Zippora Gromet-Elhanan

ΔpH and membrane potential in bacterial chromatophores

The light-induced pH rise in chromatophore suspensions is generally interpreted as an inward electrogenic translocation of protons which, as in chloroplasts, creates a difference in the proton electrochemical gradient (AFH) across the chromatophore membrane [l] . Isaev et al. [2] showed energy dependent uptake of some synthetic permeant anions, and Jackson and Crofts observed spectroscopic changes [3] suggesting the existence of a membrane potential (A+) which is positive inside. Several observations (4-6) indicate the existence of a proton concentration gradientfApH). Moreover, the observation that chromatophores are less sensitive than chloroplasts to amines and nigericin uncoupling [4,5,7-91 and the effect of several ions [lO,l l] on chromatophore reactions led to the suggestion that A

Effect of ammonium salts, amines and antibiotics on proton uptake and photophosphorylation in Rhodospirillum rubrum chromatophores.

rather than ApH provides the major contribution to the high energy state of the chromatophores. However, quantitative estimation of ApH and A

Comparison of the electrochemical proton gradient and phosphate potential maintained by Rhodospirillum rubrum chromatophores in the steady state

of these subcellular structures is still unsatisfactory, due to the uncertainty in the mode of action of several of the monitoring probes used [ 12141 and to insufficient data about the particles water volume and their buffer capacity [ 151. In the present work the determination of the osmotic volume of the chromatophores enabled the calculation of ApH and A

Relationship between light-induced quenching of atebrin fluorescence and ATP formation in Rhodospirillum Rubrum chromatophores

. The ApH was calculated from

Differences in sensitivity to valinomycin and nonactin of various photophosphorylating and photoreducing systems of Rhodospirillum rubrum chromatpohores

Abstract 1. NH4Cl inhibits the light-induced proton uptake but not photophosphorylation in chromatophores prepared from Rhodospirillum rubrum. This selective inhibition of proton uptake was also obtained with various amines which are well-known uncouplers in chloroplasts. 2. Addition of ion-transporting antibiotics like valinomycin or nonactin resulted in the inhibition of photophosphorylation with NH4Cl but not with the other amines. The synergistic inhibition with valinomycin and NH4Cl was abolished in the presence of K+. 3. Similar results were found also in subchloroplast particles. Both in chromatophores and in subchloroplast particles valinomycin was required to induce the inhibition of photophosphorylation with ammonium nitrate, bicarbonate and acetate as well as chloride. 4. The effect of the ion-transporting antibiotics can be attributed to an enhanced permeability of NH4+ in their presence. The resulting inhibition of photophosphorylation can be explained by the initiation of an energy-dependent cyclic cation transport involving an active influx of protons coupled with a passive efflux of NH4+ facilitated by the antibiotics. The results can also be explained according to the chemiosmotic theory, if it is assumed that all the anions tested, namely Cl−, NO3−, HCO3− and acetate are impermeable in chromatophores as well as in subchloroplast particles.

Microsomes as sites of α-amylase synthesis in the rat-paratid gland

Abstract A technique for the estimation of light-induced membrane potential in chromatophores is described. It is based on measurement of light-induced enhancement in fluorescence of 8-anilinonaphthalene sulfonic acid, which is calibrated by known K + diffusion potentials. The electrochemical proton gradient (Δ μ H+ − ) formed during lightinduced electron transport in Rhodospirillum rubrum chromatophores amounts to 250 mV, which is almost equally distributed between the membrane potential and the pH gradient as measured by changes in the fluorescence of anilinonaphthalene sulfonate and 9-amino acridine. Addition of the permeant anion, NaSCN, or of NH 4 Cl reduces the overall Δ μ H+ − by less than 20% but changes its distribution between the pH gradient and the membrane potential so that with NaSCN it is composed mainly of the first and with NH 4 Cl mainly of the second. Initiation of phosphorylation causes a drop of about 50 mV in the measured Δ μ H+ − . In the absence of salts, the drop is observed in both components, although two-thirds of it are reflected in the membrane potential. In the presence of NaSCN or NH 4 Cl the 50-mV drop is exclusively recorded in the pH gradient or in the membrane potential, respectively. The steady-state phosphate potential maintained during electron transport was found to change in parallel to the Δ μ H+ − , but exceeded it by 60 to 80 mV when based on a stoichiometry of two protons translocated per ATP synthesized.

[53] Selective extraction and reconstitution of F1 subunits from Rhodospirillum rubrum chromatophores

Membrane-bound fluorochromes silch as 8-anilinol-naphthalenesulfonic acid [l] have been employed as probes of the energization of mitochondria and submitochondrial membranes [2]. Energy-dependent uncouplerinhibited fluorescence changes have been observed [361, which could be accounted for by changes in binding of the probe, induced by charge changes of the membrane [6] . Recently a new fluorescent probe, atebrin, which is by itself an uncoupler [7] has been introduced to study the energized state in chloroplasts [8]. A quenching of the atebrin fluorescence was induced by electron transport, ATP hydrolysis or a pH gradient and the quenching decreased when the system was uncoupled. It was therefore concluded that there is a stoichiometric relationship between the generation of energy and the quenching of atebrin fluorescence in chloroplasts. In the present study the relationship between the quenching of atebrin fluorescence and ATP formation was investigated in R. rubrum chromatophores. Evidence will be provided that there is no direct correlation between the two processes, since the light-induced quenchinf of atebrin fluorescence could be completely eliminated under conditions when ATP formation was not affected.

Energy-transfer inhibitors and electron transport inhibitors in chloroplasts

Abstract 1. The effect of valinomycin and nonactin on photophosphorylation and photoreduction by Rhodospirillum rubrum chromatophores was tested in the presence of various electron carriers and donors, which differed in their sensitivity to hydroxyquinoline N -oxide. 2. The phosphorylating system with N -methylphenazonium methosulphate was resistant to both antibiotics and to hydroxyquinoline N -oxide. All the other phosphorylating systems tested were inhibited far beyond the 50% level by valinomycin and nonactin. No correlation was observed between the sensitivity to hydroxyquinoline N -oxide and to valinomycin or nonactin. 3. Both antibiotics inhibited NAD + photoreduction to the same extent as photophosphorylation. The addition of NH 4 Cl resulted in an increased inhibition of both photoreactions. 4. The sensitivity, as well as the resistance to valinomycin and nonactin, was independent of the presence of transportable ions such as K + . 5. According to these findings the inhibition by valinomycin and nonactin cannot be an inhibition of energy transfer or an uncoupling due to their ion-transporting activity. It is, however, impossible in these systems to resolve the question whether they are electron transport inhibitors or uncouplers in a way not related to their ion-transporting ability.

Identification of subunits required for the catalytic activity of the F1-ATPase

Abstract The biosynthesis of a specific protein, α-amylase, was studied in the rat parotid gland. The cell proteins were labeled in vivo by injecting [ 14 C]amino acids. Subsequently the specific radioactivity, as well as the total counts incorporated into amylase and into the total proteins of the various cell fractions, were recorded for different time intervals. The following results were obtained. 1. 1. The specific radioactivity at short time intervals (5–15 min after the injection), was highest in the amylase of the microsomal fraction, and lowest in the zymogen fraction. A rather high 14 C-concentration was also found in the amylase of the supernatant fraction. 2. 2. A twenty-fold increase in the specific radioactivity of amylase in the zymogen granules was recorded after 120 min, while in the other cell components only a moderate rise occurred. In the microsomes there was even a drop in 14 C-content. 3. 3. The distribution of the total counts incorporated into amylase 15 min after the injection was lowest in the zymogen granules. However, after 120 min the 14 C accumulated to a rather high extent in this fraction, while its concentration in the microsomal fraction decreased. It is concluded that amylase is synthesized in the microsomes, rapidly released to to the soluble portion of the cell, and subsequently transferred to the zymogen granules, where it is concentrated and stored.

Demonstration of acid—base phosphorylation in chromatophores in the presence of a K+ diffusion potential

Publisher Summary his chapter describes the selective extraction and reconstitution of F 1 subunits from Rhodospirillurn rubrum chromatophores. The F 1 -ATPase isolated from membranes of mitochondria, bacteria, and chloroplasts is very similar, containing five nonidentical polypeptide subunits: α, β, γ, δ, and ɛ. Using R. rubrum chromatophores, two F 1 subunits—β and γ—are extracted in two consecutive steps, leaving all other F1 subunits attached to the chromatophore membrane. The procedures developed for the extractions, for purification of the isolated subunits, and for their reconstitution into the depleted chromatophores are described. The isolated β and γ subunits have no activity by themselves, although their extraction leads to complete loss of photophosphorylation and ATPase activities of the depleted chromatophores. The subunits are therefore identified during the extraction and purification procedures by their reconstitutive activity. This is their capacity to rebind to depleted chromatophores and restore their lost activities. The experimental system for measuring the reconstitutive activity involves two steps: (1) reconstitution of the isolated subunits into the depleted chromatophores, and (2) assay of the reconstituted chromatophores for restored activities.