Abstract
The role of AMP in photophosphorylation was studied using rapid mixing acid quench techniques. Fragmented spinach chloroplast membranes or subchloroplast particles were illuminated and rapidly mixed with [32P]orthophosphate and AMP at pH 7 for 10 ms to 60 s after which time perchloric acid was added to quench the reaction. ATP was found to be the primary and predominant nucleotide labeled. It was found that after illumination, an adenylate kinase-like activity carried out an AMP-dependent conversion of labeled ATP to labeled ADP which was inhibited by the presence of ADP. This reaction was characterized as being similar to chloroplast adenylate kinase in Mg2+ dependency and in sensitivity to phlorizin and tentoxin and distinct from chloroplast coupling factor 1. The small amounts of adenylate kinase activity present in fragmented well washed chloroplast membranes were found to be sufficient to carry out this rapid reaction. These results necessitated a reinterpretation of the earlier findings of Tiefert and Moudrianakis (Tiefert, M.A., and Moudrianakis, E.N. (1979) J. Biol. Chem. 254, 9500-9508) and no longer support the role of ADP as a phosphorylated intermediate in ATP synthesis.
Highlights
The role of AMP in photophosphorylationwas stud- tion experiments in which labeled ADP was detected earlier ied using rapid mixing acid quench techniques, Frag- than labeled ATP, a finding which could be considered to mented spinach chloroplast membranes or subchloro- suggest that ADP could function as a kinetic intermediate in plast particles were illuminated and rapidly mwixitehd the synthesis of ATP
If substrate ADP was added during the dark period, In 1971, Roy and Moudrianakis [1] proposed a mechanism for ATPsynthesis in oxidative and photophosphorylation
Preparation of Chloroplasts, Subchloroplast Particles,and Chloroplast Membranes-Chloroplasts were isolated according to Howell and Moudrianakis [12] and were washed twice as described therein
Summary
Preparation of Chloroplasts, Subchloroplast Particles,and Chloroplast Membranes-Chloroplasts were isolated according to Howell and Moudrianakis [12] and were washed twice as described therein. Fragmented NaC1-washed chloroplast membranes were prepared as described by Tiefert and Moudrianakis (IO). Adenylate kinase activity, measured as the rate of conversion of ADP to ATPand AMP (2 ADP + ATP + AMP), was assayed using the hexokinase-glucosemethod described by Moudrianakis and Tiefert [15]modified by adding 0.1%bovine serum albumin and 0.25 mM EDTA to the reaction mixture [16]. The reaction transferring the y-phosphoryl group of [y-'lP]ATP to AMP to yield v-32P]ADP (dismutation reaction) was assayed in a medium containing chloroplast membrane fractions or adenylate kinase, 50 m~ Tricine NaOH, pH 7,20 mM NaCl, 1 mMMgC12, 0.02 mM pyocyanine perchlorate, and 0.1%bovine serum albumin in a 4.4ml volume at 25 "C in darkness with stirring. Other chemicals were obtained as described [10]
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