Richard J. Berzborn

Intracellular coding sites of polypeptides associated with photosynthetic oxygen evolution of photosystem II

SummaryThree hydrophilic polypeptides of approximately 34, 23, and 16 kd located on the inner thylakoid surface are associated with the water-splitting activity of photosystem II. Stable transcripts for the three proteins were found only in cytosolic (polyadenylated) RNA, suggesting that they are encoded in nuclear genes. The immunologically reacting products synthesized in a rabbit reticulocyte cell-free translation system are larger in size than the authentic mature proteins by about 6–10 kd. These larger precursors are imported post-translationally into isolated, intact chloroplasts, and are processed to their mature forms during or after translocation. The imported proteins can be extracted from thylakoids by procedures used to isolate the three native proteins of the water-splitting complex, suggesting that they have assembled properly into their final destination, the inner thylakoid surface.

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

Abstract (1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.

Loss of function of biomembranes and solubilization of membrane proteins during freezing

Isolated thylakoid membranes are damaged during freezing in dilute salt solutions, as shown by the inactivation of photochemical thylakoid reactions. After freezing, a number of membrane proteins were found in the particle-free supernatant. Up to 5% of the total membrane protein was solubilized by freezing, and the pattern of released proteins as seen in sodium dodecyl sulfate gel electrophoretograms was influenced by the nature of the solutes present. Membranes protected by sucrose did not release much protein during freezing. Concentrated salt solutions caused protein release also in the absence of freezing. Among the proteins released were ferredoxin--NADP+ reductase, plastocyanin and coupling factor CF1. Subunits of CF1 were found in different proportions in the supernatants of thylakoid suspensions after freezing in the presence of different salts. Cyclic photophosphorylation was largely inactivated before significant protein release could be detected. It is suggested that protein release is the final consequence of the nonspecific suppression of intramembrane ionic interactions by the high ionic strength created in the vicinity of the membranes by the accumulation of salts during slow freezing. Salt effects on water structure and alterations of nonpolar membrane interactions by the incorporation of (protonated) lipophilic anions from organic salts into the membrane phase during freezing may also be involved.

A 16 kDa protein co-isolating with gap junctions from brain tissue belonging to the class of proteolipids of the vacuolar H+-ATPases

A 16 kDa protein from an enriched gap junction preparation was isolated from bovine brain tissues. N‐terminal amino acid microsequencing of the first 20 amino acids showed a complete homology with a recently published sequence of a proteolipid from a vacuolar H+‐ATPase from chromaffin granules. Incubation of the brain gap junction preparation with 14C‐N,N′‐dicyclohexylcarbodiimide showed a significant binding of this compound to the 16 kDa protein, indicating that a proton binding site also occurs within that particular protein. The data suggest that this 16 kDa protein, which has also been described in gap junction preparations from various other tissues, belongs to the proton transporting ATPase.

Zero-length crosslinking between subunits σ and I of the H+-translocating ATPase of chloroplasts

Abstract Treatment of spinach thylakoids with 1-ethyl-3-(dimethylaminopropyl)-carbdiimide (EDC) / N -hydroxysulfosuccinimide (sulfo-NHS) induced formation of a zero-length crosslink of an apparent molecular mass of 38 kDa. This product was shown, by immunodetection, to consist of subunit σ of CF 1 and subunit I of CF 0 . The crosslink was isolated by preparative SDS gel electrophoresis and subjected to cyanogen bromide cleavage. Electrophoretic and immunological analysis of the resulting peptides suggested that the crosslink was formed between a glutamyl or aspartyl residue at the C-terminal end of subunit I and a basic amino acid of subunit δ in the range between Val-1 to Met-165. Treatment of thylakoids with EDC/Sulfo-NHS resulted in inhibition of photophosphorylation and CF 0 CF 1 -catalyzed ATP hydrolysis without affecting formation of a proton gradient related to phenazine methosulfate mediated cyclic electron transport. Inhibition of H + transport-coupled ATP hydrolysis was more pronounced than non-coupled methanol-stimulated ATP hydrolysis. The results suggest that subunits δ and I form a connection between the partial complexes CF 1 and CF 0 in situ. Crosslinking of the subunits may impede the translocation of protons through CF 0 CF 1 .

Photophosphorylation: mechanism of reconstitution by coupling factor 1 (CF1).

Loss of photophosphorylation capacity by treatment of thylakoid membranes can be due to inhibition of ATP-synthetase, loss of ATP-synthetase, loss of an electron transport component, damage to the energy conserving reactions (e.g. H’ pump), or alteration of the membrane (e.g. uncoupling due to high H’ leakage). Since EDTA-treatment decreases ApH [l] and releases photosynthetic control [2,3], loss of photophosphorylation capacity may be due to uncoupling and not to a loss of ATP-synthetase itself. The discrepancy between total loss of phosphorylation capacity and only partial loss of CFr , measured as Ca”-ATPase, has been noticed [4]. Residual amount of ATP-synthetase cannot be measured directly, and residual ATPase activity needs trypsin or heat activation to become manifest. After optimal activation 720 pmol of ATP are hydrolyzed per mg Chl X h at 37°C by summer spinach, whereas with saturating light even at 22°C ATP synthesis up to 1400 pmol/mg Chl X h can be observed in the PMS system. Rebinding of CFr leads to a decrease in H’ efflux [5] and inhibition of electron flow [6] ; therefore residual ATP-synthetase may be activated by sufficient ApH again, and the added protein need not become catalytically active [7]. On the other hand thylakoids treated with silica tungsten acid, which depletes them entirely of the coupling factor as judged by ATPase activity and the presence of ‘knobs’, were reconstituted partially by a homogeneous preparation of soluble CF, [8] . We report herein that the degree of reconstitution of photosynthetic ATP-synthetase is indeed dependent

Isolation and properties of chloroplast particles of Scenedesmus obliquus D3 with high photochemical activity

Abstract An improved and standardized procedure for isolation of chloroplast particles from the unicellular green alga, Scenedesmus obliquus , D 3 , is described. The method is generally applicable to heterotrophically- and autotrophically-grown cells of Scenedesmus as well as to Chlamydomonas reinhardti and Chlorella sorokiniana (7-11-05) cultures. Chloroplast particles with high NADP + photoreducing capacity are obtained from heterotrophic cultures only when the cell types are random and the culture is in the logarithmic growth phase; maximal rates of 240–260 μmoles NADP + reduced/h per mg chlorophyll are achieved. Optimal conditions for separation of such chloroplast particles require the use of Tricine buffer (20 mM, pH 7.5), 50 nM EDTA, 10 mM KCl and 0.5 mM dithiothreitol in the breaking medium; for the maintenance of high photochemical activity it is necessary to store particles in a solution consisting of 0.4 M sucrose, 30 mM KCl and 1% bovine serum albumin. Optimum reaction conditions were developed and the properties of the isolated particles investigated. Maximal activities are obtained when the sucrose concentration is maintained below 0.4 M; the pH optimum with Tricine buffer is between 7.8–8.1; and at least 30 mM Cl − is required. Red actinic light (wavelength >620 nm) with an intensity of 10 6 ergs/cm 2 per s is required for saturation. Ferrodoxin and ferredoxin-NADP + oxidoreductase are lost from the particles during the preparatory procedures and maximum photochemical activity is attained only when they are added back in balanced amounts. Stimulatory effects of added plastocyanin and cytochrome c -553 are noted only with particles having an initially low photochemical activity.

Quantitative immunochemical evidence for identical topography of subunits CF0II and CF0I within the photosynthetic ATP-synthase of spinach chloroplasts

Monospecific polyclonal antisera against the 16 kDa and the 18 kDa polypeptides II and I from the CF0 moiety of the photosynthetic ATP‐synthase in spinach were produced. Inhibition, agglutination and absorption experiments were combined with quantitative determinations in ELISA of residual antibodies and of amounts of CF0 and CF1. It follows that subunits CF0II and CF0I are membrane anchored, extend into the matrix space, and are partially shielded underneath or within CF1. These results on the structure suggest that CF0II and CF0I are homologous to each other, and in function both homologous to E. coli F0b; thus CF0II together with CF0I would be part of the ‘stalk’.

Immunological and fluorescence studies with the coupling factor ATPase from Rhodospirillum rubrum

1. Purification of the coupling factor ATPase from Rhodospirillum rubrum has been achieved by a combination of a previously described procedure with chromatography on DEAE-Sephadex A50. 2. Identification of the coupling factor ATPase during purification, and estimation of the relative amount of the enzyme in each fraction was greatly simplified by utilization of its unusual fluorescence. 3. Preparations of R. rubrum coupling factor ATPase injected into rabbits yielded antisera which were suitable for following the course of purification. 4. Judged by immunoelectrophoretic analysis and polyacrylamide gel electrophoresis the final preparation was pure. Under standardized conditions, apparently pure preparations showed fluorescence ratios at 300/350 nm of 3-6, which are considerably higher than those reported for pure CF1 from chloroplasts. 5. The enzyme lost its activity and changed its immunological identity during prolonged storage and by treatment with urea. Antisera against urea-treated enzyme showed the presence of two distinct antigens in the modified preparations.

Electroimmunodiffusion — a Powerful Tool for Quantitative Determinations of Both Soluble and Membrane Bound Chloroplast ATPase, CF1

1. Electroimmunodiffusion can be used for routine specific quantitative microdeterminations of photosynthetic ATP-synthase. The method has been worked out for samples of both the dissociated (CF1) and the membrane bound enzyme (CF0CF1). 2. Only a monospecific antiserum against CF1 , is required for relative determinations of CF1 in any fraction. If beyond this an absolute determination is intended of either the separated or the membrane bound ATPase, in addition a pure CF1 , is needed for calibration. 3. A varying CF1/chlorophyll ratio between 1/700 and 1/1200 has been found for thylakoids of spinach plants grown under various light intensities. 4. Since the amount of CF1 , was the limiting factor of photophosphorylation capacity in the electron transport system used (PMS), from simultaneous measurement of CF1 , content and photophosphorylation capacity a turn over time of the ATPsynthase of 4 ms /ATP /CF0CF1 could be recalculated.