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A chlorophyll (a + b)--protein complex associated with photosystem I (PSI) was isolated from a larger PSI complex (CPIa) produced by electrophoresis of barley thylakoids solubilized with 300 mM octyl glucoside. It had an apparent Mr of 35,000-43,000 on 7.5% and 10% acrylamide gels respectively, and a chlorophyll a/b ratio of 2.5 +/- 1.5. Denaturation released four polypeptides migrating between 21-24 kDa. They were well separated from the polypeptides of the two photosystem II chlorophyll a + b antenna complexes: LHCII (25-27 kDa) and CP29 (28-29 kDa). In order to study the PSI antenna complex, antibodies were raised against highly purified CPIa. The antigen appeared to be pure when electrophoresed, blotted and reacted with its antiserum, i.e. anti-CPIa detected only the 64-66-kDa CPI apoprotein and the four 21-24 kDa antenna polypeptides. However, when blotted against the whole spectrum of thylakoid proteins, it cross-reacted with both LHCII and CP29 apoproteins. Removal of anti-CPI activity from the anti-CPIa did not affect these cross-reactions, showing that they were not due to antibodies directed against CPI. To show that the same antibody population was reacting with both the photosystem I and photosystem II antenna polypeptides, anti-CPIa was adsorbed onto highly purified CPIa on nitrocellulose. The bound antibody was eluted and used again in a Western blot against whole thylakoid proteins. This selected antibody population showed the same relative strength of reaction with photosystem I and photosystem II antenna polypeptides as the original antibody population had. Similar observations have been made with antibodies to the two photosystem II antenna complexes. We therefore conclude that there are antigenic determinants in common among the chlorophyll a + b binding polypeptides, and predict that there could be amino acid sequence similarities.

Isolated chloroplasts of Acetabularia incorporate radioactive amino acids into more than 30 polypeptides in the light, including the apoprotein of the P700-chlorophyll a protein complex, the reaction centre core of photosystem I [Biochim. Biophys. Acta, 609. 107-120 (1980)]. In this paper it is shown that the apoproteins of the two minor chlorophyll a complexes, thought to be part of photosystem II reaction centre core, are also synthesized by isolated chloroplasts. Furthermore, they are integrated correctly into the thylakoid membrane in the absence of any cytoplasmic contribution, such that they can be isolated as chlorophyll-protein complexes indistinguishable from those already in the membrane. In contrast, the minor chlorophyll a + b complex 'CP 29' [Camm, E. L. and Green, B. R. (1980) Plant Physiol. 66, 428-432] and its dimers are not synthesized by isolated chloroplasts. In this they resemble the other chlorophyll a + b complex, the light-harvesting complex (LHC). It may be significant that the LHC, which is not essential for photosynthetic activity, is under nuclear control, while the reaction centre polypeptides, cytochrome b559, and cytochrome f, are synthesized on chloroplast ribosomes.

1. Wheat chloroplast coupling factor (CF1) was extracted with a modification of the chloroform extraction method of Younis et al. (J. Biol. Chem. 252, 1814--1818, 1977). A one-step purification on an 8--25% sucrose gradient yielded a CF1 which was at least 98% pure as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. 2. Inclusion of proteolysis inhibitors during extraction and purification consistently gave a CF1 containing all five subunits. Selective loss of the sigma and epsilon subunits was observed when proteolysis inhibitors were omitted. 3. Proteolysis inhibitors prevented the release of wheat CF1 from the thylakoid by the low-ionic-strength wash method of Strotmann et al. (Biochim. Biophys. Acta, 314, 202--210, 1973). The enzyme extracted with chloroform and low ionic strength were compared by electrophoresis and no evidence of a difference in molecular weight of any subunit was observed. This suggests that a proteolytic event is not required for release of wheat CF1 by the low-ionic-strength method, even though release is inhibited by proteolysis inhibitors. 4. The gamma subunit of wheat CF1 probably contains at least one internal disulfide bridge, as the electrophoretic mobility of this subunit is lower in the presence of reducing agent than in its absence. 5. Wheat CF1 was viewed by the electron microscope after negative staining. Discrete particles, many appearing hexagonal, were observed at high magnifications. Markham rotational analysis confirmed that the enzyme has sixfold symmetry in at least one of its orientations.

Polypeptides of the three major chlorophyll a + b protein complexes were detected in a chlorophyll-b-less barley mutant (chlorina f2) using immunological techniques. Antibodies to CP Ia, a photosystem I complex containing both the reaction center (CP I) and the chlorophyll a + b antenna (LHCI), detected substantial amounts of LHCI polypeptides in mutant thylakoids. Some polypeptides of the two photosystem-II-associated chlorophyll a + b complexes, CP 29 and LHCII, were also detected using antibodies raised against these complexes. The CP 29 apoprotein and the minor 25-kDa polypeptide of LHCII were present in amounts that could be seen by Coomassie blue staining. In contrast, the two major polypeptides of LHCII were greatly diminished in amount, and one of them may be completely absent. These data suggest that the absence of chlorophyll b may have differing effects on the synthesis, processing or turnover of the various chlorophyll a + b binding polypeptides. They also show that these polypeptides can be inserted into thylakoids in the absence of Chl b, and that significant amounts of some of them are accumulated in the mutant thylakoids.