Development and use of chlorotetracycline fluorescence as a measurement assay of chloroplast envelope-bound mg

Experiments were conducted to develop chlorotetracycline (CTC) fluorescence as an assay of Mg²⁺ bound to the envelope of the intact chloroplast. This assay technique has been widely used to measure envelope associated divalent cations in animal cell and subcellular systems, but has not been used with chloroplasts. Chloroplast envelope-associated Mg²⁺ was altered by pretreatment with Mg²⁺ and divalent cation chelating agents and by additions of Mg²⁺ to the CTC assay medium. Results indicated that for a given chloroplast preparation, relative changes in envelope-associated Mg²⁺ can be effectively monitored with CTC fluorescence. It was concluded that the limitations of this assay system are: (a) chlorophyll strongly quenches CTC fluorescence signal, so a constant chlorophyll concentration must be maintained, (b) measurements must be made quickly, and (c) use of the technique to compare different chloroplast preparations may not be valid. Studies with ²⁸Mg²⁺ confirmed our interpretation of the fluorescence results, and also suggested that the chloroplast envelope is fairly impermeable to Mg²⁺. It was concluded that changes in Mg²⁺ associated with the chloroplast due to incubation of plastids in solutions containing up to 5 millimolar Mg²⁺ may be exclusively due to increased envelope-associated Mg²⁺. The CTC assay was used in experiments to demonstrate that increases in chloroplast envelope-associated Mg²⁺ inhibit photosynthetic capacity. This inhibition can be partially overcome by the presence of K⁺ in the photosynthetic reaction media.