CONVERSION OF TIGHTLY BOUND ADP TO BOUND ATP ON CHLOROPLAST MEMBRANES AND PARTIAL CHARACTERIZATION OF SPINACH CHLOROPLAST BINDING SITES.
Item
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Title
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CONVERSION OF TIGHTLY BOUND ADP TO BOUND ATP ON CHLOROPLAST MEMBRANES AND PARTIAL CHARACTERIZATION OF SPINACH CHLOROPLAST BINDING SITES.
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Identifier
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AAI8401959
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identifier
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8401959
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Creator
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SZYJEWICZ, JERRY.
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Contributor
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A. Lukton
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Date
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1983
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, General
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Abstract
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{lcub}('3)H{rcub}-Adenine nucleotides were exchanged into the binding sites of spinach chloroplasts. A maximum of 3.7 nmoles of {lcub}('3)H{rcub}-nucleotides/mg chlorophyll remained bound to the membranes following repeated washings (centrifugation and resuspension in fresh medium) of the labeled chloroplasts. Passage of similarly labeled chloroplasts through a Sephadex G-50 column removed additional bound {lcub}('3)H{rcub}-nucleotides; 1.98 nmoles of {lcub}('3)H{rcub}-nucleotides/mg chlorophyll remained bound to the membranes. Two types of binding sites were identified in these chloroplasts: (1) the exchangeable site(s)--these sites slowly shed their nucleotides in the dark (0.87 nmoles of {lcub}('3)H{rcub}-nucleotides/mg chlorophyll dissociated from the chloroplasts in the dark 30 min following elution from the Sephadex G-50 column). An additional 0.14 nmoles of {lcub}('3)H{rcub}-nucleotides were lost from the membranes in the dark following the addition of free ADP or ATP or upon excitation of the chloroplasts in the absence of added nucleotides; (2) the non-exchangeable site(s)--0.89 numoles of bound {lcub}('3)H{rcub}-nucleotides/mg chlorophyll remain uneffected by the conditions that brought about the release of nucleotides from the exchangeable site(s). These nucleotides exchanged with medium nucleotides, ATP or ADP, only following excitation of the chloroplasts (t(, 1/2) = 0.2-0.5 s). This exchange was inhibited by 20 mM NH(,4)Cl or by 1 mM NBD.Cl and showed a requirement of MG('++).;{lcub}('3)H{rcub}-ADP at the non-exchangeable site was converted to {lcub}('3)H{rcub}-ATP which remained firmly attached to the chloroplasts when the chloroplasts were illuminated in the presence of inorganic phosphate but in the absence of added nucleotides. Formation of bound {lcub}('3)H{rcub}-ATP from bound {lcub}('3)H{rcub}-ADP was also observed when labeled chloroplasts that were passed through Sephadex G-50 were incubated in the dark with ATP. ATP mediated phosphorylation of bound {lcub}('3)H{rcub}-ADP was inhibited by free ADP, the ATP analogue AMPPNP, and by the uncouplers NH(,4)Cl (20 mM) and NBD.Cl (1 mM). Conversion of bound ADP to ATP was catalyzed by a chloroplast membrane componant, possibly CF(,1), and not by enzymes such as adenylate kinase (AK). The rate of phosphorylation of bound {lcub}('3)H{rcub}-ADP on the non-exchangeable site (T(, 1/2) (TURN) 5.0 s) and the rate of exchange of labeled nucleotides from this site with free nucleotides were too slow to serve as intermediates in photophosphorylation.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biochemistry
