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1.
Clin Microbiol Infect ; 16(2): 126-31, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19456835

ABSTRACT

Eighty-six carbapenem non-susceptible Pseudomonas aeruginosa isolates collected in the National Institute of Respiratory Diseases of Mexico City were screened for the presence of metallo-beta-lactamase (MBL) activity using both E-test strips and a microbiological assay with EDTA-imipenem. Genomic comparisons and sequence analyses conducted with these isolates revealed the presence of bla(VIM-2) in two clonally related isolates, and bla(IMP-15) in a clonally unrelated isolate. Both genes were found to be carried by class 1 integrons, and bla(IMP-15) was additionally present on a broad host-range plasmid. This is the first report of co-existing P. aeruginosa strains producing different MBLs in a Mexican hospital, highlighting the necessity of appropriate surveillance to prevent dissemination of carbapenem resistance.


Subject(s)
Bacterial Proteins/biosynthesis , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/isolation & purification , beta-Lactamases/biosynthesis , Bacterial Proteins/genetics , DNA, Bacterial/genetics , Hospitals , Humans , Integrons , Mexico , Microbial Sensitivity Tests/methods , Plasmids , beta-Lactamases/genetics
2.
Arch Biochem Biophys ; 361(2): 202-6, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9882447

ABSTRACT

Incubation of bovine liver mitochondrial rhodanese in dilute, reducing solutions at temperatures ranging between 30 and 45 degreesC conduced to a rapid loss of enzymatic activity. This inactivation was substantially reduced in the presence of millimolar concentrations of alkali metal ions, divalent cations (including Mg2+, Ca2+, and Ba2+) were ineffective. The extent of protection afforded by monovalent cations was highly dependent on their ionic radii, with K+ and Na+ ions being the most effective protective agents. The protection afforded by a number of anions, including thiosulfate, could be totally ascribed to the presence of the accompanying monovalent cation. The overall results indicate that K+ and Na+, at concentrations and temperatures within the physiological range, substantially contribute to the stabilization of the functional structure of rhodanese.


Subject(s)
Hot Temperature , Metals, Alkali/pharmacology , Mitochondria, Liver/drug effects , Mitochondria, Liver/enzymology , Thiosulfate Sulfurtransferase/metabolism , Animals , Cattle , Enzyme Activation/drug effects , Enzyme Stability/drug effects , Potassium/pharmacology , Salts/pharmacology , Sodium/pharmacology , Thiosulfates/pharmacology
3.
Protein Expr Purif ; 14(2): 275-82, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9790891

ABSTRACT

Using an Escherichia coli strain (RF101) in which the endogenous chromosomal groESL operon was removed, we overexpressed the GroEL and GroES chaperonins cloned from the photosynthetic bacterium Chromatium vinosum. The identities of these proteins were confirmed by immunological and N-terminal sequence analyses. The native molecular masses of GroEL and GroES, as determined by size-exclusion chromatography, were 830 and 74 kDa, respectively. This suggests a tetradecameric structure for GroEL and a heptameric structure for GroES. C. vinosum GroEL catalyzed a K+-stimulated ATP hydrolysis with a specific activity at 25 degreesC of 50.2 +/- 3.8 nmol Pi released min-1 mg protein-1. GroEL ATPase was inhibited by GroES, reaching about 50% inhibition at a ratio GroES-7mer/GroEL-14mer of 1 in the presence of 10 mM KCl. The ATPase Vmax increased almost fivefold in the 25 to 65 degreesC temperature range; higher temperatures led to a rapid inactivation of this activity. The chaperone activity of the C. vinosum GroEL/GroES system was characterized by its effect on the refolding of guanidinium chloride-unfolded rhodanese. In the presence of ATP and GroES, C. vinosum GroEL assisted rhodanese refolding. The heterologous combination C. vinosum GroEL/E. coli GroES or E. coli GroEL/C. vinosum GroES was as effective as the homologous complexes. In summary, this strategy allowed the purification at high yields of fully functional, homogenous C. vinosum GroEL and GroES chaperonins from E. coli.


Subject(s)
Bacterial Proteins/genetics , Chaperonin 10/chemistry , Chaperonin 60/chemistry , Chaperonins/genetics , Chromatium/chemistry , Recombinant Proteins/chemistry , Adenosine Triphosphatases/metabolism , Bacterial Proteins/chemistry , Enzyme Activation/physiology , Enzyme Stability/physiology , Escherichia coli/genetics , Gene Expression/genetics , Kinetics , Operon/genetics , Protein Conformation , Protein Denaturation , Protein Folding , Sequence Analysis , Temperature , Thiosulfate Sulfurtransferase/chemistry
4.
Cell Mol Biol (Noisy-le-grand) ; 44(6): 891-7, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9763192

ABSTRACT

The presence of Hsp70 and Hsp60 molecular chaperones in amyloplasts isolated from cultured sycamore cells was analyzed by immunoblotting. Hsp70 homologs were located in both amyloplast envelope and stromal fractions, but no Hsp60 homologs were detected in any of the different suborganellar fractions. Incubation of whole amyloplasts or their envelope fraction with Mg2+ gamma-32P-ATP resulted in a rapid phosphorylation of the envelope-associated Hsp70 homolog, which constitutes a major target of phosphorylation in these plastids.


Subject(s)
HSP70 Heat-Shock Proteins/metabolism , Plant Proteins/metabolism , Trees/chemistry , Cell Membrane/metabolism , Cells, Cultured , Chaperonin 60/immunology , Chaperonin 60/metabolism , Immunoblotting , Organelles/metabolism , Phosphorylation , Plant Proteins/immunology , Plastids/metabolism , Sequence Homology, Amino Acid , Trees/cytology
5.
J Infect Dis ; 177(5): 1308-13, 1998 May.
Article in English | MEDLINE | ID: mdl-9593017

ABSTRACT

A random-amplified polymorphic DNA assay using partially degenerate oligonucleotides as primers was used for the characterization of 78 epidemiologically related and unrelated clinical isolates of Streptococcus agalactiae belonging to different serotypes. Thirty distinct amplification profiles were obtained among 52 unrelated S. agalactiae isolates assigned to nine groups by serotyping (including 3 nontypeable strains), uncovering the extent of genomic heterogeneity existent within serotypes. This method was particularly useful in providing evidence for or against vertical transmission of a given clone of this microorganism, as well as for relapsing or reinfection in related cases, and suggested clonal relatedness between unrelated S. agalactiae isolates associated with some invasive infections. Thus, this simple methodology represents a suitable tool for the epidemiologic study of S. agalactiae infections.


Subject(s)
Genetic Variation , Random Amplified Polymorphic DNA Technique , Streptococcal Infections/virology , Streptococcus agalactiae/genetics , Base Sequence , DNA Primers , DNA, Bacterial/chemistry , DNA, Bacterial/isolation & purification , Genotype , Humans , Polymerase Chain Reaction/methods , Serotyping , Streptococcus agalactiae/classification , Streptococcus agalactiae/isolation & purification
6.
Eur J Biochem ; 251(3): 724-8, 1998 Feb 01.
Article in English | MEDLINE | ID: mdl-9490045

ABSTRACT

The DnaK system is required for the productive folding of pea chloroplast ferredoxin-NADP+ reductase (FNR) expressed in Escherichia coli. The formation of a mature active enzyme was severely impaired in E. coli dnaK, dnaJ or grpE mutants expressing either the cytosolic precursor of the reductase (preFNR) or the mature apoenzyme, and these forms aggregated extensively in these cells. Coexpression of dnaK from a multicopy plasmid in the dnaK-null mutants restored preFNR processing and folding of FNR, rendering a mature-sized active enzyme. Overexpression of GroESL chaperonins failed to prevent preFNR aggregation, but it restored productive folding of FNR in dnaK-null mutants expressing the mature enzyme. Expression of preFNR in OmpT-protease-deficient E. coli cells resulted in the accumulation of the unprocessed precursor in the soluble fraction of the cells. The interaction of this soluble preFNR, but not the mature reductase, with DnaK and GroEL was evidenced by immunoprecipitation studies. We conclude that, in addition to the GroE chaperonins [Carrillo, N., Ceccarelli, E. A., Krapp, A. R., Boggio, S., Ferreyra, R. G. & Viale, A. M. (1992) J. Biol. Chem. 267, 15537-15541], the DnaK chaperone system plays a crucial role in the folding pathway of FNR.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/physiology , Ferredoxin-NADP Reductase/biosynthesis , Ferredoxin-NADP Reductase/chemistry , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Operon , Pisum sativum/enzymology , Protein Folding , Alleles , Bacterial Proteins/genetics , Binding Sites , Chaperonins , Chloroplasts/enzymology , Cloning, Molecular , Escherichia coli/genetics , HSP40 Heat-Shock Proteins , HSP70 Heat-Shock Proteins/genetics , Heat-Shock Proteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry
7.
Eur J Biochem ; 248(3): 848-55, 1997 Sep 15.
Article in English | MEDLINE | ID: mdl-9342238

ABSTRACT

We have studied the in vivo requirements of the DnaK chaperone system for the folding of recombinant ribulose-bisphosphate carboxylase/oxygenase in Escherichia coli. Expression of functional dimeric or hexadecameric ribulose-bisphosphate carboxylase from different bacterial sources (including purple bacteria and cyanobacteria) was severely impaired in E. coli dnaK, dnaJ, or grpE mutants. These enzymes were synthesized mostly in soluble, fully enzymatically active forms in wild-type E. coli cells cultured in the temperature range 20-42 degrees C, but aggregated extensively in dnaK null mutants. Co-expression of dnaK, but not groESL, markedly reduced the aggregation of ribulose-bisphosphate carboxylase subunits in dnaK null mutants and restored the enzyme activity to levels found in isogenic wild-type strains. Ribulose-bisphosphate carboxylase expression in wild-type E. coli cells growing at 30 degrees C promoted an enhanced synthesis of stress proteins, apparently by sequestering DnaK from its negative regulatory role in this response. The overall results indicate that the DnaK chaperone system assists in vivo the folding pathway of ribulose-bisphosphate carboxylase large subunits, most probably at its very early stages.


Subject(s)
Escherichia coli Proteins , Escherichia coli/enzymology , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Protein Folding , Ribulose-Bisphosphate Carboxylase/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chaperonins/genetics , Chaperonins/metabolism , Cyanobacteria/enzymology , Dimerization , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Gene Expression Regulation, Bacterial/genetics , HSP70 Heat-Shock Proteins/genetics , Immunoblotting , Molecular Chaperones/genetics , Mutation , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribulose-Bisphosphate Carboxylase/metabolism , Temperature
8.
Plant Mol Biol ; 35(6): 763-75, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9426597

ABSTRACT

We cloned and characterized a genomic locus encoding a distinct member of the DnaK/Hsp70 family of molecular chaperones, dnaK1, from the halotolerant cyanobacterium Aphanothece halophytica. Co-expression of dnaK1 with a plant plastocyanin precursor in Escherichia coli resulted in a dramatic increase in the solubility of the plant protein. This indicates that A. halophytica dnaK1 encodes a functional protein possessing functions assigned to DnaK/Hsp70 chaperone members. The A. halophytica dnaK1 locus also encompasses grpE and dnaJ homologue genes in the order grpE-dnaK1-dnaJ. The transcript content of dnaK1 increased strongly upon subjecting cyanobacterial cells to heat stress. Northern analyses using specific probes indicated transcript species of 2.8, 2.2, 1.3, and 0.7 kb, which comprised grpE-dnaK1, dnaK1, dnaJ, and grpE, respectively. This indicates the presence of different terminators and/or heat stress promoters in this locus. Both dnaK1 transcript and protein levels increased in cyanobacterial cells transferred to hyperosmotic environments, suggesting a role of DnaK1 in the protection and/or recovery of A. halophytica from this particular stress.


Subject(s)
Cyanobacteria/genetics , Escherichia coli Proteins , Genes, Bacterial , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/isolation & purification , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cyanobacteria/physiology , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Genome, Bacterial , HSP70 Heat-Shock Proteins/biosynthesis , HSP70 Heat-Shock Proteins/chemistry , Hot Temperature , Hypertonic Solutions , Molecular Sequence Data , Phylogeny , Recombinant Proteins/biosynthesis , Sodium Chloride/pharmacology
10.
Medicina (B Aires) ; 55(6): 681-4, 1995.
Article in English | MEDLINE | ID: mdl-8731579

ABSTRACT

Epidemiological studies of Streptococcus agalactiae strains have been limited by the lack of sensitive and discriminatory methods for comparing clinical isolates. Serotyping, albeit a widely used methodology, has been shown to possess low capability to distinguish between epidemiologically related and unrelated isolates. We have employed here a random amplification of polymorphic DNA (RAPD) assay, using degenerate oligonucleotides as primers, to characterize S. agalactiae isolates from related or unrelated clinical samples. Epidemiologically-related isolates (mother-infant pairs) showed identical profiles by this methodology. On the contrary, 12 epidemiologically-unrelated isolates (classified into 5 different serotypes) resulted in 11 distinct RAPD patterns. This suggests that the proposed modified RAPD assay provides a highly discriminatory tool for the analysis of genomic diversity among isolates from pathogenic organisms.


Subject(s)
Random Amplified Polymorphic DNA Technique , Streptococcus agalactiae/isolation & purification , DNA Primers , Female , Genome, Bacterial , Humans , Infant, Newborn , Polymerase Chain Reaction , Pregnancy , Serotyping , Streptococcus agalactiae/classification , Streptococcus agalactiae/genetics
11.
Medicina [B Aires] ; 55(6): 681-4, 1995.
Article in English | BINACIS | ID: bin-37151

ABSTRACT

Epidemiological studies of Streptococcus agalactiae strains have been limited by the lack of sensitive and discriminatory methods for comparing clinical isolates. Serotyping, albeit a widely used methodology, has been shown to possess low capability to distinguish between epidemiologically related and unrelated isolates. We have employed here a random amplification of polymorphic DNA (RAPD) assay, using degenerate oligonucleotides as primers, to characterize S. agalactiae isolates from related or unrelated clinical samples. Epidemiologically-related isolates (mother-infant pairs) showed identical profiles by this methodology. On the contrary, 12 epidemiologically-unrelated isolates (classified into 5 different serotypes) resulted in 11 distinct RAPD patterns. This suggests that the proposed modified RAPD assay provides a highly discriminatory tool for the analysis of genomic diversity among isolates from pathogenic organisms.

12.
Int J Syst Bacteriol ; 44(3): 527-33, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7520741

ABSTRACT

The essential GroEL proteins represent a subset of molecular chaperones ubiquitously distributed among species of the eubacterial lineage, as well as in eukaryote organelles. We employed these highly conserved proteins to infer eubacterial phylogenies. GroEL from the species analyzed clustered in distinct groups in evolutionary trees drawn by either the distance or the parsimony method, which were in general agreement with those found by 16S rRNA comparisons (i.e., proteobacteria, chlamydiae, bacteroids, spirochetes, firmicutes [gram-positive bacteria], and cyanobacteria-chloroplasts). Moreover, the analysis indicated specific relationships between some of the aforementioned groups which appeared not to be clearly defined or controversial in rRNA-based phylogenetic studies. For instance, a monophyletic origin for the low-G+C and high-G+C subgroups among the firmicutes, as well as their specific relationship to the cyanobacteria-chloroplasts, was inferred. The general observations suggest that GroEL proteins provide valuable evolutionary tools for defining evolutionary relationships among the eubacterial lineage of life.


Subject(s)
Bacteria/classification , Bacteria/genetics , Bacterial Proteins/genetics , Biological Evolution , Heat-Shock Proteins/genetics , Bacteria/chemistry , Base Composition , Chaperonin 60 , Chlamydia/classification , Chlamydia/genetics , Cyanobacteria/classification , Cyanobacteria/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Helicobacter pylori/classification , Helicobacter pylori/genetics , Phylogeny , Porphyromonas gingivalis/classification , Porphyromonas gingivalis/genetics , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Sequence Homology, Nucleic Acid , Species Specificity , Spirochaetales/classification , Spirochaetales/genetics
13.
FEBS Lett ; 341(2-3): 146-51, 1994 Mar 21.
Article in English | MEDLINE | ID: mdl-7907991

ABSTRACT

The heat-shock 60 proteins (Hsp60) constitute a subset of molecular chaperones essential for the survival of the cell, present in eubacteria as well as in eukaryotic organelles. Here, we have employed these highly conserved proteins for the inferences of the origins of the organelles. Hsp60s present in mitochondria from different eukaryotic lineages formed a clade, which showed the closest relationship to that of the Ehrlichia/Rickettsia cluster among the alpha-Proteobacteria. This, in addition to phenotypic characteristics, suggests that these obligate intracellular parasites and the lineage that generated the mitochondrion shared last common ancestry. In turn, Hsp60s present in chloroplasts from plants and a red alga, respectively, clustered specifically with those of the cyanobacteria, suggesting that all plastids derive exclusively from this eubacterial lineage.


Subject(s)
Biological Evolution , Organelles/physiology , Proteins/physiology , Animals , Bacteria , Chaperonin 60 , Chaperonins , Heat-Shock Proteins/physiology , Humans , Organelles/classification , Plastids
14.
J Bacteriol ; 175(5): 1514-23, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8444812

ABSTRACT

A recombinant lambda phage which was able to propagate in groE mutants of Escherichia coli was isolated from a Chromatium vinosum genomic DNA library. A 4-kbp SalI DNA fragment, isolated from this phage and subcloned in plasmid vectors, carried the C. vinosum genes that allowed lambda growth in these mutants. Sequencing of this fragment indicated the presence of two open reading frames encoding polypeptides of 97 and 544 amino acids, respectively, which showed high similarity to the molecular chaperones GroES and GroEL, respectively, from several eubacteria and eukaryotic organelles. Expression of the cloned C. vinosum groESL genes in E. coli was greatly enhanced when the cells were transferred to growth temperatures that induce the heat shock response in this host. Coexpression in E. coli of C. vinosum groESL genes and the cloned ribulose bisphosphate carboxylase/oxygenase genes from different phototrophic bacteria resulted in an enhanced assembly of the latter enzymes. These results indicate that the cloned DNA fragment encodes C. vinosum chaperonins, which serve in the assembly process of oligomeric proteins. Phylogenic analysis indicates a close relationship between C. vinosum chaperonins and their homologs present in pathogenic species of the gamma subdivision of the eubacterial division Proteobacteria.


Subject(s)
Bacterial Proteins/genetics , Chaperonins/genetics , Chromatium/genetics , Heat-Shock Proteins/genetics , Amino Acid Sequence , Base Sequence , Biological Evolution , Cloning, Molecular , DNA, Bacterial , Escherichia coli , Escherichia coli Proteins , Genes, Bacterial , Genetic Complementation Test , Molecular Sequence Data , Restriction Mapping , Sequence Homology, Amino Acid , Temperature
15.
J Biol Chem ; 267(22): 15537-41, 1992 Aug 05.
Article in English | MEDLINE | ID: mdl-1353496

ABSTRACT

We have recently reported the expression in Escherichia coli of an enzymatically competent ferredoxin-NADP+ oxidoreductase from cloned pea genes encoding either the mature enzyme or its precursor protein (Ceccarelli, E. A., Viale, A. M., Krapp, A. R., and Carrillo, N. (1991) J. Biol. Chem. 266, 14283-14287). Processing to the mature form by bacterial protease(s) and FAD assembly occurred in the bacterial cytosol. Expression of ferredoxin-NADP+ reductase in chaperonin-deficient (groE-) mutants of E. coli resulted in partial reductase assembly at permissive growth temperatures (i.e. 30 degrees C), and in total breakdown of holoenzyme assembly, and accumulation as aggregated inclusion bodies at non-permissive temperatures (i.e. 42 degrees C). Coexpression in these mutants of a cloned groESL operon from the phototrophic bacterium Chromatium vinosum resulted in partial or total recoveries of ferredoxin-NADP+ reductase assembly. The overall results indicate that bacterial chaperonins are required for the productive folding/assembly of eucaryotic ferredoxin-NADP+ reductase expressed in E. coli.


Subject(s)
Bacterial Proteins/metabolism , Chaperonins/metabolism , Chromatium/genetics , Escherichia coli/genetics , Ferredoxin-NADP Reductase/genetics , Operon , Bacterial Proteins/genetics , Chaperonin 10 , Chaperonin 60 , Chromatium/enzymology , Cloning, Molecular , Escherichia coli/metabolism , Escherichia coli Proteins , Gene Expression , Heat-Shock Proteins/genetics , Plasmids
16.
J Bacteriol ; 173(16): 5224-9, 1991 Aug.
Article in English | MEDLINE | ID: mdl-1907267

ABSTRACT

An open reading frame, rbcR, was identified 226 bp upstream of rbcAB, i.e., the ribulose 1,5-bisphosphate carboxylase genes expressed in the phototrophic purple bacterium Chromatium vinosum. Several features reveal that rbcR encodes a member of the LysR family of transcriptional regulators, in which an anomalous content of lysine and arginine residues (Lys/Arg anomaly) was found. The expression of rbcR in Escherichia coli as a protein fused to the N-terminal region of beta-galactosidase led to reduced expression of rbcAB. Thus, rbcR is likely to encode a trans-acting transcriptional regulator of rbcAB expression in C. vinosum.


Subject(s)
Bacterial Proteins , Chromatium/genetics , Gene Expression Regulation, Bacterial/genetics , Genes, Regulator/genetics , Transcription Factors/genetics , Amino Acid Sequence , Arginine/genetics , Base Sequence , Chromatium/enzymology , Escherichia coli/metabolism , Lysine/genetics , Molecular Sequence Data , Open Reading Frames/genetics , Recombinant Fusion Proteins/biosynthesis , Ribulose-Bisphosphate Carboxylase/genetics , Sequence Alignment
17.
J Biol Chem ; 266(22): 14283-7, 1991 Aug 05.
Article in English | MEDLINE | ID: mdl-1907276

ABSTRACT

The flavoprotein ferredoxin-NADP+ reductase (FNR) catalyzes the final step of the photosynthetic electron transport chain, i.e. the reduction of NADP+ by ferredoxin. A cloned FNR cDNA from a pea library (Newman, B., and Gray, J. (1988) Plant Mol. Biol. 10, 511-520) was used to construct plasmids which express the apoenzyme in Escherichia coli. Two recombinant vectors were prepared, one containing the sequence corresponding to the mature enzyme and another including, in addition, the sequence of the transit peptide that directs FNR to the chloroplast. These proteins were expressed as fusion products to the NH2-terminal portion of beta-galactosidase. In both cases, a 35-kDa immunoreactive polypeptide was the major product, suggesting that the proteins were processed in vivo. NH2-terminal sequence determination of the purified recombinant proteins indicate cleavage at positions -1/-2 with respect to the normal processing site in chloroplasts. The processed enzymes showed enzymatic activities and spectral properties that were similar or identical to those of native plant FNR. When a La protease-deficient E. coli strain was used as a host, the expressed FNR precursor was found to be poorly processed, associated to bacterial pellets, and showed no detectable FNR activity. The overall results indicate that acquisition of the native enzyme conformation and assembly of the prosthetic group takes place in the bacterial host, generating an enzyme that is, as far as studied, indistinguishable from plant FNR.


Subject(s)
Fabaceae/enzymology , Ferredoxin-NADP Reductase/genetics , Plants, Medicinal , Protein Precursors/genetics , Protein Processing, Post-Translational , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Ferredoxin-NADP Reductase/metabolism , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Molecular Sequence Data , Plasmids , Protein Precursors/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , beta-Galactosidase/genetics
18.
FEBS Lett ; 287(1-2): 62-6, 1991 Aug 05.
Article in English | MEDLINE | ID: mdl-1879538

ABSTRACT

Structurally intact and metabolically competent mitochondria isolated from liquid-culture cells of sycamore (Acer pseudoplatanus L.) were shown to incorporate ADPglucose. Employing the double silicone oil layer filtering centrifugation method, we examined the kinetic properties of the uptake of various adenylates as well as the inhibitory effects exerted by carboxyatractyloside, atractyloside and bongkrekic acid, known specific inhibitors of the mitochondrial adenylate translocator. Immunoblot patterns of peptides derived from the partial proteolytic digestion of the mitochondrial and plastid adenylate translocators were shown to be essentially the same. We conclude that the molecular entities engaged in the adenylate transport system operating in two different organelles, mitochondria and amyloplasts, are very similar.


Subject(s)
Adenine Nucleotides/metabolism , Mitochondria/enzymology , Mitochondrial ADP, ATP Translocases/metabolism , Plants/ultrastructure , Adenosine Diphosphate Glucose/metabolism , Atractyloside/analogs & derivatives , Atractyloside/pharmacology , Biological Transport , Bongkrekic Acid/pharmacology , Cells, Cultured , Immunoblotting , Kinetics , Mitochondrial ADP, ATP Translocases/antagonists & inhibitors
19.
Plant Physiol ; 96(4): 1142-9, 1991 Aug.
Article in English | MEDLINE | ID: mdl-16668311

ABSTRACT

Incubation of amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.) with [gamma-(32)P]ATP resulted in the rapid phosphorylation (half-time of 40 seconds at 25 degrees Celcius) of organellar polypeptides. The preferred substrate for amyloplast protein kinases was Mg(2+). ATP, and recovery of only [(32)P]serine after partial acid hydrolysis indicated the predominance of protein serine kinases in the organelle. These activities were located in the envelope and stromal fractions of the plastid, which showed different specificities toward exogenous protein substrates and distinct patterns of phosphorylation of endogenous polypeptides. A 66-kilodalton polypeptide, inaccessible to an exogenously added protease, was one of the major phosphorylated products found in intact amyloplasts at low [gamma-(32)P] adenosine triphosphate concentrations. This polypeptide represented the major phosphoprotein observed with the isolated envelope fraction. The patterns of polypeptide phosphorylation found in intact amyloplasts and chloroplasts from cultured cell lines of sycamore were clearly distinguishable. The overall results indicate the presence of protein phosphorylation systems unique to this reserve plastid present in nonphotosynthetic tissues.

20.
Proc Natl Acad Sci U S A ; 88(13): 5769-73, 1991 Jul 01.
Article in English | MEDLINE | ID: mdl-11607196

ABSTRACT

Starch biosynthesis has been studied by using amyloplasts isolated from cultured cells of sycamore trees (Acer pseudoplatanus L.). Highly purified intact amyloplasts, free from mitochondria and starch granules derived from broken amyloplasts, were isolated from a Percoll step gradient. Subsequently, the double silicone oil layer centrifugation technique was used to study adenylate transport in the amyloplasts. An adenylate-specific carrier was found to be active in the uptake of ATP, ADP, AMP, and most importantly, ADPglucose (ADP-Glc). Kinetic analyses showed that the uptake of these adenylates was mutually competitive with each other. In contrast to the mitochondrial adenylate carrier, in amyloplasts only ATP and ADP-Glc uptake were inhibited by carboxyatractyloside. Evidence is presented that the ADP-Glc transported into the amyloplast stroma can be used in starch synthesis catalyzed by starch synthase (ADP-Glc:1,4-alpha-D-glucan 4-alpha-D-glucosyltransferase, EC 2.4.1.21). We propose that starch biosynthesis in amyloplasts is tightly coupled with the direct transport of ADP-Glc synthesized in the cytosol by sucrose synthase (ADP-Glc:D-fructose 2-alpha-D-glucosyltransferase, EC 2.4.1.13)

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