Chlorophyll-proteins of thylakoids from wild-type and mutants of barley (Hordeum vulgare L.)

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Sonication of barley thylakoids in a high salt buffer released three polypeptides of M r 32,000, 23,000, and 13,500 which were purified to homogeneity by chromatofocusing.Highly purified inside-out photosystem II preparations were obtained by French Press treatment or by Triton X-100 fractionation of stacked lamellar systems. Both preparations are composed of pairs of appressed membrane sheets. In the French Press preparation, the majority of these membrane pairs are sealed whereas they are predominantly unsealed in the Triton X-100 preparation. Both preparations are able to evolve oxygen and show reversed proton pumping. The oxygen evolving capacity and variable fluorescence of both preparations were lost upon washing with high salt buffer. This treatment also removed three polypeptides at M r 32,000, 23,000, and 13,500. The inactivated preparations were reconstituted with respect to oxygen evolution and variable fluorescence by rebinding of the isolated M r 23,000 polypeptide. The M r 32,000 and 13,500 polypeptides had no effect on reconstitution.Abbreviations: Chl = chlorophyll; chl-P = chlorophyll-protein; DCCD = dicyclohexyl carbodiimide: DCMU = 3-(3,4-dichlorophenyl)-l,l-dimethylurea; DCPIP = 2,6-dichlorophenol indophenol; EPR = electron paramagnetic resonance; Hepes = N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid; PPBQ = phenyl-p-benzoquinone: PS = photosystem; SDS-PAGE = sodium dodecylsulfate polyacrylamide gel electrophoresis; TMBZ = 3.Y,5,5'-tetramethylbenzidine; Tricine = N-(2-hydroxy-l,l-bis(hydroxymethyl)ethyl)glycine: Tris = tris-(hydroxymethyl)-amino methane 0105/1938/83/0048/0161/$05.00

Section: Characteristics and Function Of The Isolated M R 32000 Polymentioning

confidence: 99%

A thylakoid polypeptide involved in the reconstitution of photosynthetic oxygen evolution

Møller

Høj

1983

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“…The mutant chlorina-f2 was grown under the same conditions. Mutant and wild type thylakoids were prepared for SDS-PAGE as described in (25). Grana membranes were prepared using a modification of the Triton X-100 method of BERTHOLD et al (7) as described in (30).…”

Section: Biological Preparationsmentioning

confidence: 99%

“…The gels were run at 4 ~ using buffer system II (25). The samples were solubilized using 10 mg dodecyl-13,D-maltoside per mg Chl (final concentration 1%) and the SDS concentration in the gels was lowered to 0.03% to minimize pigment loss.…”

Section: Electrophoresismentioning

confidence: 99%

Isolation of the photosystem II reaction center complex from barley. Characterization by circular dichroism spectroscopy and amino acid sequencing

Hinz

1985

A photosystem II (PSII) reaction center complex containing Chla-protein 2, Chlo-protein 3, cytochromc b~,, the herbicide-binding polypeptide and the 33 kD polypeptide from the oxygen-evolving site of PSII was isolated by sucrose gradient centrifugation of the dodecyl-13,D-maltoside extract of grana membranes highly enriched in PSII. The reaction center complex contained ca 19 lipid molecules per reaction center and sedimented slightly faster than catalase. It mediated light-induced, DCMU-sensitive electron transport from 1,5-diphenylcarbazide to dichlorophenolindophenol.The N-terminal amino acid sequence of a proteolytic fragment of ChL-protein 2 was determined. Eighteen out of 20 amino acid residues were identical with the deduced amino acid sequence of the 51 kD ChL-apoprotein from spinach.In contrast to grana membranes, which had a 77 K fluorescence emission maximum at 685 nm with a pronounced shoulder at 695 nm, the PSII reaction center complex had a single peak at 685 nm. Circular dichroism measurements in the visible range showed that the pigments associated with the isolated PSII reaction center complex and the light-harvesting complex (LHC) were highly oriented. The secondary structure of the PSII reaction center complex and LHC was investigated by circular dichroism measurements in the ultraviolet range. Both had a high ct-helix content, suggesting that the chlorophyll molecules may be oriented between transmembrane helices in a similar way as in bacterial reaction centers and light harvesting systems. INTRODUCrlONOver the last seven years many different PSII preparations with varying degrees of complexity have been described. Preparations of grana membranes highly enriched in PSII by phase partitioning (l, 20) or 30) made it possible to study the function of PSII in a system which was close to the in vivo situation and to investigate the lateral distribution of proteins and lipids in the thylakoid membrane. Additionally, grana membranes were fractionated by extraction with various detergents folAbbreviations: Chl = chlorophyll; DCMU = 3-(3,4-dichlorophenyl)-l,l-dimethylurea; DCPIP = 2,6-dichlorophenolindopbenol; DPC = 1,5-diphenylcarbazide; HEPES = N-(2-hydroxyethyl)-piperazine-N'-2-ethane sulfonic acid; LHC = light harvesting complex; LHCII = LHC of photosystem II; PAGE = polyacrylamide gel electrophoresis; PS = photosystem; PTH = phenylthiohydantoin; SDS = sodium dodecyl sulfate; tricine = N-(tris(hydroxymethyl)methyl)glycine; Tris = tris-(hydroxymethyl)aminomethane.Springer-Verlag 0105 -1938/85/0050/0285/$02.80

“…Sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out using linear gradient gels (7.5 to 15%) as previously described (18). After electrophoretic fractionation the gels were stained with tetramethylbenzidine (TMBZ) (14,26) and subsequently with the protein stain Coomassie Blue R250.…”

Section: Electrophoretic Analysesmentioning

confidence: 99%

Reactivation of photosynthetic oxygen evolution in tris-inactivated inside-out photosystem II vesicles from spinach

Henry

Møller

Andersson

et al. 1982

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Purified inside-out photosystem II vesicles with retained capacity for oxygen evolution and a simple polypeptide composition were found to be more sensitive to washing with tris(hydroxymethyl)aminomethane (Tris) at alkaline pH than right-side out thylakoids. This suggests that the thylakoid membrane limits the penetration of the active unprotonated form of Tris.The inhibition of photosynthetic oxygen evolution by alkaline Tris-washing (pH 8.5) coincided with the release of a polypeptide with a molecular weight of 32,000 from the inside-out vesicles. Only this polypeptide was released with concentrations of Tris up to 200 mM, while higher concentrations removed additional polypeptides with a molecular weight of 34,000 and 56,000. About 70 96 of the membrane bound manganese is lost by washing the vesicles with 200 mM-Tris. Oxygen evolution was not inhibited nor were polypeptides released with Tris buffered at pH 6.5. From right-side out thylakoids the 32,000 molecular weight polypeptide could not be extracted into the ambient medium by Tris-washing.Restoration of oxygen evolution of Tris-inactivated inside-out and right-side out vesicles was possible with the aid of reduced dichlorophenol indophenol but without the addition of the 32,000 molecular weight polypeptide or manganese.Abbreviations: Chl = chlorophyll; DCMU = 3-(3,4-dichlorophenyl)-l,l-dimethyl urea; DCPIP = 2,6-dichlorophenol indophenol; Hepes = N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid; TMBZ = tetramethylbenzidine; Tris = tris(hydroxymethyl)aminomethane.