Electron paramagnetic resonance spectrometry of photosystem I mutants in barley

Monoclonal antibodies have been produced to the two putative 18.3 and 15.2 kD iron-sulphur centre proteins in barley. When photosystem I particles, containing only chlorophyll a-protein 1 and the 18.3 and 15.2 kD proteins, were used as the antigen, thirteen independent clones secreting either IgG or IgM antibodies against the 15.2 kD protein were formed. No hybridomas secreting antibodies to the 18.3 kD protein were detected. When a delipidated polypeptide preparation was the immunogen, we obtained one hybridoma, secreting antibodies of the IgM class to the 18.3 kD protein.These antibodies were used in immune-blot assays. Neither the 18.3 nor 15.2 kD putative iron-sulphur centre proteins were detected in etioplast membranes. However, in the chloroplast membranes of the photosystem I mutant viridis-zb ~' we found small amounts of these proteins.The two proteins were purified by electroelution and then tested by immune-blot assay. Fractions with positive reaction were collected and the amino acid compositions determined. There were significant differences in the amounts ofserine, glycine and histidine between the two proteins.

Section: Introductionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Monoclonal antibodies used for the characterization of the two putative iron-sulphur centre proteins associated with photosystem I

Høyer‐Hansen

Hønberg

Simpson

1985

“…Unique sets of polypeptides characterize the grana membranes containing photosystem II (4,10,27,28,38,39,72) and the stroma lamellae containing photosystem I together with the coupling factor (9,27,40,41,42). There are indications that the individual pigments and acyl lipids of the thylakoids are likewise specifically associated with certain membrane fractions and individual membrane proteins.…”

Section: Introductionmentioning

confidence: 99%

Pigment and acyl lipid composition of photosystem I and II vesicles and of photosynthetic mutants in barley

Henry

Mikkelsen

Møller

1983

Self Cite

The pigment and acyl lipid composition ofphotochemically active stroma lamellae and inside-out photosystem II vesicles have been investigated. Mechanical disruption followed by phase partitioning in an aqueous two phase system resulted in a clean separation of the photosystems. A qualitative and quantitative determination of the pigment content was achieved using reversed-phase high performance liquid chromatography. A single chromatographic separation provided adequate resolution of neoxanthin, violaxanthin, lutein, chlorophyll b, chlorophyll a, and 13-carotene and was completed in 22 min. For each molecule of P700, the stroma lamellae contained 166 molecules of chlorophyll a, 14 molecules of chlorophyll b, 0 molecules of neoxanthin, 3 molecules of violaxanthin, 7 molecules of lutein, and 13 molecules of 13-carotene. For each molecule of P680 the photosystem II vesicles contained 250 molecules of chlorophyll a, 100 molecules of chlorophyll b, 7 molecules of neoxanthin, 7 molecules of violaxanthin, 22 molecules of lutein, and 4 molecules of l~-carotene. The pigment content of three photosynthetic mutants chlorina-[2, viridis-zb63 and viridis-zd69 was determined. On a chlorophyll basis, these mutants generally showed a reduced carotenoid content, except that chlorina-f2 had an increased level ofl3-earotene and viridis-zd69 an unaltered level of lutein.The acyl lipid composition of stroma lamellae, photosystem II vesicles and lamellar systems was also determined. Although the photochemical activities of the stroma lamellae and photosystem II vesicles were enriched compared to the parent lamellar system, more than 50% of the acyl lipids in the subchloroplastic membranes were degraded or severely modified resulting in a drastically reduced content of monogalactosyldiglyceride and digalactosyldiglyceride in the stroma lamellae and photosystem II vesicles. Due to the lipolytic degradation the level of saturation in the gatactolipids was higher in stroma lamellae and photosystem II vesicles compared with the lamellar system. Stroma lamellae do not contain the light-harvesting chlorophyll a/b-protein, but were enriched in phosphatidylglycerol and trans-3-hexadecenoic acid.Abbreviations: Chl = chlorophyll; ChI-P = chlorophyll-protein; DG = digalactosyldiglyceride; GLC = gas-liquid chromatography; HPLC = high-performance liquid chromatography; MG = monogalactosyldiglyceride; PC = phosphatidylcholine; PG = phosphatidylglycerol; PI = phosphatidylinositol; SL = sulfoquinovosyldiglyceride; TLC = thin layer chromatography.

“…The nuclear gene mutant viridis-zb 63 has been shown to completely lack PSI activity (14) and the associated EPR signals (32). In the polypep-tide pattern of the mutant ( 14, 21, 31) Figure 5, lanes 1 and 3) showed a decrease of translated polypeptides in the 25-15 kD range in the mutant.…”

Section: Presence Of Transcripts Of the 152 Kd Polypeptide In The Psmentioning

confidence: 98%

Probing in vitro translation products with monoclonal antibodies to a 15.2 kD polypeptide subunit of photosystem I

Høyer‐Hansen

Hønberg

Høj

1985

Three monoclonal antibodies to the 15.2 kD polypeptide subunit of a barley photosystem I (PSI) particle have been characterized. Immune-blot assays showed that there are at least two different antigenic sites on the polypeptide. The antibodies were employed for immunological identification ofpolypeptides translated in vitro in an m RNA-dependent cell-free rabbit reticuloeyte lysate. The IgG's, CMpI5.2:I and CMp 15.2:2, precipitated a polypeptide of molecular weight 23 kD from in vitro translates primed with poly A*RNA but not when chloroplast RNA from greening barley was used. No 23 kD precipitation band was found, if these antibodies were mixed with a PSI particle preparation before they were added to the translate. We conclude that the putative iron-sulphur centre binding 15.2 kD PSI subunit is synthesized on cytoplasmic ribosomes as a 23 kD precursor, and is coded by nuclear DNA. Transcripts for the 15.2 kD polypeptide were also found among the poly A § of the PSI mutant viridis-zb 6~.