Preparation of Antibodies against Specific Chloroplast Membrane Polypeptides Associated with the Formation of Photosystems I and II in Chlamydomonas reinhardi y-1

Abstract

The major Chlamydomonas reinhardi y-l chloroplast membrane polypeptides-I + II, IV, V(a + b)-have been isolated by use of preparative sodium dodecyl sulfate-gel electrophoresis.Rabbit antisera prepared against these polypeptides interact with intact membranes as well as membrane fractions containing these specific antigens. Antisera against polypeptides I + II partially inactivate photosystem I. Antisera against polypeptide IV prevent inactivation of the Hill reaction and inhibit photooxidation of diphenylcarbazide with dichlorophenolindophenol as an electron acceptor. The protection of the Hill reaction by IV antiserum, measured with whole membranes, is partially hindered by the presence of V antiserum, which, by itself, causes inhibition of diphenylcarbazide photooxidation.We concluded that parts of the primary structure of these polypeptides are exposed in situ and can be recognized in whole membranes by antisera prepared against sodium dodecyl sulfate-denatured antigens. Polypeptides I and II, and polypeptides IV, Va, and Vb are associated with photosystem I and photosystem II, respectively.It has been shown that during development of photosynthetic membranes in Chlamydomonas reinhardi y-1, polypeptides of cytoplasmic origin are required for the formation of the membrane structure, although not sufficient for establishing photosynthetic activity. In order to allow photosynthetic electron flow, polypeptides of chloroplastic origin also must be synthesized and integrated into the membranes (20). In addition, the different membrane polypeptides were found to be associated with either PSI or PSII (2).Availability of antibodies specific to these polypeptides should be useful for the study of the synthesis and transport of these various polypeptides to their final location, and the process of their integration into the developing membrane.In the present work, an attempt was made to determine whether antibodies prepared against SDS-isolated membranespecific polypeptides can recognize the primary structure of the antigen in the intact membrane and affect the photosynthetic activities in which these specific polypeptides participate. MATERIALS AND METHODSCultivation of Cells and Preparation of Membranes. C. reinhardi y-I cells were grown in the light as described by Ohad et al. (21). Cell harvesting and preparation of chloroplast membrane for assays of photosynthetic activities were carried out as previously described (23). For electrophoretic separation, the membranes were further purified by centrifugation on sucrose gradients (11).Preparative Gel Electrophoresis. Fractionation of the photosynthetic membrane into different components was carried out by preparative SDS-polyacrylamide gel electrophoresis. Membrane polypeptides (25 mg) were separated on a 6-cm diameter gel, according to Lagoutte and Duranton (14). Chlorophyllprotein complexes were obtained using the methods described by Thornber and Highkin (CPI,3 CPll [26]), or Laemmli (PSI, PSII [13]). In the latter case, however, the membranes were dissol...

Presence of Polypeptides of Cytoplasmic and Chloroplastic Origin in Isolated Photoactive Preparations of Photosystems I and II in Chlamydomonas reinhardi y-1

“…Photosystem I complex was considered to consist of higher mol wt polypeptides (> 50,000) mostly of chloroplastic origin (8,13). The chlorophyll-protein I and II complexes, prepared according to Thornber and Highkin (22), have polypeptide compositions similar to those of the PSI and PSII fractions, prepared by digitonin or Triton X-100 (3,21). 3When asolectin and deoxycholate, in concentrations used for fractionation, were added to whole membranes during the activity assay, photoreduction rate of methyl viologen was slightly enhanced, while that of DCIP was reduced to 25%.…”

White Light Effects on the mRNA for the Light-Harvesting Chlorophyll a/b-Protein in Lemna gibba L. G-3

“…The Chl a/b-protein precursor polypeptide was identified in translation products coded for by mRNA isolated from L. gibba by using the antiserum to this protein from Chlamydomonas (21). A single band of mol wt 32,000 was precipitated from the translation products of L. gibba poly(A) RNA (Fig.…”

Contribution to the structural characterization of eucaryotic PSI reaction centre ? II. Characterization of a highly purified photoactive SDS-CP1 complex

“…Incubation with/3-mercaptoethanol after heating reveals a weaker and very diffuse band of 62kD, just below the main 65kD component [20]. The conditions of dissociation of this complex (and also the procedure of its isolation) seems to affect the number and MW of the polypeptides comprised in it [4,5,8,15,21,28,31,32]. There are also disturbing results concerning the action of reducing agents (like DTT).…”