Héctor A. Lucero

Inhibition of spinach chloroplasts photophosphorylation by the antibiotics leucinostatin and efrapeptin.

The ATPases that catalyze the synthesis of ATP in oxidative and photosynthetic phosphorylation are rather similar with respect to molecular weight, subunit distribution, amino acid composition and coupling activity (for a review see [ 11). However, they are immunologically different and do not replace each other as coupling factors. They also differ in their sensitivity towards some inhibitors. For instance, I.ardy and coworkers have introduced several antibiotics as specific inhibitors of oxidative phosphorylation [2]. The best known and more widely used are oligomycin and aurovertin [3,4]. Although both antibiotics inhibit oxidative phosphorylation their binding sites are different and only aurovertin inhibits the ATPase activity of soluble F1 [4]. On the other hand, they do not affect photophosphorylation except for a weak uncoupling by high concentrations of oligomycin [5]. Recently, Lardy et al; [2] have postulated four mitochondrial binding sites for antibiotics that inhibit phosphoryl transfer. Two of them are in F, and correspond to aurovertin and to efrapeptin. The other two are located on the membrane component of the ATPase complex; one of them is the oligomycin site and the fourth corresponds to leucinostatin. Since photophosphorylation is not affected by oligomycin and aurovertin, we thought it would be interesting to test the effects of leucinostatin and efrapeptin on spinach chloroplasts.This paper shows that both antibiotics inhibited photophosphorylation although by different mechanisms: leucinostatin

Sulphydryl groups in photosynthetic energy conservation III. Inhibition of photophosphorylation in spinach chloroplasts by CdCl2

Abstract Low concentrations of CdCl2 inhibited cyclic and non-cyclic photophosphorylation in spinach chloroplasts and depressed coupled electron transport to the basal level. The basal electron transport and the electron flow stimulated by several uncouplers, except arsenate, were not affected. The inhibitory effects of cadmium were reversed by dithiols like dithioerythritol or 2,3-dimercaptopropanol. The light-induced pH rise of chloroplast suspensions and the trypsin-activated Ca-ATPase were not affected by cadmium at the concentrations that inhibited ATP synthesis but the latter was affected by higher concentrations.

Inhibition of energy conservation reactions in chromatophores of Rhodospirillum rubrum by antibiotics.

Abstract The antibiotics efrapeptin and leucinostatin inhibited photosynthetic and oxidative phosphorylation and related reactions such as the dark and light ATP-Pi exchange reactions and the Mg-ATPase in Rhodospirillum rubrum chromatophores. Higher concentrations of leucinostatin were required for inhibition of the phenazine methosulfate-catalyzed photophosphorylation and light ATP-Pi exchange reaction than for the endogenous or succinate-induced photophosphorylation and dark ATP-Pi exchange reaction. Efrapeptin and leucinostatin inhibited the ATP-driven transhydrogenase while only the latter inhibited the light-driven transhydrogenase, proton gradient formation, and NAD+ reduction by succinate in chromatophores. Efrapeptin, but not leucinostatin, inhibited the soluble Ca-ATPase activity of the coupling factor obtained from chromatophores. The inhibition was competitive with ATP. It is concluded that efrapeptin is an effective energy transfer inhibitor whose site of action may be localized in the soluble coupling factor, while the effects of leucinostatin are more complex and cannot be explained as a simple uncoupling.

Protein phosphorylation in the photosynthetic bacterium Rhodospirillum rubrum

Endogenous protein phosphorylation in cellular fractions from Rhodospirillum rubrum was manifested after exposure to [γ‐32P]ATP. At least six phosphorylated protein bands of 90, 86, 64, 31, 13 and 11 kDa were found in the cell‐free extract. Treatment of the 64‐kDa band with V8 protease yielded smaller radioactive bands. Phosphoserine, phosphothreonine and phosphotyrosine were detected after acid hydrolysis of the phosphorylated fractions. Protein phosphorylation in all the fractions was insensitive to cAMP, did not recognize exogenous protein substrates and was rapidly reverted upon elimination of the excess of [γ‐32P]ATP. The chlorophyll‐anthena apoprotein from R. rubrum chromatophores overlapped the 13‐kDa phosphorylated band during gel filtration by high‐pressure liquid chromatography suggesting that it is one of the substrates of the protein kinase(s) of R. rubrum.

Involvement of an essential arginyl residue in the coupling activity of Rhodospirillum rubrum chromatophores.

Abstract The arginine reagents phenylglyoxal and 2,3-butanedione in borate buffer completely inhibited photophosphorylation and Mg-ATPase of Rhodospirillum rubrum chromatophores. The inactivation rates followed apparent first order kinetics. Oxidative phospho-rylation and the light-dependent ATP-P i exchange reactions of R. rubrum chromatophores and the Ca-ATPase activity of the soluble coupling factor were similarly inhibited by 2,3-butanedione in borate buffer. The apparent order of reaction with respect to inhibitor concentrations for all these reactions gave values of near 1 suggesting that inactivation was the consequence of modifying one arginine per active site. ATP synthesis and hydrolysis by R. rubrum chromatophores were strongly protected against inactivation by ADP and ATP, respectively, and by other nucleotides that are substrates of the reactions but not by the products. Similarly, the Ca-ATPase of the soluble coupling factor was protected by ATP but not by ADP. Inactivation of chromatophores reactions by butanedione in borate buffer was more rapid in the light than in the dark. The results suggest that the catalytic sites for ATP synthesis and hydrolysis on the chromatophore coupling factor are different and both contain an essential arginine.

Evidence of tyrosine kinase activity in the photosynthetic bacterium Rhodospirillum rubrum

The photosynthetic bacterium Rohodospirillum rubrum evidenced tyrosine protein phosphorylation under photoautotrophic conditions in the presence of [32P]Pi. The stability to alkaline treatment of the [32P] bound to the cell-free extract proteins suggested that tyrosine residues were carrying the labelling. One- and two-dimensional high voltage paper electrophoresis analysis revealed that such extracts do contain [32P]-phosphotyrosine residues. Furthermore, the association of alkali stable [32P] bound to specific proteins of the cell-free extract was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with KOH treatment of the gel. A definite argument in favor of protein kinase(s) phosphorylating tyrosine residues in R.rubrum proteins was obtained by partial purification of a tyrosine kinase activity from cell-free extract capable of phosphorylating synthetic peptides that only contain a single tyrosine residue as phosphate acceptor.

Phosphorylation of serine residues in endogenous proteins of thylakoids and subthylakoid particles in the dark under nonreducing conditions

Abstract Isolated thylakoids, Photosystem I and Photosystem II particles were phosphorylated with [γ-32P]ATP at high specific radioactivity in the dark under nonreducing conditions and in the light in the absence of electron acceptor. The resulting phosphoproteins were compared by gel electrophoresis and autoradiography. Phosphorylation of thylakoids in the dark and in the light rendered distinct patterns of phosphoproteins. Some of the dark-phosphorylated proteins in thylakoids were diminished or not detected in the light-phosphorylated membranes. Phosphorylation of subthylakoid particles was insensitive to light and most of the phosphoproteins in these membranes were also observed in the dark-phosphorylated thylakoids. Dark phosphorylation rendered mostly phosphoserine in individual proteins of thylakoids, subthylakoid particles and lysine-rich histone phosphorylated by the particles. Conversely, phosphothreonine was prevalent in light-phosphorylated thylakoids. The results are consistent with the presence of a protein serine kinase activity that is distributed homogeneously within the thylakoid regions, is more active in the dark, does not require reducing conditions for activity and phosphorylates a number of endogenous substrates most of which belong to the stroma membranes.

Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: modification of a tyrosine protected by phosphate

Abstract Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg2+-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca2+-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F1) was inactivated by NBD-Cl with kinetics resembling those described for Mg2+-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F1 fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca2+-ATPase activity of R. rubrum F1 were fully protected by 5 mM Pi against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca2+-ATPase activity by Pi indicated that NBD-Cl and Pi are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F1 underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.