Antibodies to the photosystem I chlorophyll a+b antenna cross-react with polypeptides of CP29 and LHCII

Abstract

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 M , of 35000-43000 on 7.5% and 10% acrylamide gels respectively, and a chlorophyll a/b ratio of 2.5 f 1.5. Denaturation released four polypeptides migrating between 21 -24 kDa. They were well separated from the polypeptides of the two photosystem I1 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 I1 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 I1 antenna polypeptides as the original antibody population had.Similar observations have been made with antibodies to the two photosystem I1 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.All the chlorophyll (Chl) in thylakoid membranes is bound to specific proteins [I] to form chlorophyll-protein (CP) complexes. There are three Chl-a-containing complexes known as CPI, CP47 and CP43. They are closely associated with reaction centers and their polypeptides are synthesized in the chloroplast [2-61. There are also at least three Chla+b-containing complexes known as LHCII, CP29 and the PSI antenna complex or LHCI (see [7 -91) antenna (Chl a/b = 3-4) associated with PSI [S, 161. There are several versions of LHCI (or PSI antenna) containing between one and four polypeptides, depending on the isolation method used [9,[17][18][19][20][21][22][23][24]. In this paper, a simple method requiring only octyl glucoside solubilization and gel electrophoresis is used to isolate a PSI antenna-CP complex containing four polypeptides in the 21 -24-kDa range. It is isolated from CPIa, a PSI complex which consists of CPI (the P700 reaction center complex of PSI) and the PSI antenna complex. Polyclonal antibodies to this complex were found to cross-react with polypeptides of the two PSII Chl a + b protein complexes, LHCII and CP29. MATERIALS AND METHODSPreparation and deter...

Polypeptides belonging to each of the three major chlorophyll a + b protein complexes are present in a chlorophyll-b-less barley mutant

“…Rabbit polyclonal antibodies to the purified barley CPs were raised as previously described [25,271. All samples used for Western blotting were heat-denatured in 2% SDS, 50 mM dithiothreitol, 65 mM Tris/HCI, pH 7.4 at 65°C for 30 min before electrophoresis.…”

“…Bonanza) and chlorina f2 barley were grown in a greenhouse in potting soil for 7 -10 days. Washed thylakoids were prepared from leaves as previously described [25].…”

“…Gels were electroblotted onto nitrocellulose, blocked using 3% (v/v) fish skin gelatin (Norland Hipure), incubated with the appropriate antiserum, washed and then incubated in affinity-purified goat anti-rabbit serum conjugated to alkaline phosphatase [16]. After further washing, blots were developed in naphthol AS MX phosphate, disodium salt (O.loh w/v) and fast red TR salt (0.2% w/v) which produced pink bands [25]. Silver staining of polyacrylamide gels after blotting did not show any remaining protein.…”

“…The PSI antenna polypeptides were detected with antisera prepared to the PSI complex CP I a purified by two or more electrophoretic cycles (see [25]). Purified CP I a contained only CP I and the PSI antenna polypeptides [25].…”

Probing in vitro translation products with monoclonal antibodies to chlorophylla/b-binding proteins of barley thylakoids

“…There have been reported polyclonal antibodies to LHCI that react with LHCII (6) and monoclonal antibodies raised to LHCII that reacts with LHCI (5). There have also been raised both mono-and polycional antibodies to LHCI, that react with Chla/h-Pl and vice versa (14,26).…”