tlrc transport process may be formally divided into at least two separate steps: binding of the substrate and translocation of the substrate through the membrane. On the basis of observations in this study, the following interpretation is advanced: (a) Upon the addition of ablastin and after time is allowed for association with the trypanosomes, the translocation of sugar is decelerated, but substrate binding remains unaffected. Such a deceleration could be brought about by any general change in the architecture of the membrane affecting in some way the mobility of the carrier proteins. (b) Upon transfer to exposing medium containing normal serum, the rapid dissociation of ablastin results in acceleration of the translocation process. This interpretation explains transport inhibition by ablastin of such different substrates as carbohydrates and thymidine. The effect of ouabain on T. leu;isi was studied in order to find out whether known transport inhibitors had an ablastic effect (Patton 1972b). The inhibition by ouabain on reproduction of T. leurisi in axenic cultures is concentration dependent. The incorporation of 14C-TdR by the parasites in axenic culture is inhibited by ouabain, but is reversed in the presence of excess K’. Other workers have reported similar findings for phytohemagglutininstimulated lymphocytes (Quastel and Kaplan 1968). The physiological action of ouabain is inhibition of the sodium pump in many organisms (Post et uZ. 1960; Schoner et al. 1968; Skou 1960, 1962, 1965). This system has not been looked for in trypanosomes. A preparation of ATPase from T. Zewisi following the method of Skou (1962) catalyzed the hydrolysis of ATP in the presence of Mgz+, and the activity of the enzyme was stimulated by Na+ + K’. These results have been reported elsewhere (Patton 1970, 1972b). The concentrations of ouabain that inhibited reproduction of T.

L.

PATTON

lelcisi in axcnic culture also inhibited tlrc Na+ + K+ stimulation of the ATPase. IgG enriched fractions of serum from the blood of rats infected for 10 days reduced the Na+ + K+ stimulation of the ATPase activity by two- to four-fold (Patton 1970). In experiments by many workers on the ouabain-sensitive membrane ATPase (Glynn 1964; Post et al. 1960; Schoner et al. 1968; Skou 1960, 1962, 1965), the data are interpreted as involving a Na+ stimulated phosphorylation of the enzyme system by ATP followed by a K+ dependent phosphate reaction. The addition of K+ inhibits and partially dissipates the binding of 3Houabain to the phosphorylated intermediate. From these observations the following conclusions may be advanced: (a) Ablastin arrests cell division; (b) glucose uptake is a carrier mediated, stereospecific process; (c) the Km value for glucose transport in reproducing trypanosomes is undistinguished from the Km value for ablastininhibited trypanosomes; (d) Vmax values for glucose transport are reduced in ablastin-inhibited trypanosomes, which indicates a reduction in the number of transport sites; (e) ablastic serum directly affects transport in that it inhibits the outward flow of 2-deoxy-1*C-glucose in the presence of glucose; (f) the cardioglycoside, ouabain, has an ablastic effect on axenic cultures of T. Zewisi at 37” and also inhibits, as does ablastin, a Na+ + K’ stimulated ATPase. Ouabain sensitive ATPase in other biological systems is associated with active transport. Thus, inhibition of membrane transport leads indirectly to inhibition of reproduction of T. Zewisi. By extension, a direct inhibition by ablastin of translocation of carbohydrates, thymidine, and possibly other substrates into T. Zewisi results secondarily in the reversible arrest of reproduction of the parasite. Changes in membrane function appear among the earliest events when T. Zewisi reacts to the addition of ablastin.

INHIBITION

OF MEMBRANE

ACKNOWLEDGMENTS This investigation was supported in part by U.S.P.H.S. Training Grant AI 00192 when the author was at The Rockefeller University and by U.S.P.H.S. research grant AI 10245 from NIAD, NIH. The author was the recipient of a U.S.P.H.S. Career Development Award K 0470512. REFERENCES ANRAKU, Y. 1967. Reduction and restoration of galactose transport in osmotically shocked cells of Escherichia coli. Journal of Biological Chemistry 242, 793-800. ANRAKU, Y. 1968a. Transport of sugars and amino acids in bacteria. I. Purification and specificity of the galactose- and leucine-binding proteins. Journal of Biological Chemistry 243, 3116-3122. ANRAKU, Y. 1968b. Transport of sugars and amino acids in bacteria. II. Properties of galactoseand leucine-binding proteins. Journal of Biological Chemistry 242, 3123-3127. ANRAKU, Y. 1968c. Transport of sugars and amino acids in bacteria. III. Studies on the active transport. Journal of Biological Chem%try

243, 3128-3135. ASHCROFT, M., BURTT, E., AND FAIRBAIRN, H. 1959. The experimental infection of some African wild animals with Trypanosoma rhodesiense, T. brucei and T. congolense. Annals of Tropical Medicine and Parasitology

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