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The response of diphosphoinositide and triphosphoinositide to perturbations of the adenylate energy charge in cells of Saccharomyces cerevisiae
Biochimica et Biophysics Acta, 306 (1973) 412-421 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BBA 56271
THE RESPONSE OF DIPHOSPHOINOSITIDE AND TRIPHOSPHOINOSITIDE TO PERTURBATIONS OF THE ADENYLATE ENERGY CHARGE IN CELLS OF SACCHAROMYCES CEREVISIAE
R. T. TALWALKAR
and ROBERT L. LESTER
Department of Biochemistry, College of Medicine, University of Kentucky, Ky. Lexington, 40506 (U.S.A.) (Received December z7th, 1972)
SUMMARY
In cells of Saccharomyces cerevisiae uniformly labeled with 32P the levels of ATP, ADP, AMP, diphosphoinositide and triphosphoinositide were measured simultaneously after chromatographic separation. When a respiratory deficient mutant strain was removed from complete growth medium, there resulted an immediate and large drop in the levels of diphosphoinositide, triphosphoinositide, and ATP as well as the adenylate energy charge. Addition of glucose to starved cells in buffer resulted in a rapid increase in the concentrations of ATP, diphosphoinositide and triphosphoinositide. a-Deoxyglucose caused a rapid decrease of the adenylate energy charge to intermediate values; again, the levels of diphosphoinositide and triphosphoinositide fell rapidly but not to the same extent as with simple starvation. The close association between the adenylate energy charge and the-concentration of these polyphosphoinositides herein demonstrated is discussed in relation to the well known turnover of phosphomonoester groups of these lipids.
INTRODUCTION
Di- and triphosphoinositide have been long known’-3 as significant lipid constituents of nervous tissue, with smaller amounts occurring in other animal tissues4-6. More recently these lipids have been found in fungi’**. Interest in the biological role of these compounds has centered on the observations that in animal tissues’-” and yeast l6 there is a rapid turnover of the monoester phosphate groups of these compounds. In several broken cell systems the 7-P of ATP could be shown to be a precursor of the monoesterd groups of di- and triphosphoinositide, reactions mediated by kinases acting on phosphatidylinosito11’-19. Specific phosphohydrolases are thought to catalyse the conversion of di- and triphosphoinoThus action of the kinases and the phosphohydrositide to phosphatidylinositol 5*20~21. lases are thought to account for the turnover of the phosphomonoester groups. The biologically significant factors governing the action of these enzymes which result in the regulation of the cellular levels of di- and triphosphoinositide are not known. Aside
POLYPHOSPHOINOSITIDES
AND ADENYLATE
ENERGY CHARGE
413
from post-mortem decreases of these inositides observed in brain22*23, to our knowledge, no systematic in viva studies have been carried out to examine the regulation of the cellular concentrations of the polyphosphoinositides. In this paper we describe rapid changes in the di- and triphosphoinositide content of yeast associated with changes in the adenylate energy charge of the cell. METHODS
Growth of yeast
A respiratory deficient mutant, RDA3, spontaneously derived from the strain (A3 I) of Saccharomyces cerevisiae used in earlier studies7S’6 was employed throughout these experiments except where specifically noted. The defined Yeast Nitrogen Base medium (Difco Laboratories) supplemented with 4 % (w/v) glucose, 0.048 M sodium succinate, pH 5, and carrier-free 0.2 Ci/l 32Pi (New England Nuclear Corporation) was used for growing labeled cells which were harvested in log phase. Yeast Nitrogen Base medium contains orthophosphate as the sole source of phosphorus at a concentration of 7.4 mM. Cells were grown aerobically at 30 “C in a rotary shaker. Under these standard growth conditions the cellular doubling time was 3.3 h. Growth was monitored by following the turbidity at 650 nm, r-cm path. One absorbance unit = 180 pg dry wt of cells = I ml of culture with an absorbance of 1.0 = approximately 9oo pg wet wt. Extraction and chromatography of lipids The lipids were extracted as described earlier’6*24 with several changes to
enable simultaneous processing of the acid soluble nucleotides. r-ml aliquots from various incubation mixtures were mixed with 0.2 ml chilled 35% HCIO, in screwcapped centrifuge tubes. After remaining at o “C for 15 min non-radioactive carrier cells were added24, and the cells were rapidly frozen and thawed twice and centrifuged at 2500 rev./min in the cold. The supematant was saved for the nucleotide estimations. The pellet was washed twice with 5 ml water to remove acid and then was extracted with 2 ml of ethanol-water-diethylether-pyridine (I 5 : 15 : 5 : I, by vol.) for I 5 min at 60 “C followed by immediate centrifugation at low speed. In the typical experiment 50-~1 aliquots of the supernatant extract (equivalent to approximately 20 pg dry wt cells) were chromatographed on EDTA-treated, silica gel-impregnated paper in two dimensions as described25, except that the second dimension solvent was changed to chloroform-methanol-acetic acid-water (I 5 : 6 : 4: 2, by vol.), and 0.0 I 2 % butylated hydroxyanisole (Nutritional Biochemicals Corp.) was added to the first dimension solvent. Before spotting the lipids, 5 ,ul of a 1.5 % methanolic solution of butylated hydroxyanisole was applied to the origin to prevent the oxidation of lipids while drying. The phospholipid spots were located by autoradiography, cut out and counted in toluene-Triton X-100 (2:1, by vol.) counting fluid containing 0.4 % 2-(4’-tertbutylphenyl)-5-(4’-biphenyl)-1,3,4-oxidazole, 0.03 % 2-(4’-biphenyl)-6-phenylbenzoxazole plus o. I 17 vol. of water, using a Packard TriCarb liquid scintillation spectrometer. The molar quantity of each phospholipid was calculated from the specific activity of 32Pi in the growth medium and the radioactivity in each individual spot.
414
R. T. TALWALKAR,
R, L. LESTER
Extraction aPld~~~ornato~ap~y of adenine nu~~eotide~ The HCIO, extract (above) was quickly neutralised with 2.6 M KOH in presence of internally added chlorphenol red indicator, and the precipitate removed by centrifugation. A known amount of a mixture of tritiated adenine nucleotides ([8-3H]adenosine mono-, di- and triphosphates, Schwarz/Mann) was added to each sample and mixed thoroughly before charcoal adsorption in order to monitor and precisely correct for losses of adenylates during analysis. Darco G-60 charcoal (Fisher Scientific Co.) was purifiedz6 and washed twice with warm IO mM sodium EDTA adjusted to pH 7.5 with ammonia followed by several water washes. In a typical experiment IOO mg of this purified charcoal was added to each extract, representing approx. 700 pg cells, allowed to stand for 30 min with intermittent shaking and the suspension was filtered in a small column through a o.5-cm layer of Celite 545 (Fisher Scientific Co.). The column was washed with 5 ml of 0.05 M potassium phosphate buffer, pH 6.8, and then with 5 ml water. The ‘H standards were quantitatively adsorbed on the charcoal. The nucleotides were eluted with 2 ml of pyridinium acetate, pH 5.3 (pyridine-3 M acetic acid (3 : 7, v/v)). Eluted in this small volume were about 70 “/oof the added ADP and ATP counts and about 85 % of the AMP. One fourth of the eluate was evaporated to dryness and dissolved in 0.1 ml of water, of which 25 ~1 were spotted on MN Polygram CEL 300 PEI (Brinkamn Instruments Inc.) sheet along with carrier nonradioactive adenine nucleotides. The aliquot spotted represented about 40 pg dry weight of cells. Chromatography was carried out according to the procedure described by Nazar et aL27 except that the concentrations of both LiCl and acetic acid in the first solvent were reduced to 0.8 M. The adenine nucleotides were located with an ultraviolet lamp and by autoradiography, to eliminate any nearby 32P con~minants. The spots were cut out an placed in scintillation vials containing I ml 2 M LiCl solution. After standing 6 h at 37 “C, 15 ml of counting fluid (as above but without water) was added. From the internal standard ‘H counts, the recovered 32Pi counts and the specific activity of phosphate, the molar amount of each nucleotide in the original sample was calculated. RESULTS
The aim of the present work was to see if adenylate energy charge was related to pol~hosphoinosi~de concentration. Experiments were carried out with cells labeled unifo~ly with “Pi of known specific activity which enabled ovulation of the amounts of phospho~pids and nucleotides after chromato~ap~c separation. Logarithmically growing cells were transferred from growth medium to sodium succinate buffer after thorough washing, Several minutes later lipids were extracted from the cells and the total extract was chromatographed in two dimensions (Fig. IA and IB). It can be seen that under these conditions di- and triphosphoinositide, stand out as lipids which decreased significantly. With cells grown in the presence of 32Pi and [3H]inositol, it was found that the presumed phosphoinositides got labeled with the expected relative 32P/3H ratios (Table I). This is a reliable approach to check the identity and isotopic purity of inositol phospholipids as it has been shownz4 in an earlier report from this laboratory that [3H]inositol is not catabolised by yeast. Effects on the cellular levels of the major inositol phospho~pids and adenine nucleotides of the respiratory mutant and the parental strain, when transferred from
POLYPHOSPHOINOSITIDES
AND ADENYLATE
ENERGY
CHARGE
415
Fig. I. Chromatograms showing decrease in the di- and triphosphoinositide content of yeast upon removal from growth medium. Cells were grown in the presence of 32Pi to AsroDm = 4.1. After 1.0 ml was taken out for lipid extraction (A), the rest were harvested on a Miilipore filter (0.22 pm), washed once with IO ml 0.048 M sodium succinate buffer, pH 5.0, resuspended in the same buffer to AssOnm = 4.0 and shaken at 30 “C for 4 min, at which time (B) 1.0 ml was taken out for the extraction of lipids. Extraction and chromatography of the lipids was carried out as described in Methods; the first solvent is the vertical dimension. A and B, autoradiograms of lipids from growing and starved cells, respectively; ~,ceramide(inositol)~-~~-manno~; 2,triphosphoinositide; 3,diphosphoinositide; q,phosphatidylinositol.
the growth medium to sodium succinate buffer, were examined as a function of time (Table II). The levels of tri- and diphosphoinositide in the respiratory deficient mutant drop sharply within a minute after transfer to the buffer solution. In contrast, the major inositol-cont~ning ~ycerophospho~pid, phosphatidylinositol, and the major inositol-~ont~ning sphingo~pidz5~‘*, ceramide~inositol)~~phosphate)~-mannose were unchanged. After transfer to buffer, the composition of the adenine nucleotide pool changed drastically. A so-fold drop in the ATP level was observed, matched by an increase in the AMP concentration. Adenylate energy charge, as defined by Atkinson and Waltonz9, decreased about IO-fold in the respiratory deficient cells transferred to buffer. As is evident from Table I, after IO min in the buffer there was a slight increase in the concent~tion of both ~phosphoinosi~de and ATP. This increase might possibly result from the mobilization of endogenous energy reserves. The results of a similar “starvation” experiment carried out with the parental strain (Table II) showed a qualitatively similar pattern to that with the mutant but differed significantly in certain quantitative aspects. The concentrations of the polyphosphoinositides, ATP, and adenylate energy charge in the parental strain did not drop as far as with the mutant and started recovery of their original levels at a faster rate. Presumably, the wild type cells can mobilize their endogenous energy reserves more effectively. For this reason, and as well as to restrict ATP generation to non-mitochondrial, glycolytic reactions, it was deemed useful to employ a respiratory-deficient mutant throughout this inves-
R. T. TALWALKAR,
416
R. L. LESTER
tigation. The cellular concentration of the phospholipids and nucleotides varied over anarrow range when measured during an exponential growth span of A650nm = 0.86.0. TABLE I RADIOACTIVITY IN MAJOR FROM CELLS GROWN WITH PAPER CHROMATOGRAPHY
INOSITOL-CONTAINING PHOSPHOLIPIDS, DERIVED [z-3H]MYOINOSITOL AND 32P, AND SEPARATED BY
Cells were grown as described in Methods in a medium supplemented with 32P1(0.2 Ci/l) and [2-3H])myoinositol(o.01 Ci/l) to ACIcnm = 4.0. Growth was stopped by adding trichloroacetic acid to a final concentration of 5 ‘A (w/v). The cells were washed once with 5 oAtrichloroacetic acid, once with water and lipids extracted and chromatographed as described in Methods. After autoradiography appropriate spots (Fig. IA) were cut out and counted. Abbreviations: PI, phosphatidylinositol; DPI, diphosphoinositide; TPI, triphosphoinositide; CZ, ceramide(inositol),-P,-mannose. Lipid
PI DPI TPI Cl
cpmlspot -P
3H
Relative 3aP/3H (PI = I) Expected Found
102220 3670 1390 20435
39785 730 165 7860
1.0 2.0 3.0 1.0
1.0 I.9 3.2 1.0
TABLE II CELLULAR CONTENT OF INOSITOL PHOSPHATIDES AND ADENINE NUCLEOTIDES OF YEAST CELLS TRANSFERRED FROM COMPLETE GROWTH MEDIUM TO SUCCINATE BUFFER ro-ml cultures of cells were grown in presence of 32PI to an absorbance of 4.2 (RDA3) and 5.7 (A~I), filtered through a Millipore filter (0.22 pm). The cells were washed with IO ml 0.048 M sodium succinate, pH 5, resuspended in IO ml of the same buffer to A 630nm= 4.2 and incubated with stirring at 30 “C. From growing cells (zero time) and at time intervals indicated, r.o-ml aliquots were removed and processed for the estimation of lipids and nucleotides as described in Methods. The r-min time point includes about 30 s of washing with buffer on the filter at room temperature and about 30 s of resuspension and incubation, both at 30 “C. Abbreviations: TPI, triphosphoinositide; DPI, energy charge, diphosphoinositide; PI, phosphatidylinositol; C Z, ceramide(inositol),-P2_mannose; (ATP + r/2 ADP)/(ATP + ADP + AMP). nmolesjabsorbance unit G
ATP
ADP
AMP
Energy charge
Respiratory mutant (strain RDA3) 0 10.5 76.0 3.63 I 3.1 35.7 3.71 5 2.0 14.3 3.70 IO I.7 8.3 3.58 15 I.4 17.1 3.59 30 1.1 21.2 3.64
1.00 0.99 I .02 0.92 I .03 0.99
1.112 0.020 0.020 0.015 0.022 0.024
0.312 0.141 0.135 0.116 0.158 0.145
0.073 1.051 I .041 I .004 0.891 0.823
0.847 0.075 0.073 0.064 0.095 0.097
Wild type (strain A~I) 0 12.3 74.9 I 8.8 43.7 5 4.8 20.5 30 8.3 42.7
1.04 0.94 0.94 0.92
I .006 0.063 o.o92 0.279
0.183 0.204 0.19o o-303
0.055 0.662 0.685 0.420
0.88 0.18 0.19 0.43
Time after pmoleslabsorbance unit transfer (min) TPI DPI
PI
2.90 2.64 2.59 2.52
POLYPHOSPHOiNOSiTlDES
AND ADENYLATE
ENERGY
CHARGE
417
Fig. 2. Kinetics of the changes in the cellular content of di- and triphosphoinositide and adenine nucleotides when glucose is added to starved yeast cells. Cells were grown in 25 ml growth medium containing 4.95 mCi 3zP for ~4 h to AsseDm = 2.43. At this point a I.O-ml aliquot was removed for estimations representing iog growth (A). The rest was filtered through a Miiipore f%lter (0.22 pm), washed once with IO nil 0.048 M sodium succinate, pH 5.0, and then suspended in the same buffer to Assorrm= 4.95 and incubation continued with shaking at 30 “C. Less than 2 ruin ensued between the time of harvest and return to the shaker. After 4 min of incubation, an aliquot was removed (B) and the rest of cell suspension was transferred (zero time) to another flask containing glucose to give final con~ntration of I % (w/v); at the times indicated r-o-ml aliquots were removed for estimation of lipids and nucleotides as described in Methods.
We next observed the effect of adding glucose to respiratory deficient cells which had been in buffer for several minutes. It can be seen (Fig. 2) that after a small lag ATP, ADP, AMP and energy charge returned to levels found in the growing cells within z min. Tri- and ~~osphoinositi~ also returned to levels found in cells in growth medium afkr addition of glucose at approximately the rate observed for ATP, however, with a slightly larger lag. The above results showed a correlation in the rates of increase in the adeuylate energy charge and the polyphosphoinositides. We therefore sought another experimental condition which would cause a rapid decrease in energy charge. The experi-
R. T. TALWALKAR,
418
R. L. LESTER
mental starvation conditions (Table II, Fig. 2) involved filtration and resuspension steps, the timing of which might be a source of experimental variation. 2-Deoxyglucose has been used by others30*31 to deplete the ATP pool of yeast. We therefore added 2-deoxyglucose to cells which had been transferred to buffer and fed glucose for 5 min (Fig. 3). Addition of 2-deoxyglucose also caused a prompt drop in the levels of triand diphosphoinositide at a rate definitely slower than observed for ATP. As in the case of ATP and energy charge, the relative decrease in the polyphosphoinositide levels was less than that observed for the cells incubated in buffer.
I
I
I
I
I.0 _
I
I
I
l NP
-
+z-do*
__-
-2-dOlC
\
0
ADP
A
AYC
1.0
5 3 1.3. t
. 1.0 -
0 cl go.7 c
_
P
I
I
0
I
I
IUE
men
I
I
K)
0
oEoxwLucoQ
I lo
AoolmNwM
Fig. 3. Changes in di- and triphosphoinositide and adenine nucleotide contents of glucose-fed cells by addition of z-deoxyglucose. The cells were grown in 23 ml medium containing 3.0 mCi 32P, for a4 h to Assonm = 2.64. The cells were harvested, suspended in the buffer to Aesonm = 4.1 I and starved for 4 min as described in the legend to Fig. 2. At this point, glucose was added to a final concentration of I ok (w/v) and incubation continued for 4 min, at the end of which (zero time) 2-deoxyglucose was mixed with part of the cell suspension (to the final concentration of I %) and at times indicated, I .o ml cells removed for the estimation of lipids and nucleotides as described in Methods.
POLYPHOSPHOINOSITIDES
AND ADENYLATE
ENERGY CHARGE
419
DISCUSSION
In examining the biological meaning of the turnover of the phosphomonoester groups of the polyphosphoinositides, both stoichiometric and catalytic mechanisms come to mind. A useful process may be stoichiometrically coupled to either the formation or breakdown of the polyphosphoinositides. Binding and release of Ca*+ or transport of other ions have been considered and reviewed by Hawthorne and coworkers32S33; thus, some of the energy released from ATP hydrolysis in one turnover cycle (phosphatidylinositol + [di- and triphosphoinositide] --, phosphatidylinositol) would be conserved for driving such processes. In this regard attention was focused on neural processes because of the relatively high levels of the polyphosphoinositides in brain. The widespread occurrence of the polyphosphoinositides in other animal tissues4-‘j as well as in fungi’s8 directs attention to biological roles for these lipids that are not unique to nerve tissue. In addition to functioning stoichiometrically di- or triphosphoinositide may act catalytically such as exerting an allosteric effect on the activity of a membrane enzyme. These lipids could also have an intrinsic catalytic role. Strategically located in a membrane, the phosphate groups of these lipids could combine with protons or other cations to produce molecular species of lowered net charge which could facilitate the diffusion of such ions across the membrane. In this manner these lipids could act, in a sense as physiological uncoupling agents, to regulate the magnitude of the electrochemical gradient and in turn regulate metabolite flow insofar as such gradients were significant to metabolite flow. In the general case of a catalytic role, the turnover of the phosphomonoester groups can be viewed as an attempt to achieve the lipid concentrations appropriate to the metabolic state of the cell. Whatever one might speculate to be the role of the polyphosphoinositides, it is apparent that there must be some means of regulating the formation and hydrolysis of these lipids or else a futile cycle would result. In yeast, we find that the levels of di- and triphosphoinositide respond promptly to changes in the cellular ATP concentration and/or the adenylate energy charge. Since we cannot effectively vary the in vivo cellular ATP concentration independent of the adenylate energy charge, it cannot be said which might be the more important. The increase of di- and triphosphoinositide (Fig. 2) appears to occur as fast as the increase of ATP when glucose is added to starved cells in buffer. When ATP is caused to fall abruptly, a similar rate of decrease of di- and triphosphoinositides is seen (Fig. 3). If these rates of synthesis and breakdown apply to turnover during growth, this would correspond roughly to a half-life of I min. Earlier data based on a pulse-chase experiment16 suggested a considerably longer half-life, however, it was clearly recognized that the turnover rate might be underestimated. The maximal levels of di- and triphosphoinositide observed either during growth (Table II) or by adding glucose to starved cells in buffer (Fig. 2), represent only a few percent of the total phosphatidylinositol pool. These maximum values are less than IO % of the cellular ATP concentration and would thus not appear to be simple equilibrium values, although a balanced rate of synthesis and breakdown cannot be ruled out altogether. A specific pool of phosphatidylinositol earmarked for di- and triphosphoinositide formation and/or specifically regulated enzymes responsible for their synthesis and degradation, appear to be equally plausible possibilities. There may also be more than one pool of di- and triphosphoinositide since in spite of
420
R. T. TALWALKAR,
R. L. LESTER
a jo-fold depletion of the ATP pool we have rarely seen more than a ro-fold drop in the levels of the polyphosphoinositides (Table II. Fig. 2). %Deoxyglucose has been used by others30*31 to deplete the ATP pool of yeast, presumably as a result of its conversion to a non-metabolisable 6-phosphate derivative or by inhibiting glucose entry into the cells 34. In any case, it can be seen (Fig. 3) that the drop in ATP achieved when 2-deoxyglycose is added, although quite rapid, does not fall to the values obtained by simple starvation in buffer. It can also be seen that the fall in di- and triphosphoinositide is also not as great with the 2-deoxyglucose perturbation as with simple starvation. Thus we have some evidence that intermediate values for adenylate energy charge and/or ATP concentration are associated with intermediate values of the polyphosphoinositides. The values obtained for the yeast adenine nucleotide pool agree well with published values3’. The enzymes reponsible for the synthesis and degradation of diphosphoinositide are membrane-bound in animal tissues studied33 and a recent note3’ indicates that phosphatidylinositol kinase activity in S. cerevisiue is associated with membrane fragments. Although these studies have thus far not shed too much light on the specific mechanism that regulates the cellular levels of polyphosphoinositides, they make it seem likely that the locus of regulation is membrane associated. Dawson and Eichberg22, as well as Wells and Dittmerz3 have shown that the di- and triphosphoinositide levels of rat brain fall rapidly after decapitation. The lipid data of Dawson and Eichberg22 bears a striking resemblance to the data obtained by Lowry et aZ.36 for the ATP concentration of mouse brain which also falls rapidly a couple of minutes after decapitation. Thus it seems likely that in brain also the level of the polyphosphoinositides is related to the cellular ATP concentration. These studies with yeast, however, represent the first evidence for rapid and reversible in vivo changes in the cellular concentrations of di- and triphosphoinositide. ACKNOWLEDGEMENTS
We are indebted to Mr Wallace W. Angus for his advice concerning the measurements of inositol phosphatides. We also thank Mr. G. W. Wells for his assistance. Supported in part by Grant NBo8323 from the U.S. Public Health Service. REFERENCES I Folch, J. (1949) J. Biol. Chem. 177, 497-504 Brockerhoff, H. and Ballou, C. E. (1961) J. Biol. Chem. 236, 1907-1911 3 Dittmer, J. C. and Dawson, R. M. C. (1961) Biochem. J. 81, 535-540 4 H&hammer, L., H&l, J. and Wagner, H. (1961) Nuturwissenschuften 48, 103 5 Lo Chang, T. and Sweeley, C. C. (1963) Biochemistry 2, 592-604 6 Hawthorne, J. N. and Michell, R. H. (1966) in Cyclitob and Phosphoinositides (Kindl, H., ed.), pp. 49-55, Pergamon, London 7 Lester, R. L. and Steiner, M. (1968) J. Biol. Chem. 243,4889-4893 8 Prottey, C., Seidman, M. M. and Ballou, C. E. (1970) Lipids 5, 463-468 9 LeBaron, F. N., Kistler, J. B. and Hauser, G. (1960) Biochim. Biophys. Actu 44, 170-172 IO Wagner, H., Lissau, A. and H&hammer, L. (1961) Biochem. Z. 335, 312-314 II Brockerhoff, H. and Ballou, C. E. (1962) J. Biol. Chem. 237, 49-52 12 Sheltawy, A. and Dawson, R. M. C. (1969) Biochem. J. III, 147-154 13 Santiago-Calvo, E., Mule, S., Redman, C. M., Hokin, M. R. and Hokin, L. E. (1964) Biochim. Biophys. Acta 84, 550-562 2
POLYPHOSPHOINOSITIDES I4 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
AND ADENYLATE
ENERGY
CHARGE
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Andrade, F. and Huggins, C. G. (1964) Biochim. Biophys. Acta 84, 681-693 Schneider, R. P. and Khschner, L. B. (1970) Biochim. Biophys. Acta 202, 283-294 Steiner, S. and Lester, R. L. (1972) Biochim. Biophys. Acta 260, 82-87 Hayashi, K., Yagihara, Y., Nakamura, I., Katagirl, A., Arakawa, Y. and Yamazoe, S. (1967) J. Biochem. Tokyo 62, 15-20 Colodzin, M. and Kennedy, E. P. (1965) J. Biol. Chem. 240, 3771-3780 Kai, M., Salway, J. G. and Hawthorne, J. N. (1968) Biochem. J. 106, 791-801 Thompson, W. and Dawson, R. M. C. (1964) Biochem. J. 91, 233-236 Salway, J. G., Kai, M. and Hawthorne, J. N. (1967) J. Neurochem. 14, 1013-1024 Dawson, R. M. C. and Eichberg, J. (1965) Biochem. J. 96, 634-643 Wells, M. A. and Dittmer, J. C. (1965) Biochemistry 4, 2459-2468 Angus, W. W. and Lester, R. L. (1972) Arch. Biochem. Biophys. 151,483-495 Steiner, S. and Lester, R. L. (1972) J. Bucteriol. 109, 81-88 Plaisted, P. M. and Reggio, R. B. (1963) Contrib. Boyce Thompson Inst. Plant Res. 22 (I), 71-80 Nazar, R. N., Lawford, H. G. and Tze-Fei Wong, J. (1970) Anal. Biochem. 35, 305-313 Steiner, S., Smith, S., Waechter, C. J. and Lester, R. L. (1969) Proc. Nutl. Acad. Sci. U.S. 64, 1042-1048 Atkinson, D. E. and Walton, G. M. (1967) J. Biol. Chem. 242, 3239-3242 Maitra, P. K. and Estabrook, R. W. (1967) Arch. Biochem. Biophys. 121, 129-139 Eddy, A. A., Backen, K. and Watson, G. (1970) Biochem. J. 120, 853-858 Buckley, T. J. and Hawthorne, J. N. (1972) J. Biol. Chem. 247, 7218-7223 Kai, M. and Hawthorne, J. N. (1969) Ann. N.Y. Acud. Sci. 165, 761-773 Heredia, C. F., de la Fuente, G. and Sols. A. (1964) Biochim. Biophys. Acta 86, 216-223 Wheeler, G. E., Michell, R. M. and Rose, A. H. (1972) Biochem. J. 127, 64P Lowry, 0. H., Passonneau, J. V., Hasselberger, F. X. and Schulz, D. W. (1964) J. Biol. Chem. 239. 18-30
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BBA 56271
THE RESPONSE OF DIPHOSPHOINOSITIDE AND TRIPHOSPHOINOSITIDE TO PERTURBATIONS OF THE ADENYLATE ENERGY CHARGE IN CELLS OF SACCHAROMYCES CEREVISIAE
R. T. TALWALKAR
and ROBERT L. LESTER
Department of Biochemistry, College of Medicine, University of Kentucky, Ky. Lexington, 40506 (U.S.A.) (Received December z7th, 1972)
SUMMARY
In cells of Saccharomyces cerevisiae uniformly labeled with 32P the levels of ATP, ADP, AMP, diphosphoinositide and triphosphoinositide were measured simultaneously after chromatographic separation. When a respiratory deficient mutant strain was removed from complete growth medium, there resulted an immediate and large drop in the levels of diphosphoinositide, triphosphoinositide, and ATP as well as the adenylate energy charge. Addition of glucose to starved cells in buffer resulted in a rapid increase in the concentrations of ATP, diphosphoinositide and triphosphoinositide. a-Deoxyglucose caused a rapid decrease of the adenylate energy charge to intermediate values; again, the levels of diphosphoinositide and triphosphoinositide fell rapidly but not to the same extent as with simple starvation. The close association between the adenylate energy charge and the-concentration of these polyphosphoinositides herein demonstrated is discussed in relation to the well known turnover of phosphomonoester groups of these lipids.
INTRODUCTION
Di- and triphosphoinositide have been long known’-3 as significant lipid constituents of nervous tissue, with smaller amounts occurring in other animal tissues4-6. More recently these lipids have been found in fungi’**. Interest in the biological role of these compounds has centered on the observations that in animal tissues’-” and yeast l6 there is a rapid turnover of the monoester phosphate groups of these compounds. In several broken cell systems the 7-P of ATP could be shown to be a precursor of the monoesterd groups of di- and triphosphoinositide, reactions mediated by kinases acting on phosphatidylinosito11’-19. Specific phosphohydrolases are thought to catalyse the conversion of di- and triphosphoinoThus action of the kinases and the phosphohydrositide to phosphatidylinositol 5*20~21. lases are thought to account for the turnover of the phosphomonoester groups. The biologically significant factors governing the action of these enzymes which result in the regulation of the cellular levels of di- and triphosphoinositide are not known. Aside
POLYPHOSPHOINOSITIDES
AND ADENYLATE
ENERGY CHARGE
413
from post-mortem decreases of these inositides observed in brain22*23, to our knowledge, no systematic in viva studies have been carried out to examine the regulation of the cellular concentrations of the polyphosphoinositides. In this paper we describe rapid changes in the di- and triphosphoinositide content of yeast associated with changes in the adenylate energy charge of the cell. METHODS
Growth of yeast
A respiratory deficient mutant, RDA3, spontaneously derived from the strain (A3 I) of Saccharomyces cerevisiae used in earlier studies7S’6 was employed throughout these experiments except where specifically noted. The defined Yeast Nitrogen Base medium (Difco Laboratories) supplemented with 4 % (w/v) glucose, 0.048 M sodium succinate, pH 5, and carrier-free 0.2 Ci/l 32Pi (New England Nuclear Corporation) was used for growing labeled cells which were harvested in log phase. Yeast Nitrogen Base medium contains orthophosphate as the sole source of phosphorus at a concentration of 7.4 mM. Cells were grown aerobically at 30 “C in a rotary shaker. Under these standard growth conditions the cellular doubling time was 3.3 h. Growth was monitored by following the turbidity at 650 nm, r-cm path. One absorbance unit = 180 pg dry wt of cells = I ml of culture with an absorbance of 1.0 = approximately 9oo pg wet wt. Extraction and chromatography of lipids The lipids were extracted as described earlier’6*24 with several changes to
enable simultaneous processing of the acid soluble nucleotides. r-ml aliquots from various incubation mixtures were mixed with 0.2 ml chilled 35% HCIO, in screwcapped centrifuge tubes. After remaining at o “C for 15 min non-radioactive carrier cells were added24, and the cells were rapidly frozen and thawed twice and centrifuged at 2500 rev./min in the cold. The supematant was saved for the nucleotide estimations. The pellet was washed twice with 5 ml water to remove acid and then was extracted with 2 ml of ethanol-water-diethylether-pyridine (I 5 : 15 : 5 : I, by vol.) for I 5 min at 60 “C followed by immediate centrifugation at low speed. In the typical experiment 50-~1 aliquots of the supernatant extract (equivalent to approximately 20 pg dry wt cells) were chromatographed on EDTA-treated, silica gel-impregnated paper in two dimensions as described25, except that the second dimension solvent was changed to chloroform-methanol-acetic acid-water (I 5 : 6 : 4: 2, by vol.), and 0.0 I 2 % butylated hydroxyanisole (Nutritional Biochemicals Corp.) was added to the first dimension solvent. Before spotting the lipids, 5 ,ul of a 1.5 % methanolic solution of butylated hydroxyanisole was applied to the origin to prevent the oxidation of lipids while drying. The phospholipid spots were located by autoradiography, cut out and counted in toluene-Triton X-100 (2:1, by vol.) counting fluid containing 0.4 % 2-(4’-tertbutylphenyl)-5-(4’-biphenyl)-1,3,4-oxidazole, 0.03 % 2-(4’-biphenyl)-6-phenylbenzoxazole plus o. I 17 vol. of water, using a Packard TriCarb liquid scintillation spectrometer. The molar quantity of each phospholipid was calculated from the specific activity of 32Pi in the growth medium and the radioactivity in each individual spot.
414
R. T. TALWALKAR,
R, L. LESTER
Extraction aPld~~~ornato~ap~y of adenine nu~~eotide~ The HCIO, extract (above) was quickly neutralised with 2.6 M KOH in presence of internally added chlorphenol red indicator, and the precipitate removed by centrifugation. A known amount of a mixture of tritiated adenine nucleotides ([8-3H]adenosine mono-, di- and triphosphates, Schwarz/Mann) was added to each sample and mixed thoroughly before charcoal adsorption in order to monitor and precisely correct for losses of adenylates during analysis. Darco G-60 charcoal (Fisher Scientific Co.) was purifiedz6 and washed twice with warm IO mM sodium EDTA adjusted to pH 7.5 with ammonia followed by several water washes. In a typical experiment IOO mg of this purified charcoal was added to each extract, representing approx. 700 pg cells, allowed to stand for 30 min with intermittent shaking and the suspension was filtered in a small column through a o.5-cm layer of Celite 545 (Fisher Scientific Co.). The column was washed with 5 ml of 0.05 M potassium phosphate buffer, pH 6.8, and then with 5 ml water. The ‘H standards were quantitatively adsorbed on the charcoal. The nucleotides were eluted with 2 ml of pyridinium acetate, pH 5.3 (pyridine-3 M acetic acid (3 : 7, v/v)). Eluted in this small volume were about 70 “/oof the added ADP and ATP counts and about 85 % of the AMP. One fourth of the eluate was evaporated to dryness and dissolved in 0.1 ml of water, of which 25 ~1 were spotted on MN Polygram CEL 300 PEI (Brinkamn Instruments Inc.) sheet along with carrier nonradioactive adenine nucleotides. The aliquot spotted represented about 40 pg dry weight of cells. Chromatography was carried out according to the procedure described by Nazar et aL27 except that the concentrations of both LiCl and acetic acid in the first solvent were reduced to 0.8 M. The adenine nucleotides were located with an ultraviolet lamp and by autoradiography, to eliminate any nearby 32P con~minants. The spots were cut out an placed in scintillation vials containing I ml 2 M LiCl solution. After standing 6 h at 37 “C, 15 ml of counting fluid (as above but without water) was added. From the internal standard ‘H counts, the recovered 32Pi counts and the specific activity of phosphate, the molar amount of each nucleotide in the original sample was calculated. RESULTS
The aim of the present work was to see if adenylate energy charge was related to pol~hosphoinosi~de concentration. Experiments were carried out with cells labeled unifo~ly with “Pi of known specific activity which enabled ovulation of the amounts of phospho~pids and nucleotides after chromato~ap~c separation. Logarithmically growing cells were transferred from growth medium to sodium succinate buffer after thorough washing, Several minutes later lipids were extracted from the cells and the total extract was chromatographed in two dimensions (Fig. IA and IB). It can be seen that under these conditions di- and triphosphoinositide, stand out as lipids which decreased significantly. With cells grown in the presence of 32Pi and [3H]inositol, it was found that the presumed phosphoinositides got labeled with the expected relative 32P/3H ratios (Table I). This is a reliable approach to check the identity and isotopic purity of inositol phospholipids as it has been shownz4 in an earlier report from this laboratory that [3H]inositol is not catabolised by yeast. Effects on the cellular levels of the major inositol phospho~pids and adenine nucleotides of the respiratory mutant and the parental strain, when transferred from
POLYPHOSPHOINOSITIDES
AND ADENYLATE
ENERGY
CHARGE
415
Fig. I. Chromatograms showing decrease in the di- and triphosphoinositide content of yeast upon removal from growth medium. Cells were grown in the presence of 32Pi to AsroDm = 4.1. After 1.0 ml was taken out for lipid extraction (A), the rest were harvested on a Miilipore filter (0.22 pm), washed once with IO ml 0.048 M sodium succinate buffer, pH 5.0, resuspended in the same buffer to AssOnm = 4.0 and shaken at 30 “C for 4 min, at which time (B) 1.0 ml was taken out for the extraction of lipids. Extraction and chromatography of the lipids was carried out as described in Methods; the first solvent is the vertical dimension. A and B, autoradiograms of lipids from growing and starved cells, respectively; ~,ceramide(inositol)~-~~-manno~; 2,triphosphoinositide; 3,diphosphoinositide; q,phosphatidylinositol.
the growth medium to sodium succinate buffer, were examined as a function of time (Table II). The levels of tri- and diphosphoinositide in the respiratory deficient mutant drop sharply within a minute after transfer to the buffer solution. In contrast, the major inositol-cont~ning ~ycerophospho~pid, phosphatidylinositol, and the major inositol-~ont~ning sphingo~pidz5~‘*, ceramide~inositol)~~phosphate)~-mannose were unchanged. After transfer to buffer, the composition of the adenine nucleotide pool changed drastically. A so-fold drop in the ATP level was observed, matched by an increase in the AMP concentration. Adenylate energy charge, as defined by Atkinson and Waltonz9, decreased about IO-fold in the respiratory deficient cells transferred to buffer. As is evident from Table I, after IO min in the buffer there was a slight increase in the concent~tion of both ~phosphoinosi~de and ATP. This increase might possibly result from the mobilization of endogenous energy reserves. The results of a similar “starvation” experiment carried out with the parental strain (Table II) showed a qualitatively similar pattern to that with the mutant but differed significantly in certain quantitative aspects. The concentrations of the polyphosphoinositides, ATP, and adenylate energy charge in the parental strain did not drop as far as with the mutant and started recovery of their original levels at a faster rate. Presumably, the wild type cells can mobilize their endogenous energy reserves more effectively. For this reason, and as well as to restrict ATP generation to non-mitochondrial, glycolytic reactions, it was deemed useful to employ a respiratory-deficient mutant throughout this inves-
R. T. TALWALKAR,
416
R. L. LESTER
tigation. The cellular concentration of the phospholipids and nucleotides varied over anarrow range when measured during an exponential growth span of A650nm = 0.86.0. TABLE I RADIOACTIVITY IN MAJOR FROM CELLS GROWN WITH PAPER CHROMATOGRAPHY
INOSITOL-CONTAINING PHOSPHOLIPIDS, DERIVED [z-3H]MYOINOSITOL AND 32P, AND SEPARATED BY
Cells were grown as described in Methods in a medium supplemented with 32P1(0.2 Ci/l) and [2-3H])myoinositol(o.01 Ci/l) to ACIcnm = 4.0. Growth was stopped by adding trichloroacetic acid to a final concentration of 5 ‘A (w/v). The cells were washed once with 5 oAtrichloroacetic acid, once with water and lipids extracted and chromatographed as described in Methods. After autoradiography appropriate spots (Fig. IA) were cut out and counted. Abbreviations: PI, phosphatidylinositol; DPI, diphosphoinositide; TPI, triphosphoinositide; CZ, ceramide(inositol),-P,-mannose. Lipid
PI DPI TPI Cl
cpmlspot -P
3H
Relative 3aP/3H (PI = I) Expected Found
102220 3670 1390 20435
39785 730 165 7860
1.0 2.0 3.0 1.0
1.0 I.9 3.2 1.0
TABLE II CELLULAR CONTENT OF INOSITOL PHOSPHATIDES AND ADENINE NUCLEOTIDES OF YEAST CELLS TRANSFERRED FROM COMPLETE GROWTH MEDIUM TO SUCCINATE BUFFER ro-ml cultures of cells were grown in presence of 32PI to an absorbance of 4.2 (RDA3) and 5.7 (A~I), filtered through a Millipore filter (0.22 pm). The cells were washed with IO ml 0.048 M sodium succinate, pH 5, resuspended in IO ml of the same buffer to A 630nm= 4.2 and incubated with stirring at 30 “C. From growing cells (zero time) and at time intervals indicated, r.o-ml aliquots were removed and processed for the estimation of lipids and nucleotides as described in Methods. The r-min time point includes about 30 s of washing with buffer on the filter at room temperature and about 30 s of resuspension and incubation, both at 30 “C. Abbreviations: TPI, triphosphoinositide; DPI, energy charge, diphosphoinositide; PI, phosphatidylinositol; C Z, ceramide(inositol),-P2_mannose; (ATP + r/2 ADP)/(ATP + ADP + AMP). nmolesjabsorbance unit G
ATP
ADP
AMP
Energy charge
Respiratory mutant (strain RDA3) 0 10.5 76.0 3.63 I 3.1 35.7 3.71 5 2.0 14.3 3.70 IO I.7 8.3 3.58 15 I.4 17.1 3.59 30 1.1 21.2 3.64
1.00 0.99 I .02 0.92 I .03 0.99
1.112 0.020 0.020 0.015 0.022 0.024
0.312 0.141 0.135 0.116 0.158 0.145
0.073 1.051 I .041 I .004 0.891 0.823
0.847 0.075 0.073 0.064 0.095 0.097
Wild type (strain A~I) 0 12.3 74.9 I 8.8 43.7 5 4.8 20.5 30 8.3 42.7
1.04 0.94 0.94 0.92
I .006 0.063 o.o92 0.279
0.183 0.204 0.19o o-303
0.055 0.662 0.685 0.420
0.88 0.18 0.19 0.43
Time after pmoleslabsorbance unit transfer (min) TPI DPI
PI
2.90 2.64 2.59 2.52
POLYPHOSPHOiNOSiTlDES
AND ADENYLATE
ENERGY
CHARGE
417
Fig. 2. Kinetics of the changes in the cellular content of di- and triphosphoinositide and adenine nucleotides when glucose is added to starved yeast cells. Cells were grown in 25 ml growth medium containing 4.95 mCi 3zP for ~4 h to AsseDm = 2.43. At this point a I.O-ml aliquot was removed for estimations representing iog growth (A). The rest was filtered through a Miiipore f%lter (0.22 pm), washed once with IO nil 0.048 M sodium succinate, pH 5.0, and then suspended in the same buffer to Assorrm= 4.95 and incubation continued with shaking at 30 “C. Less than 2 ruin ensued between the time of harvest and return to the shaker. After 4 min of incubation, an aliquot was removed (B) and the rest of cell suspension was transferred (zero time) to another flask containing glucose to give final con~ntration of I % (w/v); at the times indicated r-o-ml aliquots were removed for estimation of lipids and nucleotides as described in Methods.
We next observed the effect of adding glucose to respiratory deficient cells which had been in buffer for several minutes. It can be seen (Fig. 2) that after a small lag ATP, ADP, AMP and energy charge returned to levels found in the growing cells within z min. Tri- and ~~osphoinositi~ also returned to levels found in cells in growth medium afkr addition of glucose at approximately the rate observed for ATP, however, with a slightly larger lag. The above results showed a correlation in the rates of increase in the adeuylate energy charge and the polyphosphoinositides. We therefore sought another experimental condition which would cause a rapid decrease in energy charge. The experi-
R. T. TALWALKAR,
418
R. L. LESTER
mental starvation conditions (Table II, Fig. 2) involved filtration and resuspension steps, the timing of which might be a source of experimental variation. 2-Deoxyglucose has been used by others30*31 to deplete the ATP pool of yeast. We therefore added 2-deoxyglucose to cells which had been transferred to buffer and fed glucose for 5 min (Fig. 3). Addition of 2-deoxyglucose also caused a prompt drop in the levels of triand diphosphoinositide at a rate definitely slower than observed for ATP. As in the case of ATP and energy charge, the relative decrease in the polyphosphoinositide levels was less than that observed for the cells incubated in buffer.
I
I
I
I
I.0 _
I
I
I
l NP
-
+z-do*
__-
-2-dOlC
\
0
ADP
A
AYC
1.0
5 3 1.3. t
. 1.0 -
0 cl go.7 c
_
P
I
I
0
I
I
IUE
men
I
I
K)
0
oEoxwLucoQ
I lo
AoolmNwM
Fig. 3. Changes in di- and triphosphoinositide and adenine nucleotide contents of glucose-fed cells by addition of z-deoxyglucose. The cells were grown in 23 ml medium containing 3.0 mCi 32P, for a4 h to Assonm = 2.64. The cells were harvested, suspended in the buffer to Aesonm = 4.1 I and starved for 4 min as described in the legend to Fig. 2. At this point, glucose was added to a final concentration of I ok (w/v) and incubation continued for 4 min, at the end of which (zero time) 2-deoxyglucose was mixed with part of the cell suspension (to the final concentration of I %) and at times indicated, I .o ml cells removed for the estimation of lipids and nucleotides as described in Methods.
POLYPHOSPHOINOSITIDES
AND ADENYLATE
ENERGY CHARGE
419
DISCUSSION
In examining the biological meaning of the turnover of the phosphomonoester groups of the polyphosphoinositides, both stoichiometric and catalytic mechanisms come to mind. A useful process may be stoichiometrically coupled to either the formation or breakdown of the polyphosphoinositides. Binding and release of Ca*+ or transport of other ions have been considered and reviewed by Hawthorne and coworkers32S33; thus, some of the energy released from ATP hydrolysis in one turnover cycle (phosphatidylinositol + [di- and triphosphoinositide] --, phosphatidylinositol) would be conserved for driving such processes. In this regard attention was focused on neural processes because of the relatively high levels of the polyphosphoinositides in brain. The widespread occurrence of the polyphosphoinositides in other animal tissues4-‘j as well as in fungi’s8 directs attention to biological roles for these lipids that are not unique to nerve tissue. In addition to functioning stoichiometrically di- or triphosphoinositide may act catalytically such as exerting an allosteric effect on the activity of a membrane enzyme. These lipids could also have an intrinsic catalytic role. Strategically located in a membrane, the phosphate groups of these lipids could combine with protons or other cations to produce molecular species of lowered net charge which could facilitate the diffusion of such ions across the membrane. In this manner these lipids could act, in a sense as physiological uncoupling agents, to regulate the magnitude of the electrochemical gradient and in turn regulate metabolite flow insofar as such gradients were significant to metabolite flow. In the general case of a catalytic role, the turnover of the phosphomonoester groups can be viewed as an attempt to achieve the lipid concentrations appropriate to the metabolic state of the cell. Whatever one might speculate to be the role of the polyphosphoinositides, it is apparent that there must be some means of regulating the formation and hydrolysis of these lipids or else a futile cycle would result. In yeast, we find that the levels of di- and triphosphoinositide respond promptly to changes in the cellular ATP concentration and/or the adenylate energy charge. Since we cannot effectively vary the in vivo cellular ATP concentration independent of the adenylate energy charge, it cannot be said which might be the more important. The increase of di- and triphosphoinositide (Fig. 2) appears to occur as fast as the increase of ATP when glucose is added to starved cells in buffer. When ATP is caused to fall abruptly, a similar rate of decrease of di- and triphosphoinositides is seen (Fig. 3). If these rates of synthesis and breakdown apply to turnover during growth, this would correspond roughly to a half-life of I min. Earlier data based on a pulse-chase experiment16 suggested a considerably longer half-life, however, it was clearly recognized that the turnover rate might be underestimated. The maximal levels of di- and triphosphoinositide observed either during growth (Table II) or by adding glucose to starved cells in buffer (Fig. 2), represent only a few percent of the total phosphatidylinositol pool. These maximum values are less than IO % of the cellular ATP concentration and would thus not appear to be simple equilibrium values, although a balanced rate of synthesis and breakdown cannot be ruled out altogether. A specific pool of phosphatidylinositol earmarked for di- and triphosphoinositide formation and/or specifically regulated enzymes responsible for their synthesis and degradation, appear to be equally plausible possibilities. There may also be more than one pool of di- and triphosphoinositide since in spite of
420
R. T. TALWALKAR,
R. L. LESTER
a jo-fold depletion of the ATP pool we have rarely seen more than a ro-fold drop in the levels of the polyphosphoinositides (Table II. Fig. 2). %Deoxyglucose has been used by others30*31 to deplete the ATP pool of yeast, presumably as a result of its conversion to a non-metabolisable 6-phosphate derivative or by inhibiting glucose entry into the cells 34. In any case, it can be seen (Fig. 3) that the drop in ATP achieved when 2-deoxyglycose is added, although quite rapid, does not fall to the values obtained by simple starvation in buffer. It can also be seen that the fall in di- and triphosphoinositide is also not as great with the 2-deoxyglucose perturbation as with simple starvation. Thus we have some evidence that intermediate values for adenylate energy charge and/or ATP concentration are associated with intermediate values of the polyphosphoinositides. The values obtained for the yeast adenine nucleotide pool agree well with published values3’. The enzymes reponsible for the synthesis and degradation of diphosphoinositide are membrane-bound in animal tissues studied33 and a recent note3’ indicates that phosphatidylinositol kinase activity in S. cerevisiue is associated with membrane fragments. Although these studies have thus far not shed too much light on the specific mechanism that regulates the cellular levels of polyphosphoinositides, they make it seem likely that the locus of regulation is membrane associated. Dawson and Eichberg22, as well as Wells and Dittmerz3 have shown that the di- and triphosphoinositide levels of rat brain fall rapidly after decapitation. The lipid data of Dawson and Eichberg22 bears a striking resemblance to the data obtained by Lowry et aZ.36 for the ATP concentration of mouse brain which also falls rapidly a couple of minutes after decapitation. Thus it seems likely that in brain also the level of the polyphosphoinositides is related to the cellular ATP concentration. These studies with yeast, however, represent the first evidence for rapid and reversible in vivo changes in the cellular concentrations of di- and triphosphoinositide. ACKNOWLEDGEMENTS
We are indebted to Mr Wallace W. Angus for his advice concerning the measurements of inositol phosphatides. We also thank Mr. G. W. Wells for his assistance. Supported in part by Grant NBo8323 from the U.S. Public Health Service. REFERENCES I Folch, J. (1949) J. Biol. Chem. 177, 497-504 Brockerhoff, H. and Ballou, C. E. (1961) J. Biol. Chem. 236, 1907-1911 3 Dittmer, J. C. and Dawson, R. M. C. (1961) Biochem. J. 81, 535-540 4 H&hammer, L., H&l, J. and Wagner, H. (1961) Nuturwissenschuften 48, 103 5 Lo Chang, T. and Sweeley, C. C. (1963) Biochemistry 2, 592-604 6 Hawthorne, J. N. and Michell, R. H. (1966) in Cyclitob and Phosphoinositides (Kindl, H., ed.), pp. 49-55, Pergamon, London 7 Lester, R. L. and Steiner, M. (1968) J. Biol. Chem. 243,4889-4893 8 Prottey, C., Seidman, M. M. and Ballou, C. E. (1970) Lipids 5, 463-468 9 LeBaron, F. N., Kistler, J. B. and Hauser, G. (1960) Biochim. Biophys. Actu 44, 170-172 IO Wagner, H., Lissau, A. and H&hammer, L. (1961) Biochem. Z. 335, 312-314 II Brockerhoff, H. and Ballou, C. E. (1962) J. Biol. Chem. 237, 49-52 12 Sheltawy, A. and Dawson, R. M. C. (1969) Biochem. J. III, 147-154 13 Santiago-Calvo, E., Mule, S., Redman, C. M., Hokin, M. R. and Hokin, L. E. (1964) Biochim. Biophys. Acta 84, 550-562 2
POLYPHOSPHOINOSITIDES I4 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
AND ADENYLATE
ENERGY
CHARGE
421
Andrade, F. and Huggins, C. G. (1964) Biochim. Biophys. Acta 84, 681-693 Schneider, R. P. and Khschner, L. B. (1970) Biochim. Biophys. Acta 202, 283-294 Steiner, S. and Lester, R. L. (1972) Biochim. Biophys. Acta 260, 82-87 Hayashi, K., Yagihara, Y., Nakamura, I., Katagirl, A., Arakawa, Y. and Yamazoe, S. (1967) J. Biochem. Tokyo 62, 15-20 Colodzin, M. and Kennedy, E. P. (1965) J. Biol. Chem. 240, 3771-3780 Kai, M., Salway, J. G. and Hawthorne, J. N. (1968) Biochem. J. 106, 791-801 Thompson, W. and Dawson, R. M. C. (1964) Biochem. J. 91, 233-236 Salway, J. G., Kai, M. and Hawthorne, J. N. (1967) J. Neurochem. 14, 1013-1024 Dawson, R. M. C. and Eichberg, J. (1965) Biochem. J. 96, 634-643 Wells, M. A. and Dittmer, J. C. (1965) Biochemistry 4, 2459-2468 Angus, W. W. and Lester, R. L. (1972) Arch. Biochem. Biophys. 151,483-495 Steiner, S. and Lester, R. L. (1972) J. Bucteriol. 109, 81-88 Plaisted, P. M. and Reggio, R. B. (1963) Contrib. Boyce Thompson Inst. Plant Res. 22 (I), 71-80 Nazar, R. N., Lawford, H. G. and Tze-Fei Wong, J. (1970) Anal. Biochem. 35, 305-313 Steiner, S., Smith, S., Waechter, C. J. and Lester, R. L. (1969) Proc. Nutl. Acad. Sci. U.S. 64, 1042-1048 Atkinson, D. E. and Walton, G. M. (1967) J. Biol. Chem. 242, 3239-3242 Maitra, P. K. and Estabrook, R. W. (1967) Arch. Biochem. Biophys. 121, 129-139 Eddy, A. A., Backen, K. and Watson, G. (1970) Biochem. J. 120, 853-858 Buckley, T. J. and Hawthorne, J. N. (1972) J. Biol. Chem. 247, 7218-7223 Kai, M. and Hawthorne, J. N. (1969) Ann. N.Y. Acud. Sci. 165, 761-773 Heredia, C. F., de la Fuente, G. and Sols. A. (1964) Biochim. Biophys. Acta 86, 216-223 Wheeler, G. E., Michell, R. M. and Rose, A. H. (1972) Biochem. J. 127, 64P Lowry, 0. H., Passonneau, J. V., Hasselberger, F. X. and Schulz, D. W. (1964) J. Biol. Chem. 239. 18-30