Divalent cations and inorganic phosphate metabolism in starved Tetrahymena

554

Biochimica et Biophysica Acta, 338 ( 1 9 7 4 ) 5 5 4 - - 5 6 0 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 2 7 3 4 3

DIVALENT CATIONS AND INORGANIC PHOSPHATE METABOLISM IN STARVED TETRAHYMENA

E D N A S. K A N E S H I R O * , S A R I S C O T T a n d R O B E R T L. C O N N E R

Department of Biology, Bryn Mawr College, Bryn Mawr, Pa. 19010 (U.S.A.) ( R e c e i v e d A u g u s t 31st, 1 9 7 3 )

Summary

Tetrahymena pyriformis maintained under starvation conditions release orthophosphate into the suspension medium. Ca 2÷ and/or Mg 2÷ addition reduced the a m o u n t of orthophosphate excreted during a 3-h period. Cellular orthophosphate levels were n o t altered by divalent cation supplements; however, an increase in the pyrophosphate c o n t e n t was observed which was equivalent in a m o u n t to the reduction in orthophosphate efflux. These observations suggest that divalent cations are i m p o r t a n t n o t only in the acquisition of phosphorus during growth but also in the conservation of this element during starvation. Introduction Cultures of Tetrahymena pyriformis grown in a chemically defined nutrient medium that includes Mg2÷, Ca 2~ and orthophosphate contain large numbers of pyrophosphate granules that are not associated with mitochondria [ 1,2]. These membrane-enclosed complexes disappear when orthophosphate is removed from the medium and reappear when orthophosphate is reintroduced. Orthophosphate deprivation is accompanied by Ca 2÷ , and later, Mg 2÷ efflux. When the medium contains orthophosphate and only one of the divalent cations, a markedly slower uptake of orthophosphate and lessened deposition of granules yesults. Cultures of Tetrahymena grown in a peptone-based medium and transferred to a non-nutrient buffer excrete h y p o x a n t h i n e , uracil, and orthophosphate [3--5]. The a m o u n t of purine and pyrimidine bases and orthophosphate found in the suspending fluid is related to a decline in the cellular RNA c o n t e n t

* Present address: D e p a r t m e n t of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221, U.S.A.

555 [3,5]. Cline [6] demonstrated that Mg 2÷ reduced the orthophosphate loss and suggested that the cation stabilized RNA. Koroly and Conner [5], however, showed that Mg2÷ had little influence on the level of the nucleic acid in starved cells and noted that the decrease in orthophosphate efflux was accompanied by an increase in the size of the cellular acid-soluble phosphorus pool. The nature of the accumulated material was n o t ascertained. The present investigation was undertaken to determine if the reduction in orthophosphate excretion and the increase in the cellular acid-soluble pool observed during starvation in the presence of Mg2÷ was due to an accumulation of cellular pyrophosphate. It has been shown that Mg2÷ and/or Ca 2÷ reduces the loss of orthophosphate from starved Tetrahymena while the cellular orthophosphate c o n t e n t remains at a constant value. The decrement observed in orthophosphate excretion is paralleled by a stoichiometric increase in the cellular pyrophosphate content. Materials and Methods Cultures of Tetrahymena pyriformis W were grown in peptone--yeast extract medium supplemented with an iron chelate and 0.5% (w/v) glucose [7]. 500 ml log phase cultures grown for 19 h at 28.5 °C were concentrated by continuous-flow centrifugation [8] and washed with Tris HC1 buffer (70 mM, pH 7.5) to give a final dilution with regard to culture fluid of 1:3100. 15 min after the harvesting procedure was begun the cells were resuspended in the buffer at a density of approx. 3 • 10 s ciliates/ml, and when desired, with CaC1 and/or MgSO4 to give a final concentration of each of 1 mM. The ciliates were incubated at 28.5 ° C. Cell numbers were estimated at each time point by use of a Coulter particle counter (Model A) equipped with 200-pm orifice. Cell volumes were determined by measurements of the length and width of osmiumfixed (1% OsO4, w/v, final concentration) specimens in a Sedgewick--Rafter chamber. The formula for a prolate spheroid was used for the calculations [9]. Cells were removed from the suspension medium by filtration after various periods of incubation [5]. The orthophosphate c o n t e n t of the cellular suspension and of the cell-free suspension fluid was estimated by the m e t h o d of Mozersky et al. [10]. The absorbance of the unreduced p h o s p h o m o l y b d a t e complex was determined at 313 nm with a Hitachi Perkin--Elmer 139 spectrophotometer. Intracellular orthophosphate amounts were calculated by subtraction of the incubation fluid values from those obtained for the cellular suspensions. 5 or 10 pg orthophosphate phosphorus were added to one-half of the replicate sets of assay mixtures as an internal standard. Pyrophosphate was estimated as orthophosphate released by an excess of inorganic yeast pyrophosphatase (EC 3.6.1.1; Worthington Biochemical Corp.) under standard conditions [11] from the deproteinized material, using appropriate controls, blanks, and standards [10,12]. The intracellular pyrophosphate c o n t e n t was calculated by subtraction of the extracellular orthophosphate and pyropho~vhate, as well as the cellular orthophosphate amounts~from the total. Absorbance measurements of aliquots of cell-free suspension fluid were made at 260 nm to determine purine and pyrimidine efflux [5].

556 TABLE I C O M P A R I S O N OF S E V E R A L ASSAY P R O C E D U R E S FOR THE I N T R A - AND E X T R A C E L L U L A R O R T H O P H O S P H A T E C O N T E N T OF TERTAHYMENA SUSPENSIONS

T. p y r i f o r m i s W c u l t u r e s w e r e g r o w n at 2 8 . 5 ° C for 21 h, harvested and i n c u b a t e d for 3 h in 'I~is~-HCl o ( 7 0 m M , p H 7.5, 2 8 . 5 C). Ciliate d e n s i t y was 3 . 4 - 1 0 ' ; - - 4 . 5 " 1 0 s ceUslml. V a l u e s are e x p r e s s e d in # g P ] 1 0 7 cells ± S,E. N u m b e r o f d e t e r m i n a t i o n s are in p a r e n t h e s e s . Mg 2+ was a d d e d t o give a final c o n c e n t r a t i o n of 1 raM. Procedure Martin and Dory [13] Cellular Control Mg2+-supplemented

Fiske a n d S u b b a R o w [ 1 4 ]

3 0 . 3 ± 2.2 ( 9 ) 35.6 ± 3 . 2 ( 1 0 )

Cell-free s u s p e n s i o n fluid Control 45.3 +_ 2.8 ( 9 ) Mg 2+ s u p p l e m e n t e d 3 0 . 3 _ 1 . 9 ( 1 0 )

49.2 + 3.3 ( 1 0 ) 32.2 + 3.1 ( 1 0 )

Recovery of internal s t a n d a r d (%)

97.1 + 1.4 ( 1 6 )

1 1 1 . 5 _+ 3.1 ( 3 7 )

M o z e r s k y et al [ 1 0 ]

14.1 ± 1.7 13.7 ± 1.5

8) 7)

4 0 . 6 __+1.7 29.6 ± 1.2

8) 8)

1 0 7 . 4 +__1.4 ( 3 0 )

Results

Table I lists the data obtained for intracellular and extracellular orthophosphate when various analytical procedures were employed [10,13,14]. The technique of Mozersky et al. [ i 0 ] produced lower values for cellular orthophosphate than that of Martin and Doty [13], while the extracellular values were similar. Recovery of an internal standard was complete by all procedures employed. It appears that the Mozersky et al. procedure results in less destruction of acid-labile phosphoryl esters, and therefore is preferable for the estimation of the cellular orthophosphate content. The intracellular orthophosphate c o n t e n t of ciliates suspended in Tris--HC1 buffer remained relatively constant at 20 pg orthophosphate/107 ciliates over a 6-h period and was n o t altered by 1 mM Mg 2÷ . The average volume of freshly harvested cells was 34 pl which decreased to 27 pl after 90 min and to 24 pl after 180 min incubation in the Tris--HC1 buffer. Since the size of the cells decreased throughout the incubation, the intracellular concentration of orthophosphate increased from 2.0 mM to 3.1 mM after 3 h of starvation. The orthophosphate concentration in the suspension fluid after 3 h starvation was 0.16 mM when divalent cations were omitted and, thus, was lower than the estimated cellular concentration at all times. The delay in the appearance of orthophosphate in the suspension medium with Mg2* supplementation, as reported earlier [4,5], was observed. Cells starved for 3 h in the presence of Mg 2÷ , Ca 2÷ , or the combination of cations, revealed that either ion independently reduced orthophosphate efflux, but did not alter the intracellular content (Fig. l a and lb). Some summation of the ion effect on efflux was noted. Pyrophosphate was detected in small amounts in the suspension fluid. The a m o u n t of pyrophosphate in the cells did n o t change significantly over a 3-h

557

D

30 20 ~'~k'5040

¢ontr01

O

/

control

3C~

Mg

20

Ca

Cot-Mq 10

0

180

Incubatlon time (mini Fig. 1. The effect o f the a d d i t i o n o f 1 m M Mg 2+, 1 m M Ca 2+, and the c o m b i n a t i o n o f Ca 2+ and Mg 2+ to a non-nutrient, phosphate-free medium, on the e f f l u x (a) and intzacellulcr content (b) o f o r t h o p h o s p h a t e phosphorus in Tetrahyrnena over a 3-h Period. Each value represents 6--16 determinations with a S.E. o f -+ 10%. Recovery o f the internal standard was 99.8 +- 1.2% (S.E.) in 131 determinations.

T A B L E II I N O R G A N I C P H O S P H O R U S V A L U E S (/L/g P / 1 0 7 C E L L S ) F O R T E T R A H Y M E N A I N C U B A T E D IN NON-NUTRIENT, PHOSPHATE-FREE SUSPENSION WITHOUT DIVALENT CATIONS AND WITH 1 m M Ca 2+ A N D 1 m M M g 2+ O V E R A 3-h P E R I O D Pi = o r t h o p h o s p h a t e p h o s p h o r u s ; PPi = p y r o p h o s p h a t e P h o s p h o r u s ; I n t = i n t r a c e l l u l a x c o n t e n t ; E x t = e x t r a c e n u l a x c o n t e n t ( e f f l u x ) ; /k ( C a 2+ + Mg 2 + ) _ c o n t r o l values. All v a l u e s i n ~{g P / 1 0 7 cells ,+ S.E.; n u m b e r s i n p a r e n t h e s e s i n d i c a t e n u m b e r o f e x p e r i m e n t a l values. Fraction

Addition

Pi ( I n t )

None Ca 2+ + Mg 2+

Pi(Ext)

None C a 2 + + M g 2+ /~

PPi ( I n t )

None Ca 2+ + Mg 2+ /~

PPi ( E x t )

None C a 2 + + Mg 2+

Initial

Starvation period (hi 1.5

3.0

2 0 . 5 + 2.7 (5) 1 8 . 7 _+ 2 . 2 (5)

2 0 . 5 _+ 1 . 5 (5) 2 3 . 4 _+ 4 . 6 (5)

--

9 . 8 _+0.8 ( 1 0 ) 9.5-+0.4(9) 0.3 +1.2

1 4 . 2 + "0.8 (8) 9.5+ 0.9(8) - - 4 . 8 + 1,7

39.4_+ 1 . 6 (8) 12.3_+ 1 . 2 ( 9 ) - - 2 7 . 1 ,+ 2 . 8

+

7 . 3 -+ 4 . 6 ( 5 ) 9 . 3 _+ 4.7 ( 5 ) 2.0+9.3

6 . 9 + 6.1 ( 5 ) 2 2 . 8 + 8 . 5 (5) + 15.9_+14.5

1 1 . 2 ± 4 . 0 (5) 3 9 . 3 ,+ 8.1 (5) + 28.2±12.1

0 . 9 _+ 1 . 4 ( 5 ) 0.8-+1.0 (5)

1.6 ,+ 1 . 5 (5) 2.0_+ 1.7 (5)

1 . 9 ,+ 3 . 2 (5) 3 . 4 - + 2 . 6 (5)

19.8 + 2.4 (5) 1 8 . 5 _+2.4 ( 5 )

558 T A B L E lIl ESTIMATE OF RNA DEGRADATION C A L C U L A T E D F R O M 2 6 0 nrn A B S O R B A N C E MENTS OF THE CILIATE SUSPENSION FLUID OVER A 3-h-STARVATION PERIOD

MEASURE-

All v a l u e s are given in b/g c e l l u l a r R N A d e g r a d e d / 3 - 105 cells [ 5 ] .

Expt

1 2

Initial

Starvation period (1,5 h)

S t a r v a t i o n p e r i o d (3 h)

Control

Ca : + + Mg : +

Control

Ca : + + Mg : +

Control

Ca : + + Mg 2+

3.9 2.1

3.2 2.0

23.5 18.3

25.8 18.4

34.2 25.9

35.4 27.4

period unless the cells were supplemented with Ca 2÷ , Mg 2÷, or the combination of cations. Mg:÷ addition led to an increase in intracellular pyrophosphate of 196%, and Ca :÷ of 304% the a m o u n t found in unsupplemented cells. A balance sheet is given in Table II for ciliates incubated in the presence of both divalent cations. An equivalence is seen between the decrease in orthophosphate efflux and the increase in the intracellular pyrophosphate. Table III lists values for two experiments in which the efflux of purines and pyrimidines was followed by absorbance measurements at 260 nm. These values are listed as RNA equivalents [5]. The addition of Ca 2÷ and Mg 2÷ to the suspension fluid did not alter the a m o u n t of purine and pyrimidine excreted and, presumably, did n o t influence RNA levels. Discussion The intracellular concentration of orthophosphate in Tetrahymena was found to be lower than previously noted [1,2,4]. Cline and Conner [4,7] employed the Martin--Doty orthophosphate assay and reported a concentration of 5.0 mM + 10% while in the present investigation, with the same technique, a value of 4.4 mM -+ 7% was noted. When the procedure of Mozersky et al. [10] was used a value of 2.0 + 12% was found. Since the two methods give comparable results for known standards and for the extracellular orthophosphate assays, the higher intracellular orthophosphate values found with the Martin- Doty technique may be due to more extensive degradation of acidlabile phosphoryl esters. Stoner and Dunham [15] concluded that orthophosphate was a small fraction of total cellular solutes and therefore n o t important in osmoregulation. These investigators measured cellular osmolarity of cell extracts by a freezing point depression technique and noted that Na ÷, K ÷, C1- and free amino acids accounted for most of the osmotically active components. Our estimation of cellular orthophosphate (2.0 mM) indicates that this ion contributes no more than 2% of the total cellular solutes of Tetrahymena (123 mM) and thus would reinforce this conclusion. Cells supplemented with Mg 2+ and/or Ca 2÷ excreted less orthophosphate than non-supplemented cells after 3 h incubation in a non-nutrient medium. This excretion differential is accounted for by an increase in the cellular pyrophosphate content. These results indicate that during the step-down nutritional

559 s t a t e in which RNA catabolism provides increased amounts of orthophosphate [3,5], the cell maintains a constant level of this ion and excretes the excess into the suspension fluid. Presumably this allows the acid--base balance and osmotic properties of the ciliate to be conserved. When Ca 2÷ and/or Mg 2÷ are provided, orthophosphate is retained, condensed to pyrophosphate which, in turn, presumably forms an insoluble complex with the cations to give rise to the granules. Munk and Rosenberg [1] found that the presence of orthophosphate and the t w o divalent cations in the medium was an absolute requirement for deposition of Ca- Mg- -pyrophosphate granules. The uptake of the two cations and phosphate by the cells was demonstrated to be coupled [2]. Rosenberg [6] reported that the granules contained approximately equimolar amounts of the cations and phosphorus; however, Coleman et al. [17] employing electron microprobe analysis of granules in situ found Ca 2÷ to be present in amounts of 1.5 times greater than Mg 2+. The effectiveness of either divalent cation in stimulating p y r o p h o s p h a t e formation in the present experiments suggests that even greater latitude may be possible in the composition of the granular complex. An alternative explanation for the observed action of either cation would be that both ions are not required for granule deposition, b u t are necessary for orthophosphate entry into the cells. Intracellular orthophosphate arising from R N A catabolism, thus, would bypass the entry process. A second possibility is that the newly formed p y r o p h o s p h a t e is not deposited into granules when only one cation is present. Polyphosphates are reported to be absent in Tetrahymena [ 1 8 ] , and thus, p y r o p h o s p h a t e appears to be the major phosphorus storage form. This mechanism of phosphate conservation during nutritional imbalance or starvation may be related to the increased percentage of successful conjugations in paramecia populations after the cells have been starved in a solution containing orthophosphate and Ca 2+ [ 1 9 ] . Conjugation as well as cytokinesis are periods in which food vacuoles are not formed by the cells and the intake of nutrients is restricted. Rosenberg [16] noted that pyrophosphate granules in Tetrahymena are associated with the dividing nucleus during mitosis which suggests that these elements may be necessary for the successful accomplishment of nuclear events associated with mitosis as well as with meiosis. Acknowledgements These investigations were supported by awards from the National Science Foundation, GB-32944, and the U.S. Public Health Service, HL-12872. E.S.K. was a Postdoctoral Fellow (National Science Foundation Biochemistry Development Grant GB-3181). (Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221.) S.S. was an Undergraduate Research Participant (National Science Foundation, Grant GY-8867). References 1 M u n k , N. a n d R o s e n b e r g , H. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 1 7 7 , 6 2 9 - - - 6 4 0 2 R o s e n b e r g , H. a n d M u n k , N. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 1 8 4 , 1 9 1 - - 1 9 7 3 L e b o y , P.S., Cline, S.G. a n d C o n n e r , R . L . ( 1 9 6 4 ) J. P r o t o z o o l . 11, 2 1 7 - - 2 2 2

560 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Cline, S.G. and Conner, R.L. (1966) J. Cell. Physiol. 68, 149--156 Koroly, M.J. and Conner, R . L . J . Protozool. Vol. 21, in t he press Cline, S.G. (1966) J. Cell. Physiol. 68, 157--163 Conner, R.L. and Cline, S.G. (1964) J. Protozool. 1 1 , 4 8 6 - - 4 9 1 Conner, R.L., Cline, S.G., Koroly, M.J. and Hamilton, B. (1966) J. Protozool. 13, 377--379 Thormar, H. (1962) Exptl. Cell Res. 27, 585--586 Mozersky, S.M., Pettinati, J.D. and Kolmar, S.D. (1966) Anal. Chem. 38, 1182--1187 Kuntz, M. (19'52) J. Gen. Physiol. 35, 4 2 3 - - 4 5 0 Kushmerick, M.J. (1972) Anal. Biochem. 46, 129--134 Martin, J.B. and Doty, D.M. (1949) Anal. Chem. 2 1 , 9 6 5 - - 9 6 7 Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem. 6 6 , 3 7 5 - - 4 0 0 Stoner, L.C. and Dunham, P.B. (1970) J. Exp. Biol. 5 3 , 3 9 1 - - 3 9 9 Rosenberg, H. (1966) Exp. Cell Res. 41, 397---410 Coleman, J.R., Nilsson, J.R., Warner, R.R. and Batt, P. (1972) Exp. Cell Res. 7 4 , 2 0 7 - - 2 1 9 Hill, D.L., Judd, J. and VanEys, J. (1965) Comp. Biochem. Physiol. 14, 1--10 Dryl, S. (1959) J. Protozool. 6 (Suppl.), 25