SIp NMR studies on microorganisms have been carried out with the cells embedded in agarose gel. The novel use of the gel for the NMR studies has ...
Bioscience Reports i, 141-150 (1981) Printed in Great Britain
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Improved technique for investigation of c e l l m e t a b o l i s m by 31p NMR spectroscopy Lev 3ACOBSON and 3ack S. COHEN Developmental Pharmacology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205, U.S.A. (Received 16 January 1981)
SIp NMR studies on microorganisms h a v e been c a r r i e d out with the cells embedded in agarose gel. The novel use of the gel for the NMR studies has advantages over t h e u s u a l l i q u i d s u s p e n s i o n s in t e r m s of i m p r o v e d reproducibility of data and cell v i a b i l i t y , with no n e t loss of s p e c t r a l q u a l i t y . P o l y p h o s p h a t e formation in E s c h e r i c h i a c o l i was monitored continuously for up to 24 h and metabolic changes in yeast for 6 h. Changes of the intracellular pH during glycolysis in y e a s t w e r e d e t e r m i n e d from the chemical shift of the internal Pi. NMR t i t r a t i on curves of Pi in t h e p r e s e n c e of Mg ~+ indicate uncertainties in internal pH values estimated by this technique. Nuclear magnetic resonance spectroscopy is now widely applied as a tool to study metabolism in living t i s s u e s and c e l l s ( B u r r e t a l . , 1979). U s ua l l y f o r NMR s t u d i e s of m i c r o o r g a n i s m s the ceils are re-suspended a f t e r centrifugation. They tend to s e t t l e down to t h e b o t t o m of t h e NMR t u b e , where because of the rapid depletion of oxygen and nutrients they begin to die. One s trat agem taken to avoid this happening is to re-suspend the cells by bubbling oxygen; but this spoils t h e m a g n e t i c f i e l d h o m o geneity. To partially overcome this problem a sophisticated spectrom e t e r interlock has been used (Navon e t a l . , 1977; Ogawa e t a l . , 1 9 7 8 a) ; but this is t i m e - c o n s u m i n g in terms of the acquisition of spectra, and does not prevent i n t e r m i t t e n t deterioration of the sample. In addition, many cells are sensitive to variations in dissolved oxygen tension. In s e a r c h of an a l t e r n a t i v e approach we a t t e m p t e d to set up a continuous flow-through system, using oxygenated m e d i u m . But t hi s 'perfusion' is much less successful for ceils than for organs, since the cells block most filters. We now des c r i be a simple but viable al t ernat i ve which keeps the ceils alive over a long period, namely to embed them in agarose gel s u p p l e m e n t e d with the desired nutrients. Agarose has the virtue of allowing normal diffusion of oxygen and nut ri ent s, and cei l s can be evenly suspended in it at any desired concentration. In fact, growth of microorganisms in agarose gel is a w i d e l y used m i c r o b i o l o g i c a l technique. To e n s u r e t h e c o n t i n u i n g a v a i l a b i l i t y of nutrients and oxygen, a liquid culture m e d i u m above the agarose b l o c k is b u b b l e d through with either oxygen or air. 9
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In this manner we have obtained Sip NMR spectra of E s c h e r i c h i a and yeast in relatively low concentrations in gel suspension. The spectra obtained are comparable to those from liquid suspensions, with the added advantages of i m p r o v e d r e p r o d u c i b i l i t y of t h e d a t a and i n c r e a s e d long-term cell viability. We dem onst rat e these advantages by observation of p o l y p h o s p h a t e a c c u m u l a t i o n in E . c o l i and on glycolysis in yeast over extended periods of time. From the position of the internal Pi peak it is possible to e s t i m a t e the intracellular pH (Moon & Richards, 1973; Salhany e t a l . , 1975; Navon e t a l . , 1979), and we discuss the limitations of the accuracy of the intracellular pH determination. coli
Methods Cell preparations Yeast. Saccharomyces cerevisiae (AN 33) were grown from slant in YPGE (1% Yeast Extract, 296 Bacto-peptone, 3% gyucerol, and 2% ethanol) on a rotary shaker at 30~ Cells, from either the early or the lathe exponential phase of growth, w e r e h a r v e s t e d by l o w - s p e e d c e n t r i f u g a t i o n and w a s hed t w i c e with mineral salt solution (8 mM MgSO4~ 2 mM NaCl~ and 7 mM K-phosphate, pH 7.0), always in the cold. S a m p l e s e m b e d d e d in a g a r o s e block w e r e p r e p a r e d in the following m anner : a washed pellet of a known amount of yeast ceils Was resuspended in mineral salt solution and warmed to 37~ and 0.5 ml of t h e suspension was mixed with 0.5 ml of the same salt solution supplemented with 0.5 agarose (Sigma), also prewarmed to 37~ The final mixing was done in the 10-mm NMR t u b e , w h i c h was i m m e d i a t e l y i m m e r s e d i nt o i ced w a t e r . S e v e r a l ml of liquid mineral medium of the same composition~ but supplemented with glucose, was c a r e f u l l y l a y e r e d on top of the agarose block and a Pasteur pipette was immersed approximately 1 cm below t h e liquid~ t h r o u g h which oxygen was bubbled at the rate of 15 cc/min. E. c o l i . K-12 'wild-type' s t r a i n ( A T C C 23716) was grown in peptone broth supplemented with 0.5% glucose on a rotary shaker at 37~ Cells f r o m t h e m i d d l e e x p o n e n t i a l phase of g r o w t h w e r e harvested by low-speed centrifugation and washed twice with ice-cold isotonic KCI-NaCI. The washed pellet was resuspended in sulfur-less medium (3acobson, unpublished results) containing l0 mM K-phosphate buffer, pH 7.0, warmed up to 37~ mixed with the same sulfur-less m e d i u m , supplemented with 0.5% agarose, and solidified as described for the yeast sample. The liquid layer on t h e t op of t h e a g a r o s e block contained sulfur-less m e d i u m s u p p l e m e n t e d with 0.1 M K-phosphate buffer, pH 7.0. Orthophosphate titrations
10 mM solutions of NaH2PO 4 and Na~HPO 4 each containing 0.1 M KCI were mixed together to give final pH values between # and 8.5. The resulting solutions were each divided into four samples, and MgCI 2 (3 M stock solution) was added to t hree of those to give 1:3, 1=2, and 3:2 Mg/P r a t i o s , r e s p e c t i v e l y . Chemical shifts relative to internal trimethylphosphate w e r e m e a s u r e d a t 25 + 0 . 3 ~ In a s e p a r a t e
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experiment the trimethylphosphate Mg2*-independent.
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shift was shown to be pH- and
NMR spectroscopy
31p NMR s p e c t r a w e r e o b t a i n e d at 109.3 MHz; 1000-3000 scans (0.25 sec) each at the t e m p e r a t u r e specified were a c c u m u l a t e d i n t o #1< d a t a p o i n t s w i t h 1 0 0 - m s e c d e l a y s . T h e RF p u l s e width was adjusted to 60 ~ (15 IJsec). C h e m i c a l shifts, with or without agarose, w e r e r e f e r e n c e d to i n t e r n a l t e t r a e t h y l m e t h y l e n e d i p h o s p h o n a t e (Alfa C h e m i c a l Co.). The c h e m i c a l shift of the l a t t e r d o e s not depend on pH, is s u f f i c i e n t l y soluble in water, and has the a d v a n t a g e t h a t its Slp r e s o n a n c e lies s u f f i c i e n t l y downfield f r o m p h o s p h a t e m e t a b o l i t e s so t h a t it d o e s not o b s c u r e the resonances of i n t e r e s t . H o w e v e r , for c o n v e n i e n c e c h e m i c a l shifts are quoted r e l a t i v e to t r i m e t h y l p h o s p h a t e . C h e m i c a l s h i f t s of p a r t i a l l y o v e r l a p p e d Pi r e s o n a n c e s of m i c r o organisms w e r e o b t a i n e d by c u r v e fitting using t h e N i c o l e t s u p p l i e d N T C C A P S u b r o u t i n e i m p l e m e n t e d on the NIC 1180 c o m p u t e r . The e r r o r in the c h e m i c a l shift d e t e r m i n a t i o n by this m e t h o d depends upon t h e a m o u n t of noise present in the s p e c t r u m but in no case was ~t g r e a t e r than +3 Hz. This would give a m a x i m u m e r r o r in t h e pH d e t e r m i n a t i o n - o f -J 20 0 Q_
0
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TI/~E (HRS) Fig. 2. Inorganic polyphosphate accumulation in resting E. coli cells subjected to sulfur-starvation. The polyphosphate content was determined by integration of the resonances in Fig. 1 and is e x p r e s s e d relative to the value after 22.3 h taken as i00.
r e s o n a n c e f r o m t h e i n t e r n a l o r t h o p h o s p h a t e ( P i n ) a l m o s t does not change. These results a r e e s s e n t i a l l y in c o n f o r m i t y w i t h t h o s e of Navon e e a.Z., (1979), e x c e p t t h a t our use of the gel allowed us to m o n i t o r m e t a b o l i c c h a n g e s in y e a s t over a m u c h longer t i m e . In a n o t h e r s e t of e x p e r i m e n t s y e a s t cells w e r e h a r v e s t e d in the very e a r l y e x p o n e n t i a l phase of growth. This leads to the u n e x p e c t e d observation t h a t t h e r e is no p o l y p h o s p h a t e p r e s e n t initially in t h e s e cells ( f r o m the a b s e n c e of r e s o n a n c e s at -10.5 and -27 ppm in Fig. 4A). This is c o n s i s t e n t , h o w e v e r , with the n u m e r o u s o b s e r v a t i o n s t h a t p o l y p h o s p h a t e is not a n o r m a l c o n s t i t u e n t of m i c r o o r g a n i s m % b u t is rapidly a c c u m u l a t e d by t h e m under conditions of n u t r i t i o n a l i m b a l a n c e or in aged c u l t u r e s ( H a r o l d , t 9 6 6 ) . The a c c u m u l a t i o n oi p o l y p h o s p h a t e u n d e r the conditions of the NMR e x p e r i m e n t (Fig. 4) r e a c h e s a b o u t 50% of the t o t a l cellular p h o s p h a t e c o n t e n t , w h i l e f o r c o m parison this value is a b o u t 60% in y e a s t grown in l i q u i d c u l t u r e and h a r v e s t e d in m i d - to l a t e - e x p o n e n t i a l phase ( 3 a c o b s o n , 1979). In order to d e t e r m i n e t h e i n t r a c e l l u l a r pH f r o m the c h e m i c a l shift of t h e Pi r e s o n a n c e it is n e c e s s a r y to use an a p p r o p r i a t e t i t r a t i o n curve ior calibration. We h a v e p r e v i o u s l y shown ( P o l l a r d el: a 2 . , 1979) t h a t t h e NMR t i t r a t i o n c u r v e ( 8 - p H ) of t h e y - p h o s p h a t e of ATP in f r e e solution c a n n o t be used as an a d e q u a t e c a l i b r a t i o n c u r v e for i n t r a g r a n u l a r pH m e a s u r e m e n t s , p a r t l y due to the l a r g e e f f e c t of Mg 2+ on this t i t r a t i o n curve. It was n e c e s s a r y to t i t r a t e the i n t r a g r a n u l a r ATP to obtain an a d e q u a t e c a l i b r a t i o n c u r v e ( P o l l a r d e t a 2 . , 1979), a n d a s i m i l a r t i t r a t i o n of i n t e r n a l Pi has been c a r r i e d out in m i t o chondria ( O g a w a ee a 2 . , 197gb). D e s p i t e s o m e a s s e r t i o n s to t h e
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Fig. 3. 31p NMR spectra of yeast (S. cerevisiae) from the late exponential phase of growth. (A) Packed Cells (approx. 108 cells/ml) in the mineral salt solution. (B-D) Cells embedded in 0.25% agarose (approx. 10 7 c e l l s / m l ) as described in 'Methods'. Spectra were recorded at 30~ after 15 min (B), 1 h (C), and 5 h (D) from the onset of oxygen bubbling. Assignments are: SP, sugar phosphates, with a possible contribution from nucleotide monophosphates; Pex, orthophosphate in the external medium; Pin, intracellular orthophosphate; EP, end-phosphates of short-chain polyphosphates; NPP~ a broad region around -15 ppm which includes resonances from such P~P'-pyrophosphate diesters as NAD/NADH, ADP-glucose~ etc; MPI~ middle phosphates of short-chain polyphosphates; MP2, middle phosphates of long-chain polyphosphates.
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F i g , 4, 31p NMR s p e c t r a o f yeast (S. cerevisiae) from t h e e a r l y e x p o n e n t i a l phase o f g r o w t h , Cells were embedded i n agarose at approx, 107 e e l l s / m l a.~ described in 'Methods' S p e c t r a were r e c o r d e d at 30~ from I0 min (A), 2 h (B), 3 h (C), and 4.5 h (D) after the onset of the oxygen bubbling. The spectral a s s i g n m e n t s are d e s c r i b e d in the legend to Fig. 3 with the exception of the resonance at 19 ppm~ which is of t e t r a e t h y l m e t h y l e n e d i p h o s p h o n a t e added as an internal standard.
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pH Fig. 5. 31p N M R - p H titration of I0 orthophosphate in 0.1 M NaCI either alone presence of 3 mM MgCI2 (1:3 Mg:P) (+), (1:2 Mg:P) (*), or 15 mM MgCI 2 (3:2 Mg:P)
mM sodium (x), or in 5 mM MgCI 2 (O).
contrary (Burt et a l . , 1979), Mg~+ and other divalent metal ions are known to complex with orthophosphate (Tabor & Hastings) 1943), and do have a measurable effect upon the 8-pH titration curve (3acobson & Cohen, i981). For example, in Fig. 5 are shown r e p r e s e n t a t i v e titration curves of Pi in the presence of increasing amounts of Mg2+. It is clear that both the apparent pK and the chemical s h i f t are affected. This latter effect presumably arises from the shielding of the phosphorus nucleus in the Mg2+ complex relative to t h a t in the free phosphate ion. The 8-pit t i t r a t i o n curves in Fig. 5 clearly demonstrate the ambiguity in the measurement of i n t r a c e l l u l a r pH from the measured Pi chemical shift, since between pH 5 and 7.5 this can lead to an error of as much as 0.5 pH unit, depending upon the degree of Mg2+ -Pi compJexation. In determining the intracellular pH value from the chemical shift of Pi in the yeast experiments described in Fig. 3, the simple (without Mg2+) titration curve of Pi was used (Fig. 5, solid line), as has been the case previously (Ogawa ee a l . , 1978a; Ugurbil e t a l . , 197g; Cohen et a i . , 1978). Our particular choice of a calibration curve is not, however, completely arbitrary. The fact that the position of the intracellular Pi peak is almost constant strongiy suggests that there is no appreciable change in the availability of Mg 2+ in this case. In fact, most Mg2+ in mature yeast cells is either strongly bound to the soluble polyphosphate or immobilized in the volutin granules (3acobson, 1979; 3acobson et a l . , 1981). By computer deconvolution of the two
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p a r t i a l l y o v e r l a p p e d Pi peaks in Fig. 3 it was possible to d e t e r m i n e t h e i r c h e m i c a l shifts and to c a l u l a t e c h a n g e s in ApH (pHin - p H e x ) o v e r a p e r i o d of s e v e r a l h o u r s . T h e r e s u l t s s h o w t h a t while the i n t r a c e l l u l a r pH did not c h a n g e a p p r e c i a b l y f r o m its value of 6.9, t h e e x t e r n a l pH d e c r e a s e d f r o m 7.1 (A = -0.2) b e f o r e glucose addition and oxygen bubbling, to pH 6.8 (A = 0.1) a f t e r 5 h incubation. The f a c t that the acidification of t h e e x t e r n a l m e d i u m was less than t h a t o b s e r v e d by Navon e t a l . (1979) is e v i d e n t l y due to the much thinner c e l l s u s p e n s i o n and the additional b u f f e r r e s e r v o i r a b o v e the a g a r o s e block in our e x p e r i m e n t s . The d e g r e e of u n c e r t a i n t y of the i n t r a c e l l u l a r pH d e t e r m i n a t i o n is d e m o n s t r a t e d by the b e h a v i o r of the internal and e x t e r n a l Pi r e s o n a n c e s in the e x p e r i m e n t r e p r e s e n t e d in Fig. #. While the position of t h e Pex is shifting upfield, indicating the e x p e c t e d a c i d i f i c a t i o n , t h e r e s o n a n c e of the Pin also m o v e s , but in the o p p o s i t e direction. While one c a n n o t rule out with c e r t a i n t y t h e p o s s i b i l i t y t h a t t h e c y t o s o l a c t u a l l y b e c o m e s m o r e basic, an a l t e r n a t i v e , m o r e plausible, e x p l a n ation is t h a t the a c c u m u l a t i o n of p o l y p h o s p h a t e r e m o v e s f r e e Mg 2+ f r o m t h e c y t o s o l , w h e r e it w o u l d o t h e r w i s e c o m p l e x to Pi. This, a c c o r d i n g to the t i t r a t i o n c u r v e s in Fig. 5, would lead to an a p p a r e n t a l k a l i n i z a t i o n of the cytosol. Conclusion The use of a g a r o s e gel as a m e d i u m for the i n v e s t i g a t i o n of cell m e t a b o l i s m by 31p NMR s p e c t r o s c o p y is described. It should e n a b l e an e x t e n s i o n of t h i s t e c h n i q u e to studies of s m a l l e r c o n c e n t r a t i o n s of cells and to l a r g e r m a m m a l i a n cells which are more sensitive to e n v i r o n m e n t a l conditions. Such f a c t o r s as the i-elative c o n c e n t r a t i o n s of divalent m e t a l ions and i o n - c o m p l e x i n g m e t a b o l i t e s m u s t be t a k e n i n t o a c c o u n t in a c c u r a t e l y d e t e r m i n i n g the i n t r a c e l l u l a r pH by NMR methods. References Burt CT, Cohen SM, & Barany M (1979) Ann. Rev. Biophys. Bioeng. 8, 1-25. Cohen SM, Ogawa S, Rottenberg H, Glynn P, Yamane T, Brown TR & Shulman RG (1978) Nature 273, 554-556. Harold FM (1966) Bacteriol. Rev. 30, 772-794. Jacobson L (1979) Ph.D. Thesis, Weizmann Institute, Israel. Jaeobson L & Cohen JS (1981) in Non-Invasive Probes of Tissue Metabolism, Cohen JS (ed.), Wiley, New York, in press. Jacobson L, Halmann M, & Yariv J (1981) Biochem. J. in press. Moon RB & Richards JH (1973) J. Biol. Chem. 248, 7276-7278. Navon G, Ogawa S, Shulman RG, & Yamane T (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 888-891. Navon G, Shulman RG, Yamane T, Eccleshall TR, Lam KB, Baronofsky JJ, & Marmur J (1979) Biochemistry 18, 4487-4499. Ogawa S, Shulman RG, Glynn P, Yamane T, & Navon G (1978a) Biochim. Biophys. Acta 502, 45-50. Ogawa S, Rottenberg H, Brown TR, Shulman RG, Castillo CL, & Glynn P (1978b) Proc. Natl. Acad. Sci. U.S.A. 75, 1796-1800.
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Pollard H, Shindo H~ Creutz CE 9 Pazoles CJ~ & Cohen JS (1979) J. Biol. Chem. 254, 1170-1179. Roberts JKM~ Ray PM~ Wade-Jardetzky N, & Jardetzky O (1980) Nature 283, 870-872. Salhany JM, Yamane T, Shulman RG, & Ogawa S (1975) Proc. Natl. Ncad. Sci. U.S.A. 72, 4966-4970. Tabor H & Hastings AB (1943) J. Bio2. Chem. 148, 627-632. Ugurbil K, Rottenberg H, Glynn P, & Shulman RG (1978) Proc. Natl. Ncad. Sci. U.S.A. 75, 2244-2248.
