Studies on the Hydrolysis of Adenosine Triphosphate by Spinach Chloroplasts

The incorporation of water oxygens into ATP made by photophosphorylation is known to be increased markedly when either Pi or ADP concentration is lowered. The present studies show a similar increase in oxygen exchange when light intensity is lowered even with ample ADP and Pi present. The number of reversals of bound ATP formation prior to release increases about 1 to about 27 in the presence of dithiothreitol and to 5 in its absence. The equilibrium of the bound reactants still favors ATP at low light intensity, as shown by measurement of the amount of bound ATP rapidly labeled from [32P]Pi during steady-state photophosphorylation. Changes observed in the interconversion rate in the absence of added thiol are likely involved in the regulation of the dark ATPase activity in the chloroplast. The interconversion rate of bound ATP to bound ADP and Pi in the presence of thiol is about the same at low and high light intensities. This rate of bound ATP formation is not sufficient, however, to account for the maximum rate of photophosphorylation. Thus, when adequate protonmotive force is present, the rate of conversion of bound ADP and Pi to bound ATP, and possibly that of bound ATP to bound ADP and Pi, must be increased, with proton translocation being completed only when bound ATP is present to be released. These observations are consistent with the predictions of the binding change mechanism with sequential participation of catalytic sites and are accommodated by a simplified general scheme for the binding change mechanism that is presented here.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Resultsmentioning

confidence: 99%

Catalytic and regulatory effects of light intensity on chloroplast ATP synthase

Stroop¹,

Boyer²

1987

“…Mg2+ and Ca2+ have been reported to exert different effects on the thylakoid membrane bound CF0F, ATP synthase-ATPase complex. Thylakoids carry out Mg-dependent ATP synthesis (Whatley et al, 1959) and hydrolysis (Petrack et al, 1965), and the Mg-ATPase is coupled to H+ translocation (Carmeli, 1970). Ca2+ on the other hand maintains only ATP hydrolysis (Whatley et al, 1959;Avron, 1962), which has recently been characterized as not coupled to H+ translocation (Pick & Weiss, 1988).…”

Section: Discussionmentioning

confidence: 99%

Regulation of .DELTA.~.mu.H + -coupled ATP synthesis and hydrolysis: role of divalent cations and of the F0F1-.beta. subunit

Gromet-Elhanan¹,

Weiss²

1989

The divalent cation specificity of ATP-linked reactions catalyzed by the H+-translocating F0F! ATP synthase-ATPase complex has been followed in the Rhodospirillum rubrum chromatophore bound complex. In the presence of Mg2+ and Mn2+ the complex catalyzes ATP synthesis and hydrolysis as well as ATP-driven H+ translocation, but in the presence of Ca2+ it catalyzes only ATP hydrolysis, which is not coupled to H+ tanslocation. The inability of Ca2+ to maintain the coupling process is not due to opening of a proton leak in its presence nor to any release of p! from the membrane, because (a) an identical light-induced H+ translocation is observed in the absence or presence of Ca-ATP and (b) the Ca-ATPase, as well as the Mg-and Mn-ATPase activities, is blocked by specific F0 inhibitors. These results indicate that the divalent cations play an important role in the regulation of H+-coupled ATP synthesis and hydrolysis by the F0Fj complex. Further tests suggest that their site of action is located on the Fr/3 subunit. The isolated ß subunit of the R. rubrum FqF, has been reported to contain two nucleotide binding sites, a Mg-independent and a Mg-dependent site [Gromet-Elhanan, Z., & Khananshvili, D. (1984) Biochemistry 23, 1022-1028]. Addition of Mn2+ also enables the binding of 2 mol of ATP/mol of this isolated ß subunit. But under identical conditions, Ca2+ does not enable ATP binding to this cation-dependent site and inhibits its binding in the presence of Mg2+ or Mn2+. In light of these results we propose that the binding of Mg-ATP or -ATP, but not of Ca-ATP, to the R. rubrum ¥3-ß subunit forms the trigger that opens the pathway for H+ translocation through the FqF] complex during catalysis.Electron-transport coupled ATP synthesis in respiratory and photosynthetic organisms is catalyzed by a reversible prot Supported in part by grants from the Minerva Foundation, Munich, West Germany, and from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. ton-translocating ATP synthase-ATPase complex that is composed of two distinct structures (Senior & Wise, 1983; Merchant & Selman, 1985): a readily solubilized catalytic ATPase sector, Fb and an intrinsic membrane sector that is involved in proton fluxes, F0. The F,-ATPase from many different sources has a 3ß3• ( subunit structure and contains 0006-2960/89/0428-3645S01.50/0 © 1989 American Chemical Society

“…In all instances where it has been examined, the enzyme appears to contain five nonidentical subunits, although the stoichiometry of the subunit composition is still disputed (Baird & Hammes, 1979). In most cases, with the exception of the E. gracilis enzyme (Chang & Kahn, 1966), the ATPase activity of the enzyme is latent and is only expressed after activation by either proteolysis, heat treatment, or incubation of the enzyme in the presence of a high concentration of thiol reducing agents (Farron & Racker, 1970;Petrack et al, 1965;Vambutas & Racker, 1965). The soluble enzyme is usually assayed as a CaATPase (McCarty & Racker, 1968), although Nelson et al (1972) have described a MgATPase activity for the enzyme isolated from spinach when measured in bicarbonate or maleate buffers.…”

mentioning

confidence: 99%

Isolation, purification, and characterization of coupling factor 1 from Chlamydomonas reinhardi

Selman-Reimer¹,

Merchant²,

Selman³

1981

Chloroplast thylakoid particles were prepared from wild-type Chlamydomonas reinhardi by gentle sonication. These particles catalyzed phenazine methosulfate dependent photophosphorylation with rates ranging from 300 to 700 mumol of adenosine 5'-triphosphate (ATP) formed (mg of chlorophyll)-1h-1. Photophosphorylation was not sensitive to tentoxin but was sensitive to an anticoupling factor 1 (CF1) antiserum preparation made against spinach CF1. The C. reinhardi chloroplast CF1 was isolated from thylakoid particles by either chloroform or ethylenediaminetraacetic acid extraction. The former enzyme appeared to be missing the gamma subunit and did not reconstitute with partially resolved thylakoid particles. The latter enzyme reconstituted with partially resolved particles and had a specific activity at 37 degrees C of 2-5 umol of ATP hydrolyzed (mg of protein)-1 min-1. The enzyme utilized both MnATP and MgATP. CaATP was a poor substrate, and SrATP was not hydrolyzed. The enzyme was not activated by heat or proteolysis but was stimulated approximately 2-fold by 50 mM dithiothreitol. Alcohols reversibly stimulated the ATPase activity of the enzyme 5-25-fold. Ethanol, 20%, dramatically lowered the temperature optimum from approximately 75 to approximately 45 degrees C and slightly lowered the pH optimum from 8.5 to 8.2. Ethanol had no effect on the activation energy of the ATPase reaction (17 +/- 1.7 kcal/mol). The kinetics of the ATPase reaction catalyzed by the C. reinhardi enzyme are complex. Both free divalent cations and divalent cation ATP inhibited the activity of the enzyme. The apparent Km for MgTAP (55 uM free Mg2+) was approximately 0.2 mM.