Two possible roles of bacteriorhodopsin; a comparative study of strains of Halobacterium halobium differing in pigmentation.

The light-induced changes in pH and ATP level were compared for cell suspensions between strains of Halobacterium halobium differing in pigmentation after growth under the same conditions. Upon illumination, red cells which contained no detectable amount of bacteriorhodopsin showed only a pH increase, which, in the case of purple cells containing bacteriorhodopsin, was followed by a spontaneous pH decrease during illumination. Pre-incubation of cells at 75° for 5 min depressed the pH increase in both cells. Pre-illumination of cells with hydroxylamine depressed the pH decrease in purple cells. Whenever the pH increase was observed, the cellular ATP level increased. The presence of a bacteriorhodopsin different from that in the purple membrane is postulated.     

Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H⁺ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H⁺ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10.     

The ATP synthase of Halobacterium salinarium (halobium) is an archaebacterial type as revealed from the amino acid sequences of its two major subunits.

The head piece of the A-type ATP synthase in an extremely halophilic archaebacterium, namely Halobacterium salinarium (halobium), is composed of two kinds of subunit, alpha and beta, and is associated with ATP-hydrolyzing activity. The genes encoding these subunits with hydrolytic activity have been cloned and sequenced. The putative amino acid sequences of the alpha and beta subunits deduced from the nucleotide sequences of the genomic DNA consist of 585 and 471 residues, respectively. The amino acid sequence of the alpha subunit of the halobacterial ATPase is 63 and 49% identical to the alpha subunits of ATPases from two other archaebacteria, Methanosarcina barkeri and Sulfolobus acidocaldarius, respectively. The sequence of the beta subunit is 66 and 55% identical to the beta subunits from these respective organisms. The homology between the alpha and beta subunits is around 30%. In contrast, the sequences of the halobacterial ATPase is less than 30% identical to F1 ATPase when any combination of subunits is considered. However, they are greater than 50% identical to a eukaryotic vacuolar ATPase when alpha and a, beta and b combinations are considered. These data fully confirm the first demonstration of this kind of relationship which was achieved by immunoblotting with an antibody raised against the halobacterial ATPase. We concluded that the archaebacterial ATP synthase is an A-type and not an F-type ATPase. This classification is also demonstrated by a "rooted" phylogenetic tree where halobacteria locate close to other archaebacteria and eukaryotes and distant from eubacteria.     

Chemical Modification of the Membrane-bound ATP Synthetase of Spinach Chloroplasts with Pyridoxal Phosphate in Light

Photosynthetic activities of the thylakoid membranes modifiedwith pyridoxal phosphate (PLP) and sodium borohydride in lightwere studied and compared with those modified in the dark. PLPmodified the membrane-bound chloroplast coupling factor 1 (CF₁)and inhibited photophosphorylation. Only PLP modification inlight stimulated basal electron transport. This stimulationof electron transport was prevented by the presence of ATP orcarbonylcyanide m-chlorophenylhydrazone in the modificationmixture. Magnesium ion was required for PLP modification. Theextent of lightinduced proton uptake was decreased by PLP modificationin light. N,N'-Dicyclohexylcarbodiimide lowered the stimulatedelectron transport to the basal level of unmodified chloroplastsand restored proton uptake. When chloroplasts were modified with 4 mM PLP in light and dark,11.6 and 11.0 mol of PLP were incorporated into mol of CF₁,respectively. ATP could bind with high affinity to CF₁ isolatedafter PLP modification in light. The results indicate that PLP modifies membrane-bound CF₁ whichhas a conformation altered by energization of the thylakoidsin light, and causes an apparent uncoupling of phosphorylation(stimulation of basal electron transport). The results suggestthat this uncoupling is induced by the loss of the regulatoryfunction of CF₁ for proton translocation after PLP modificationin light. ¹ Presented at the ISRACON on Control Mechanisms in Photosynthesis.Aug. 31-Sept. 4, 1980, Acre, Israel (Received June 22, 1981; Accepted August 28, 1981)     

Inhibition and uncoupling of the ADP-regulated electron transport in isolated chloroplasts

The effects of energy transfer inhibitors, electron transport inhibitors and uncouplers on the ADP-regulated and ADP-independent activity of ferricyanide reduction in isolated spinach chloroplasts were studied. Phlorizin and sulfate did not affect the ADP-independent ferricyanide reduction. In the ADP-regulated reduction, these reagents did not affect the ADP inhibition process but inhibited the activity restoration process due to phosphorylation. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and linolenic acid depressed both ADP-regulated and ADP-independent activity of ferricyanide reduction. Gramicidin S and 2-amino-1-butanol depressed ADP-regulated activity and stimulated ADP-independent activity. The decrease in the ADP-regulated ferricyanide-reducing activity (restoration) due to (incomplete) uncoupling paralleled the decrease in phosphorylation activity (P/Δe=1).     

The gamma-subunit of ATP synthase from spinach chloroplasts. Primary structure deduced from the cloned cDNA sequence

cDNA clones encoding the gamma-subunit of chloroplast ATP synthase were isolated from a spinach library using synthetic oligonucleotide probes. The predicted amino acid sequence indicated that the mature chloroplast gamma-subunit consists of 323 amino acid residues and is highly homologous (55% identical residues) with the sequence of the cyanobacterial subunit. The positions of the four cysteine residues were identified. The carboxyl-terminal region of the chloroplast gamma-subunit is highly homologous with those of the gamma-subunits from six other sources (bacteria and mitochondria) sequenced thus far.     

Comparative studies on the properties of spinach chloroplasts prepared by media containing choline chloride or sucrose

Spinach chloroplasts were isolated and stored in a medium containing0.5 M choline chloride. The properties of these chloroplastswere compared with those of chloroplasts prepared in an ordinarysucrose medium. In marked contrast to sucrose-prepared chloroplasts, choline-preparedchloroplasts did not show "conventional uncoupling" (stimulationof ferricyanide reduction paralleling the inactivation of phosphorylation)after transient warming in the temperature range between 20?and 55?C. The thermal stability of the oxygen evolving system, the vulnerabilityof the lipophilic structure in the thylakoids to alcohol duringthe warming treatment, and the effects of amine on photophosphorylationwere similar in both chloroplast preparations. After the warming treatment, the degree of swelling in chloroplastswas larger and the intermediate electron transport systme (fromreduced 2,6-dichlorophenolindophenol to methyl viologen) wasmore stable against uncoupling, in choline-prepared chloroplaststhan in sucrose-prepared ones. The presence and absence of "conventional uncoupling" in twochloroplast preparations were ascribed to differences in thermalstability. In choline-prepared chloroplasts the uncoupling temperatureof the electron transport system was higher than the inactivationtemperature of the oxygen evolving system, but it was lowerin the sucrose-prepared ones. (Received February 13, 1974; )     

Lipophilic interaction between thylakoid membranes and aliphatic compounds

Spinach chloroplasts pre-incubated at various temperatures, changed their photosynthetic activities (measured at 15°C) as the incubation temperature was raised; these activities showed characteristic activitytemperature profiles as follows:

1. The profiles were shifted to lower temperatures if various aliphatic compounds were present in the incubation mixture.
2. In general, the extent of the shift was proportional to the product of the concentration and the partition coefficient of the compound, i.e. to the concentration of the compound partitioned in the lipophilic chloroplast phase.

The combined effects of heat and the aliphatic compounds tested indicated that the lipophilic groups in these compounds play a determinative role in the heat denaturation of thylakoids. The consequence of such structure alterations is inactivation of photosynthetic functions. The lipophilic properties of the spinach thylakoid membrane as compared with certain artificial and natural membranes are described.     

Purification and properties of a peroxidase from Halobacterium halobium L-33

A peroxidase was purified from Halobacterium halobium L-33 to an electrophoretically homogeneous state and some of its properties were studied. The enzyme showed an absorption peak at 406 nm in the oxidized form and peaks at 440, 558, and 591 nm in the reduced form. The difference spectrum, reduced + CO minus reduced, of the enzyme showed peaks at 425, 538, and 577 nm and troughs at 444, 562, and 596 nm. These spectral properties were apparently similar to those of "cytochrome a1" except for the occurrence of the peak at 558 nm in the reduced form. The molecular weight of the enzyme was 110,000 and the enzyme possessed one unit of protoheme in the molecule. The activity to oxidize guaiacol in the presence of H2O2 of the peroxidase was about one-twentieth of that of horseradish peroxidase. The enzyme also showed a catalase-activity one-fourth as active as that of liver catalase. The reactions catalyzed by the enzyme were strongly inhibited by KCN.