Studies on the Reconstitution of O₂-Evolution of Chloroplasts

Abstract

Extraction of spinach (Spinacia oleracea L.) chloroplasts with cholateasolectin in the absence of Mge+ results in the rapid and selective inactivation of 02 evolution and a partial (30 to 40%) loss of photosystem II (PSII) donor activity without extraction of thylakoid bound Mn (-5 to 6 Mn per 400 Chlorophyll). Inclusion of ethylene glycol in the extractions inhibits loss of 02 evolution and results in quantitative and qualitative differences in proteins solubilized but does not significantly inhibit the partial loss of PSII donor activity. Similarly, in two stage experiments (extraction followed by adtion oforganic solvent and solubilized thylakold protein), 02 evolution (V and V..) of extracted chlorplasts is enhanced approximately 2.5-to 8-fold. However, PSII donor activity remains unaffected. This reversal of cholate inactivation of 02 evolution can be induced by solvents including ethanol, methanol, 2-propanol, and dinethyl sulfoxide.Such enhancements of 02 evolution specificaly required cholate-solubilized proteins, which are insensitive to NH2OH and are only moderately heat-labile. NH2OH extraction of chloroplasts prior to cholate-asolectin extraction abolishes reconstitutablilty of 02 evolution. Thus, the protein(s) affecting reconstitution is unlike those of the 02 .M enzyme. eThe specific activity of the protein fraction effecting reconstitution of 03 evolution is greatest in fractions depleted of the reported Mn-containg, 65-kilodalton, and the Fe-heme, 232-kilodalton (58-kilWalton monomer), proteins. Divalent (-3 millimlar) and monovalent (-30 illimhr) cations do not affect reconstitution of PSII donor activity but do affect reconstitution of 02 evolution by decreasing the protein(s) concentration required for reconstitution of 02 evolution in nonfractionated, cholate-asolectin extractions. The data indicate a reconstitution of the PSII segment inkig the PSII secondary donor(s) to 0revolving centers.Considerable information is available concerning the kinetic aspects of 02 evolution (37) and the intermediates linking the Sstates of 02 evolution to P60 (6). Additionally, much evidence has accumulated which strongly suggests that thylakoid-bound Mn is essential in the 02-evolving reactions (37) and that such Mn undergoes oxidation-reduction during cycling of the S-states (14,40,51).By and large, however, this segment of the electron transport sequence of photosynthesis, in contrast to PSI, has been rather intractable to biochemical approaches aimed at the isolation and characterization of the proteinaceous catalysts involved (37). Such information is vital to the basic understanding of this biologically unique sequence of reactions, namely, the photosynthetic production of 02-Recently, two different approaches have been used in attempts to gain such insights: analyses of polypeptide composition and thylakoid Mn abundance of mutants blocked specifically on the oxidizing side of PSII (32, 33, 41); and detergent extraction and reconstitution analyses of chloroplasts with the use of artificial lipo...

Relocating a gene for herbicide tolerance: A chloroplast gene is converted into a nuclear gene

“…Sci. USA 8S (1988) or after import of the proteins into the organelle (11 and the coding region for the transit peptide (M) and two amino acids of the mature SSU (S) (see also ref. 5).…”

“…Thylakoid membranes were isolated from tobacco plants (11). The membrane proteins were electrophoresed at 4°C in a NaDodSO4/polyacrylamide gel (12.5%) and electrotransferred to nitrocellulose paper (12).…”

A thylakoid polypeptide involved in the reconstitution of photosynthetic oxygen evolution

“…Although the results of this study could not be repeated (42,43), it is interesting to note that an M r 23,000 polypeptide constitutes a major component during the initial purification steps of the M r 65,000 polypeptide. Its presence as a minor impurity in the preparation used for the rebinding experiments may have resulted in the reported reconstitution.…”

“…SPECTOR and WINGET (45) reported the isolation of a cholate-extractable Tris-sensitive manganese-containing polypeptide of M r 65,000 which reconstituted oxygen evolution when added to the cholate-extracted thylakoid material after its incorporation into artificial liposomes. SAYRE and CHENIAE (42,43) were unable to isolate such a manganese-containing polypeptide.…”

Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed inEscherichia coli

“…Miranda) and spinach (Spinacia oleracea L. cv. Monatoi; Baywa, Mfinchen, FRG) thylakoids were isolated following the procedure of Sayre and Cheniae (1982). Thylakoids were solubilized at 4 ~ C by adding 100 mM Tris-HC1 (pH 9), 5 mM 6-aminocaproic acid, I mM benzamidine, 12.5% sucrose, 2% sodium dodecyl sulfate (SDS) at an SDS :Chl weight ratio of 10:1.…”