[Effects of two methods of heat therapy on the acute vascular response and hemodynamics in healthy subjects]. / Efectos de dos métodos de termoterapia sobre la respuesta vascular aguda y parámetros hemodinámicos en un grupo de sujetos sanos.

OBJECTIVE: Recently, non-pharmacological resources to relieve pain like hot packs and ultrasound (US) have become common in clinical practice. However, little experimental evidence is available about the possible mechanisms through which these methods bring about pain relief. We aimed to determine the effects of hot packs and US on the acute vascular response and on hemodynamic parameters in healthy subjects. MATERIALS AND METHODS: We conducted an experimental study in 20 healthy subjects (10 men and 10 women; mean age, 22.54±1.70 years). The two interventions were randomly applied: a) hot packs (n=10): 15min at 60°C and b) US (n=10): 15min at 1Mhz. Before and after each intervention, the following vascular parameters were measured in the brachial artery using Doppler ultrasonography with a 7MHz probe: arterial compliance, elastic modulus, beta stiffness index, systolic and diastolic arterial diameters, systolic flow velocity and diastolic flow velocity, systolic/diastolic ratio, resistance index, and pulsatility index. The following hemodynamic parameters were monitored: heart rate and blood pressure (systolic, diastolic, and mean). RESULTS: After the application of hot packs, we observed changes in diastolic flow velocity and in the pulsatility index (P<05). After the application of US, we observed changes in diastolic flow velocity, systolic/diastolic ratio, resistance index, and arterial compliance (P<05). No changes in hemodynamic parameters were observed after either intervention. CONCLUSIONS: Applying hot packs or US modifies the physiology of the vascular system but does not affect hemodynamic parameters in healthy subjects.

Biochemical characterization of the Arabidopsis biotin synthase reaction. The importance of mitochondria in biotin synthesis.

Biotin synthase, encoded by the bio2 gene in Arabidopsis, catalyzes the final step in the biotin biosynthetic pathway. The development of radiochemical and biological detection methods allowed the first detection and accurate quantification of a plant biotin synthase activity, using protein extracts from bacteria overexpressing the Arabidopsis Bio2 protein. Under optimized conditions, the turnover number of the reaction was >2 h(-1) with this in vitro system. Purified Bio2 protein was not efficient by itself in supporting biotin synthesis. However, heterologous interactions between the plant Bio2 protein and bacterial accessory proteins yielded a functional biotin synthase complex. Biotin synthase in this heterologous system obeyed Michaelis-Menten kinetics with respect to dethiobiotin (K(m) = 30 microM) and exhibited a kinetic cooperativity with respect to S-adenosyl-methionine (Hill coefficient = 1.9; K(0.5) = 39 microM), an obligatory cofactor of the reaction. In vitro inhibition of biotin synthase activity by acidomycin, a structural analog of biotin, showed that biotin synthase reaction was the specific target of this inhibitor of biotin synthesis. It is important that combination experiments using purified Bio2 protein and extracts from pea (Pisum sativum) leaf or potato (Solanum tuberosum) organelles showed that only mitochondrial fractions could elicit biotin formation in the plant-reconstituted system. Our data demonstrated that one or more unidentified factors from mitochondrial matrix (pea and potato) and from mitochondrial membranes (pea), in addition to the Bio2 protein, are obligatory for the conversion of dethiobiotin to biotin, highlighting the importance of mitochondria in plant biotin synthesis.

Is plant biotin holocarboxylase synthetase a bifunctional enzyme?

Holocarboxylase synthetases (HCSs) catalyse the biotinylation of biotin-dependent carboxylases in both prokaryotes and eukaryotes. In Escherichia coli and Bacillus subtilis, the protein also acts as a transcriptional repressor that regulates the synthesis of biotin. Previously, we isolated and characterized a cDNA encoding an Arabidopsis thaliana HCS and subsequently assigned this enzyme form to the chloroplast compartment. To investigate whether or not the Arabidopsis protein may function as a regulator in E. coli, we have expressed the functional plant HCS in a birA-derepressed mutant strain of E. coli devoid of the corresponding E. coli protein and carrying a promoter-less LacZ gene marker inserted into the biotin operon, such that the bio promoter drives the synthesis of beta-galactosidase. Our data demonstrate that although the expressed plant HCS efficiently complemented the function of apo-carboxylase biotinylation in E. coli, it proved unable to regulate the expression of the biotin biosynthetic genes.

Purification and properties of the chloroplastic form of biotin holocarboxylase synthetase from Arabidopsis thaliana overexpressed in Escherichia coli.

Holocarboxylase synthetases (HCSs) are key enzymes in biotin utilisation in both prokaryotes and eukaryotes. In a previous study, we demonstrated that, in plants, HCS activity is localised in cytosol, chloroplasts and mitochondria. We also described the cloning and sequencing of a full-length cDNA encoding an Arabidopsis thaliana HCS isoform with a putative organelle-transit peptide. In the study reported here, this cDNA was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated using an efficient three-step purification procedure. Polyclonal antibodies raised against pure HCS were produced to elucidate the subcellular localisation of this protein. Immunodetection carried out by Western blotting of isolated pea leaf subcellular compartments specifically revealed a single polypeptide that was ascribed to the chloroplast compartment. Immunocytochemistry of thin-cut sections from tobacco leaves, transformed by the complete coding sequence of A. thaliana HCS cDNA via Agrobacterium tumefaciens, confirmed that the enzyme encoded by this cDNA is the chloroplastic isoform. Moreover, physicochemical, biochemical and kinetic properties of the pure recombinant HCS were determined. The native recombinant enzyme is a 37-kDa monomer. In contrast to the major part of HCS activity measured in leaf extracts, the recombinant chloroplastic enzyme did not require addition of Mg2+ to be fully active, but was substantially inhibited by EDTA. This suggested that the chloroplastic HCS may contain a tightly-bound divalent cation required for enzyme activity. The recombinant enzyme was able to biotinylate efficiently apo-biotin carboxyl carrier protein (BCCP) from E. coli and apo-methylcrotonoyl-CoA carboxylase (MCCase) from A. thaliana. Apparent Km values for the enzyme substrates D-biotin, ATP and apo-MCCase were found to be 130 nM, 4.4 microM and 32 microM, respectively. Steady-state kinetic analyses of the HCS-catalysed reaction were investigated with respect to reaction mechanism and inhibition by AMP, one of the end-products of the enzyme-catalysed reaction. Substrate interaction and product inhibition patterns indicated that ATP and D-biotin bind sequentially, in an ordered manner, to the enzyme and that ATP or D-biotin and apo-BCCP bind in ping-pong fashion.

Evidence for multiple forms of biotin holocarboxylase synthetase in pea (Pisum sativum) and in Arabidopsis thaliana: subcellular fractionation studies and isolation of a cDNA clone.

The intracellular compartmentation of biotin holocarboxylase synthetase has been investigated in pea (Pisum sativum) leaves, by isolation of organelles and fractionation of protoplasts. Enzyme activity was mainly located in cytosol (approx. 90% of total cellular activity). Significant activity was also identified in the soluble phase of both mitochondria and chloroplasts. Two enzyme forms were separated by anion-exchange chromatography. The major form was found to be specific for the cytosol compartment, whereas the minor form was present in mitochondria as well as in chloroplasts. We also report the isolation and DNA sequence of a cDNA encoding an Arabidopsis thaliana biotin holocarboxylase synthetase. This cDNA was isolated by functional complementation of a conditional lethal Escherichia coli birA (biotin ligase gene, which regulates biotin synthesis) mutant. This indicated that the recombinant plant protein was able to biotinylate specifically an essential apoprotein substrate in the bacterial host, that is a subunit of acetyl-CoA carboxylase called biotin carboxyl carrier protein. The full-length nucleotide sequence (1534 bp) encodes a protein of 367 amino acid residues with a molecular mass of 41172 Da and shows specific regions of similarity to other biotin holocarboxylase synthetase genes as isolated from bacteria and yeast, and with cDNA species from human. A sequence downstream of the first translation initiation site encodes a putative peptide structurally similar to organelle-targeting pre-sequences, suggesting a mitochondrial or chloroplastic localization for this isoform.

Biotin synthesis in higher plants: purification and characterization of bioB gene product equivalent from Arabidopsis thaliana overexpressed in Escherichia coli and its subcellular localization in pea leaf cells.

Biotin synthase catalyses the final step in the biotin biosynthetic pathway and is encoded by the bioB gene in Escherichia coli. To investigate the conversion of dethiobiotin to biotin in the plant kingdom, the cDNA encoding the bioB gene product equivalent from Arabidopsis thaliana was used to construct an E. coli overexpression strain. The purified A. thaliana bioB gene product is a homodimer (100 kDa) with a reddish color and has an absorbance spectrum characteristic of protein with [2Fe-2S] clusters. Its intracellular compartmentation in pea leaves discloses a unique polypeptide of 39 kDa within the matrix of mitochondria.

Kinetic studies on two isoforms of acetyl-CoA carboxylase from maize leaves.

The steady-state kinetics of two multifunctional isoforms of acetyl-CoA carboxylase (ACCase) from maize leaves (a major isoform, ACCase1 and a minor isoform, ACCase2) have been investigated with respect to reaction mechanism, inhibition by two graminicides of the aryloxyphenoxypropionate class (quizalofop and fluazifop) and some cellular metabolites. Substrate interaction and product inhibition patterns indicated that ADP and P(i) products from the first partial reaction were not released before acetyl-CoA bound to the enzymes. Product inhibition patterns did not match exactly those predicted for an ordered Ter Ter or a random Ter Ter mechanism, but were close to those postulated for an ordered mechanism. ACCase2 was about 1/2000 as sensitive as ACCase1 to quizalofop but only about 1/150 as sensitive to fluazifop. Fitting inhibition data to the Hill equation indicated that binding of quizalofop or fluazifop to ACCase1 was non-cooperative, as shown by the Hill constant (n(app)) values of 0.86 and 1.16 for quizalofop and fluazifop respectively. Apparent inhibition constant values (K' from the Hill equation) for ACCase1 were 0.054 microM for quizalofop and 21.8 microM for fluazifop. On the other hand, binding of quizalofop or fluazifop to ACCase2 exhibited positive co-operativity, as shown by the (napp) values of 1.85 and 1.59 for quizalofop and fluazifop respectively. K' values for ACCase2 were 1.7 mM for quizalofop and 140 mM for fluazifop. Kinetic parameters for the co-operative binding of quizalofop to maize ACCase2 were close to those of another multifunctional ACCase of limited sensitivity to graminicide, ACC220 from pea. Inhibition of ACCase1 by quizalofop was mixed-type with respect to acetyl-CoA or ATP, but the concentration of acetyl-CoA had the greater effect on the level of inhibition. Neither ACCase1 nor ACCase2 was appreciably sensitive to CoA esters of palmitic acid (16:0) or oleic acid (18:1). Approximate IC50 values were 10 microM (ACCase2) and 50 microM (ACCase1) for both CoA esters. Citrate concentrations up to 1 mM had no effect on ACCase1 activity. Above this concentration, citrate was inhibitory. ACCase2 activity was slightly stimulated by citrate over a broad concentration range (0.25-10 mM). The significance of possible effects of acyl-CoAs or citrate in vivo is discussed.

Induction of beta-methylcrotonyl-coenzyme A carboxylase in higher plant cells during carbohydrate starvation: evidence for a role of MCCase in leucine catabolism.

Induction of beta-methylcrotonyl-coenzyme A carboxylase (MCCase) activity was observed during carbohydrate starvation in sycamore cells. In mitochondria isolated from starved cells, we noticed a marked accumulation of the biotinylated subunit of MCCase, of which the apparent molecular weight of 74000 was similar to that of the polypeptide from mitochondria of potato tubers. Our results provide evidence for a role of MCCase in the catabolic pathway of leucine, a branched-chain amino acid which transiently accumulates in carbon-starved cells in relation to a massive breakdown of proteins. Furthermore, when control sycamore cells were incubated in the presence of exogenous leucine, this amino acid accumulated in the cells and no induction or accumulation of MCCase was observed, indicating that leucine is not responsible for the induction of its catabolic machinery. Finally, MCCase is proposed as a new biochemical marker of the autophagic process triggered by carbohydrate starvation.

Protein biotinylation in higher plants: characterization of biotin holocarboxylase synthetase activity from pea (Pisum sativum) leaves.

Biotin holocarboxylase synthetase was partially purified from pea leaves by a sequence of ammonium sulphate fractionation and DEAE 52-cellulose chromatography. Enzyme activity was assayed using apo-(biotin carboxyl carrier protein) from an Escherichia coli bir A mutant affected in biotin holocarboxylase synthetase activity. Conditions for optimal catalytic activity and biochemical parameters of the plant enzyme were determined. This is the first direct evidence of the existence of biotin holocarboxylase synthetase activity in plants.

Isolation and Characterization of Biotin Carboxylase from Pea Chloroplasts.

Pea (Pisum sativum L.) leaf acetyl-coenzyme A carboxylase (ACCase) exists as two structurally different forms: a major, chloroplastic, dissociable form and a minor, multifunctional enzyme form located in the leaf epidermis. The dissociable form is able to carboxylate free D-biotin as an alternate substrate in place of the natural substrate, biotin carboxyl carrier protein. Here we report the purification of the biotin carboxylase component of the chloroplastic pea leaf ACCase. The purified enzyme, free from carboxyltransferase activity, is composed of two firmly bound polypeptides, one of which (38 kD) is biotinylated. In contrast to bacterial biotin carboxylase, which retains full activity upon removal of the biotin carboxyl carrier component, attempts to dissociate the two subunits of the plant complex led to a complete loss of biotin carboxylase activity. Steady-state kinetic studies of the biotin carboxylase reaction reveal that addition of all substrates on the enzyme is sequential and that no product release is possible until all three substrates (MgATP, D-biotin, bicarbonate) are bound to the enzyme and all chemical processes at the active site are completed. In agreement with this mechanism, bicarbonate-dependent ATP hydrolysis by the enzyme is found to be strictly dependent on the presence of exogenous D-biotin in the reaction medium.

Kinetics of the two forms of acetyl-CoA carboxylase from Pisum sativum. Correlation of the substrate specificity of the enzymes and sensitivity towards aryloxyphenoxypropionate herbicides.

Steady-state kinetics of the 220-kDa form of acetyl-CoA carboxylase (ACC220), as purified from mature pea seeds, have been investigated with respect to the substrate specificity and inhibition by quizalofop, a herbicide of the aryloxyphenoxypropionate type. The enzyme showed a dual specificity, being able to carboxylate propionyl-CoA at a maximal rate approximately 20% that measured in the presence of the acetyl-CoA substrate. These two reactions occur at separate sites on the enzyme. One site binds either acetyl-CoA (Km = 226 microM) or propionyl-CoA (Km = 38 microM) and is strongly inhibited by quizalofop (Ki = 25 microM and 9.3 microM for the acetyl-CoA and propionyl-CoA substrates, respectively). The other is specific for acetyl-CoA (Km = 11 microM) and is much less inhibited by quizalofop (Ki = 256 microM). Owing to the existence of these two catalytically different sites, the enzyme obeyed Michaelis-Menten kinetics with propionyl-CoA, but exhibited kinetic co-operativity in the presence of acetyl-CoA. Also, kinetics of propionyl-CoA carboxylase activity of ACC220 exhibited hyperbolic inhibition in the presence of quizalofop, but co-operative inhibition when following the ACC activity of the enzyme. The results suggest that the higher the substrate specificity, the lower the quizalofop sensitivity of the active site. Similar kinetic behaviour was observed with ACC220 purified from pea leaves. Also, the apparent correlation between the substrate specificity and the sensitivity of ACC towards quizalofop was confirmed by kinetic analyses of the low-molecular-mass form of ACC present in chloroplasts of young pea leaves. This enzyme was insensitive to quizalofop inhibition and was not able to carboxylate propionyl-CoA. No other propionyl-CoA carboxylase activity, different from that catalysed by ACC220, could be detected from either reproductive or vegetative organs of pea plants at any stage of development.

Localization and characterization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides.

Young pea leaves contain two structurally different forms of acetyl-CoA carboxylase (EC 6.4.1.2; ACCase). A minor form, which accounted for about 20% of the total ACCase activity in the whole leaf, was detected in the epidermal tissue. This enzyme was soluble and was purified to homogeneity from young pea leaf extracts. It consisted of a dimer of two identical biotinyl subunits of molecular mass 220 kDa. In this respect, this multifunctional enzyme was comparable with that described in other plants and in other eukaryotes. A predominant form was present in both the epidermal and mesophyll tissues. In mesophyll protoplasts, ACCase was detected exclusively in the soluble phase of chloroplasts. This enzyme was partially purified from pea chloroplasts and consisted of a freely dissociating complex, the activity of which may be restored by combination of its separated constituents. The partially purified enzyme was composed of several subunits of molecular masses ranging from 32 to 79 kDa, for a native molecular mass > 600 kDa. One of these subunits, of molecular mass 38 kDa, was biotinylated. This complex subunit structure was comparable with that of microorganisms and was referred to as a 'prokaryotic' form of ACCase. Biochemical parameters were determined for both ACCase forms. Finally, both pea leaf ACCases exhibited different sensitivities towards the grass ACCase herbicide, diclofop. This compound had no effect on the 'prokaryotic' form of ACCase, while the 'eukaryotic' form was strongly inhibited.

Developmental patterns of free and protein-bound biotin during maturation and germination of seeds of Pisum sativum: characterization of a novel seed-specific biotinylated protein.

Mature dry pea seeds contain three major biotinylated proteins. Two of these of subunit molecular mass about 75 kDa and 200 kDa are associated with 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4) and acetyl-CoA carboxylase activities (EC 6.4.1.2) respectively. The third does not exhibit any of the biotin-dependent carboxylase activities found in higher organisms and represents the major part of the total protein-bound biotin in the seeds. This novel protein has been purified from a whole pea seed extract. Because in SDS/polyacrylamide gels the protein migrates with an apparent molecular mass of about 65 kDa, it is referred to as SBP65, for 65 kDa seed biotinylated protein. The molecular mass of native SBP65 is greater than 400 kDa, suggesting that the native protein assumes a polymeric structure, resulting from the association of six to eight identical subunits. The results of CNBr cleavage experiments suggest that biotin is covalently bound to the protein. The stoichiometry is 1 mol of biotin per 1 mol of 65 kDa polypeptide. The temporal and spatial pattern of expression of SBP65 is described. SBP65 is specifically expressed in the seeds, being absent from leaf, root, stem, pod and flower tissues of pea plants. The level of SBP65 increases dramatically during seed development. The protein is not detectable in very young seeds. Its accumulation pattern parallels that for storage proteins, being maximally expressed in the mature dry seeds. SBP65 disappears at a very high rate during seed germination. The level of free biotin has also been evaluated for various organs of pea plants. In all proliferating tissues examined (young developing seeds, leaf, root, stem, pod and flower tissues), free biotin is in excess of protein-bound biotin. Only in the mature dry seeds is protein-bound biotin (i.e. that bound to SBP65) in excess of free biotin. These temporal expression patterns, and the strict organ specificity for expression of SBP65, are discussed with regard to the possibility that in plants, as in mammals, biotin plays a specialized role in cell growth and differentiation.

Purification and Characterization of 3-Methylcrotonyl-Coenzyme A Carboxylase from Higher Plant Mitochondria.

3-Methylcrotonyl-coenzyme A (CoA) carboxylase was purified to homogeneity from pea (Pisum sativum L.) leaf and potato (Solanum tuberosum L.) tuber mitochondria. The native enzyme has an apparent molecular weight of 530,000 in pea leaf and 500,000 in potato tuber as measured by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate disclosed two nonidentical subunits. The larger subunit (B subunit) is biotinylated and has an apparent molecular weight of 76,000 in pea leaf and 74,000 in potato tuber. The smaller subunit (A subunit) is biotin free and has an apparent molecular weight of 54,000 in pea leaf and 53,000 in potato tuber. The biotin content of the enzyme is 1 mol/133,000 g of protein and 1 mol/128,000 g of protein in pea leaf and potato tuber, respectively. These values are consistent with an A4B4 tetrameric structure for the native enzyme. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8 to 8.3 and at 35 to 38[deg]C in the presence of Mg2+. Kinetic constants (apparent Km values) for the enzyme substrates 3-methylcrotonyl-CoA, ATP, and HCO3- were: 0.1 mM, 0.1 mM, and 0.9 mM, respectively, for pea leaf 3-methylcrotonyl-CoA carboxylase and 0.1 mM, 0.07 mM, and 0.34 mM, respectively, for potato tuber 3-methylcrotonyl-CoA carboxylase. A steady-state kinetic analysis of the carboxylase-catalyzed carboxylation of 3-methylcrotonyl-CoA gave rise to parallel line patterns in double reciprocal plots of initial velocity with the substrate pairs 3-methylcrotonyl-CoA plus ATP and 3-methylcrotonyl-CoA plus HCO3- and an intersecting line pattern with the substrate pair HCO3- plus ATP. It was concluded that the kinetic mechanism involves a double displacement. Purified 3-methylcrotonyl-CoA carboxylase was inhibited by end products of the reaction catalyzed, namely ADP and orthophosphate, and by 3-hydroxy-3-methylglutaryl-CoA. Finally, as for the 3-methylcrotonyl-CoA carboxylases from mammalian and bacterial sources, plant 3-methylcrotonyl-CoA carboxylase was sensitive to sulfhydryl and arginyl reagents.

Localization of free and bound biotin in cells from green pea leaves.

Cytosol and vacuoles from protoplasts, chloroplasts, and mitochondria from green pea (Pisum sativum) leaves were purified and examined for their biotin content. The bulk of free biotin was shown to be exclusively associated with the cytosolic fraction at a concentration of about 4 pmol/mg protein and no bound biotin was detected. The bulk of bound biotin (biotin-containing carboxylases) was associated with the soluble fraction of chloroplasts and mitochondria at a concentration of about 1.2 and 13 microM, respectively. No free biotin was detected in these organelles. Western blot analysis of total, chloroplastic, and mitochondrial polypeptides, using horseradish peroxidase-labeled streptavidin, revealed three biotin-containing polypeptides with molecular mass of 220,000, 76,000 and 34,000. All were detected in the total pea leaf extract, but the M(r) 76,000 and the M(r) 34,000 biotinylated polypeptides were only detected in mitochondria and chloroplasts, respectively. 3-Methylcrotonyl-coenzyme A carboxylase and acetyl-coenzyme A carboxylase activities were measured in these two compartments, respectively. Previously, it has been shown that the M(r) 76,000 polypeptide was the biotinylated subunit of the mitochondrial 3-methylcrotonyl-coenzyme A carboxylase. In this paper, the origin and putative function of free biotin located in cytosol are discussed.

Immunization of rabbits with proteins reacted with malonic dialdehyde (MDA): kinetics and specificity of the immune response.

MDA-modified casein, lysozyme or polylysine (MC, ML and MP respectively), was intradermically injected to rabbits in the presence of complete Freund's adjuvant (cFA). Two other animal sets received either cFA alone, or MDA alone. MDA, cFA and MP did not induce any antibody response. Both ML and MC produced an increase of antibody reactivity towards ML, but reactivity towards native lysozyme (L) was increased only by ML and not by MC. According to these results, it was concluded that the epitopes recognized by antibodies reacting with ML and not with L are AIP bridges and possibly the two surrounding aminoacyl (especially lysyl) residues.