Chloroplasts proton uptake

Other optional experiments may be completed if time allows. For example, the effectiveness of various redox dyes may be analyzed. In addition to those listed in the text, FMN, ferricyanide, and dichlorophenolindophe-nol may be tested (Neumann and Jagendorf, 1964). It has been shown that NH4C1 and amines stimulate proton uptake. If a potassium ion-specific electrode is available, the light-induced efflux of K+ from spinach chloroplasts may be studied (Dilley, 1972). 

Experimental arrangement for measuring proton uptake by illuminated chloroplasts. See text for details. 

When chloroplasts are pre-illuminated in the absence of a phosphorylation component under conditions in which massive proton uptake occurs, ATP synthesis can be observed in the dark in the post-illumination period when the missing component is added [17]. The amount of ATP synthesized is a function of the number of protons stored in the inner thylakoid compartment during the pre-illumination period, which is in turn a function of the intrathylakoid buffer capacity, and the pH gradient sustained [14]. 

The respiratory and photosynthetic electron-transfer pathways are proton pumps operating with the same polarity as does the A TP synthase when hydrolyzing A TP Since it is difficult to detect protons circulating in the steady-state, Mitchell and Moyle [19] studied the transient extrusion of protons when a small amount of oxygen is injected into an anaerobic incubation of mitochondria in the presence of substrate. Prior to this, Neumann and Jagendorf [20] had observed a light-dependent proton uptake into chloroplast thylakoid membranes. 

The digitonin preparation of PSI-RC has also been demonstrated to be able to catalyze a light-induced proton uptake when incorporated in phospholipid liposomes and illuminated in the presence of ascorbate and phenazine methosulphate [47] incorporation of chloroplast ATPase in the same system yielded the reconstitution of photophosphorylation in a model system. The PSI-RC preparation therefore seems to possess all the functional features of PSI for the vectorial transmembrane electron transfer [48] (see Fig. 4.7). 

Pea plants were grown in a growth chamber at 20-25 C, in an 8/16 hour light/dark regime. The illuminance was about 50 /imol quanta.m . s . Class C chloroplasts were prepared as already described [4] and stored in liquid nitrogen in resuspension medium plus 5% dimethylsulphoxide, or kept on ice for more immediate use. There was little difference in results from either source of chloroplasts. bf complex was prepared by a modification [5] of the method of Hurt and Hauska [6]. Measurements of proton uptake, cytochrome kinetics and carotenoid bandshift were as in [7]. 

FIGURE 4- Relative extent of proton uptake which beceme undetectible upon mild extraction of four-subunit CF1 by EDTA as function of the medium pH. Circles (spinach) and squares (pea chloroplasts) experimental, curves calculated for a hexacooperative buffer according to the following equation ... 

When outer membrane proteins were modified by trypsin treatment, the plastoquinone acceptor Q became accessible to externally added ferricyanide (G.Renger, 1976). From this a "proteinaceous shield" covering the reducing side of PSII was apparent. Since tryptic attack predominantly modified the DCMU-binding 32 kD protein, Mattoo et al.(l98l) speculated that this protein might fullfill the requirements of the diffusion barrier. However, proton uptake was not observed in trypsinated chloroplasts (G.Renger, R.Tiemann,1979) It remained to be documented whether the diffusion barrier for ferricyanide was identical with the diffusion barrier for proton uptake or not. 

F-ATPases (including the H+- or Na+-translocating subfamilies F-type, V-type and A-type ATPase) are found in eukaryotic mitochondria and chloroplasts, in bacteria and in Archaea. As multi-subunit complexes with three to 13 dissimilar subunits, they are embedded in the membrane and involved in primary energy conversion. Although extensively studied at the molecular level, the F-ATPases will not be discussed here in detail, since their main function is not the uptake of nutrients but the synthesis of ATP ( ATP synthase ) [127-130]. For example, synthesis of ATP is mediated by bacterial F-type ATPases when protons flow through the complex down the proton electrochemical gradient. Operating in the opposite direction, the ATPases pump 3 4 H+ and/or 3Na+ out of the cell per ATP hydrolysed. 

Fig. 3. Primary carbon metabolism in a photosynthetic C3 leaf. An abbreviated depiction of foliar C02 uptake, chloroplastic light-reactions, chloroplastic carbon fixation (Calvin cycle), chloroplastic starch synthesis, cytosolic sucrose synthesis, cytosolic glycolysis, mitochondrial citric acid cycle, and mitochondrial electron transport. The photorespiration cycle spans reactions localized in the chloroplast, the peroxisome, and the mitochondria. Stacked green ovals (chloroplast) represent thylakoid membranes. Dashed arrows near figure top represent the C02 diffusion path from the atmosphere (Ca), into the leaf intercellular airspace (Ci), and into the stroma of the chloroplast (Cc).SoHd black arrows represent biochemical reactions. Enzyme names and some substrates and biochemical steps have been omitted for simplicity. The dotted line in the mitochondria represents the electron transport pathway. Energy equivalent intermediates (e.g., ADP, UTP, inorganic phosphate Pi) and reducing equivalents (e.g., NADPH, FADH2, NADH) are labeled in red. Membrane transporters Aqp (CO2 conducting aquaporins) and TPT (triose phosphate transporter) are labeled in italics. Mitochondrial irmer-membrane electron transport and proton transport proteins are labeled in small case italics.

There are several lines of argument to support the notion of the nonuniform distribution of transmembrane pH gradients. One of them arises from the ApH measurements, while another one is from the comparison between the time-courses of proton accumulation inside the thylakoids during cyclic and noncyclic electron transport. The kinetic study of protons and TA uptake enables us to discriminate between the events associated with loading the protonic pools of grana- and stroma-exposed thylakoids. Comparing the uptake of protons and spin label TA by chloroplasts suspended in the media with different osmomolarity (and thus having different internal volumes), we... 

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