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Title: Protein methylation reactions in intact pea chloroplasts

Abstract

Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ({sup 3}H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented.

Authors:
 [1]
  1. (Univ. of Wisconsin, Madison (USA))
Publication Date:
OSTI Identifier:
6957857
Resource Type:
Journal Article
Resource Relation:
Journal Name: Plant Physiology, Supplement; (USA); Journal Volume: 89:4
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PEAS; PHYSIOLOGY; PROTEINS; METHYLATION; CHLOROPLASTS; LABELLING; TRACER TECHNIQUES; TRITIUM COMPOUNDS; CELL CONSTITUENTS; CHEMICAL REACTIONS; FOOD; HYDROGEN COMPOUNDS; ISOTOPE APPLICATIONS; ORGANIC COMPOUNDS; PLANTS; VEGETABLES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Niemi, K.J. Protein methylation reactions in intact pea chloroplasts. United States: N. p., 1989. Web.
Niemi, K.J. Protein methylation reactions in intact pea chloroplasts. United States.
Niemi, K.J. 1989. "Protein methylation reactions in intact pea chloroplasts". United States. doi:.
@article{osti_6957857,
title = {Protein methylation reactions in intact pea chloroplasts},
author = {Niemi, K.J.},
abstractNote = {Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ({sup 3}H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented.},
doi = {},
journal = {Plant Physiology, Supplement; (USA)},
number = ,
volume = 89:4,
place = {United States},
year = 1989,
month = 4
}
  • The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with ({sup 3}H-methyl)-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. Onemore » methylinkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile ({sup 3}H)methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the ({sup 3}H)methyl group.« less
  • Amino acid incorporation into protein by cholorplasts from primary leaves of Phaseolus vulgaris L., var. Black Valentine is only partially inhibited by 400 ..mu..g/ml ribonuclease. The rate of incorporation, in the presence of ribonuclease, is progressively inhibited with time and ceases after about half an hour. Preincubation of chloroplasts at 25/sup 0/, in the absence of ribonuclease, increases the inhibitory effect of ribonuclease on the initial rate of incorporation of amino acid into protein. Examination of electron micrographs of freshly prepared chloroplast suspensions shows that chloroplasts are largely intact. However, after incubation at 25/sup 0/ for 1 hour the chloroplastsmore » are disrupted, as indicated by loss of their stroma contents. It is concluded that the intact chloroplast membrane is relatively impermeable to ribonuclease. Amino acid incorporating activity probably becomes inhibited as the inside of the chloroplast is made accessible to ribonuclease by breakage of membranes during incubation at 25/sup 0/. 17 references, 5 figures, 2 tables.« less
  • The time course of light-induced O/sub 2/ exchange by isolated intact chloroplasts and cells from spinach was determined under various conditions using isotopically labeled O/sub 2/ and a mass spectrometer. In dark-adapted chloroplasts and cells supplemented with saturating amounts of bicarbonate, O/sub 2/ evolution began immediately upon illumination. However, this initial rate of O/sub 2/ evolution began immediately upon illumination. However, this initial rate of O/sub 2/ evolution was counterbalanced by a simultaneous increase in the rate of O/sub 2/ uptake, so that little net O/sub 2/ was evolved or consumed during the first approx. 1 minute of illumination. Aftermore » this induction (lag) phase, the rate of O/sub 2/ evolution increased 3- to 4-fold while the rate of O/sub 2/ uptake diminshed to a very low level. Inhibition of the Calvin cycle had negligible effects on the initial rate of O/sub 2/ evolution or O/sub 2/ uptake; both rates were sustained for several minutes, and about balanced so that no net O/sub 2/ was produced. Uncouplers had an effect similar to that observed with Calvin cycle inhibitors, except that rates of O/sub 2/ evolution and photoreduction were stimulated 40 to 50%. These results suggest that higher plant photosynthetic preparations which retain the ability to reduce CO/sub 2/ also have a significant capacity to photoreduce O/sub 2/. With near-saturating light and sufficient CO/sub 2/, O/sub 2/ reduction appears to take place primarily via a direct interaction between O/sub 2/ and reduced electron transport carriers, and occurs principally when CO/sub 2/ fixation reactions are suboptimal. The inherent maximum endogenous rate of O/sub 2/ reduction is approximately 25 to 50% of the maximum rate of noncyclic electron transport coupled to CO/sub 2/ fixation. Although the photoreduction of O/sub 2/ is coupled to ion transport and/or phosophorylation, this process does not appear to supply significant amounts of ATP directly during steady-state CO/sub 2/ fixation in strong light.« less
  • Incubation of intact spinach (Spinacia oleracea L.) chloroplasts in the presence of {sup 35}SO{sub 4}{sup 2{minus}} resulted in the light-dependent formation of a chloroform-soluble sulfur-containing compound distinct from sulfolipid. The authors have identified this compound as the most stable form (S{sub 8}) of elemental sulfur (S{sup 0}, valence state for S = O) by mass spectrometry. It is possible that elemental sulfur (S{sup 0}) was formed by oxidation of bound sulfide, i.e. after the photoreduction of sulfate to sulfide by intact chloroplasts, and released as S{sub 8} under the experimental conditions used for analysis.
  • Addition of exogenous Mg/sup 2 +/ (millimolar) to illuminated intact spinach (Spinacia oleracea L.) chloroplasts caused acidification of the stroma and a 20% decrease in stromal K/sup +/. Addition of K/sup +/ (10-50 millimolar) reversed both stromal acidification and K/sup +/ efflux from the chloroplast caused by Mg/sup 2 +/. These data suggested that Mg/sup 2 +/ induced reversible H/sup +//K/sup +/ fluxes across the chloroplast envelope. Ca/sup 2 +/ and Mn/sup 2 +/ (2 millimolar) were as effective as 4 millimolar Mg/sup 2 +/ in causing K/sup +/ efflux from chloroplasts and inhibition of O/sub 2/ evolution. In contrast,more » 10 millimolar Ba/sup 2 +/ indicated that the effects of divalent cations such as Mg/sup 2 +/ cannot be attributed to generalized electrostatic interactions of the cation with the chloroplast envelope. With the chloroplasts used in this study, stromal acidification caused by 2 millimolar Mg/sup 2 +/ was small (0.07 to 0.15 pH units), but sufficient to account for the inhibition of O/sub 2/ evolution (43%) induced by Mg/sup 2 +/. Oligomycin was the only ATPase inhibitor which specifically inhibited photosynthesis in the presence of Mg/sup 2 +/ + K/sup +/, but had little or no effect in the absence of these cations. In the presence of oligomycin much higher concentrations (50 millimolar) of exogenous K/sup +/ were required to reverse Mg/sup 2 +/-induced acidification and inhibition of O/sub 2/ evolution than in its absence. Oligomycin (in the absence of divalent cations) increased the inhibition of photosynthesis caused by sodium acetate. In addition, the chloroplast envelope ATPase was inhibited 45% by oligomycin. These results suggested that H/sup +/ fluxes across the chloroplast envelope are regulated by two mechanisms: an active, oligomycin-sensitive H/sup +/ efflux and a reversible, Mg/sup 2 +/-dependent, oligomycin-insensitive H/sup +//K/sup +/ exchange.« less