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This report describes the isolation and preliminary characterization of a new pigment-protein complex from the chromophyte alga, Ochromonas danica. The pigment-protein complex was obtained by extracting a thylakoid membrane preparation with the zwitterionic detergent lauryldimethylamine oxide followed by ultracentrifugation on sucrose gradients. The pigment-protein complex has been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, absorption spectroscopy, and low temperature (77 Kelvin) chlorophyll fluorescence spectroscopy. A polypeptide with a monomeric molecular weight of 31,000 as determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was the major constituent of this pigment-protein complex. The major pigment in this complex was chlorophyll a, although an as yet unidentified carotenoid was also present. There was no evidence for the presence of chlorophyll c.

The phosphorylation of thylakoid membranes in the Chromophyte alga Ochromonas danica was studied in whole cells and in vitro. Protein kinase activity was observed in the thylakoid fraction, and several membrane-bound polypeptides were found to be phosphorylated. The thylakoid protein kinase demonstrated several unusual regulatory properties. Both the polypeptides that were phosphorylated and the rate of protein phosphorylation were independent of illumination. Protein kinase activity was also unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron. The kinase activity was inhibited under strong reducing conditions. Whole cells labeled with (32)PO(4) (3-) were converted to light states I and II by pre-illumination favoring photosystem I or photosystem II, respectively. Analysis of the phosphoproteins from cells in state I and state II showed that no changes in phosphorylation accompanied the change in energy redistribution.

The assimilation of CO 2 by etiolated Hordeum vulgare seedlings during an illumination period indicates a conversion of the organisms to autotrophy. After 1 hour illumination, increases in the photo-assimilation of CO 2 are observed and the distribution of C 14 in the soluble fraction of the plants is predominantly in intermediates of the Calvin cycle.

The chlorophyll-containing lamellae within higher plant chloroplasts are the site of the light reactions and associated electron transport reactions of the photosynthetic process (12, 19). Investigations on the chemical composition of purified spinach chloroplast lamellae have shown that they are about 50 % protein and 50 % lipid (13). Of the 2 fractions, the lipid has been studied the most extensively owing to the considerable interest in the photosynthetic pigments. and the majority of the fraction can be accounted for by known compounds (8, 11). The protein fraction contains the 2 cytochromes, f anid b,; and the transitioln metals iron (nonheme), manigalnese and copper (13). This fraction has received very little direct attention, presumably owing to the extreme insolubility of the material at phvsiological pH and the lack of appropriate enzymological tools to cope with the problem. Davenport and Hill (3) purified cytochromle f from an ammoniacal ethanolic extract of fresh parsley leaves by ammonium sulfate fractionation and calcium phosphate gel adsorption. The pure protein was shown to be MW = 110,000 and contained 2 hemes. Indirect evidence concerning the properties of the protein fraction comes from work primarily designed to investigate the nature of the chlorophyll-protein link in attempts to elucidate the state of chlorophyll in vivo (14,15). For this purpose a wide variety of detergents and organic solvents have been used to solubilize the membranes of chloroplasts. It was decided that a similar use of detergents for the removal of lipid from the lamelflae and solubilizationi of the protein would be a fruitful approach to the studv of the chloroplast protein fraction. These methods have also been shown to be of great value in the study of the mitochonidrion where similar problems of protein insolubility prevail (5). This report describes the preparation and some physical properties of the protein fraction of chlloroplast lamellae in an attempt to derive informiiationl pertinent to the molecular architecture of the photosynthetic membrane.

Reactions of photosynthetic electron transport and photophosphorylation were studied in preparations from the blue-green alga, Phormidium luridum. Osmotic lysis of protoplasts proved to be a superior technique for the production of cell-free preparations with high enzymatic activity. Such lysed protoplasts sustain high rates of photophosphorylation coupled to the photo-reduction of NADP(+) or ferricyanide. P/2e(-) ratios close to unity were routinely observed. The same preparations, and also those prepared by grinding the cells in solutions containing sucrose or ethylene glycol, are active in cyclic photophosphorylation mediated by phenazine methosulfate or dichloro-phenolindophenol. The particles prepared by grinding the cells are, however, inactive in non-cyclic photophosphorylation.Extensive washing of the membranes with solutions containing sucrose removes the majority of the residual soluble fraction of the algal cell which includes cytochromes C(554) and C(549) and phycocyanin. Cyclic photophosphorylation activity is unimpaired by this treatment, but is abolished when the membranes are washed with very dilute buffers. This activity is restored by the addition of a soluble protein which is not a known redox constituent such as cytochrome C(554) or plastocyanin, and may be a coupling factor.Analysis of the well-washed membranes by low temperature (77 degrees K) difference spectrophotometry reveals the presence of cytochrome b(6) and a bound form of cytochrome C(554) in proportions similar to that found in higher plant chloroplasts. The concentration of the membrane-bound cytochrome C(554), relative to cytochrome b(6) is not altered by extensive washing, sonication or treatment with 1% digitonin. This indicates that this cytochrome is an integral component of the cytoplasmic lamellae and we suggest that it is of functional significance. The soluble form of cytochrome C(554), which is present in concentrations about 3-fold higher than the bound form, depending upon growth conditions, is not essential for cyclic photophosphorylation. The concentration of cytochrome b(6): chlorophyll a was found to be 1:500.Under the conditions employed, we were unable to detect a bound form of the low potential cytochrome C(549).