Reactivation of the Hill reaction of Tris-washed chloroplasts

Yamashita, Takashi; Tsuji, J. S.; Tomita, Giiti

doi:10.1093/oxfordjournals.pcp.a074594

Cited by 56 publications

(33 citation statements)

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“…Manganese was extracted from the chloroplast membranes either by magnesium exchange according to the procedure of Chen and Wang (1 974) or by washing in Tris-acetone according to the procedure of Yamashita and Tomita (1974). For extraction by magnesium exchange, chloroplasts were incubated in the isolation media containing MgClz at various [Mg]/[chl] ratios (0; 167: 332; 667; 2500; 10 000) for 2 h at 4 "C. The chloroplasts were then centrifuged and resuspended in the isolation medium to a final concentration of 3 mg of chl/mL.…”

Section: Methodsmentioning

confidence: 99%

“…The homogenate was filtered through four layers of cheesecloth and a 10 y m mesh nylon cloth, centrifuged, and finally resuspended in the complete medium. Chlorophyll was determined by the method of Arnon (194Y), and the suspensions were adjusted to a final concentration of 3 mg of chlorophyll (chl)/mL.Manganese was extracted from the chloroplast membranes either by magnesium exchange according to the procedure of Chen and Wang (1 974) or by washing in Tris-acetone according to the procedure of Yamashita and Tomita (1974). For extraction by magnesium exchange, chloroplasts were incubated in the isolation media containing MgClz at various [Mg]/[chl] ratios (0; 167: 332; 667; 2500; 10 000) for 2 h at 4 "C. The chloroplasts were then centrifuged and resuspended in the isolation medium to a final concentration of 3 mg of chl/mL.…”

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confidence: 99%

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Nuclear magnetic relaxation by the manganese in aqueous suspensions of chloroplasts

Wydrzynski¹,

Marks²,

Schmidt³

et al. 1978

Biochemistry

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Proton and oxygen-17 N M R relaxation rate (T1-l and T2-l) data are presented for aqueous suspensions of dark-adapted chloroplasts. It is concluded from the dependence of the proton relaxation rates (PRR) upon Mn concentration that T1-l and T2-l are determined largely by the loosely bound Mn present in the chloroplast membranes. The frequency and temperature dependences of the PRR are characteristic of Mn(I1). The effects of oxidants (e.g., ferricyanide) and reductants (e.g., tetraphenylboron) on the PRR indicate that only about one-third to one-fourth of the loosely bound Mn is present in the dark-adapted chloroplasts as T h e involvement of manganese ions in photosynthesis was first suggested in the early studies of Pirson (1937). Much later, the site of its activity was assigned to the oxygen-evolving mechanism of photosystem I1 (Kessler et al., 1957; Spence and Possingham, 1961;Cheniae and Martin, 1966;Homann, 1968). Subsequently, Kok et al. (1 970) proposed a model for oxygen evolution in which a photochemically activated intermediate accumulates up to 4 oxidizing equiv before reacting with water to produce oxygen. Manganese can exist in several oxidation states and thus it could play a central role in the charge accumulation (Cheniae, 1970; Olson, 1970;Renger, 1970;Earley, 1973). However, until recently there has been no direct experimental evidence to show that manganese changes its oxidation state during photosynthesis (Wydrzynski et al., 1976a,b).The current view is that manganese is complexed to some protein bound to the chloroplast membrane. However, attempts to isolate a manganese protein and to reconstitute oxygen evolution have failed (Cheniae and Martin, 1966; Lagoutte and Duraton, 1975;Henriques and Park, 1976). One possible exception is the stimulation of oxygen evolution by a low-molecular-weight manganese polypeptide isolated from Phormidium luridium (Tel-Or and Avron, 1974). In any event, if one wishes to study the role of manganese in oxygen evolution, it is necessary to use an experimental approach which responds to changes in situ of the manganese.The unpaired electrons of Mn(I1) can, in favorable instances, give rise to a detectable electron paramagnetic reso- Mn(II), the remainder being in a higher oxidation state@), probably Mn(II1). The frequency dependence of the PRR for the chloroplast suspensions was fitted by a simplified form of the Solomon-Bloembergen-Morgan equations, and the following parameters were obtained:s; and B = (0.9 f 0.09) X 1019. The oxygen-17 T1 and T2 data for suspensions before and after treatment with a detergent are consistent with the location of the manganese in the interior of the thylakoids. An analysis of the relaxation rates shows that the average lifetime of a water molecule inside a thylakoid is > 1 ms.nance (EPR') spectrum. However, so far there has been only one report of EPR spectra from Mn bound to chloroplasts (Siderer et al., 1977). Another possibility is that neighboring magnetic nuclei are perturbed enough by the large magnetic mom...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Nuclear magnetic relaxation by the manganese in aqueous suspensions of chloroplasts

Wydrzynski¹,

Marks²,

Schmidt³

et al. 1978

Biochemistry

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“…The numbers along the traces indicate the rate of O2 evolution expressed in ,umoles 02/mg Chl hr. (25), although O2 evolution is not completely inhibited by these treatments (27). While inhibition of electron flow by tris treatment results in a mild, reversible denaturation of components (27), (17).…”

Section: Methodsmentioning

confidence: 90%

“…(25), although O2 evolution is not completely inhibited by these treatments (27). While inhibition of electron flow by tris treatment results in a mild, reversible denaturation of components (27), (17). This suggests that digitonin may release plastocyanin from its site in the chloroplast membrane without impairing the ability of plastocyanin to participate in photosystem I electron transfer reactions.…”

Section: Methodsmentioning

confidence: 97%

The Effects of Digitonin on Photochemical Activities of Isolated Chloroplasts

Robinson

Wiskich²

1975

Plant Physiol.

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The addition of digitonin to chloroplasts stimulated the rate of oxygen evolution followed by a gradual inhibition. The effect of digitonin was dependent on the digitonin to chlorophyll ratio and on temperature and time. The initial stimulation of oxygen evolution appeared to be a result of uncoupling as digitonin did not stimulate oxygen evolution by uncoupled chloroplasts. The stimulatory effect occurred more rapidly at high digitonin to chlorophyll ratios but the extent of stimulation was low and inhibition occurred soon after addition of the detergent. The inhibition of electron flow by digitonin was due to a site of action near photosystem II which resembled the inhibition reported for tris buffer and resulted in photobleaching. However, digitonin inhibition could not be recovered by washing with reducing agents and was only partially recovered by the addition of artificial electron donors to photosystem II. Electron flow mediated by photosystem I was unaffected by the addition of digitonin but was decreased when the chloroplasts were separated by subsequent centrifuging. This suggested that digitonin solubilizes photosystem I components which remain active in the soluble form.

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“…The inactive Tris(pH 8.8)-treated chloroplasts washed with reduced DCIP were then subjected to light reactivation [8] ; the inactive chloroplasts were suspended in a medium containing 15 mM Tris(pH 7.8), 20 mM NaCl, 4 mM MgCI,, 2 mM Ca-acetate, 50 /.LM Mn-acetate and 0.5 mM dithiothreitol and exposed to weak white light (70 pW/cm') at room temperature for 20 min. The illuminated chloroplasts showed about 35% of the initial activity.…”

Section: Methodsmentioning

confidence: 99%