A differential effect of pH on cell growth and respiration

A DIFFERENTIAL

EFFECT AND

OF PH ON CELL

RESPIRATION

13.W’.W11,SOU,O.E.LIl’E:TOU,T.I,..~-\liNar~tiS.H.I,F:\ Department

GROWTfi

of Zoology, L’niversity

t’L)-\llI,

qf CaQiirnin,

Kereived lhxernher

Los Angeles, CaliJ,

1 -.S. 4.

29, 1958

(‘-,LLI.S utilize carbon sources for J)oth energy and growth. Changes in the external pH have been she\\-n h\- both growth and respiration cxlxrimcnts to affect the utilization of ionizable carbon sources by Euglenrr, and othcl closely related flagellates [3,f, 7,9, 10, 111. Ho\verer, in general, both measurcments have not been performcti in a given experiment, anti as a result a relation bet\\-ten the respiration anti growth of protozoa has been implitd, but not tested. Van Dach [l 71, using dsfrrsirr lon!gfr, a colorless flagellate, found an absence of respiration in the presence of certain Krcbs cycle intermediates at pH 5.X. Danforth [4], as a part of his study showing the presence of a Krebs cycle in Euglrnn, investigated the effect of pH on the rate of respiration due to various ionizable carbon sources. An increase in the respiration rates at pH values more acid than pH 7.0 suggested that the cells xverc more permcablr to the free acid than to the corresponding ion of these substrates. Certain effects of pH on the respiration rate of acetate adapted Erl!glencr lead Danforth and FVilson to suggest that an ion transport mechanism for acetate existed in these cells [a]. The implications of these respiration responses have not Jjeen investi~atrd in terms of qw\\-th. Differing experimental techniques have resulted in tiifl’erent reports of the optimum pH for growth of Euglenrr [3, i, 9, 101. Pklwrimedium show ments in this report, conducted 011 the defined Cramer-Myers that the optimal pH for growth of the cells varies tvith the substrate [1X]. In short, there appears to J)e little data available to determine if chanjies in the rate of respiration induced by pH mill be reflected in changes in the rate of growth. These studies were aided by financial assistance from the following: (1) A contract between Office of Naval Research, Dept. of the Navy and the University of California, Los Angeles, NR 120-336, (2) California Cancer Grant 498, and (3) NSF Grant G5666. Experimental

Cell Research 18

Differential

MATERIALS

effect of pH

AND

METHODS

Experimental

and stock cultures of a streptomycin-bleached strain, SM-Ll, of var. bacillaris, were grown axenically on Cramer-Myers medium [3], with 2.5 g,‘l or 5.0 g/l of acetate, fumarate, malate, succinate or citrate as the sole carbon source. No growth occurred upon citrate, a normal constituent of the medium, at any acidity tested. The media were adjusted to acidities ranging from pH 3.5 to pH 7.0. Stock cultures were grown with acetate as the sole carbon source at pH 6.8 to pH 7.0. All growth and respiration studies were carried out at 25°C. Growth studies-one-tenth ml of the stock culture, in the logarithmic growth phase, was inoculated into 125 x 20 mm screw-cap tubes containing 10 ml of autoclaved medium, carbon source, and added vitamins B, and B,,. The tubes were shaken in the dark in a constant temperature water bath. No additional aeration was applied. Growth was measured turbidimetrically with a Coleman Jr. spectrophotometer by changes in the optical density of the cultures at 570 mp. Several experiments showed that optical density was proportional to cell number throughout the logarithmic growth phase. Growth rates were calculated in terms of generation times, and are reported as fractions of the optimal generation time of cells grown on acetate at pH 7.0 and 25°C. Respiration studies.-Euglena cells are capable of adapting to acetate, and such an adaptation involves changes in the rate and pH sensitivity of the respiration [5]. For this reason all respiration studies were conducted with cells grown upon the substrate under investigation, so that the cells, as well as the conditions of the respiration studies, closely approximated the cells and conditions of the growth studies. Cells from an acetate stock culture, in a concentration of l/l0 ml cells to 10 ml media, were transferred to 300 or 500 ml of medium containing the desired substrate, and grown at 25°C. The cells were harvested during the logarithmic growth phase, centrifuged several times, and resuspended in a small volume of distilled water. In other experiments resuspension of the cells in basal Cramer-Myers medium at pH 7.0 resulted in respiration measurements compatible with those observed in this study. The respiration rates, in the presence of the basal growth medium, were determined by standard Warburg techniques [15]. Vitamins B, and B,, were not added to the respiration flasks since “carryover” amounts of these vitamins were considered sufficient for the duration of the respiration experiments [14]. After the cells were tipped into the substrate, readings were taken at twenty-minute intervals up to 240 minutes. An endogenous control, containing medium but no carbon source was included in each series. Respiration rates were measured in PL O,/hr/lOe cells and are reported as per cent of the endogenous respiration. Each value reported is the average of at least three experiments. The pH of each flask was checked at the conclusion of each experiment. The respiration rates were linear, and the optimal generation time of Euglena attained in this medium is near twenty hours [a], a period long compared to the short duration of the respiration experiments. Therefore, little growth could have occurred in the vessels, and no correction to the respiration measurements was necessary, even though a medium capable of supporting growth was present. Euglena

gracilis

Experimental

Cell Research 18

RESULTS

The data in Fig. 1 are cornpilcci frotn c~slwrimcnts \vith su~c,in:~l~,-~:ro\\-i1 cells. III growth csprriments the crlls uwc’ gro\vn at tlic inclicntwl l)II. ‘I’ht~ rtspiration of cells grown at pH 5.0 u-as then inrestigatcd in scparatc c,xlwimclnls at pH 7.0, 3.0, anti 11.5. The growth rate of the cells upon slic*c*inatv was independent of pH within the precision of the c~spri~ncnt. ‘1’11~rcsljiration results sho\v that at pH 7.0, whrrc gro\vth u’as optimal, thv respiration rate was \-cry lo\\-, hcing only 10 pr cent above the cntlogcnous. .It I~OIY acid conditions the respiration rate of the cells row to ahout 100 lwr ccii1 ahorc the c~nciogenous rate. l’rtliminary stutlics \\-ith l)H 5.0-grou-n cells shouxd that the respiralion of thcsc cells at pH 7.0, ;,.(I, and 3.5 is the sanl( tlnta as that ol)tainrcl with pH 7.0~gromm cells. ‘I’hc go\\-th and rcsl)iralioii anti wsl)irntion indicate that thcrc is no obvious wlationship lwt\vetln gi~~~vth of Rwglenff in the presence of succinatc. Fig. 2 prcscnts the data tiescrihing the behavior of funlaratc-gro~~tl ~~~11s. ‘I’hc lo\\-cr the pH the faster \lras the ralc of gro\\-th. l’hcrr \vas no apprc~ciablr growth at pH 7.0, cmtl at pH 3.3 the go\\-th rate \\-as HO lwr cent that of A(*-7.Cgro\vn cvlls. l’lic rate of oxygen consumption of the I=um-.j.O-~ro\\-n SUCCINATE

T -.1

5.0 -

50

3.5

-

-

GROWTH

Fig. l.-Effect of pH on the rate of growth Respiration rates from succinate-7.0-grown Experimental

T -.1

1

I 6.0

-.:

T -.T1 -.-

Ceil Research 18

3.5 -

-

RESPIRATION

and the rate of respiration cells.

of Eugkncc on succinate.

Differential

457

effect of pH

cells followed somewhat the same course as that obtained for growth, increasing at more acid pH values. There was little respiration above the endogenous upon fumarate at pH 7.0 where there was little, if any, growth. Examination of the cells after nine days showed them still viable at this pH. However, changes in the growth rate at other pH values were more marked than were changes in the rate of respiration. Thus, with fumarate utilization, changes in pH seem to affect the growth and respiration processes in the same general manner, but again, there is no direct correspondence.

I -.1 35 GROWTH

Fig. ‘L.-Effect of pH on the rate of growth Respiration rates from fumarate-5.0-grown

and the rate of respiration cells.

of Eugkna

on fumarate.

The data for the respiration and growth of Euglena upon malate is given in Fig. 3. There was no growth upon malate at pH 7.0, and extremely poor growth at pH 3.5. The growth rate at pH 5.0 and pH 6.0 was 50 to 60 per cent that of Ac-7.0-grown cells. Oxygen consumption of Mal-5.0-grown cells was low at pH 7.0, where there was no growth, and rose to about 170 per cent of the endogenous rate at pH 5.0 and pH 3.5. (A single experiment revealed a respiration rate at pH 6.0 similar to that found at pH 5.0.) The situation with malate at pH 3.5 is opposite to that seen for cells utilizing succinate at pH 7.0. In the case of malate, the respiration rate was high, and the rate of growth was negligible, while with smcinate. the growth rate was high and there was very low respiration. Viability tests showed that the high malate respiration was not due to injury, for cells incubated in malate media at pH 3.5 were still living after nine days, as were cells incubated in malate at pH 7.0. 29 - 5937OIl

Ezperimentd

Cell Research 18

c5x

Ij. IV. \Vilwl,

1). E. 13urtorn, 7’. L. ./cthn curd 13. II. Lrv~dtrlrl

Fig. 4 presents data sho\ving Ihal on acetate, at pH 5.0, thca gro\vth rate of the cells was lower than it was at pH 5.0, but that the c’onv~rw \\‘;is true of the respiration rate. The cells \vc‘r(’ viable after nine tlars of incuJ)aiion on acetate at pH 5.0.4t the more acid pH of 3.3,growth is absent. Other stuclics have sho\vn [4] that with the concentrations of acctatc used, the respiration MALATE

RESPIRATION

GROWTH

Fig. 3.-Effect of pH on the rate of: growth and the rate of respiration Respiration rates from malate-5.0-grown cells.

of Euglena on malate.

ACETATE

-i-

_.I

-.- T 1

-p-

T -a 1

5.0

I

GROWTH

1

i.0

3-5

Fig. 4.-Effect of pH on the rate of growth Respiration rates from acetate-7.0-grown from Danforth [4]. Experimental

Cell Research 18

END

6.0 -

5.0 -

3.5 D

RESPlRATlON

and the rate of respiration of Euglena on acetate. cells. The lack of respiration on acetate at pH 3.5

Differential

459

effect of pH

rate would be severely inhibited below pH 4.5. It was also noted that no cells were viable at pH 3.5 after two days. A comparison of the growth rates of the cells on malate and acetate favors the theory that the growth processes are more sensitive to pH changes, or perhaps, free acid concentrations, than arc the respiration processes. However, with the ranges of pH and substrate concentrations used in this study only acetate and malate-grown cells showed any inhibition of growth or respiration at acid pH. DISCUSSION

The processes of growth as defined by synthesis of new material require energy which is supplied to the cells by the oxidation of exogenous substrate. Therefore, it is plausible to expect some linkage between cellular oxidations and cell growth as has been discussed by Swann [15]. If the cell oxidative systems are directly linked to the growth systems, changes in the rate of energy production should be reflected in changes in the rate of growth, as long as no other substance in the medium is limiting. The results in this paper do not indicate the presence of a simple relationship between growth and respiration in Euglena. The effect of pH depends upon the substrate, and varies, sometimes inhibiting growth but not respiration (acetate-pH 5.0, malate-pH 3.5), sometimes inhibiting respiration but not growth (succinate-pH 7.0), and sometimes affecting both processes in an analogous manner (fumarate-pH 7.0 to pH 3.5). This differential effect of pH need not signify the lack of any relationship between cell growth and respiration at any single pH. Growth was measured by optical density changes, here an estimate of the cell number, and may not be proportional to other criteria of growth, such as the dry weight of the cells. Secondly, a change in the extent of oxidation, the ratio of oxidation to assimilation, of the various substrates with pH might account for the results. However, Wilson and Danforth [19], in respiration experiments, using a buffered medium lacking a nitrogen sourc,e, have shown the extent of oxidation of acetate and ethanol to be unaffected by changes in pH from pH 5.5 to pH 7.0 in a similar strain of Euglena. Studies concerning the extent of oxidation of Krebs cycle substrates in the presence of a nitrogen source are now in progress. The respiration rates of the cells are lower at pH 7.0 than at more acid pH values for all weak acid substrates tested, as previously described by Danforth [4]. Hunter and Lee [8] recently reported the same behavior on a Experimental

Cell Research 18

‘I’hcsr~ \veII clocumented rcspiralion wsults, as \\.cll as carlivr gro\\ th sll~tliw on other organisms, haye suggcslcti 14, 9 that ionizable cB:ir.t)ou ~ou~~~~~~~ arc mow readily a\-ailable to the cvll at lo\vc~r pH values. This has 1)(,(&nclplained on the basis that the ccl1 membrane is more pvrmeabk IO unclissoriatetl ~noleculcs than to inns, and that the proportion of ~lndissoc.ialctl molecules in the external medium increases with increasing hyclrogcn ion concentration. Such an explanation has long been used for the toxicit!, of acids anti alkaloids in certain pH ranges [ll]. Table I lists lhc per cent of each substrate undissociated, and the concentration of the undissociatcd species present at the acidities w~ti in this study. Jacobs, in a review in 1940 ClZ], considered the permeability of a cell to monobasic acids as a function of pH. He showed that if one assumes merely that the membrane is permeable to undissociateti acids and impermeable to ions, and that the usual la\vs of difl’nsion apply, so that the equilibrium van‘~AISLI;

I. Free mid amounts at different pH urrlues.

Substrate

PH

Per cent acid’ undissociated

Acetalc (pK= 4.7)

7.0 G.0 5.0 3.5 7.0 6.0 5.0 3.5 7.0 G.0 5.0 3..i 7.0 6.0 5.0 3.5

0.385 5.56 3l.G 95.0 5 % lo-” 0. 1 10.6 83.0 10-4 3.2 x 10-S 0.23 22.6 2.8 x 1 o-4 2.4 x 1OF 1.3 43.G

Succinate (ph’, = 1.2) (PIP = 5.5) Fumarate (pK, = 3.0) (pli, = 4.5) Malate (pK, = 3.4) (pK, = 5.0)

Maximum concentration’ undissociated acid ‘2.16 :i 1W2 mJI 2.06 11.6 35.0 9.25 x 1UP mJ2 7.4 >: 10-z m.II 1.96 15.3 4 ‘: 10-e IllJl 1.38 x 10-3 mN 10.5 : 10 ~2 9.7 1.03 x 1ov m31 8.9 x 10-a 0.47 1 li

1 From dissociation constants K, and K, at 25X, assuming dissociations occur independentlgper cent undissociated acid is then the reciprocal of 1 -I- K,/(H + ) + k, k, ‘(H + )” (6). 2 Total substrate added-5 g [l]. Experimental

Cell Kesearch 18

Differential

effect of pH

461

centration of undissociated acid inside and outside is the same, the concentration of total acid inside may be equal to, greater than, or less than that on the outside, according to the equation: a. C,=

1 + lOPHi-*K C 1 +

~oPH.-PK

0

where C, and C, are the total acid concentrations inside and outside, respectively, pH, and pH, are the pH values inside and outside, respectively, and pK is the pK value of the weak acid under consideration. ,4n inspection of this equation shows that depending upon the magnitude of the pH, and the pK of the acid under consideration, total acid may accumulate, may be equal to, or may be lower than the acid concentration in the external medium at any given external pH, assuming that the internal pH remains constant. In view of these long known principles, it is obviously difficult to draw conclusions regarding whether the ionized form of the acid does or does not penetrate a cell membrane, when the data concerning growth, respiration, or some other metabolic activity, is not accompanied by measurements of cytoplasmic pH. The only way to interpret the results is to assume some value or values for the cytoplasmic pH and to draw tentative conclusions in the hope that these will lead to indirect methods of proof. If the internal pH of Euglena is assumed to he near 7.0 and only the first ionization constant of the acids in this study are considered, application of the Jacobs equation serves to unify part of the results. By this procedure it is possible to explain the beneficial growth effect of malate as the pH is decreased from 7.0 to 6.0 and to 5.0 on the basis of greater accumulation of substrate and therefore greater availability of useful material. Growth inhibition at pH 3.5 could result from excessive accumulation resulting in inhibition but not in lethality, as evidenced from the viability studies. Likewise the inhibitory effec,t of acetate on growth at all pH values belovv 7.0 and on respiration below pH 5.0 could be explained on this basis of excessive accumulation. An additional factor is involved if we assume that some of the active metabolic sites within the cell are separated from the continuous portion of the cytoplasm by a semipermeable membrane, as is known to occur for mitochondria [l, 61. There would be another equilibrium set up between the concentration at this active site and the site of any other activity localized in the continuous cytoplasm, and again the Jacobs equation could be applied. Therefore, all cell activities need not be affected in the same manner by external substrates, not only because cell processes may differ in their sensitivity to substrates, but also because the concentrations of the weak acids may be Experimental

Cell Research 18

greatlv dilfcrcnt al various sites \\.ilhin lhtb ccl1 nicrclv on Ilit, t)ahis 01’ Ilic, equilibrium cqnations of difl’usion. On this basis \ve co~lltt explain the lack of an inhibitory clfwt 01‘ tnalatc, on respiration at pH 3.5 \vhtrc thcrc is an inhibition of gro\\.th sirnI)ly b! assuming that accumulation at levels necessary for inhibition 1)~.malatc tlow not occur at the site of respiration. The fact that respiration is ccntcrctl in mitochondria, and the probability that the pH difference bct\vcrn cytoplasm and mitochondria is much less than the difference between outside medium and cytoplasm, makes this an attractive possibility for speculation. Escept for succinatc, this explanation would apply to all the weak acids studied, since growth on acetate, malatc, and fumarate appears some\\-hat mow scnsitive to pH changes than does respiration. The authors are aware that application of the Jacobs equation and the use of only the first dissociation constant for the dibasic acids used represent an oversimplified and naive approach. However, this approach has been discussed to shoxv that the ideas of simple difTusion can account qualitativel! for the results. When the Jacobs equation is generalized to include both dissociation constants for the dibasir acids under consideration, the following cquation is obtained: c.=l+fo 1

pHi - PK, +

1 .+ ~()PH.

-DIG +

1 ()!2pHi I()ZPHO-‘PK,

CpK, .I PK,) VPK,)

c,.

Application of this equation to the data shows that the calculated internal accumulated concentration of a dibasic acid can be much higher than that calculated for a monobasic acid. For example, the internal concentration of malate is computed to be 160,000 times that of the external, \vhen the csternal pH is 3.5 and the internal pH is assumed to be 7.0, obviously a yuestionable result. Therefore, it seems probable that factors other than simple passive diffusion may be operative. The diffusion theory as applied here takes into consideration such factors as molecular size, lipoid solubility, and steric considerations. In addition to these, other factors can play an important role namely, membrane specificity, the in determining the final concentration; rate of utilization of substrate, and the total binding capacity of the cytoplasm to the acid under consideration. The viewpoint of purely passive permeability has been modified rccentl! Danforth and Wilson L-5’ \vith respect to the acetate respiration of Euglena. presented evidence for an adaptive ion transport mechanism for ac‘ctate, which increases the rate and lowers the pH sensitivity of the acetate rrsI)iralion of acetate-grown cells upon ethanol. Experimentnl

Cell Research 18

Differential

effect of pH

The results of the present study might be interpreted to entail a further revision of the ideas concerning the rate of acid permeability in Euglena. For example, the optimal growth rate of the cells on succinate at pH 7 .O where succinate is more than 99 per cent ionized indicates the inadequacy of the idea that less than 1 per cent of the material is available to the cell. If the internal pH were known to be well above 7.0, the above arguments for passive accumulation of succinate within the cell could apply. However, if the internal pH were known to be below pH 7.0 it would be necessary to assume some type of an active process for transport of the succinate ion. Table I contains data that allow the possibility that some ion transport mechanism for succinate might be present. The maximum concentration of undissociated acid present at pH 7.0 was only 0.25 X 10-4 mM, seemingly too low to support optimal growth. Secondly, the growth rates of the cells on succinate were relatively unaffected by pH over a range of maximum free acid concentration of 9.25 x 10-d to 15.3 mM, and the respiration rates were essentially constant from pH 5.0 to pH 3.5 where the free acid concentration increased from 1.96 to 15.3 mM. The real dilemma, however, is the fact that the rate of growth is optimal on succinate at pH 7.0, whereas the rate of respiration is only 10 per cent above the endogenous. If we assume, as is true of acetate [19], that the endogenous respiration of these cells continues relatively unchanged in the presence of exogenous substrate, it is apparent that a low rate of aerobic energy production can support rapid growth. The minimum amount of energy necessary to support a given amount of growth remains to be determined [15]. If we assume that the amount of energy required for growth is more than that available from the aerobic processes measured as respiration and known to include the tricarboxylic acid cycle in Euglena [4], the next question is whether there are alternate pathways for succinate which do not require oxygen. Such a mechanism could explain the present results, but no data are avail.able concerning its existence. Presumably such an anaerobic system would be in the cytoplasm rather than in the mitochondria, so that it would be more subject to the etfects of acid accumulation than would the normal tricarboxylic acid cycle, and could furnish energy for growth under conditions (e.g., pH 7.0) which do not accelerate respiration. It is apparent that the pH for optimal growth or respiration varies with the substrate used. Only fumarate-grown cells exhibited maximal growth and respiration at the same pII. In the present experiments the fastest growth rates were observed using acetate and succinate. The growth rates of the cells on fumarate and malate reached only 50 to 60 per cent of the rates observed vvith Experimental

Cell Resrurch 18

acetate ant1 suc’c’inatc , anal \vcrt’ optinlal in lhc acid range. (;ross aricl .I:ihrl [7] using a different basal medium reported optimal gro\\-th of thv saint xlrain of Euglenn at pH 4.3 to pH 7.3 using acetate as a carbon sourct’. Cramer and hlyers [3 in\cstigatrtl tlic growth of scvvral grcvn strains 01’ Eugleno, at several values of pH in lhc (lark and in the light. ‘I’hcst~ authors reported optimal growth on suvcinatc at pH 1.5 for green cells of the hnc~ill~rris strain in the dark. Experiments comparing the eflects of pH on the growth of green and streptomycin-bleached Euglenn might reveal significant dil‘t’crcnccs. If the permeability of the cells to the ionic and undissociated species of thtb substrates are factors in the regulation of these carbon sources, it is difficull to visualize holy a single permeability barrier surrounding the cell could lead tleto the differential effects of pH on the gro\\-th and respiration processw scribed here. The presence of more than one permeability barrier \vilhin the cell [C,, 131 is one of many untested unifying explanations for these results. A cell is a multidimensional system with its enzymatic process distributed in localized regions. One would expect environmental stresses to manifest themselves in more than one \\-a!-. Investigating the grolvth and respiration processes of the cell concurrently seems to yield dif‘ferent interpretations and perhaps more information than a study of the two processes separately. Ideally, both gro\vth and respiration would be measured simultancouslv, and such experiments are now in progress. Howerer, if interpreted with caution, it would appear experiments such as these assist in evaluating concepts initially formulated from single-valued experimental approaches. Slultivaluecl experimental designs, though difficult to interpret, may \vell prove signifkant in unraveling the complexities of ccl1 regulation. SUMMARY The pH of the growth medimrl has different effects on the growth and the respiration processes of a colorless strain of Euqlena yracilis var. bacilloris when succinate, fumarate, malate, citrate and acetate are used as sole carbon sources. The responses of the cells \vere studied at pH 7.0, 6.0, 5.0, and 3.5 in a manner such that the conditions of the respiration studies approximated the conditions of the growth studies. Respiration, but not growth, was inhibited on succinate at pH 7.0. Growth, but not respiration, was inhibited on acetate at pH 5.0 as well as on malate at pH 3.5. The respiration rates of the cells on fumarate responded to changes in pH in a manner similar to that of the observed growth rates. No growth occurred on citrate at any of the pH’s tested. Experimentul

Cell Research 18

Differential

465

effect of pH

The fact that succinate supported optimal growth at pH 7.0, where the substrate is essentially totally ionized and the growth rate of the cells on succinate is independent of pH, implies the presence of a transport mechanism for succinate ion in Euglena. The evidence is not sufficient to permit postulation of similar systems for the malate or fumarate substrates. Evidence is also presented to support the idea that the growth processes for Euglena utilizing acetate, malate, and possibly fumarate, are more sensitive to pH than are the respiration processes. Some implications of these findings on the relationship between energy production and growth, and between differential permeability of ions and free acids are discussed. The possible accumulation of weak acids within cells as a result of lower external pH values of the medium is also considered. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19.

BARTLEY, W. and DAVIES, R. E., Biochem. J. 57, 37 (1954). BUETOW, D. E. and LEVEDAHL, B. H., Arch. Biochem. Riophys. 73, 273 (1958). CRAMER, M. and MYERS, J., Arch. Microbiot. 17, 384 (1952). DANFORTH, W. F., Arch. Biochem. Biophys. 46, 164 (1953). DANFORTH, W. F. and WILSON, B. W., J. Protozool. 4, 52 (1957). DIXON, M. and WEBB, E. C., The Enzymes. Longmans, London, 1958. GROSS, J. A. and JAHN, T. L., J. Protozool. 5. 126 (1958). HUNTER, F. R. and LEE, J. W., J. ProfozooL’5, suppl. 15 (1958) abs. HUTNER, S. H. and PROVASOLI, L., in Lwoff A., The Biochemistry and Physiology of Protozoa, Vol. 1, p. 27. Academic Press, Inc., New York, 1957. HUTNER, S. H. and PROVASOLI, L., in Hutner, S. H. and Lwoff. A.. The Biochemistrv and Physiology of Protozoa, Vol. 2, p. 17. Academic Press, Inc:, New York, 1955. ” JAHN, T. L., Cold Spring Harbor Symposia on Quant. Riot. 167 (1934). JACOBS, M. H., Cold Spring Harbor Symposia on Quant. Riot. 8, 30 (1940). RACKER, E., Harvey Lecfures 51, 143 (1957). ROBBINS, W. S., HERVEY, A. and STEBBINS, M. E., Ann. N.Y. Acad. Sci. 56, 818 (1953). SWANN, M. M., Cancer Research 17, 727 (1957). UMBREIT, W. W., BURRIS, R. H. and STAUFFER, J. F., Manometric Techniques. Burgess Publishing Co., Minneapolis, Minnesota, 1957. VON DACH, T., Riot. Bull. 82, 356 (1942). WILSON, B. W., BUETOW, D. E., LEVEDAHL, B. H. and JAHN, T. L., J. Protozoot. 5, suppl. 15 (1958) abs. WILSON, B. W. and DANFORTH, W. F., J. Gen. Microbiot. 18, 535 (1958).

Experimental

CelZ Reseurch 18