Spinach chloroplast envelope

Malherbe, A., Block, M.A., Douce, R. and Joyard, J. (1995) Solubilisation and biochemical properties of phosphatidate phosphatase from spinach chloroplast envelope membranes. Plant Physiol. Biochem. 33, 149-161... 

UPD-QALACTOSE-INDEPENDENT SYNTHESIS OF MONO-GALAC-TOSYLDIACYLGLYCEROL (MGDG). AN ENZYMATIC ACTIVITY OF THE SPINACH CHLOROPLAST ENVELOPE... 

Miquel M., Block M.A., Joyard J., Dome A.J., Dubacq J.P,, Kader J.C.and Douce R. (1987) - Protein mediated transfer of phosphatidyl -choline from liposomes to spinach chloroplast envelope membranes. Biochim. Biophys. Acta 937, 219-228. 

Table 1. Enzymatic reactions of the galactolipid metabolism of spinach chloroplast envelope membranes. 

Fig. 1. Lipid pattern of spinach chloroplast envelope membranes, obtained from thermolysin-treated (left) and untreated (right) chloroplasts. Chromatograms are shown obtained by HPLC analysis only non-acidic lipids were eluted from the aminopropyl-modified silica column (18). Peak labels 1, carotenoids and diacylglycerol 2, unknown 3, MGDG 4, PC 5-6, DGDG 7, PG and sulfolipid (overload) 8, TGDG. Note the small peak 1, the high MGDG peak, and the absence of TGDG in the thermolysin-treated membranes.

CHARACTERIZATION OF GALACTOSYLTRANSFERASES IN SPINACH CHLOROPLAST ENVELOPE MEMBRANES... 

MGDG SYNTHASE OF SPINACH CHLOROPLAST ENVELOPE PROPERTIES OF THE SUBSTRATE BINDING SITES... 

Covis, J., Joyard, J., and Douce R. Lipid requirement and kinetic studies of solubilized UDP-galactose diacylglycerol galactosyltransferase activity from spinach chloroplast envelope membranes. Proc. Natl. Acad. Sci. USA 1988 85, 4966-4970... 

Marechal E, Block MA, Joyard J, Douce R. Purification of UDP galactose 1.2-diacylglycerol galactosyltransferase from spinach chloroplast envelope membranes. C. R. Acad. Sci. Paris, t. 313, Sene III, 1991 521-528. 

Spinach chloroplast envelope membranes, incubated in vitro in the presence of [y PJATP, incorporated labelled phosphate mainly in three proteins exhibiting Mr of 67, 26 and 14 kDa. Two of them, the 26 and 14 kDa phosphoproteins, are pools of LHCIIb and Rubisco small subunit tightly bound to envelope membranes [1] and are phosphorylated by a unique Ca -dependent serine protein kinase [2]. The 67 kDa phosphoprotein is likely to be autophosphorylated its P-labelling would take place via its own phosphoglucomutase activity [2-4]. The aim of this investigation is to modulate envelope protein kinase activities following incubation for various times with the phospholipase C (PLC) from Bacillus cereus or the lipase from Rhizopus arrhizus (LRa). 

Spinach chloroplast envelope membranes were prepared according to [5]. Envelope protein samples (30 pg) were phosphorylated in the presence of 33 nM [y PjATP as described in [2]. The envelope protein kinase activities were stopped after incubation at 23°C for 1.5 min by adding one volume of SDS-PAGE sample buffer. The incorporated [ Pjphosphate into proteins was quantified by the Cerenkov procedure. Lipid extraction and thin layer chromatography (TLC) were carried out as described in [2] and [6]. Immediately after charring, the separated lipids on TLC plates were quantified by laser densitometry (Bioimage Millipore). 

Bovet L, Mtlller MO, Siegenthaler PA. The 26 and 14 kDa phosphoproteins associated with spinach chloroplast envelope membranes are distinct membrane-bound pools of LHCII and of Rubisco small subunit. Planta 1994 (in press)... 

Siegenthaler PA, Dumont N. Characteristics of spinach chloroplast envelope, Thylakoid and stroma polypeptides as revealed by Triton X-114 phase partition. Plant Cell Physiol 1990 31 1101-1108. 

A (Mg2+-Ca2+)-STIMULATED ATPase IN SPINACH CHLOROPLAST ENVELOPES ISOLATION BY CALMODULIN AFFINITY CHROMATOGRAPHY... 

Spinach chloroplast envelopes were prepared according to Douce et al. (1973) and resuspended in buffer A (50 mM Tris-HCl, pH 7.8, 300 mM sucrose). Envelopes were solubilized with 5 mg Triton X-lOO/mg protein at 4°C for 15 min, then 1 mM MgCl2 and 0.1 mM CaCl2 were added. The unsolubilized material was pelleted at 100 000 x g for 30 min at 4°C. 

TABLE I. Activation of the membrane-bound and partially purified ATPase from spinach chloroplast envelopes... 

In conclusion, the use of calmodulin-Sepharose affinity chromatography enabled to isolate quickly a protein fraction enriched in ATPase activity. This partially purified enzyme had properties which were similar to those of the envelope-bound ATPase, in particular both were stimulated by Ca " " and Mg ions and by calmodulin. In addition to the two RuBP carboxylase subunits and the phosphate translocator, the present ATPase seems to be the fourth protein identified in spinach chloroplast envelopes. Due to its special properties, this enzyme might well modulate the exchanges of ions, metabolites and/or proteins between the chloroplast and the cytosol. 

The chloroplast envelope plays a central role in mediating transport of metabolites and proteins between cytoplasm and the stroma (Heldt and Heber, 1981). The role, if any, of a membrane ATPase in these processes is as yet unclear. Sabnis et al. (1970) first demonstrated the presence of a Mg2+-dependent ATPase in the chloroplast envelope of Pisurn tendril cells. Joyard and Douce (1975) characterized an ATPase activity from spinach chloroplast envelopes. The activity hydrolyzed a broad range of nucleoside triphosphates, was insensitive to N,N dicyclohexylcarbodiimide (DCCD) and was not stimulated by monovalent cations. The purpose of the present study is to characterize the ATPase activity found on the chloroplast envelope of pea and to establish its location with respect to the inner and outer chloroplast envelope membranes. 

These results establish the presence of a Mg " "-dependent ATP hydrolyzing activity on the inner chloroplast envelope membrane of Pisum sativum. Cytochemical evidence for an envelope ATPase in pea was reported by Sabnis et al. (1970). Joyard and Douce (1975) have characterized an ATPase of similar specific activity from spinach chloroplast envelope membranes. 

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