Sodium-dependent succinate uptake in purple bacterium Ectothiorhodospira shaposhnikovii

91

Biochimica et Biophysica Acta, 598 (1980) 91--99 © Elsevier/North-Holland Biomedical Press

BBA 78682

SODIUM-DEPENDENT SUCCINATE UPTAKE IN PURPLE BACTERIUM ECTOTHIORHODOSPIRA SHAPOSHNIKO VII

V.V. KARZANOV and R.N. IVANOVSKY

Department of Microbiology, Moscow State University, Moscow 117234 (U.S.S.R.) (Received March 23rd, 1979) (Revised manuscript received September 11th, 1979)

Key words: Succinate transport; Na÷; Symport; Artificial gradient; (E. shaposhnikovii)

Summary Succinate, malate and fumarate uptake in purple sulfur bacterium Ectothiorhodospira shaposhnikovii, strain 1 K MSU, obligatorily depends on the presence of Na*. Other monovalent cations such as K+, Li÷, NH~ could not replace Na ÷. Experiments with energy
Introduction According to Mitchell's chemiosmotic theory [1--3] electrochemical potential gradients of cations play a major role in energization of uphill transport processes in living cells. Most possible cosubstrates for symport are H÷ and Na ÷ as they are actively extruded by cells by appropriate mechanisms thus creating inwardly directed ApH and ApNa. There are many examples of solutes symport with protons in bacteria (for review see Ref. 4). It was shown, for instance, that succinate, malate and fumarate transport in Escherichia coli is carried out in symport with protons down ApH [5]. In eukaryotic cells sodium gradients formed by (Na÷ + K÷)-ATPase are the driving force for accumulation of different substrates via symport systems Abbreviation: CCCP,carbonyl

cyanide

m-chlorophenylhydrazone.

92 Na÷/substrate [6,7]. Lately the role of Na ÷ in bacterial transport has been studied extensively by many investigators. Frank and Hopkins [8] demonstrated that the presence of Na ÷ is required for glutamate uptake in E. coli. B. Halpern et al. [9] reported that the presence of K ÷ and Na ÷ was required for active transport of glutamate in E. coli K-12. Stevenson [10] found that glutamate uptake b y Halobacterium salinarium was stimulated b y the addition of NaC1. Recent studies of membrane vesicles of Halobacterium halobium have shown that Na ÷ gradients are involved in amino acid transport via symport [11--13]. Electrochemical gradient of Na ÷ is also the driving force for glutamate accumulation in energy
93 Methods

The cultures of E. shaposhnikovii, strain 1 K, from the collection of Microbiology department of Moscow State University were grown in Larsen medium [34] plus 0.5% NaHCO3, 0.1% NaC1 and 0.2% sodium succinate. The medium was adjusted to pH 8.0 with H3PO4. Glass flasks were completely filled with the medium to create anaerobic conditions. The cultures were incubated at 30°C for 40--42 h under two 60-W incandescence lamps. Cells were harvested by centrifugation at 4°C for 15 min at 8000 × g and washed twice with the experimental medium. In the experiments with succinate uptake energized b y artificial ApNa and A ~ , cells were energy depleted by 10 #M CCCP in the argon atmosphere for 1 h in the darkness. All experiments with energy~lepleted cells were performed in glass thermostated vessels wrapped up in light-proof paper. To create anaerobic conditions argon bubbling was conducted through the reaction medium prior to the start of the uptake experiments and during the experiments as well. The reaction medium in the vessel was continuously stirred by a magnetic stirrer. To create ApNa energy
94 In all experiments the samples of the cell suspension (1 ml) were withdrawn at the appropriate periods of time after the start of the uptake and were filtered through membrane filters (pore size 0.4 ~m). Then the filters were washed once with 5 ml of reaction medium, dried and counted for radioactivity in Mark-II (Nuclear~hicago) liquid scintillation spectrometer. Protein was measured as described by Lowry et al. [35] using bovine serum albumin as a standard. In all the assays the temperature was set at 35°C. The details of methods used are explained in legends to the figures. Chemicals. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), valinomycin and Tris were obtained from Sigma, U.S.A. Tetra-n-butylammonium hydroxide was purchased from Chemapol, Czechoslovakia. [ 2,3-14C] Succinate (1.5 Ci/mol) was purchased from Sojuzizotop, U.S.S.R. All other chemicals were reagent grade and obtained from commercial sources. Results Preliminary experiments have shown that light-induced uptake of succinate, malate and fumarate in E. shaposhnihovii at substrate concentrations below 100 /~M strongly depends on the presence of Na ÷ in reaction mixture. Na* cannot be replaced by other monovalent cations such as K ÷, Li +, NH~ or divalent cations such as Mg 2+, Mn :+, Ca:*. The uptake of succinate at maximum rate needs relatively high (more than 50 mM) NaC1 concentrations (Fig. 1). The results of the experiments with energy
95

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Fig. 1. L i g h t - i n d u c e d s u c c i n a t e u p t a k e in E. shaposhnikovii as a f u n c t i o n of NaC1 c o n c e n t r a t i o n at p H 7.5. Fig, 2. S u c c i n a t e u p t a k e e n e r g i z e d b y s o d i u m g r a d i e n t s a t p H 7.5. Cells w e r e e n e r g y d e p l e t e d b y 10 ~tM CCCP as d e s c r i b e d u n d e r M e t h o d s . e , 1 m M NaCI a n d 4 9 9 m M KCI i n t r a c e l l u l a r ; 5 0 0 m M NaCI e x t r a cellular, c r e a t i n g a LkpNa o f 5 0 0 : 1. m, 10 m M NaCI a n d 4 9 0 m M KCI i n t r a c e l l u l a r ; 5 0 0 m M NaCI e x t r a cellular, c r e a t i n g a A p N a o f 50 : 1. A 50 m M NaCI a n d 4 5 0 m M KCI i n t r a c e l l u l a r ; 5 0 0 m M NaCl e x t r a cellular, c r e a t i n g a A p N a o f 1 0 : 1. o, 5 0 0 m M NaCl i n t r a - a n d e x t r a c e l l u l a r ( c o n t r o l ) .

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Fig. 3. R o l e of m e m b r a n e p o t e n t i a l in s u c c i n a t e t r a n s p o r t e n e r g i z e d b y artificial s o d i u m gTadient ~ p N a a t d i f f e r e n t p H values. Cells w e r e e n e r g y d e p l e t e d b y 10 ~M CCCP a n d A ~ (inside n e g a t i v e ) w a s i m p o s e d as a K + d i f f u s i o n p o t e n t i a l as d e s c r i b e d u n d e r M e t h o d s . e , 10 m M NaCI a n d 4 9 0 m M KCI i n t r a e e l l u l a r ; 5 0 0 m M NaCI e x t r a c e l l u l a r , c r e a t i n g ~ p N a . a 10 m M NaCI, 4 9 0 m M KCI a n d 20/~M v a l i n o m y c i n intracellular; 5 0 0 m M NaCI a n d 2 0 /~M v a l i n o m y c i n e x t r a c e l l u l a r , c r e a t i n g ~ p N a a n d ~ . m, 5 0 0 m M NaCI intra- and extracellular (control).

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97 Light-induced succinate uptake is inhibited b y CCCP, a well-known proton c o n d u c t o r which dissipates both ApH and A~ produced b y A p H ÷ generators (Fig. 5). CCCP inhibits succinate uptake both at pH 7.0 and at pH 9.0. At the same time lipid-soluble tetra-n-butylammonium cation inhibits light-induced succinate uptake at pH 9.0 b u t increases accumulation rate b y 40% at pH 7.0. Such a difference in tetra-n-butylammonium action at different values of pH is all the more interesting because the rate of light-induced succinate uptake at pH 9.0 is much higher than that at pH 7.0 (Fig. 5). Discussion Two possible mechanisms for Na + stimulation of succinate transport were considered: (1) Na ÷ is a 'cofactor' in the activation of the transport system which involves ApH
98 (positively or negatively), because the molecular structure and actual charge of binding groups for succinate and sodium are unknown. A considerable difference in accumulation level of succinate uptake at pH 7.0 and 9.0 was rather difficult to explain when ApNa was the only driving force of the uptake (Fig. 3). The accumulation level of succinate down ApNa at pH 7.0 exceeds the level at pH 9.0 3-fold though the rate of light-induced uptake of succinate at pH 9.0 considerably exceeds the rate at pH 7.0 (Fig. 5). This discrepancy in the findings may be explained if we suggest that dissipation rate of the Na ÷ gradient increases at higher pH value (9.0). Perhaps this could be a reflection of the higher numbers of Na ÷ cotransported with succinate at pH 9.0 compared with pH 7.0. An increase in stoichiometry of Na÷/ substrate symport at higher pH for methyl-fl-D-thiogalactoside transport in S. typhimurium was supposed by Tokuda and Kaback [17]. The similar increase in stoichiometry for symport of neutral substrates and anions with protons in E. coli was shown by Ramos and Kaback [37]. The data presented in Fig. 5 indicate that A~ (inside negative) is not required for succinate uptake at pH 7.0 but is necessary for the uptake at pH 9.0. The precise point of tetra-n-butylammonium action on the uptake (transport or creation of ApNa) is not possible to be detected from these results. The method of tetra-n-butylammonium probe within wide range of pH values can be used, nevertheless, for the approximate statement of the pH regions where the uptake depends or does not depend on A~. Acknowledgements We thank Dr. A.N. Glagolev for useful suggestions during the course of the study, Professor V.P. Skulachev and Professor E.N. Kondratieva for helpful criticism during the preparation of the manuscript. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

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