Susanne Selman-Reimer

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell‐wall‐less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5‐fold higher than the ATPase activity observed in dark‐adapted cells. The light‐induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF1). This activity, ∼ 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half‐time of about 6 min at room temperature) in intact cells but only slowly decays (with a half‐time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri‐ but not ferrocyanide, suggesting that the stabilization of the activated form of CF1 is due to the reduction of the enzyme in vivo in the light.

Evidence for solvent-induced conformational changes of the soluble Dunaliella chloroplast coupling factor 1 (CF1)

The ATPase activity of the chloroplast coupling factor 1 (CF1) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca2+ ‐dependent activity. The Ca2+ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca2+ to Mg2+. Both the Ca2+ ‐dependent and Mg2+ ‐dependent ATPase activities of heat‐treated Dunaliella CF1 are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF1. However, when assayed under identical conditions, the Ca2+ ‐dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg2+ ‐dependent activity. These data are interpreted as indicating that soluble Dunaliella CF1 can exist in a variety of conformations, at least one of which catalyzes a Ca2+ ‐dependent ATPase and two or more of which catalyze an Mg2+ ‐dependent ATPase.

The partial purification of a factor from Dunaliella salina that causes the rapid in situ inactivation of light-activated chloroplast coupling factor 1 (CF1)

A factor having the expected properties of the in vivo oxidant responsible for inactivating the in vivo light‐activated chloroplast coupling factor 1 (CF1) has been partially purified from cell‐free extracts of Dunaliella salina. This factor is highly polar, weakly acidic, and relatively temperature stable. The ability of this factor to inactivate light‐activated CF1 is prevented if it is pretreated with reductants such as dithiothreitol. The factor has virtually no effect on the ethanol‐induced, Mg2+ ‐dependent ATPase activity of the isolated CF1.

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF1) to chemical modification by N-ethylmaleimide has been investigated. When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF1 ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF1 as shown by the time-course for the inhibition and the maximal extent of inhibition. Treatment of the purified, latent, D. salina CF1 with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I50 approximately 50 microM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF1 is partially reversed by either prolonged incubation at low concentrations of N-ethylmaleimide or short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF1 that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF1 are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.

The Characterization of a Potent, Naturally Occurring, ATPase Inhibitor (SIF) Isolated from the Halotolerant Algae Dunaliella

The in situ regulation of the catalytic activity of the chloroplast coupling factor (CF0•CF1) appears to be quite complex, involving protomotive force (μp)-induced conformational changes (1). These conformational changes are manifest in (i) the exposure of buried proton exchangeable groups in CF1 to the aqueous medium (2), (ii) the exposure of buried reactive amino acid residues to hydrophilic chemical modifiers (3), (iii) large magnitude changes in the dissociation constant of CF1 for its ligands (4), and (iv) an increased susceptibility of a disulfide bridge on the γ-subunit to reduction by dithiols (5). Stabilization of the in situ activated form of the ATPase is apparently achieved by the reduction of the exposed γ-subunit disulfide bridge to vicinal dithiols by dithiol reagents [e.g., dithiothreitol (DTT) in vitro (6) or thioredoxin in organello (7)], enabling on to measure an active ATPase in the subsequent dark period. In the absence of µp, reoxidation of the vicinal dithiols to the disulfide causes a rapid inactivation of the induced ATPase activity (8). Although the nature of the in vivo reductant has been postulated to be thioredoxin (7), the in vivo oxidant remains elusive (9).

A Comparison of Chloroplast Coupling Factor One, CF1, Using Polyclonal Chicken Antibodies

Antibodies directed against the chloroplast thylakoid coupling factor 1, CF1, the extrinsic membrane-bound sector of the reversible energy transducing H+-ATP synthase complex, have been widely used to study both the enzyme structure (1–3) and subunit function and accessibility (4,5). Most antibody preparations that have been employed have been monospecific polyclonal antisera or IgG enriched fractions usually isolated from rabbits (6) although more recently some studies employing mouse hybridoma monoclonal antibodies have been reported (e.g. ref. 3). Although it is relatively easy to raise antibodies in rabbits, it usually takes several months until the animals achieve peak titre, and both the immunization and subsequent bleeding processes can be rather traumatic (7). The failure of more wide spread application of monoclonal antibodies is testament to the enormous effort required to obtain clones and subsequent tumors, a process that can require a year.

A Partial Characterization of the Halotolerant, Green Algal Dunaliella bardawil CF1 ATPase and its Modification by Fluorescein Isothiocyanate

Coupling factor one (CF1) complexes have been isolated and characterized from a wide variety of photosynthetic organisms. These include bacteria (Beechy et al., 1975). thermophilic cyanobacteria (Binder, Bachofen, 1979), unicellular, eukaryotic green algae (Selman-Reimer et al., 1981), and higher (vascular) plants (Nelson, 1976). D. bardawil is a halotolerant, unicellular, eukaryotic green alga that is capable of growing under extreme environmental stress in the presence of NaCl concentrations as high as 5 M. In order to maintain its integrity, the alga accumulates massive amounts of glycerol as its osmoticum (Wegmann et al., 1980). Because the glycerol concentration in the chloroplast stroma is unlikely to vary much from that of the cytosol, it was of interest to us to compare the properties of the D. bardawil CF1 (DCF1) to other coupling factors not usually required to function under such extreme physiological conditions. In this communication, we characterize the enzymatic properties of the DCF1, identify the subunits of the ATPase as resolved by SDS-PAGE, and describe the modification of DCF1 by fluorescein isothiocyanate (FITC).