Chloroplast Integrity and Biochemical Function

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Abstract
Semi-intact chloroplasts were isolated from spinach leaves which contained grana and lamellar structures in the same conformation as in the intact leaf. However, outer membrane and stromal ground substance had been lost. The ability of these semi-intact chloroplasts to carry out the photoreduction of NADP+ and the photophosphorylation of ADP was tested. Sonic disruption as well as a more gentle aging procedure was used. Photoreduction of NADP+ was stimulated and protected from the effects of sonication by the addition of photosynthetic pyridine nucleotide reductase (PPNR). This enzyme is quickly lost from the chloroplast during its preparation. The addition of exogenous sources of electrons also enhanced photoreduction. Upon disruption by sonication, the ability to reduce NADP+ was rapidly lost. The rate of phosphate esterification in the light in the absence of any added electron carriers or enzymes was 6-10 times greater than had been observed for other isolated chloroplast preparations under similar assay conditions. This rate was stimulated by the addition of various co-factors involved in cyclic and noncyclic photophosphorylation. Although disruption of the chloroplasts by sonication or aging caused a loss of phosphorylating ability, there was a residual low activity. Phenazinemethosulfate, a stimulator of the cyclic pathway, was unique in that after short periods of sonication its activity was enhanced. Evidence is presented to indicate that 2 independent sites for photophosphorylation may be operative.