Isolated and purified plasma and thylakoid membranes of the cyanobacteriumAnacystis nidulans contain immunologically cross-reactive aa3-type cytochrome oxidase

Cell-free extracts ofAnacystis nidulans were fractionated by discontinuous sucrose density gradient centrifugation resulting in the separation of two distinct types of membranes, the heavier one containing the chlorophyll and the lighter one devoid of chlorophyll. Identity of the latter with plasma membrane was confirmed by labeling of intact cells with impermeant marker,³⁵S-diazobenzenesulfonate, prior to cell disruption. Both membrane fractions were purified individually by repeated recentrifugation on identical gradients. Purified membranes were subjected to dissociating polyacrylamide gel electrophoresis, either type of membranes yielding a distinct polypeptide pattern. After transfer of the polypeptides to nitrocellulose by Western blotting, two of the proteins, with molecular weights of approximately 55,000 and 32,000, respectively, gave strong and specifically complementary cross-reactions with antibodies raised against subunits I and II of the aa₃-type cytochrome oxidase fromParacoccus denitrificans. The findings will be discussed in terms of the presence of aa₃-type cytochrome oxidase in both plasma and thylakoid membranes ofAnacystis nidulans.     

Light-independent NADPH-protochlorophyllide oxidoreductase activity in purified plasma membrane from the cyanobacterium Anacystis nidulans

A light plasma membrane fraction corresponding to a buoyant density of 1.087 +/- 0.005 g/cm3 and devoid of chlorophyll was prepared and purified from Anacystis nidulans according to a recently published procedure (G.A.Peschek, V.Molitor, M.Trnka, M.Wastyn and W.Erber (1988) Methods Enzymol. 167, 437-449). Besides major amounts of carotenoids the plasma membranes contained a small but significant pool of chlorophyllide a and protochlorophyllide a as verified by room temperature and 77K spectrofluorimetry and analytical separation and identification by high performance liquid chromatography using authentic standards. Incubation of the plasma membranes in strict darkness in the presence of NADPH was accompanied by the gradual and stoichiometric replacement of protochlorophyllide by chlorophyllide, NADP+ effecting the reverse transition. The reaction was completely insensitive to illumination (5-20 w/m2 tungsten light) but abolished after heating of the membranes (90 degrees C, 5 min) or in the presence of 10 mM EGTA, and was specifically stimulated by calcium ions. Our results indicate the occurrence of light-independent NADPH:protochlorophyllide oxidoreductase activity in the plasma membrane of Anacystis nidulans.     

Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans

Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [35S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min-1 (mg of protein)-1, respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa3-type enzyme from the properties of its redox-active and EDTA-resistant Cu2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa3-type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa3-type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme (EC 1.9.3.1).     

Impact of salt adaptation on esterified fatty acids and cytochrome oxidase in plasma and thylakoid membranes from the cyanobacterium Anacystis nidulans

During adaptation of photoautotrophically growing fresh water cyanobacterium Anacystis nidulans to high salinity the cells showed a pronounced increase of proton-sodium antiporter activity, and of cytochrome c oxidase in isolated and purified plasma membrane. At the same time the concentrations of plasma membrane-bound EDTA-resistant copper and iron (determined by inductively coupled plasma atomic emission spectrometry) rose proportionately, accompanied by an increase in whole cell respiration. In plasma membranes from salt adapted cells lipid/protein ratios were markedly higher than in control cells, levels of esterified saturated and long-chain fatty acids being significantly higher than the respective levels of unsaturated and short-chain fatty acids which explains the higher lipid-phase transition temperatures derived from Arrhenius plots. Immunoblotting of the membrane proteins with antisera raised against the cytochrome c oxidases from Paracoccus denitrificans and A. nidulans gave two cross-reacting bands with apparent molecular weights around 50000 and 30000 (subunits I and II, respectively) which were more pronounced in plasma membranes from salt adapted cells when compared to control cells. The protein pattern of plasma membranes from salt adapted cells also showed the appearance of bands at apparent molecular weights of 44000–48000 and 54000–56000 which might stem from the proton/sodium-antiporter in this membrane.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - cyt cytochrome - DCCD N,N-dicyclohexylcarbodiimide - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - ICP-AES inductively coupled plasma atomic emission spectrometry - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - EPR electron paramagnetic resonance spectrometry     

Proton pump coupled to cytochrome c oxidase in the cyanobacterium Anacystis nidulans

Intact spheroplasts of the cyanobacterium Anacystis nidulans were found to oxidize various exogenous c-type cytochromes with concomitant proton extrusion. In the coupled state, H+/e stoichiometries close to 1 were measured, regardless of absolute reaction rates. It is concluded that the proton translocation observed is an intrinsic property of the cytoplasmic membrane-bound cytochrome c oxidase of A. nidulans.     

Evidence for plastoquinol-cytochrome f/b-563 reductase as a common electron donor to P700 and cytochrome oxidase in cyanobacteria

Membranes isolated from $\text{Nostoc}$ sp. strain MAC and $\text{Anacystis}\text{nidulans}$ displayed spectral changes in the cytochrome $\text{f}\text{b}$ region when examined by reduced $\text{minus}$ oxidized or dual wavelength spectrophotometry under physiological conditions. The same changes accompanied both light-induced (photosynthetic) and oxygen-induced (respiratory) electron transport. Physiological reduction of the cytochrome $\text{f}\text{b}$ moiety was abolished after extraction of plastoquinone but reappeared on reconstitution of the depleted membranes with authentic plastoquinone. Moreover, a mutual inhibition of photosynthetic and respiratory activities could be directly demonstrated with the isolated membranes. From the results it is concluded that the membrane-bound plastoquinol-cytochrome $\text{f}\text{b}$ reductase functions as a common electron donor to both P700 and the cytochrome oxidase in cyanobacteria.     

Changes of some physical properties of isolated and purified plasma and thylakoid membrane vesicles from the freshwater cyanobacterium Synechococcus 6301 (Anacystis nidulans) during adaptation to salinity

Photoautotrophically growing cultures of the freshwater cyanobacterium Anacystis nidulans (Synechococcus sp.) became adapted to the presence of 0.4-0.5 M NaCl in the growth medium (about seawater level) with a lag phase of 2 days after which time the growth rate resumed at 80-90% of the control. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. Plasma and thylakoid membranes were separated from crude cell-free extracts of French pressure cell-treated Anacystis by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Concentrations of copper, iron, calcium, and magnesium ions were determined by inductively coupled plasma atomic emission spectrometry with EDTA-washed and dialyzed membrane preparations; salt adaptation was found to increase (decrease) the concentration of membrane-bound calcium in plasma (thylakoid) membranes, qualitatively reciprocal results being obtained for magnesium. Levels of plasma membrane-bound copper and iron roughly tripled during the adaptation process; by contrast, corresponding effects on thylakoid membranes were negligible. The size of the membrane vesicles was measured by quasi-elastic laser light-scattering and the electric surface charge of the membranes was measured by laser Doppler velocimetry. Salt adaptation decreased the mean diameter of plasma membrane vesicles to a much higher extent than that of thylakoid membrane vesicles. Overall surface charge densities of resting vesicles were only slightly affected by the salt treatment as was also seen from titration of the electrophoretic mobility of the vesicles with electrolytes. Yet, induction of (photosynthetic or respiratory) electron transport provoked a charge separation across the membrane which was easily measurable in terms of electrophoretic mobility. The results will be discussed with particular emphasis on the stimulated cytochrome c oxidase activity of plasma (but not thylakoid) membranes from salt-adapted cells compared to control cells and also with respect to the decreased ion permeability of the plasma membrane of salt grown cells.     

Exogenous energy supply to the plasma membrane of dark anaerobic cyanobacterium Anacystis nidulans: thermodynamic and kinetic characterization of the ATP synthesis effected by an artificial proton motive force

The net synthesis of ATP in dark anaerobic cells of Anacystis nidulans subjected to acid jumps and/or valinomycin pulses was characterized thermodynamically and kinetically. Maximum initial rates of 75 nmol ATP/min per mg dry weight at an applied proton motive force of -350 mV were obtained, the flow-force relationship (rate of ATP synthesis vs applied proton motive force) being linear between -240 and -320 mV irrespective of the source of the proton motive force. The pulse-induced ATP synthesis was inhibited by uncouplers (H+ ionophores) and F0F1-ATPase inhibitors but not by KCN or CO. In order to obtain maximum rates of pulse-induced ATP synthesis both a favorable stationary delta psi (-100 mV at pHo 9, preceding the acid jumps) and a favorable stationary delta pH (+2 units at pHo 4.1, preceding the valinomycin pulse) of the plasma membrane were obligatory, the effects of delta psi and delta pH being strictly additive. Moreover, the pulse-induced ATP synthesis required a minimum total proton motive force of -200 to -250 mV across the plasma membrane; it also required low preexisting phosphorylation potentials corresponding to -400 mV in dark anaerobic, i.e., energy-depleted, cells. The results are discussed in terms of both a reversible H+-ATPase and a respiratory electron transport system occurring in the plasma membrane of intact Anacystis nidulans.