Measurement of Proton-Linked Transport Activities in Pyranine Loaded Chloroplast Inner Envelope Vesicles 1

AUTOR(ES)

Howitz, Konrad T.

RESUMO

A method has been devised for loading chloroplast inner envelope vesicles prepared from pea (Pisum sativum L. var Progress No. 9) or spinach (Spinacia oleracea L.) with 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), a membrane impermeant, fluorescent pH indicator. Two known proton-linked transport activities of the inner envelope, glycolate/H+ co-transport and phosphate/phosphoglycerate exchange have been shown to cause quenching of the internal pyranine fluorescence. This represents the first demonstration that these vesicles are sealed and competent for transport measurements. The technique, as it now stands, is essentially qualitative. It does, however, offer advantages over transport measurements with intact chloroplasts, for example compatibility with rapid mixing techniques and accessibility of the transport proteins to antibodies.

Documentos Relacionados

• Nitrite Transport in Chloroplast Inner Envelope Vesicles (I. Direct Measurement of Proton-Linked Transport).
• Proton-linked D-xylose transport in Escherichia coli.
• Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles1
• Direct Measurement of ATP-Dependent Proton Concentration Changes and Characterization of a K+-Stimulated ATPase in Pea Chloroplast Inner Envelope Vesicles.
• Assay of Proton-Coupled Glycolate and D-Glycerate Transport into Chloroplast Inner Envelope Membrane Vesicles by Stopped-Flow Fluorescence.