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Biomedical subjects

V Rubio

Publications and source records attributed to V Rubio.

At least 55 records · Page 3Linked to original sources
PubMed1996

Photoaffinity labeling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor.

UMP is a highly specific reagent for photoaffinity labeling of the allosteric inhibitor site of carbamyl phosphate synthetase (CPS) from Escherichia coli and has been found to be photoincorporated in the COOH-terminal domain of the large subunit [Rubio et al. (1991) Biochemistry 30, 1068-1075]. In the present work we identify lysine 992 as the residue that is covalently attached to UMP. This identification is based on two lines of evidence. First, [14C]UMP is found to be incorporated between residues 939 and 1006, as shown by peptide mapping and by mass estimates of [14C]UMP-peptides generated by chemical and enzymatic cleavage of CPS. Secondly, we have purified two radioactive peptides derived exclusively from those enzyme molecules (approximately 5% of the total enzyme) that had incorporated [14C]-UMP. Edman analyses show the sequences of the labeled peptides (989)LVNXVHEGRPHIQD and (989)LVNXVHE to be overlapping. Since neither a phenylthiohydantoin (Pth) derivative (in cycle 4) nor any radioactivity is released from the membrane during sequencing, we can conclude that Lys992 and [14C]-UMP form a covalent adduct that remains bound to the membrane. Formation of this adduct agrees with all of the evidence and with the finding that UMP labeling prevents trypsin cleavage at Lys992. Lysine 992 is invariant in those CPSs that are inhibited by UMP, and is located 30 residues upstream of the site whose phosphorylation in hamster CAD reduces inhibition of CAD by UTP. Multiple sequence alignment of the residues surrounding Lys992 of the E. coli enzyme and the corresponding residues of the yeast and animal enzymes supports the existence of a uridine nucleotide binding fold in this region of the protein. We conclude that sequence changes in the binding fold provide a structural basis for the different regulatory properties found among CPSs I, II, and III.

Affinity Labels↗
PubMed1996

Relative frequency of mutations causing ornithine transcarbamylase deficiency in 78 families.

Approximately 90 different mutations associated with ornithine transcarbamylase (OTC) deficiency are currently known. Thus, the majority represent private mutations. However, some of the mutations seemed to be recurrent. Our laboratories identified apparent deleterious mutations in 78 consecutive families with OTC deficiency by screening all exons and exon/intron borders using single-strand conformational polymorphism (75 families) or sequencing of the entire coding sequence (3 families). Large deletions of one or more exons were found in 8% of families and approximately 10% had small deletions or insertions of 1-5 bases. Splice site mutations were found in 18% of families. Contrary to previous reports, recurrent point mutations seemed to be equally distributed among most CpG dinucleotides rather than show prevalent mutations. No single point mutation had a relative frequency of more than 6.4%. Of the 64 families with nucleotide substitution, 24 (38%) were G to A with the next most common being C to T (16%) and A to T (11%).

Female↗
PubMed1996

Identification at strain level of Rhizoctonia solani AG4 isolates by direct sequence of asymmetric PCR products of the ITS regions.

The relatedness of nine isolates of Rhizoctonia solani, belonging to anastomosis group (AG) 4, and one isolate of AG1 was determined by comparative sequence analysis based on direct sequencing of PCR-amplified ribosomal DNA [the internal transcribed spacer (ITS) region and the 5.8 s ribosomal DNA]. The 5.8 s rDNA is completely conserved, but both ITS regions show variation among strains. AG1 was an outgroup based on anastomosis ability and RFLP analyses. Phylogenetic analyses based on the ITS sequences suggest that the analyzed AG4 strains can be divided into three groups that correlate with habitat and virulence.

Base Sequence↗
PubMed1995

Affinity cleavage of carbamoyl-phosphate synthetase I localizes regions of the enzyme interacting with the molecule of ATP that phosphorylates carbamate.

Two ATP molecules are used in the reaction catalyzed by carbamoyl-phosphate synthetase I. One molecule (ATPA) phosphorylates HCO3- and the other (ATPB) phosphorylates carbamate. Carbamoyl-phosphate synthetase I is a 160-kDa polypeptide consisting of a 40-kDa N-terminal moiety and a 120-kDa C-terminal moiety, the latter being composed of two similar halves of molecular mass 60 kDa. We showed [Alonso, E., Cervera, J., García-España, A., Bendala, E. & Rubio, V. (1992) J. Biol. Chem. 267, 4524-4532] that Fe.ATP bound at the site for ATPB catalyzes the oxidative inactivation of carbamoyl-phosphate synthetase I in a model oxidative system consisting of Fe3+, ascorbate, and O2, and we detected ATP-promoted oxidative cleavage of the enzyme. We now provide further evidence indicating that this cleavage is catalyzed by bound Fe.ATPB, and we demonstrate that the enzyme is cleaved at seven points, which we identify as residues 1002, 1064, 1083, 1128, 1200, 1242, and 1270. All these cleavage points are confined within and distributed throughout the more N-terminal 40-kDa region of the C-terminus of the 120-kDa moiety. Thus, this 40-kDa region contains the ATPB site, is folded as a globular domain with the polypeptide recurring several times towards the nucleotide, and appears to be a modular unit equivalent to carbamate kinase, with full responsibility for ATPB binding and carbamate phosphorylation. The present results and our previous demonstration [Rodríguez-Aparicio, L., Guadalajara, A.M. & Rubio, V. (1989) Biochemistry 28, 3070-3074] of the binding of N-acetyl-L-glutamate in the C-terminal 20-kDa region, strongly support the idea that each homologous half of the 120-kDa moiety of carbamoyl-phosphate synthetase I is composed of a 40-kDa ATP-binding domain and a 20-kDa domain that, in the carboxyl half, is the regulatory domain.

Adenosine Triphosphate↗
PubMed1995

Crystallization, characterization, and preliminary crystallographic studies of mitochondrial carbamoyl phosphate synthetase I of Rana catesbeiana.

Carbamoyl phosphate synthetase I (ammonia; E C 6.3.4.16) was purified from the liver of Rana catesbeiana (bullfrog). Crystals of the protein have been obtained at 22 degrees C by the hanging drop vapor diffusion technique, with polyethylene glycol as precipitant. Tetragonal crystals of about 0.3 x 0.3 x 0.7 mm diffract at room temperature to at least 3.5 A using a conventional source and are stable to X-radiation for about 12 h. Therefore, these crystals are suitable for high resolution studies. The space group is P4(1)2(1)2 (or its enantiomorph P4(3)2(1)2), with unit cell dimensions a = b = 291.6 A and c = 189.4 A. Density packing considerations are consistent with the presence of 4-6 monomers (M(r) of the monomer, 160,000) in the asymmetric unit. Amino-terminal sequence of the enzyme and of a chymotryptic fragment of 73.7 kDa containing the COOH-terminus has been obtained. The extensive sequence identity with rat and human carbamoyl phosphate synthetase I indicates the relevance for mammals of structural data obtained with the frog enzyme.

Amino Acid Sequence↗
PubMed1995

A splicing mutation, a nonsense mutation (Y167X) and two missense mutations (I159T and A209V) in Spanish patients with ornithine transcarbamylase deficiency.

Four novel mutations are identified in the ornithine transcarbamylase (OTC) gene, in four patients with OTC deficiency (an X-linked disorder). The mutations represent three different categories: missense (Ile159Thr and Ala209Val), nonsense (Tyr167Stop), and causing inefficient splicing (G-->A in the first intronic base) with associated aberrant splicing. They are located in exons 5, and 6, and in intron 3. Two of the mutations arose de novo in the patients, and only one mutation occurs at a CpG site. The nonsense and the splicing mutation cause, respectively, lethal early onset and non-lethal, delayed early onset clinical presentations in males. Our results confirm for Spain the high genotypic heterogeneity of OTC deficiency.

Base Sequence↗
PubMed1995

Demonstration of the spf-ash mutation in Spanish patients with ornithine transcarbamylase deficiency of moderate severity.

We have found in patients with ornithine transcarbamylase (OTC) deficiency from two Spanish families (A and B), replacement by A of G at the 3'-end of exon 4 of the OTC gene. The same mutation is found in the spf-ash mouse, a rodent model of mild OTC deficiency, causing a neutral R129H mutation and inefficient splicing at the 5' donor site of the exon 4-intron 4 junction, with resultant 4%-7% residual OTC activity. The mutation, detected in our patients using polymerase chain reaction (PCR) amplification of the ten OTC exons, single strand conformation polymorphism (SSCP) analysis and direct sequencing of PCR-amplified exon 4, results in the loss of a unique MspI restriction site which can be used for rapid diagnosis. The mutation was transmitted by the mother in family A and arose de novo in the patient in family B. Residual OTC activity, determined in a male and a female patient, was 1.3% and 3.5% of normal, respectively. Despite this low activity, the surviving patients have developed normally.

Adolescent↗
PubMed1995

Quantification of fibronectin in bronchoalveolar lavage fluid and in lung parenchyma: differences between smokers and non-smokers.

Fibronectin (FN) is a glycoprotein present in the epithelial surface of airways and in the interstitial parenchyma of the lung. The aim of this study was to quantify the amount of FN in human lung parenchyma, and to investigate possible differences between smokers and non-smokers, both in lung parenchyma and in bronchoalveolar lavage (BAL) fluid. BAL fluid was recovered in 21 individuals. Quantification of FN in BAL was done by a non-competitive ELISA. Twenty-six lung parenchyma samples were also obtained from lung resections. FN was extracted with urea and heparin. Quantification of FN in lung parenchyma was determined by a competitive ELISA. The mean +/- SD FN content in BAL fluid from smokers was 1.59 +/- 1.65 micrograms/mg total protein and 0.53 +/- 0.26 in non-smokers (p < 0.05). However, the amount of FN/ml BAL fluid was not statistically different between smokers (0.15 +/- 0.13 micrograms/ml) and non-smokers (0.106 +/- 0.07). The mean extractable FN in human lung tissue was 38.6 micrograms/g wet weight (31.7-45.5, 95% mean confidence interval) with no statistical difference between smokers and non-smokers. We conclude that in smokers, FN accounts for a higher proportion of the total protein in BAL fluid, and that there were no differences in FN content in lung parenchyma between smokers and non-smokers.

Bronchoalveolar Lavage Fluid↗
PubMed1994

Location of the binding site for the allosteric activator IMP in the COOH-terminal domain of Escherichia coli carbamyl phosphates synthetase.

Using UV-irradiation we cross-linked IMP, the allosteric activator of E. coli carbamyl phosphate synthetase (a heterodimer of 117.7 and 41.4 kDa subunits), to the large subunit of the enzyme. As in the native enzyme-IMP complex, the cross-linked complex was resistant to attack by trypsin. Thus, IMP is attached to its normal site and induces the normal conformational changes. Limited digestion of the [3H]IMP-labeled enzyme with V8 staphylococcal protease or with trypsin in the presence of SDS, and NH2-terminal sequencing, showed that [3H]IMP is cross-linked to the COOH-terminal 20 kDa domain of the large subunit, downstream of residue 912, supporting the proposal that this domain is specialized in effector binding and regulation.

Affinity Labels↗
PubMed1994

Crystallization, characterization and preliminary crystallographic studies of carbamate kinase of Streptococcus faecium.

Crystals of carbamate kinase (E.C.2.7.2.2) suitable for high resolution studies have been obtained, using the hanging drop vapour diffusion technique, with polyethylene glycol 8000 and NaCl as precipitants at pH 6.5 and a temperature of 4 degrees C. Crystals of about 0.3 mm x 0.2 mm x 0.2 mm in size diffract to at least 3.2 A resolution and are stable to X-radiation for more than ten hours. The space group is P2(1)2(1)2(1), with unit cell dimensions a = 84.5 A, b = 99.6 A, c = 173.3 A. Density packing considerations are consistent with the presence of four to five monomers (M(r) of the monomer = 33,000) in the asymmetric unit, two dimers or even a tetramer being favoured by the results of cross-linking experiments of the enzyme in solution.

Amino Acid Sequence↗
PubMed1993

The influence of effectors and subunit interactions on Escherichia coli carbamoyl-phosphate synthetase studied by differential scanning calorimetry.

Differential scanning calorimetry of Escherichia coli carbamoyl-phosphate synthetase and its isolated large and small subunits reveals in each case an irreversible, kinetically controlled transition, at a temperature 14 degrees C higher for the holoenzyme than for the subunits, indicating dramatic stabilization of the subunits in the heterodimer. The deletion of the COOH-terminal 171 (mutant CarB'2373) or 385 (mutant CarB2177) residues of the large subunit results in more asymmetric transitions at a temperature 7 degrees C lower than for the wild type. The allosteric effectors IMP, UMP, and ornithine induce small reversible transitions at low temperature in the endotherm for the wild-type enzyme, but not for CarB'2373, as expected if the effectors bind in the 171-residue, COOH-terminal region. In contrast, two ligands that bind outside the deleted region, Ap5A (a ligand of both ATP sites) and glycine (an analog of glutamine) decrease and increase, respectively, the stability of the two mutants and of the wild type. The stabilization by glycine requires that the subunits are associated. The results support the implication of the 20-kDa COOH-terminal domain of the large subunit in the allosteric modulation by all the effectors and are consistent with the folding of the large subunit as a pseudohomodimer of its two homologous halves.

Allosteric Regulation↗
PubMed1993

Endotoxin-induced pulmonary dysfunction is prevented by C1-esterase inhibitor.

In septic shock, hypotension, disseminated intravascular coagulation, and neutrophil activation are related to the activation of the blood coagulation contact system. This study evaluates in dogs the effect of the C1-esterase inhibitor (C1-INH), a main inhibitor of the blood coagulation contact system, on the cardiovascular and respiratory dysfunction associated with endotoxic shock. Two groups were included: controls, which received Escherichia coli endotoxin, and a C1-INH group in which C1-INH was infused before E. coli endotoxin administration. In both groups, endotoxin produced hypodynamic shock; however, the decrease in the systolic index and the ventricular systolic work indexes were greater in controls than the C1-INH group. In controls, the arterial O2 partial pressure decreased by 30% and the alveolo-arterial O2 difference increased by 625%, these parameters remained unchanged in the C1-INH group. Hypoxemia was associated with increased intrapulmonary shunt, decreased blood coagulation contact factors, and decreased C3c. In contrast, C1-INH administration prevented endotoxin-induced hypoxemia, the increase in intrapulmonary shunt, and the decrease in blood coagulation contact factors. This study shows that, in dogs with endotoxic shock, pulmonary dysfunction is associated with an activation of the blood coagulation contact phase system. An inhibition of this system by C1-INH prevented the hypoxemia induced by endotoxic shock.

Animals↗
PubMed1992

Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents.

Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+, ascorbate, and O2, but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+, and catalase have no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site for ATPA (ATPA yields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOH-terminal domains. The oxidative inactivation promoted by ATP is inhibited by Mg2+, Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337. Groups in the 60-kDa domain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPB and support the binding of ATPB in the 60-kDa domain. We also demonstrate that mercaptoethanol, reducing impurities in glycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in the oxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.

Adenosine Triphosphate↗
PubMed1992

Human growth plate development in the fetal and neonatal period.

The development of the normal human upper tibial growth plate was studied at autopsy in 46 stillborns and 79 newborns of 20-41 weeks gestational age. During this time period, the histology of this plate evolves from a highly cellular structure with relatively poor columnar organization and matrix development to the well known structure seen later in postnatal life. The thickness of the growth plate, assessed in the area surrounding the longitudinal tibial axis, decreases continuously from 1.15 mm on the 20th week to 0.6 mm on the 38th week. This decrease results from losses of both matrix and cellular components, mostly of the latter. However, the relative fraction of area occupied by the matrix significantly increased (12%) and matrix area per cell increased 1.5 times over the last half of gestation, indicating a maturation process of the plate towards a more matrix-oriented structure with age. In this maturation process the number of cells per unit area does not change and the average size of the cells appears to decrease. Plate thickness does not decrease further in the final 3 weeks of pregnancy and increases in early neonatal life; this has no apparent influence on the tibial growth rate. In the period under study the relative anatomical participation of the upper tibial growth plate decreases from approximately 4% of the radiographic length of the tibia on the 20th week to less than 1% at term. Present data will provide fetal and neonatal growth plate standards needed to obtain a better understanding of this structure during both normal and abnormal conditions.

Bone Matrix↗