A simple method for measuring the internal pH of chloroplasts during steady-state illuminations based on the stimulation of proton uptake by monofunctional amines was developed. Predictions of a mathematical derivation concerning the dependence of the stimulation on the amine concentration, the internal volume, the pK of the amine and the external pH have been verified experimentally. To circumvent uncoupling and swelling due to large internal accumulation of amines extrapolation of the stimulation to low amine concentrations was suggested and shown to lead to valid values. Alternatively, swelling could be largely reduced in a medium containing potassium aspartate and valinomycin.

Amines↗

PubMed1976

Photosynthetic activities of membrane preparations of the blue-green alga Phormidium luridum.

Techniques have been developed for the isolation of whole spheroplaste and membrane preparations from the blue-green alga Phormidium luridum which are active in photosystem-II-sensitized and photosystem-I-sensitized electron-transport reactions and in photophosphorylation. Attention is called to the necessity of supplying magnesium and phosphate throughout the preparative and assay procedures for obtaining active membranes. Selective washing of a proper membrane preparation is shown to remove a new protein factor, termed protein factor 1, which restores the ability of the preparation ot catalyze photosystem-I-sensitized reactions.

Calcium↗

PubMed1976

Measurement of transmembrane potentials in Rhodospirillum rubrum chromatophores with an oxacarbocyanine dye.

The fluorescent dye 3,3-dipentyloxacarbocyanine (OCC) can be used as a fluorescence probe to measure transmembrane potentials across Rhodospirillum rubrum chromatophore membranes. A reversible fluorescence increase is observed in the light which is sensitive to inhibitors, permeable ions and uncouplers. Partial interchangeability between the electrical potential and the proton concentration gradient has been demonstrated by measurement of the fluorescence increase with OCC and the fluorescence quenching with 9-aminoacridine. OCC fluorescence changes can be induced also in the dark by injection of permeable salts and by rapid pH changes presumably indicating diffusion potentials. Using salt-induced diffusion potentials for calibrating the light signals and with several assumptions, the light-induced potentials were estimated as 170 mV for the maximal signal and 90-110 mV at the steady state. OCC has been shown to apparently increase the electrical conductivity of the chromatophore membrane, a fact which may be relevant to the mechanism of action of this probe. A red shift in the OCC absorption spectrum occurs when mixed with chromatophores, with a difference spectrum maximum at 495 nm. The absorption changes at 495 nm taking place in the light are similar in kinetics to the fluorescence changes. The absorbance spectrum of OCC in organic solvents is red shifted and the extent of the shift depends on the hydrophobicity of the medium. The difference spectrum compared to water in sec-butyl acetate/n-hexane (3 : 1, v/v) with a dipole moment of 5 was nearly identical to that of chromatophore-associated dye. The uncoupling properties of OCC at high concentrations and some difficulties in calibration limit the usefulness of this probe for quantitative measurements of transmembrane potentials.

Bacterial Chromatophores↗

PubMed1976