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

Høyer‐Hansen, Gunilla; Hønberg, Lisbeth Skou; Bassi, Roberto

doi:10.1007/bf02904435

Cited by 4 publications

(1 citation statement)

References 22 publications

(28 reference statements)

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“…In vitro translation of the mRNA was carried out for 60 min at 30°C in rabbit reticulocyte lysate using [35S]methionine (1200 Cdmmol, 0.8 mCi/ml final). A sample of the translation product was analysed on SDSPAGE and the remainder subjected to immunoprecipitation [17] with antibodies against the 26-kDa subunit of ornithine acetyltransferase. The immunoprecipitates were analysed by SDSPAGE and phosphorimager analysis.…”

Section: Immunoprecipitation Of In Vitro Translated Yeast Proteinsmentioning

confidence: 99%

Purification and Characterization of Ornithine Acetyltransferase from Saccharomyces cerevisiae

Liu¹,

Heeswijck²,

Høj³

et al. 1995

Eur J Biochem

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Ornithine acetyltransferase has been purified 4000-fold to homogeneity from Saccharomyces cerevisiae. The enzyme catalyses the freely reversible interchange of an acetyl group between N-acetylornithine and glutamate and has a specific activity of 22 pmol . min-' . mg-' at 37°C and pH 7.5. The K , values were determined for the substrates of the forward and reverse directions to be 1 .O mM for N-acetylornithine, 7.2 mM for glutamate, 1.5 mM for omithine and 17.1 mM for N-acetylglutamate. The enzyme was localised to the mitochondria1 matrix and was found to be a 57-kDa heterodimer consisting of subunits of 31 kDa and 26 kDa. Antibodies raised against the small subunit immunoprecipitated a single in vitro translation product of approximately 57 kDa suggesting that the subunits are processed from a single precursor protein. This is supported by N-terminal sequence analysis which shows that the 26-kDa subunit exhibits 40% sequence identity (8 out of 20) with the N-terminus of ornithine acetyltransferase from Neisseria gonorrhoeae whereas the N-terminus of the 31-kDa subunit exhibits 45% identity (9 out of 20) with a sequence located in the middle of the 60-kDa N. gonorrhoeae enzyme. The N-terminal sequence of the small subunit has the potential to form an amphiphilic helix, further suggesting that the precursor protein with the small subunit at its N-terminus could be targeted to mitochondria and processed into two subunits.

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Section: Immunoprecipitation Of In Vitro Translated Yeast Proteinsmentioning

confidence: 99%

Purification and Characterization of Ornithine Acetyltransferase from Saccharomyces cerevisiae

Liu¹,

Heeswijck²,

Høj³

et al. 1995

Eur J Biochem

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Light Stress Proteins (Elips); The Intriguing Relatives of Cab Gene Family

Adamska¹

1995

Photosynthesis: From Light to Biosphere

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Partial blocks in the early steps of the chlorophyll synthesis pathway: A common feature of chlorophyll b‐deficient mutants

Falbel¹,

Staehelin²

1996

Physiologia Plantarum

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We have analyzed precursor pools in the chlorophyll (Chi) synthesis pathway for a set of eighteen well studied Chl b‐defident mutants in monocotyledonous (barley, maize and wheat) and dicotyledonous plants (Antirrhinum, Arabidopsis, soybean, tobacco and tomato) that form abnormal thylakoid membrane systems. All of these mutants have a partial block in Chl synthesis and nearly all of them accumulate protoporphyrin IX (Proto), the last porphyrin compound common to both heme and Chl synthesis. The large number of mutants at several genetic loci affecting this critical branchpoint in tetrapyrrole biosynthesis suggests that the Mg‐chelatase enzyme, catalyzing the first committed step of Chi biosynthesis, is a multimeric complex composed of the products of some of these genetic loci, and perhaps regulated by others. We hypothesize that these mutants are Chi b‐deficient and have reduced amounts of light‐harvesting antenna complexes (LHCs.) and develop abnormal thylakoid membranes as a direct result of limited Chl synthesis. The observed bottleneck in Chl synthesis can also explain the light‐intensity‐dependent and temperature‐dependent expression of the mutant phenotype. This hypothesis offers a simple explanation for the wide variety of pbenotypes that have been reported for the many Chl‐deficient mutants in the literature. Our findings are also consistent with the notion that Chl b is made from “left over” Chl a molecules and suggest that the Chi b‐deficient mutants should be considered more appropriately as leaky Chl‐deficient mutants.

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

Cited by 4 publications

References 22 publications

Purification and Characterization of Ornithine Acetyltransferase from Saccharomyces cerevisiae

Purification and Characterization of Ornithine Acetyltransferase from Saccharomyces cerevisiae

Light Stress Proteins (Elips); The Intriguing Relatives of Cab Gene Family

Partial blocks in the early steps of the chlorophyll synthesis pathway: A common feature of chlorophyll b‐deficient mutants

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