Richard Shingles

Ferrous ion transport across chloroplast inner envelope membranes

The initial rate of Fe2+ movement across the inner envelope membrane of pea (Pisum sativum) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Fe2+-sensitive fluorophore, Phen Green SK. The rate of Fe2+ transport was rapid, coming to equilibrium within 3s. The maximal rate and concentration dependence of Fe2+ transport in predominantly right-side-out vesicles were nearly equivalent to those measured in largely inside-out vesicles. Fe2+ transport was stimulated by an inwardly directed electrochemical proton gradient across right-side-out vesicles, an effect that was diminished by the addition of valinomycin in the presence of K+. Fe2+ transport was inhibited by Zn2+, in a competitive manner, as well as by Cu2+ and Mn2+. These results indicate that inward-directed Fe2+ transport across the chloroplast inner envelope occurs by a potential-stimulated uniport mechanism.

Nitrite transport in chloroplast inner envelope vesicles. I. Direct measurement of proton-linked transport

Chloroplast inner envelope membrane vesicles that are loaded with the pH-sensitive fluorophore, pyranine, show rapid internal acidification when nitrite is added. Acidification is dependent upon [delta]pH, with the inside of vesicles being alkaline with respect to the outside. The rate of vesicle acidification was directly proportional to the concentration of nitrite that was added and the imposed pH difference across the membrane. In contrast, added nitrate had no effect on vesicle acidification. Nitrite also caused acidification of asolectin vesicles. The extent of vesicle acidification is dependent on the internal volume of vesicles. Inner envelope and asolectin vesicles that were prepared by extrusion were approximately the same size, allowing them to be compared when the final extent of acidification, measured after the pH gradient had collapsed, was similar. The rate of nitrite-dependent acidification was similar in these two preparations at any single nitrite concentration. These results indicate that nitrite movement occurs by rapid diffusion across membranes as nitrous acid, and this movement is dependent on a proton gradient across the lipid bilayer. Under conditions approximating those in vivo, the rate of diffusion of nitrous acid far exceeds that of nitrite reduction within chloroplasts.

Copper Transport Across Pea Thylakoid Membranes

The initial rate of Cu2+ movement across the thylakoid membrane of pea (Pisum sativum) chloroplasts was directly measured by stopped-flow spectrofluorometry using membranes loaded with the Cu2+-sensitive fluorophore Phen Green SK. Cu2+ transport was rapid, reaching completion within 0.5 s. The initial rate of uptake was dependent upon Cu2+ concentration and saturated at about 0.6 μm total Cu2+. Cu2+ uptake was maximal at a thylakoid lumen pH of 7.0. Cu2+ transport was inhibited by Zn2+ but was largely unaffected by Mn2+ and Cu+. Zn2+ inhibited Cu2+ transport to a maximum of 60%, indicating that there may be more than one transporter for copper in pea thylakoid membranes.

Direct Measurement of Nitrite Transport Across Erythrocyte Membrane Vesicles Using the Fluorescent Probe, 6-Methoxy-N-(3-sulfopropyl) quinolinium

Nitrite was shown to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) almost twofold more than chloride. SPQ loaded inside vesicles prepared from asolectin and isolated erythrocyte ghosts allowed for the direct measurement of nitrite movement across these membranes. Movement of nitrite across asolectin occurred by diffusion as HNO2 in a pH-dependent manner. By contrast, erythrocyte ghosts had very low diffusion rates for nitrous acid. Erythrocyte ghosts preloaded with 50 mM nitrite to quench SPQ fluorescence were utilized to study heteroexchange with externally added anions. SPQ fluorescence increases (becomes unquenched) with added bicarbonate and nitrate, indicating that nitrite is moving out of the preloaded vesicles. The pH optimum for this exchange was approximately 7.6 and exchange was inhibited by N-ethylmaleimide (NEM) and dihydro-4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These data indicate that nitrite moves across erythrocyte plasma membranes as NO2- by a heteroexchange mechanism with other monovalent anions.

Direct Measurement of ATP-Dependent Proton Concentration Changes and Characterization of a K+-Stimulated ATPase in Pea Chloroplast Inner Envelope Vesicles

Inner envelope membrane vesicles prepared from pea (Pisum sativum L. var Laxtons Progress No. 9) chloroplasts have K+-stimulated ATPase activity with a pH optimum of 8.4. ATP addition to inner envelope vesicles loaded with pyranine caused a decrease in pyranine fluorescence that was consistent with internal acidification. The transmembrane pH change induced by the addition of 5 mM ATP was about 0.4 unit. Measurement of phosphate released by ATP hydrolysis paralleled the pH change, indicating that intravesicular acidification was linked to ATPase activity. Vanadate, molybdate, N-ethylmaleimide, and dithiothreitol inhibited ATP-dependent vesicle acidification completely, whereas ATPase activity was only partially inhibited. These data indicate that pea chloroplast inner envelope vesicles contain a proton translocating ATPase and that the pyranine-loading method can be utilized to study directly ATP-dependent H+ transport across these membranes.

Ion Transport Across Chloroplast Inner Envelope Vesicles

The ionic environment of chloroplasts is important for photosynthesis, nitrogen metabolism and sulfur metabolism, yet how this internal environment is regulated is still not known. Isolated chloroplast inner envelope membranes are competent for transport studies. These membranes can be manipulated to form vesicles of largely right side-out and inside-out orientation and both the intravesicular and extravesicular buffer contents can be controlled. The vesicles can be loaded with an ion-sensitive fluorophore to measure the initial rates of ion transport across the membranes using spectrofluorometric methods. We have made measurements on a proton-pumping ATPase which may be involved in forming a pH gradient to assist in the transport other ions across the chloroplast inner envelope. Several ions such a sulfate, glycolate, phosphate, iron and calcium have enhanced rates of transport when an inward-directed pH gradient is imposed. Calcium may actually move as a result of the potential gradient which is formed across the inner envelope. In addition both nitrite and bicarbonate movement is aided by the presence of a pH gradient, nitrite moving as nitrous acid and bicarbonate moving as carbon dioxide. Movement of the latter is greatly accelerated when carbonic anhydrase is present on both sides of the membrane. A summary of our results on the study of ion transport across the chloroplast inner envelope will be presented with respect to rates of transport, effectors, and inhibitors.