Vanadate-sensitive proton efflux by filamentous cyanobacteria

FEMS MicrobiologyLetters 22 (1984) 215-218 Published by Elsevier

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FEM 01743

Vanadate-sensitive proton efflux by filamentous cyanobacteria (Blue-green algae; ATP-hydrolase (H + -ATPase); cytop 1asmic membrane)

S. Scherer a n d P. B6ger * Lehrstuhlfftr Physiologieund Biochemieder Pflanzen, Universitiit Konstanz, D-7750 Konstanz, F.R.G.

Received 31 October 1983 Revision received 28 December 1983 Accepted 10 January 1984

1. S U M M A R Y

3. M A T E R I A L S A N D M E T H O D S

Light-induced proton efflux has been investigated with intact cells of Anabaena, Nostoc, Anacystis, and Aphanocapsa. The proton efflux by filamentous blue-green algae is biphasic, strongly inhibited by ortho-vanadate and insensitive to cyanide. These data are taken as evidence for a proton-pumping ATP-hydrolase present on the cytoplasmic membrane of Anabaena and Nostoc.

Anabaena variabilis Ki~tz. (American Type Culture Collection, ATCC29413) and Nostoc muscorum (Pasteur Culture Collection, PCC7119 = Anabaena, ATCC29151) were grown under nitrogen-fixing conditions, whereas the growth medium of Aphanocapsa (PCC6714 = ATCC27178) and Anacystis nidulans (Algae Culture Collection GOttingen, B 1402-1 = Synechococcus ATCC27144) was supplemented with N O 3. The organisms were cultivated as described previously [5], harvested after 1 to 3 days of growth (middle to late logarithmic phase), washed once in reaction buffer (3 mM glycylglycine; 5 m M MgCI2; 75 mM KC1; 75 mM NaC1, p H adjusted to 6.3) and stored on ice at a chlorophyll (Chl) concentration of 600 /~g/ml of suspension. Oxygen exchange was determined as described [6], proton efflux was measured in a 3-ml reaction chamber according to Mitchell [7], with a pH-electrode (GK2321C, Radiometer, Copenhagen) connected to a standard p H meter (PHM62, Radiometer, Copenhagen, Denmark). The algal suspensions (30 ~g C h l / m l ) were incubated aerobically in the reaction chamber for 5 to 10 min in the dark at 24°C and p H 6.3 _+ 0.05. Then, light was turned on (saturating red light, defined by an RG610 cut-off filter, Schott, Mainz, F.R.G,). The change

2. I N T R O D U C T I O N Proton-pumping ATP-hydrolases different from the coupling factor ATPase have been discovered in various organisms, especially in plant and fungal cytoplasmic (plasmalemma) membranes (for review see [1]). Sensitivity to ortho-vanadate is a significant feature of c y t o p l a s m i c - m e m b r a n e ATPases [2,3]. To our knowledge, there is only one report in the literature on a proton-pumping, vandate-sensitive ATPase in bacteria [4]; vanadate-sensitive H ÷-efflux has not been reported in cyanobacteria as yet. In this communication, data are given indicative of such an enzyme present in filamentous cyanobacteria. * To whom correspondence should be addressed.

0378-1097/84/$03.00 © 1984 Federation of European MicrobiologicalSocieties

216

in p H was calibrated by adding solutions of 50 mM HC1 and 50 m M N a O H after termination of the experiment. Both oxygen exchange and p H were recorded simultaneously with a two-channel recorder. During the assay, additions to the reaction medium were introduced by /~l-syringes (Hamilton) through a small hole in the screw-cap closing the reaction chamber. Ortho-vanadate (Na3VO4) was purchased from Ventron (Karlsruhe, F.R.G.; No.81104); for other chemicals see references given.

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A light-induced proton efflux has been reported first by Scholes et al. [8] for Anabaena variabilis. However, the mechanism of this process has not been investigated in detail so far. Fig. 1 shows the results of light-driven H+-efflux studies with two filamentous cyanobacteria. In Anabaena and Nostoc, H +-efflux exhibited a biphasic time course, the first phase being faster than the second one. Proton efflux started immediately after illumination (lag phase 1 - 3 s), whereas oxygen evolution started after 10 s and reached a constant rate after 40 to 60 s. Proton efflux by these species was strongly inhibited by ortho-vanadate, the first phase

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Fig. 1. Light-induced proton efflux by Anabaena uariabilis and Nostoc muscorum, followed by alkalinization of the medium. The data given are rates in/L tool H + x mg Chl i x h - 1, taking into consideration the buffer capacity of 1 m M vanadate, which is given by the calibration bars. The first (I) and second (II) phases of proton efflux are marked. Arrows: light on; upward deflection: proton efflux; downward deflection: alkalinization.

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Fig. 2. Vanadate sensitivity of the first (I) and second (II) phase of light-induced proton efflux by Anabaena variabilis (cf. Fig. 1). Vanadate was added at the beginning of incubation in the dark. The data represent means from 7 experiments. 100% corresponds to 45.5+10.8 for the first and 16+2.9 #mol H + x mg C h l - 1 x h - 1 for the second rate of proton efflux.

being less sensitive to the inhibitor than the second one, as shown in more detail for A nabaena variabilis (Fig. 2). To inhibit proton efflux, vanadate concentrations up to 1 m M were needed. The Ca 2+ATPase [9-11] or proton-pumping ATP-hydrolase [4] of other prokaryotes were found to be more sensitive towards vanadate. This is probably due to the fact that in our study intact cells were used, whereas the data in the literature cited have been obtained with isolated membranes. In contrast to the filamentous species investigated, the light-induced proton efflux by the coccoid Anacystis and Aphanocapsa had only one phase and was barely affected by ortho-vanadate. Furthermore, the kinetics of proton efflux turned out to be clearly different between Aphanocapsa

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Oxygen evolution (% of control) Fig. 3. Correlation of oxygen evolution and alkalinization of the medium, following the light-induced proton efflux by Anabaena variabilis (cf. Fig. 1). 100% corresponds to 126 + 18.4 /~mol O 2 × m g C h l - l × h 1 and 69+11.9 /~mol O H - × m g C h l - 1 × h - 1. Inhibition of oxygen evolution and alkalinization was achieved by various concentrations of cyanide (A) and D C M U (I).

and Anacystis, the proton efflux in the former being faster than in the latter. In all species investigated, the light-induced acidification was followed by an alkalinization of the medium (Fig. 1), which exhibited a linear correlation with oxygen evolution over a wide range (Fig. 3). This is in accordance with the suggestion that light-induced alkalinization (by blue-green algae [12] and submersed plants [13]) is due to an O H - efflux caused by regulation of intracellular pH. Bicarbonate is taken up and converted to CO 2 plus O H - inside the cell, O H being extruded while CO 2 is assimilated. Light-induced proton efflux by Plectonema boryanum has only one phase and is thought to be due to redox-coupled H ÷ pumping by a cytoplasmic membrane-bound respiratory chain. Unfortunately, vanadate and cyanide sensitivity were not investigated [14]. In contrast to respiratory oxygen uptake, light-induced proton efflux by the 4 species assayed here was stimulated by 1 mM

potassium cyanide, indicating that efflux is not due to electron transport driven by a respiratory chain located on the cytoplasmic membrane. The reason for this stimulation points to regulatory features of the ATPase which is under investigation (Scherer and B6ger, unpublished). While inhibition of H+-efflux by vanadate is evident (Fig. 1), neither electron transport nor ATP synthesis in the light or in the dark (experiments done with Anabaena; not shown) were affected, as determined enzymatically by the luciferin/luciferase method (cf. [15]). However, the mechanism of the vanadate-insensitive part of proton efflux remains to be elucidated. Previously, we presented indirect evidence for a proton-translocating ATP-hydrolase, active in the dark, to be located on the cytoplasmic membrane of Anabaena oariabilis [15]. The data presented in this study, together with the well-known sensitivity of several types of plasmalemma ATPases of higher plants to ortho-vanadate have to be taken as additional evidence for a proton-translocating ATP-hydrolase active on the cytoplasmic membrane of Anabaena and Nostoc. Furthermore, our data obtained with unicellular blue-green algae indicate that the mechanisms of proton translocation across the cytoplasmic membrane may be different among blue-green algal species. Whether vanadate sensitivity of proton efflux has any systematic significance is under investigation in our laboratory.

ACKNOWLEDGMENT This study was supported by the Deutsche Forschungsgemeinschaft (Bo 310/12-4). We thank Regina Grimm for skilful technical assistance.

REFERENCES [1] Spanswick, R.M. (1981) Annu. Rev. Plant Physiol. 32, 267-289. [2] Macara, 1.G. (1980) Trends Biochem. Sci. 5, 92-94. [3] Goffeau, A. and Slayman, C.W. (1981) Biochim. Biophys. Acta 639, 197-223. [4] Yoshimura, F. and Brodie, A.F. (1981) J. Biol. Chem. 256, 12239-12242.

218 [5] Scherer, S. and B6ger, P. (1982) Arch. Microbiol. 132, 329-332. [6] Scherer, S., Kerfin, W. and B6ger, P. (1980) J. Bacteriol. 144, 1017-1023. [7] Mitchell, P. and Moyle, J. (1967) J. Biochem. 104, 588-600. [8] Scholes, P., Mitchell, P. and Moyle, J. (1969) Eur. J. Biochem. 8, 450-454. [9] Lockau, W. and Pfeffer, S. (1982) Z. Naturforsch. 37c, 658-664. [10] Lockau, W. and Pfeffer, S. (1983) Biochim. Biophys. Acta 733, 124-132.

[11] Kobayashi, H., Van Brunt, J. and Harold, R.M. (1978) J. Biol. Chem. 253, 2085-2092. [12] Kaplan, A. (1981) Plant Physiol. 67, 201-204. [13] Lucas, W.3". (1983) Annu. Rev. Plant Physiol. 34, 71-104. [14] Hawkesford, M.J., Rowell, P. and Stewart, W.D.P. (1983) in Photosynthetic Prokaryotes: Cell Differentiation and Function (Papageorgiou, G.C. and Packer, L., Eds.), pp. 199-218, Elsevier, Amsterdam. [15] Scherer, S., St~rzl, E. and B6ger P . (1984) J. Bacteriol., in press