Biomedical subjects

J Garin

Publications and source records attributed to J Garin.

At least 37 records · Page 2Linked to original sources

PubMed2000

Functional and molecular identification of novel members of the ubiquitous membrane fusion proteins alpha- and gamma-SNAP (soluble N-ethylmaleimide-sensitive factor-attachment proteins) families in Dictyostelium discoideum.

The soluble N-ethylmaleimide-sensitive-factor-attachment proteins (SNAP) are eukaryotic soluble proteins required for membrane fusion. Based on their initial identification in bovine brain cytosol, they are divided in alpha/beta and gamma subfamilies. SNAPs act as adapters between N-ethylmaleimide-sensitive factor (NSF), a hexameric ATPase, and membrane SNARE proteins (SNAP receptors). Within the NSF/SNAP/SNARE complex, SNAPs contribute to the catalysis of an ATP-driven conformational change in the SNAREs, resulting in dissociation of the complex. We have constructed a Dictyostelium discoideum strain overexpressing a c-myc-tagged form of D. discoideum NSF (NSF-myc). Its immunoprecipitation from detergent-solubilized membrane extracts reveals two associated polypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36) that are absent in NSF-myc immunoprecipitates from cytosol. Analysis of trypsin-digested peptides by microsequencing and mass spectrometry and comparison with cDNA sequences identify p33 and p36 as the D. discoideum homologues of alpha- and gamma-SNAP, respectively. The alpha-/gamma-SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of gamma-SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents, for the first time, the wide distribution of the gamma subtype. Altogether, these results suggest a specific role for gamma-SNAP, distinct from that of alpha-SNAP.

Amino Acid Sequence↗

PubMed2000

Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages.

Phagolysosome biogenesis is essential for the killing and degradation of intracellular pathogens. It involves the fusion of phagosomes with various endocytic organelles, a process known to be regulated in part by Rab proteins. We generated RAW 264.7 macrophages expressing an active mutant of Rab5 (Rab5(Q79L)) to determine the role of Rab5 in phagocytosis and phagolysosome biogenesis. Our results indicate that Rab5 stimulates phagocytosis of latex beads but not Fc or C3 receptor-mediated phagocytosis. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without complete intermixing of their membrane (kiss and run). In Rab5(Q79L)-expressing macrophages, uncontrolled fusion events occurred, leading to the appearance of giant phagosomes. These phagosomes could initiate their maturation and acquire LAMP1, but failed to generate the microbicidal conditions needed to kill intracellular parasites. These results identify Rab5 as a key molecule regulating phagosome-endosome fusion and as an essential component in the innate ability of macrophages to restrict the growth of intracellular parasites.

Animals↗

PubMed1999

Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73.

Exosomes are membrane vesicles secreted by hematopoietic cells upon fusion of late multivesicular endosomes with the plasma membrane. Dendritic cell (DC)-derived exosomes induce potent antitumor immune responses in mice, resulting in the regression of established tumors (Zitvogel, L., A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza, P. Ricciardi-Castagnoli, G. Raposo, and S. Amigorena. 1998. Nat. Med. 4:594-600). To unravel the molecular basis of exosome-induced immune stimulation, we now analyze the regulation of their production during DC maturation and characterize extensively their protein composition by peptide mass mapping. Exosomes contain several cytosolic proteins (including annexin II, heat shock cognate protein hsc73, and heteromeric G protein Gi2alpha), as well as different integral or peripherally associated membrane proteins (major histocompatibility complex class II, Mac-1 integrin, CD9, milk fat globule-EGF-factor VIII [MFG-E8]). MFG-E8, the major exosomal component, binds integrins expressed by DCs and macrophages, suggesting that it may be involved in exosome targeting to these professional antigen-presenting cells. Another exosome component is hsc73, a cytosolic heat shock protein (hsp) also present in DC endocytic compartments. hsc73 was shown to induce antitumor immune responses in vivo, and therefore could be involved in the exosome's potent antitumor effects. Finally, exosome production is downregulated upon DC maturation, indicating that in vivo, exosomes are produced by immature DCs in peripheral tissues. Thus, DC-derived exosomes accumulate a defined subset of cellular proteins reflecting their endosomal biogenesis and accounting for their biological function.

Animals↗

PubMed1999

A Dictyostelium protein binds to distinct oligo(dA) x oligo(dT) DNA sequences in the C-module of the retrotransposable element DRE.

The genome of the eukaryotic microbe Dictyostelium discoideum contains some 200 copies of the nonlong-terminal repeat retrotransposon DRE. Among several unique features of this retroelement, DRE is transcribed in both directions leading to the formation of partially overlapping plus strand and minus strand RNAs. The synthesis of minus strand RNAs is controlled by the C-module, a 134-bp DNA sequence located at the 3'-end of DRE. A nuclear protein (CMBF) binds to the C-module via interaction with two almost homopolymeric 24 bp oligo(dA) x oligo(dT) sequences. The DNA-binding drugs distamycin and netropsin, which bind to A x T-rich DNA sequences in the minor groove, competed efficiently for the binding of CMBF to the C-module. The CMBF-encoding gene, cbfA, was isolated and a DNA-binding domain was mapped to a 25-kDa C-terminal region of the protein. A peptide motif involved in the binding of A x T-rich DNA by high mobility group-I proteins ('GRP' box) was identified in the deduced CMBF protein sequence, and exchange of a consensus arginine residue for alanine within the CMBF GRP box abolished the interaction of CMBF with the C-module. The current data support the theory that CMBF binds to the C-module by detecting its long-range DNA conformation and interacting with A x T base pairs in the minor groove of oligo(dA) x oligo(dT) stretches.

Amino Acid Sequence↗

PubMed1999

Some anticardiolipin antibodies recognize a combination of phospholipids with thrombin-modified antithrombin, complement C4b-binding protein, and lipopolysaccharide binding protein.

The standard enzyme-linked immunosorbent assay (ELISA) for anticardiolipin antibodies (ACA) detects a heterogenous group of antibodies against cardiolipin on its own, beta2-glycoprotein I (beta2GPI), and, potentially, other phospholipid-binding plasma proteins from bovine or human origin. In an attempt to identify new proteic targets of ACA, we selected 6 patients who possessed cofactor-dependent ACA but no antibody to human or bovine beta2GPI detectable in the beta2GPI-ELISA. Three of these samples proved to recognize beta2GPI in combination with cardiolipin, but not beta2GPI directly immobilized on gamma-irradiated polystyrene or agarose beads. In the other cases, the component required for ACA binding was purified from adult bovine serum or plasma by means of ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, diethyl aminoethyl (DEAE)-cellulose, heparin-Ultrogel, and Sephacryl S-300 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis coupled to N-terminal amino acid microsequencing identified the cofactors of patients no. 4, 5, and 6 ACA as lipopolysaccharide binding protein (LBP), complement C4b-binding protein (C4BP), and the thrombin-antithrombin (AT) complex, respectively. Adsorption of each of these cofactor preparations with cardiolipin liposomes led to suppression of ACA reactivity, concomitant with the loss of bands from SDS gels corresponding to sequenced material. Bacterial lipopolysaccharide (which forms high-affinity complexes with LBP) specifically neutralized the cofactor activity of the LBP preparation in a concentration-dependent manner. Bovine serum and plasma, as well as the C4BP preparation, optimally supported the binding of a rabbit anti-C4BP antiserum to immobilized cardiolipin. The binding of a rabbit anti-AT antiserum to solid-phase cardiolipin was sustained by the thrombin-AT preparation and bovine serum, but neither by bovine plasma nor by native AT, thus reproducing the behavior of patient no. 6 ACA. Taking advantage of the restricted recognition by the latter ACA of a cofactor from bovine origin appearing upon clotting, we studied the generation of such activity in human plasma supplemented with bovine AT or bovine prothrombin before clotting. In these conditions, patient no. 6 antibody binding to cardiolipin required the addition of bovine AT, whereas addition of bovine prothrombin alone was ineffective. We therefore concluded that those ACA targeted bovine AT once it has been modified/cleaved by thrombin. These findings underline the wide heterogeneity of ACA and the links that may exist between various coagulation pathways, inflammation and the complement system.

Acute-Phase Proteins↗

PubMed1999

Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast.

Yap1 and Skn7 are two yeast transcriptional regulators that co-operate to activate thioredoxin (TRX2) and thioredoxin reductase (TRR1) in response to redox stress signals. Although they are both important for resistance to H2O2, only Yap1 is important for cadmium resistance, whereas Skn7 has a negative effect upon this response. The respective roles of Yap1 and Skn7 in the induction of defense genes by H2O2 were analyzed by two-dimensional gel electrophoresis. Yap1 controls a large oxidative stress response regulon of at least 32 proteins. Fifteen of these proteins also require the presence of Skn7 for their induction by H2O2. Although about half of the Yap1 target genes do not contain a consensus Yap1 recognition motif, the control of one such gene, TSA1, involves the binding of Yap1 and Skn7 to its promoter in vitro. The co-operative control of the oxidative stress response by Yap1 and Skn7 delineates two gene subsets. Remarkably, these two gene subsets separate antioxidant scavenging enzymes from the metabolic pathways regenerating the main cellular reducing power, glutathione and NADPH. Such a specialization may explain, at least in part, the dissociated function of Yap1 and Skn7 in H2O2 and cadmium resistance.

DNA, Fungal↗

PubMed1999

Tissue inhibitor of metalloproteinase-2 (TIMP-2) expression is strongly induced by ACTH in adrenocortical cells.

Besides its acute and chronic effects on corticosteroid synthesis, the pituitary adrenocorticotropic hormone (ACTH) regulates diverse adrenocortical biological functions including the synthesis of a number of mitochondrial, cytoplasmic, and secreted proteins. ACTH-induced secreted proteins are candidates to act as local extracellular relays of the hormone in either an autocrine or a paracrine manner. In the present study, we report that stimulation of primary cultures of bovine adrenocortical (BAC) fasciculata cells with 10 nM ACTH for 24 h results in a mean 8 +/- 4-fold induction of the synthesis of a secreted protein presenting an apparent Mr of 21 kDa. Peptide microsequencing and Western blotting allowed us to identify this 21-kDa ACTH-induced protein as the tissue inhibitor of metalloproteinase-2 (TIMP-2). The induction of TIMP-2 by ACTH required transcription, was mimicked by 8-bromo cyclic 3'-5' adenosine monophosphate, but was not observed in response to angiotensin II, IGF-1, fibroblast growth factor-2, transforming growth factor-beta1, or cortisol treatments. ACTH stimulated TIMP-2 mRNA levels by a factor 4, whereas TIMP-1 mRNA levels were not affected and TIMP-3 mRNA remained undetectable. The biological activity of TIMP-2 varied accordingly, as we observed that the conditioned medium of ACTH-treated BAC cells was four times more potent at inhibiting gelatinolytic activity than was the conditioned medium of control cells. Because the proteolytic activity of both progelatinase-B and progelatinase-A secreted by BAC cells remained latent, whether in the presence or in the absence of ACTH, a paracrine rather than autocrine role is proposed for TIMP-2 in the adrenal cortex.

Adrenal Cortex↗

PubMed1999

Technical Advance: Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins.

Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane alpha-helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking.

Journal Article↗

PubMed1999

The S15 self-incompatibility haplotype in Brassica oleracea includes three S gene family members expressed in stigmas.

Self-incompatibility in Brassica is controlled by a single, highly polymorphic locus that extends over several hundred kilobases and includes several expressed genes. Two stigma proteins, the S locus receptor kinase (SRK) and the S locus glycoprotein (SLG), are encoded by genes located at the S locus and are thought to be involved in the recognition of self-pollen by the stigma. We report here that two different SLG genes, SLGA and SLGB, are located at the S locus in the class II, pollen-recessive S15 haplotype. Both genes are interrupted by a single intron; however, SLGA encodes both soluble and membrane-anchored forms of SLG, whereas SLGB encodes only soluble SLG proteins. Thus, including SRK, the S locus in the S15 haplotype contains at least three members of the S gene family. The protein products of these three genes have been characterized, and each SLG glycoform was assigned to an SLG gene. Evidence is presented that the S2 and S5 haplotypes carry only one or the other of the SLG genes, indicating either that they are redundant or that they are not required for the self-incompatibility response.

Alleles↗

PubMed1999

Characterization of Dictyostelium discoideum cathepsin D.

Previous studies using magnetic purification of Dictyostelium discoideum endocytic vesicles led us to the identification of some major vesicle proteins. Using the same purification procedure, we have now focused our interest on a 44 kDa soluble vesicle protein. Microsequencing of internal peptides and subsequent cloning of the corresponding cDNA identified this protein as the Dictyostelium homolog of mammalian cathepsins D. The only glycosylation detected on Dictyostelium cathepsin D (CatD) is common antigen 1, a cluster of mannose 6-sulfate residues on N-linked oligosaccharide chains. CatD intracellular trafficking has been studied, showing the presence of the protein throughout the entire endocytic pathway. During the differentiation process, the catD gene presents a developmental regulation, which is also observed at the protein level. catD gene disruption does not alter significantly the cell behaviour, either in the vegetative form or the differentiation stage. However, modifications in the SDS-PAGE profiles of proteins bearing common antigen 1 were detected, when comparing parental and catD(-) cells. These modifications point to a possible role of CatD in the maturation of a few Dictyostelium lysosomal proteins.

Amino Acid Sequence↗

PubMed1998

Cysteine oxidation in the mitogenic S100B protein leads to changes in phosphorylation by catalytic CKII-alpha subunit.

The glial-derived calcium-binding protein S100B can be secreted to act as a neurotrophic factor or a mitogen, stimulating proliferation of glial cells. The extracellular S100B activities rely on the oxidation of the protein cysteine residues (Kligman, D., and Marshak, D. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7136-7139; Winningham-Major, F., Staecker, J. L., Barger, S. W., Coats, S., and Van Eldik, L. J. (1989) J. Cell Biol. 109, 3063-3071). Here we show that oxidation of the S100B cysteine residues, Cys-68 and Cys-84, induces a conformational change in the protein structure, unmasking a canonical CKII phosphorylation site located within the typical EF-hand calcium-binding site IIbeta. Intrasubunit disulfide-bridged S100B monomer and disulfide-bonded S100B dimer are phosphorylated by the catalytic CKII-alpha subunit on Ser-62 with a Km of 0.5 microM and a Vmax of 10 pmol/min/100 pmol of S100B. Oxidized S100B is the best in vitro CKII-alpha substrate identified so far. Next we show that intrasubunit disulfide-bridged S100B monomer is the most potent S100B species to stimulate [3H]thymidine uptake by C6 glial cells in culture. In addition, the phosphorylated intrasubunit disulfide-bridged S100B monomer retains apparent mitogenic activity toward C6 glial cells, and hence, 32P-labeled S100B should be a useful probe for characterizing the mechanisms by which extracellular oxidized S100B functions. Finally, we show that formation of intrasubunit disulfide-bridged S100B monomer is stimulated by peroxynitrite anion, suggesting that production of mitogenic S100B species could be enhanced in neuropathology associated with peroxynitrite anion production.

Amino Acid Sequence↗

PubMed1998

Two-dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry: toward a human mitochondrial proteome.

Owing to the complexity of higher eukaryotic cells, characterization of a complete proteome is likely to be difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and "physiology" of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting, but other methods were also used (N-terminal microsequencing, blotting). The optimization steps in two-dimensional (2-D) electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficiencies.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1998

New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis.

Severe quantitative loss of protein is often observed in high-resolution two-dimensional electrophoresis of membrane proteins, while the resolution is usually not affected. To improve the solubility of proteins in this technique, we tested denaturing cocktails containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and zwitterionic detergents, synthesized for this purpose. Among the dozen detergents synthesized and tested, amidosulfobetaines with an alkyl tail containing 14-16 carbons proved most efficient, solubilizing previously undetected membrane proteins.

Animals↗

PubMed1998

Use of ethanolamine for sample stacking in capillary electrophoresis.

Capillary zone electrophoresis (CZE) in the presence of ethanolamine was used in a micropreparative mode. Sample volumes up to 1 microL could be loaded onto a 100 microns diameter capillary without loss in resolution. Coupled to narrow-bore reversed-phase high-performance liquid chromatography, ethanolamine-CZE allowed the collection of sufficient amounts of pure peptidic material to perform amino acid sequence analysis.

Amino Acid Sequence↗

PubMed1998

Molecular cloning and characterization of a radial spoke head protein of sea urchin sperm axonemes: involvement of the protein in the regulation of sperm motility.

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40 degrees C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form alpha-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

Amino Acid Sequence↗

PubMed1998

Unusual organization of the genes coding for HydSL, the stable [NiFe]hydrogenase in the photosynthetic bacterium Thiocapsa roseopersicina BBS.

The characterization of a hyd gene cluster encoding the stable, bidirectional [NiFe]hydrogenase 1 enzyme in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium belonging to the family Chromatiaceae, is presented. The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized (K. L. Kovacs et al., J. Biol. Chem. 266:947-951, 1991; C. Bagyinka et al., J. Am. Chem. Soc. 115:3567-3585, 1993). As an unusual feature, a 1,979-bp intergenic sequence (IS) separates the structural genes hydS and hydL, which encode the small and the large subunits, respectively. This IS harbors two sequential open reading frames (ORFs) which may code for electron transfer proteins ISP1 and ISP2. ISP1 and ISP2 are homologous to ORF5 and ORF6 in the hmc operon, coding for a transmembrane electron transfer complex in Desulfovibrio vulgaris. Other accessory proteins are not found immediately downstream or upstream of hydSL. A hup gene cluster coding for a typical hydrogen uptake [NiFe]hydrogenase in T. roseopersicina was reported earlier (A. Colbeau et al. Gene 140:25-31, 1994). The deduced amino acid sequences of the two small (hupS and hydS) and large subunit (hupL and hydL) sequences share 46 and 58% identity, respectively. The hup and hyd genes differ in the arrangement of accessory genes, and the genes encoding the two enzymes are located at least 15 kb apart on the chromosome. Both hydrogenases are associated with the photosynthetic membrane. A stable and an unstable hydrogenase activity can be detected in cells grown under nitrogen-fixing conditions; the latter activity is missing in cells supplied with ammonia as the nitrogen source. The apparently constitutive and stable activity corresponds to hydrogenase 1, coded by hydSL, and the inducible and unstable second hydrogenase may be the product of the hup gene cluster.

Amino Acid Sequence↗

PubMed1997

The in vitro phosphorylation of p53 by calcium-dependent protein kinase C--characterization of a protein-kinase-C-binding site on p53.

We show that, in vitro, Ca2+-dependent protein kinase C (PKC) phosphorylates recombinant murine p53 protein on several residues contained within a conserved basic region of 25 amino acids, located in the C-terminal part of the protein. Accordingly, synthetic p53-(357-381)-peptide is phosphorylated by PKC at multiple Ser and Thr residues, including Ser360, Thr365, Ser370 and Thr377. We also establish that p53-(357-381)-peptide at micromolar concentrations has the ability to stimulate sequence-specific DNA binding by p53. That stimulation is lost upon phosphorylation by PKC. To further characterise the mechanisms that regulate PKC-dependent phosphorylation of p53-(357-381)-peptide, the phosphorylation of recombinant p53 and p53-(357-381)-peptide by PKC were compared. The results suggest that phosphorylation of full-length p53 on the C-terminal PKC sites is highly dependent on the accessibility of the phosphorylation sites and that a domain on p53 distinct from p53-(357-381)-peptide is involved in binding PKC. Accordingly, we have identified a conserved 27-amino-acid peptide, p53-(320-346)-peptide, within the C-terminal region of p53 and adjacent to residues 357-381 that interacts with PKC in vitro. The interaction between p53-(320-346)-peptide and PKC inhibits PKC autophosphorylation and the phosphorylation of substrates, including p53-(357-381)-peptide, neurogranin and histone H1. Conventional Ca2+-dependent PKC alpha, beta and gamma and the catalytic fragment of PKC (PKM) were nearly equally susceptible to inhibition by p53-(320-346)-peptide. The Ca2+-independent PKC delta was much less sensitive to inhibition. The significance of these findings for understanding the in vivo phosphorylation of p53 by PKC are discussed.

Amino Acid Sequence↗

PubMed1997

Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase localized in mitochondria.

In pea leaves, the synthesis of 7,8-dihydropteroate, a primary step in folate synthesis, was only detected in mitochondria. This reaction is catalyzed by a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase enzyme, which represented 0.04-0.06% of the matrix proteins. The enzyme had a native mol. wt of 280-300 kDa and was made up of identical subunits of 53 kDa. The reaction catalyzed by the 7,8-dihydropteroate synthase domain of the protein was Mg2+-dependent and behaved like a random bireactant system. The related cDNA contained an open reading frame of 1545 bp and the deduced amino acid sequence corresponded to a polypeptide of 515 residues with a calculated M(r) of 56,454 Da. Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein indicated that the plant enzyme is synthesized with a putative mitochondrial transit peptide of 28 amino acids. The calculated M(r) of the mature protein was 53,450 Da. Southern blot experiments suggested that a single-copy gene codes for the enzyme. This result, together with the facts that the protein is synthesized with a mitochondrial transit peptide and that the activity was only detected in mitochondria, strongly supports the view that mitochondria is the major (unique?) site of 7,8-dihydropteroate synthesis in higher plant cells.

Amino Acid Sequence↗