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M A Block

Publications and source records attributed to M A Block.

At least 19 recordsLinked to original sources
PubMed2001

Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana.

In Arabidopsis, monogalactosyldiacylglycerol (MGDG) is synthesized by a multigenic family of MGDG synthases consisting of two types of enzymes differing in their N-terminal portion: type A (atMGD1) and type B (atMGD2 and atMGD3). The present paper compares type B isoforms with the enzymes of type A that are known to sit in the inner membrane of plastid envelope. The occurrence of types A and B in 16:3 and 18:3 plants shows that both types are not specialized isoforms for the prokaryotic and eukaryotic glycerolipid biosynthetic pathways. Type A atMGD1 gene is abundantly expressed in green tissues and along plant development and encodes the most active enzyme. Its mature polypeptide is immunodetected in the envelope of chloroplasts from Arabidopsis leaves after cleavage of its transit peptide. atMGD1 is therefore likely devoted to the massive production of MGDG required to expand the inner envelope membrane and build up the thylakoids network. Transient expression of green fluorescent protein fusions in Arabidopsis leaves and in vitro import experiments show that type B precursors are targeted to plastids, owing to a different mechanism. Noncanonical addressing peptides, whose processing could not be assessed, are involved in the targeting of type B precursors, possibly to the outer envelope membrane where they might contribute to membrane expansion. Expression of type B enzymes was higher in nongreen tissues, i.e., in inflorescence (atMGD2) and roots (atMGD3), where they conceivably influence the eukaryotic structure prominence in MGDG. In addition, their expression of type B enzymes is enhanced under phosphate deprivation.

Amino Acid Sequence↗
PubMed2000

The multigenic family of monogalactosyl diacylglycerol synthases.

Because the synthesis of monogalactosyldiacylglycerol (MGDG) is unique to plants, identified as an important marker of the plastid envelope, involved in a key step of plastid biogenesis and is the most abundant lipid on earth, MGDG synthase activity was extensively analysed at the biochemical and physiological levels. In the present paper, we present our current knowledge on the MGDG synthase's function, structure and topology in envelope membranes, and discuss possible roles in plant cell glycerolipid metabolism. The recent discovery of a multigenic family of MGDG synthases raised the possibility that multiple isoenzymes might carry out MGDG synthesis in various tissues and developmental stages.

Amino Acid Sequence↗
PubMed1999

Plant ribosome recycling factor homologue is a chloroplastic protein and is bactericidal in escherichia coli carrying temperature-sensitive ribosome recycling factor.

We have isolated a protein, mature RRFHCP, from chloroplasts of spinach (Spinacia oleracea L.) that shows 46% sequence identity and 66% sequence homology with ribosome recycling factor (RRF) of Escherichia coli. RRF recycles ribosomes through disassembly of the posttermination complex. From the cDNA analysis and from the amino-terminal sequencing of the isolated protein, the mature RRFHCP was deduced to have a Mr of 21,838 with 193 aa. It lacks the 78-aa chloroplast targeting sequence encoded by the RRFHCP cDNA sequence. The RRFHCP synthesized in vitro was imported into isolated chloroplasts with simultaneous conversion to the mature RRFHCP. Transcription of the gene coding for RRFHCP was not dependent on light, yet it was limited mostly to photosynthetic tissues in which only one transcript size was detected. Mature RRFHCP exerted a bactericidal effect on E. coli carrying temperature-sensitive RRF at the permissive temperature whereas wild-type E. coli was not affected.

Amino Acid Sequence↗
PubMed1999

Do plastid envelope membranes play a role in the expression of the plastid genome?

A unique biochemical machinery is present within the two envelope membranes surrounding plastids (Joyard et al., Plant Physiol. 118 (1998) 715-723) that reflects the stage of development of the plastid and the specific metabolic requirements of the various tissues. Envelope membranes are the site for the synthesis and metabolism of specific lipids. They are also the site of transport of metabolites, proteins and information between plastids and surrounding cellular compartments. For instance, a complex machinery for the import of nuclear-encoded plastid proteins is rapidly being elucidated. The functional studies of plastid envelope membranes result in the characterization of an increasing number of envelope proteins with unexpected functions. For instance, recent experiments have demonstrated that envelope membranes bind specifically to plastid genetic systems, the nucleoids surrounded by plastid ribosomes. At early stages of plastid differentiation, the inner envelope membrane contains a unique protein (named PEND protein) that binds specifically to plastid DNA. This tight connection suggests that the PEND protein is at least involved in partitioning the plastid DNA to daughter plastids during division. The PEND protein can also provide a physical support for replication and transcription. In addition, factors involved in the control of plastid protein synthesis can become associated to envelope membranes. This was shown for a protein homologous to the E. coli ribosome recycling factor and for the stabilizing factors of some specific chloroplast mRNAs encoding thylakoid membrane proteins. In fact, the envelope membranes together with the plastid DNA are the two essential constituents of plastids that confer identity to plastids and their interactions are becoming uncovered through molecular as well as cytological studies. In this review, we will focus on these recent observations (which are consistent with the endosymbiotic origin of plastids) and we discuss possible roles for the plastid envelope in the expression of plastid genome.

Animals↗
PubMed1999

Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG.

MGDG synthase, the enzyme that catalyzes the synthesis of the major chloroplast membrane lipid monogalactosyldiacylglycerol (MGDG), is encoded by a multigenic family. We have analyzed the biochemical properties, subcellular localization and membrane topology of a spinach chloroplast MGDG synthase, a representative member of the type A family from Spinacia oleracea (soMGD A), using a recombinant protein that was functionally overexpressed in Escherichia coli and specific polyclonal antibodies. We demonstrated that soMGD A could catalyze the synthesis of both 'prokaryotic' and 'eukaryotic' MGDG molecular species in vitro, with a selectivity for diacylglycerol similar to that of purified chloroplast envelope MGDG synthase activity. Furthermore, soMGD A was shown to be sensitive to chemical reagents (dithiothreitol, N-ethylmaleimide and o-phenanthroline) known to affect MGDG synthesis by the partially purified enzyme, as well as in isolated chloroplast envelope membranes. In spinach chloroplasts, soMGD A was localized by Western blot analysis in the inner envelope membrane. Topological studies demonstrated that soMGD A is a monotopic enzyme, embedded within one leaflet of the inner envelope membrane from spinach chloroplasts, a structure which may involve amphipathic alpha helices. We further demonstrated that in vitro, soMGD A precursor is imported and processed to its correct mature form in intact chloroplasts. These results show that soMGD A corresponds to a mature polypeptide of approximately 45 kDa. In addition, inactivation kinetics after gamma-ray irradiation strongly suggest that both native chloroplast envelope MGDG synthase and recombinant soMGD A have a functional molecular mass of 95-100 kDa, indicating that they are probably active as homodimers made of two 45-kDa subunits. This study suggests that, in spite of the growing evidence that MGDG synthesis is catalyzed by a multigenic family of enzymes, in spinach leaves both prokaryotic and eukaryotic MGDG syntheses could be attributable to a unique dimeric enzyme, provided that diacylglycerol is transported from the outer membrane to the inner membrane of the chloroplast envelope.

Amino Acid Sequence↗
PubMed1996

Is E37, a major polypeptide of the inner membrane from plastid envelope, an S-adenosyl methionine-dependent methyltransferase?

Using antibodies raised against E37, one of the major polypeptides of the inner membrane from the chloroplast envelope, it has been demonstrated that a single immunologically related polypeptide was present in total protein extracts from various higher plants (monocots and dicots), in photosynthetic and non-photosynthetic tissues from young spinach plantlets, as well as in the cytoplasmic membrane from the cyanobacteria Synechococcus. This ubiquitous distribution of E37 strongly suggests that this protein plays an envelope-specific function common to all types of plastids. Comparison of tobacco and spinach E37 amino acid sequences deduced from the corresponding cDNA demonstrates that consensus motifs for S-adenosyl methionine-dependent methyltransferases are located in both sequences. This hypothesis was confirmed using a biochemical approach. It was demonstrated that E37, together with two minor spinach chloroplast envelope polypeptides of 32 and 39 kDa, can be specifically photolabeled with [3H]-S-adenosyl methionine upon UV-irradiation. Identification of E37 as a photolabeled polypeptide was established by immunoprecipitation. Furthermore, photolabeling of the three envelope polypeptides was specifically inhibited by very low concentration of S-adenosyl homocysteine, thus providing evidence for the presence within these proteins of S-adenosyl methionine- and S-adenosyl homocysteine-binding sites that were closely associated. Taken as a whole these results strongly suggest that E37 is an ubiquitous plastid envelope protein that probably has an S-adenosyl methionine-dependent methyltransferase activity. The 32 and 39 kDa envelope polypeptides probably have a similar methyltransferase activity.

Amino Acid Sequence↗
PubMed1995

Sequence and expression of the mRNA encoding HSP22, the mitochondrial small heat-shock protein in pea leaves.

A 3 h treatment at 40 degrees C of pea (Pisum sativum var. Douce Provence) plants induces production and accumulation of a small heat-shock protein of 22 kDa apparent molecular mass, designated HSP22, in the matrix compartment of mitochondria [Lenne and Douce (1994) Plant Physiol. 105, 1255-1261]. We show here that the HSP22 precursor (i.e. the mature protein plus the transit peptide) has an apparent molecular mass of 26 kDa after in vitro translation of mRNA extracted from heat-stressed pea plants and immunodetection. We have isolated, cloned and sequenced the full-length cDNA encoding the precursor of the mitochondrial HSP22. An analysis of the amino acid sequence of the mitochondrial HSP22 reveals that this protein is a representative member of the low-molecular-mass heat shock protein (HSP) superfamily, exhibiting the specific consensus regions that are typical of the small HSPs. Most importantly, comparison of the mitochondrial HSP22 sequence with that of chloroplast small HSPs indicates that HSP22 does not contain the typical chloroplast consensus region III. We have also analysed the kinetics of HSP22 induction, and report results on the temporal expression of HSP22 at the transcriptional level. HSP22 mRNA was detected as soon as 10 min after the temperature was raised to a high temperature of 40 degrees C. Then the amount of HSP22 mRNA declined considerably even though pea plants were still submitted to the heat treatment. These results are discussed in light of the translation data previously published [Lenne and Douce (1994) Plant Physiol. 105, 1255-1261], particularly concerning the physiological behaviour of mitochondria when plants are heat-stressed. Furthermore, we have studied the dependence of HSP22 accumulation with temperature and demonstrate that the pea mitochondrial heat-shock response is only developed under extreme environmental growth conditions.

Amino Acid Sequence↗
PubMed1995

The catalytic site of monogalactosyldiacylglycerol synthase from spinach chloroplast envelope membranes. Biochemical analysis of the structure and of the metal content.

We have analyzed the structure of the active site of monogalactosyldiacylglycerol (MGDG) synthase from spinach chloroplast envelope. Since purification of this membrane-embedded enzyme yielded such low amounts of protein that analyses of the amino acid sequence were so far impossible, we used indirect strategies. Analyses of the inhibition of MGDG synthase by UDP and of its inactivation by citraconic anhydride first indicated that the enzyme contained two functionally independent and topologically distinct binding sites for each substrate. Whereas MGDG synthase binds both the nucleotidic part of UDP-Gal and the acyl chains of 1,2-diacylglycerol, UDP is a competitive inhibitor relatively to UDP-Gal, while it does not compete with 1,2-diacylglycerol for binding on the enzyme. The UDP-Gal-binding site contains lysine residues, as demonstrated for UDP-Gal-binding sites from all galactosyltransferases studied so far. Radiolabeling of MGDG synthase by sulfur labeling reagent, a 35S-labeled lysine-blocking reagent, confirmed that MGDG synthase was a polypeptide with a low molecular mass (around 20 kDa). The 1,2-diacylglycerol-binding site contains reduced cysteines and at least one metal. The divalent cation(s) associated to apo-MGDG synthase was not unambiguously identified, but the results suggest that it could be zinc. Therefore, MGDG synthase presents some structural features in common with diacylglycerol-manipulating enzymes, such as protein kinase C and 1,2-diacylglycerol kinase, which are characterized by the presence of a ubiquitous Cys6His2 domain involved in zinc coordination in their 1,2-diacylglycerol-binding domains.

Binding Sites↗
PubMed1995

The outer membrane protein E24 of spinach chloroplast envelope: cloning of a cDNA and topological insertion of the protein in the membrane.

A cDNA coding for the outer membrane protein E24 of spinach chloroplast envelope has been obtained by screening an expression library of spinach cDNA with polyclonal antibodies. Analysis of the protein sequence and comparison with the thermolysin susceptibility of E24 in the chloroplast in situ suggest that E24 is a transmembrane protein and show that its NH2 terminal end is located on the cytosolic side of chloroplasts.

Amino Acid Sequence↗
PubMed1994

Comparison of the kinetic properties of MGDG synthase in mixed micelles and in envelope membranes from spinach chloroplast.

We have applied the 'membrane partition' kinetic modelling approach proposed by Heirwegh et al. [(1988) Biochem. J. 254, 101-108] to MGDG synthase in isolated envelope vesicles. Comparison of the kinetic parameters obtained for MGDG synthase assayed in purified envelope membranes and in mixed-micelles demonstrates that the latter are relevant to the situation in envelope membranes and that MGDG synthase has a very high affinity for dilinoleoylglycerol. Our results provide additional evidence for the hypothesis that the high affinity of the envelope MGDG synthase for dilinoleoylglycerol could be responsible for the presence of C18 fatty acids at both the sn-1 and sn-2 position of the glycerol backbone in MGDG.

Cell Fractionation↗
PubMed1994

Kinetic properties of monogalactosyldiacylglycerol synthase from spinach chloroplast envelope membranes.

We have investigated the functioning of monogalactosyldiacylglycerol (MGDG) synthase activity partially purified from spinach chloroplast envelope membranes, using mixed micelles containing diacylglycerol (the substrate for MGDG synthase), CHAPS (3-[(cholamidopropyl)dimethylammonio]-1- propanesulfonic acid), and phosphatidylglycerol. The presence of this anionic phospholipid was essential for optimal MGDG synthase activity because it strongly improves diacylglycerol solubilization by CHAPS. We have demonstrated that the "surface dilution" kinetic model proposed by Deems et al. (Deems, R.A., Eaton, B.R., and Dennis, E.A. (1975) J. Biol. Chem. 250, 9013-9020) is valid for MGDG synthase assayed in mixed micelles within a narrow range of CHAPS concentration. However, the experimental conditions we have set up in this study led to the description of defined equilibrium and kinetic parameters of the interaction of the envelope MGDG synthase with diacylglycerol. Two-substrate kinetic studies were performed with varied UDP-galactose molar concentrations and varied dioleoylglycerol surface concentrations. The families of reciprocal plots obtained were shown to intersect at a single point of the 1/[substrate] axis thus demonstrating that MGDG synthase is a sequential, either random or ordered, bireactant system. Therefore, MGDG synthase possesses two distinct and independent substrate-binding sites, a hydrophilic one for UDP-galactose and a hydrophobic one for diacylglycerol. The dependence of kinetic parameters on the diacylglycerol mol fraction allows a comparison of the affinity of the enzyme for a wide range of diacylglycerol molecular species. The Km values obtained were ranging between 0.0089 mol fraction (52 microM) for dilinoleoylglycerol (18:2/18:2) to 0.0666 mol fraction (416 microM) for distearoylglycerol (18:0/18:0), but the differences observed were not really related to the unsaturation of the molecule since the Km value for dilinoleoylglycerol was much lower than that (0.040 mol fraction) for dilinoleoylglycerol (18:3/18:3). The Km values for dioleoylglycerol (18:1/18:1) and for the diacylglycerol molecular species synthesized within chloroplasts, i.e. containing 18:1/16:0, were in the average range, i.e. lower than 0.030 mol fraction (around 170 microM).

Chloroplasts↗
PubMed1992

Feedback inhibition of phosphatidate phosphatase from spinach chloroplast envelope membranes by diacylglycerol.

Because the envelope phosphatidate phosphatase plays a pivotal role in chloroplast glycerolipid metabolism, we have analyzed whether diacylglycerol could be a regulatory factor of the enzyme. Using isolated envelope membranes in which the level of diacylglycerol was modified by thermolysin treatment of intact chloroplasts to destroy the galactolipid:galactolipid galactosyltransferase, we have demonstrated that phosphatidate phosphatase activity was reduced when the membrane was enriched in diacylglycerol. All 1,2-diacylglycerol molecular species assayed were demonstrated to inhibit the enzyme to about the same extent. Kinetic studies with envelope from thermolysin-treated chloroplasts were performed in the absence and presence of diacylglycerol, and diacylglycerol was shown to be a powerful competitive inhibitor of the reaction. Finally, using isolated intact spinach chloroplasts, we have demonstrated that in situ phosphatidate phosphatase activity can be modulated by the level of diacylglycerol present in the membrane. The relevance of phosphatidate phosphatase inhibition by diacylglycerol in the regulation of chloroplast glycerolipid biosynthesis is discussed.

Cell Fractionation↗
PubMed1992

Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether-like herbicides.

Plant protoporphyrinogen oxidase is of particular interest since it is the last enzyme of the common branch for chlorophyll and heme biosynthetic pathways. In addition, it is the target enzyme for diphenyl ether-type herbicides, such as acifluorfen. Two distinct methods were used to investigate the localization of this enzyme within Percoll-purified spinach chloroplasts. We first assayed the enzymatic activity by spectrofluorimetry and we analyzed the specific binding of the herbicide acifluorfen, using highly purified chloroplast fractions. The results obtained give clear evidence that chloroplast protoporphyrinogen oxidase activity is membrane-bound and is associated with both chloroplast membranes, i.e. envelope and thylakoids. Protoporphyrinogen oxidase specific activity was 7-8 times higher in envelope membranes than in thylakoids, in good agreement with the number of [3H]acifluorfen binding sites in each membrane system: 21 and 3 pmol/mg protein, respectively, in envelope membranes and thylakoids. On a total activity basis, 25% of protoporphyrinogen oxidase activity were associated with envelope membranes. The presence of protoporphyrinogen oxidase in chloroplast envelope membranes provides further evidence for a role of this membrane system in chlorophyll biosynthesis. In contrast, the physiological significance of the enzyme associated with thylakoids is still unknown, but it is possible that thylakoid protoporphyrinogen oxidase could be involved in heme biosynthesis.

Chloroplasts↗
PubMed1991

Purification and characterization of E37, a major chloroplast envelope protein.

We have purified to homogeneity E37, the second major polypeptide of the inner membrane of the chloroplast envelope. The protein was retained on a Mono S column at pH 7, indicating it is a basic protein. After cyanogen cleavage, the protein was partially sequenced at 2 different sites. The sequence is compared with the deduced amino acid sequence of a cDNA coding for a 37 kDa envelope polypeptide recently published by Dreses-Werringloer et al. (Eur. J. Biochem. (1991) 195, 361-368.)

Amino Acid Sequence↗
PubMed1991

Avoiding reoperation for indeterminate thyroid nodules identified as malignant after surgery.

Thyroid nodules that are indeterminate as carcinoma by needle biopsy before surgery and by study of frozen sections at the time of surgery are occasionally identified to be malignant in later studies. If only a lobectomy has been performed, the advisability of reoperation to remove the remaining thyroid tissue to facilitate radioactive iodine therapy may cause concern. To obviate this difficulty as well as to reduce the occurrence of nodules later in a preserved contralateral thyroid lobe and to provide additional thyroid tissue for study, contralateral subtotal or near total lobectomy has been performed for indeterminate thyroid nodules. The small remnant of remaining thyroid tissue can later be ablated by radioactive iodine if desired. Of 37 patients with indeterminate thyroid nodules, none required reoperation, although the diagnosis of carcinoma was established after surgery for eight patients, three of whom were treated with radioactive iodine.

Biopsy, Needle↗
PubMed1991

Isolation, transcription, and inactivation of the gene for an atypical alkaline phosphatase of Synechococcus sp. strain PCC 7942.

The alkaline phosphatase of Synechococcus sp. strain PCC 7942 is 145 kDa, which is larger than any alkaline phosphatase previously characterized and approximately three times the size of the analogous enzyme in Escherichia coli. The gene for the alkaline phosphatase, phoA, was cloned and sequenced, and the protein that it encodes was found to have little similarity to other phosphatases. Some sequence similarities were observed between the Synechococcus sp. strain PCC 7942 alkaline phosphatase, the alpha subunit of the ATPase from bacteria and chloroplasts, and the UshA sugar hydrolase of E. coli. Also, limited sequence similarity was observed between a region of the phosphatase and a motif implicated in nucleotide binding. Interestingly, although the alkaline phosphatase is transported across the inner cytoplasmic membrane and into the periplasmic space, it does not appear to have a cleavable signal sequence at its amino terminus. The half-life of the mRNA encoding the alkaline phosphatase, measured after inhibition of RNA synthesis, is approximately 5 min. Similar kinetics for the loss of alkaline phosphatase mRNA occur upon the addition of phosphate to phosphate-depleted cultures, suggesting that high levels of this nutrient inhibit transcription from phoA almost immediately. The phoA gene also appears to be the first gene of an operon; the largest detectable transcript that hybridizes to a phoA gene-specific probe is 11 kb, over twice the size needed to encode the mature protein. Other phosphate-regulated mRNAs are also transcribed upstream of the phoA gene. Insertional inactivation of phoA results in the loss of extracellular, phosphate-regulated phosphatase activity but does not alter the capacity of the cell for phosphate uptake.

Alkaline Phosphatase↗