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Experimental Section
- The New Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contraction, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease

8.1. Isolation of Thylakoid Membranes

Thylakoid membranes were isolated from spinach (Spinacia oleracea) leaves purchased from the local market by the method of Tripathy and Mohanty [88], as described earlier [11]. Briefly, spinach leaves were purchased and kept refrigerated in darkness to maintain chloroplast activity. Leaves were first deveined and a total of about 50 g of spinach leaves were washed with distilled water, kept at 4 oC, soaked on filter paper and then homogenized in a Waring blender for 40 s together with the isolation buffer (Sorbitol 0.4 M, Tris 0.05 M, EDTA 1mM, MgCl2 1 mM, adjusted to the pH 7.3) in the ratio of 1:8 w/v of spinach leaves to isolation buffer. The mixture obtained was filtered through 8 layers of cheesecloth and one layer of miracloth. The filtrate obtained was centrifuged at 1170 g for 7 min at 4 oC. The supernatant obtained was discarded and the pellet which contained chloroplast, broken cell wall, starch grains was suspended in hypotonic TE buffer (Tricine 0.01 M and EDTA 1 mM, pH 7.5) in the ratio of 5:1 w/v of spinach leaves taken initially and kept in ice for 15 min. The suspension was again centrifuged at 4650 g for 5 min at 4 oC. The supernatant was carefully discarded and the pellet containing thylakoid membrane along with cell debris and starch layer was dissolved in a minimal volume of hypertonic suspension buffer (Sorbitol 0.4 mM, Tris 0.05 M, EDTA 1 mM and MgCl2 1 mM, pH 7.5) in 5:1 w/v of spinach leaves and finally stored at -80 oC until used (for a maximum of 24 h). The whole procedure was carried out in the dark.

8.2. Estimation of Chlorophyll Content

The chlorophyll content was measured according to the method of Arnon [89]. The thylakoid membrane suspension (100 μL) was suspended in 80 % (v/v) acetone (20 mL) and filtered in the dark through Whatman no.1 filter paper. The optical density of the filtrate was determined at 652 nm against a blank of 80 % (v/v) acetone in a quartz cuvette and multiplied by a factor of 5.8 to give the chlorophyll concentration in mg/mL. The chlorophyll concentration was adjusted to 0.5 mg/mL with suspension buffer and was used for carrying out phosphorylation as described next.

8.3. Measurement of ATP Synthesis

The estimation of rate of ATP synthesis was carried out by phosphorylation of ADP by the technique of ‘acid-base’ transition [90]. The method was based on microcolorimetric estimation [91] of removal of phosphorus from the mixture, which has utilized for the formation of ATP from ADP. The classical, ‘acid –base’ transition scheme involves two steps: acidic stage and basic stage. In acid stage (AS) 0.5 mL of thylakoid membranes were incubated with 0.9 mL of acid (pH 4.0) for the desired time in order to accumulate the acid of interest. This was then transferred to 0.9 mL basic stage (BS) buffer (Tricine 100 μmoles, adenosine diphosphate 0.2 μmoles, KH2PO4 2.0 μmoles, MgCl2 5.0 μmoles, and NaOH 19.6 μmoles, pH 8.3) by 1:1 dilution, consequently allowing diffusion through the membrane from inside to out according to the concentration gradient of the species for catalytic synthesis of ATP via the ATP synthase complex. The reaction was terminated by the addition of 200 μL of 20 % (w/v) trichloroacetic acid (TCA). For the control, TCA was added in the base stage (prior to incubation with thylakoid membranes). The whole suspension (2.0 mL) was taken in two different tubes (1 mL each) and centrifuged for 10 min at 7270 g. Transfer of thylakoid membranes was carried out with 1 mL glass syringe attached with 20-gauge cannula, and the entire procedure was carried out in complete darkness. Inhibition studies were carried out with the inhibitor 4,4’-diisothiocyanostilbene- 2,2’-disulphonic acid (DIDS) added during the preparation of base stage buffer at different concentrations required.

8.4. Estimation and Calculation of Rates of ATP Synthesis

50 μl aliquot from each reaction mixture after centrifugation was mixed with 500 μl of colorimetric reagent (prepared freshly) as given earlier [91]. The OD at 660 nm was measured against sample blank for each reading. The decrease in phosphate concentration in test samples to that of control measured the ATP produced. The rate of ATP synthesis was calculated in μmoles of ATP produced (mg of chlorophyll)-1 min-1. Selected readings were also verified using ATP kit and luciferin-luciferase assay.

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