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V Rubio

Publications and source records attributed to V Rubio.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1992

Experimental absence seizures: potential role of gamma-hydroxybutyric acid and GABAB receptors.

We have investigated whether the pathogenesis of spontaneous generalized non-convulsive seizures in rats with genetic absence epilepsy is due to an increase in the brain levels of gamma-hydroxybutyric acid (GHB) or in the rate of its synthesis. Concentrations of GHB or of its precursor gamma-butyrolactone (GBL) were measured with a new GC/MS technique which allows the simultaneous assessment of GHB and GBL. The rate of GHB synthesis was estimated from the increase in GHB levels after inhibition of its catabolism with valproate. The results of this study do not indicate significant differences in GHB or GBL levels, or in their rates of synthesis in rats showing spike-and-wave discharges (SWD) as compared to rats without SWD. Binding data indicate that GHB, but not GBL, has a selective, although weak affinity for GABAB receptors (IC50 = 150 microM). Similar IC50 values were observed in membranes prepared from rats showing SWD and from control rats. The average GHB brain levels of 2.12 +/- 0.23 nmol/g measured in the cortex and of 4.28 +/- 0.90 nmol/g in the thalamus are much lower than the concentrations necessary to occupy a major part of the GABAB receptors. It is unlikely that local accumulations of GHB reach concentrations 30-70-fold higher than the average brain levels. After injection of 3.5 mmol/kg GBL, a dose sufficient to induce SWD, brain concentrations reach 240 +/- 31 nmol/g (Snead, 1991) and GHB could thus stimulate the GABAB receptor. Like the selective and potent GABAB receptor agonist R(-)-baclofen, GHB causes a dose-related decrease in cerebellar cGMP. This decrease and the increase in SWD caused by R(-)-baclofen were completely blocked by the selective and potent GABAB receptor antagonist CGP 35348, whereas only the increase in the duration of SWD induced by GHB was totally antagonized by CGP 35348. The decrease in cerebellar cGMP levels elicited by GHB was only partially antagonized by CGP 35348. These findings suggest that all effects of R(-)-baclofen are mediated by the GABAB receptor, whereas only the induction of SWD by GHB is dependent on GABAB receptor mediation, the decrease in cGMP being only partially so. Taken together with the observations of Marescaux et al. (1992), these results indicate that GABAB receptors are of primary importance in experimental absence epilepsy and that GABAB receptor antagonists may represent a new class of anti-absence drugs.

4-Butyrolactone↗
PubMed1992

Diminished anticoagulant and fibrinolytic activity following liver transplantation.

This study analyzed the coagulation changes in twenty patients after orthotopic liver transplantation. The procoagulant, anticoagulant, and fibrinolytic systems were studied during the first two postoperative weeks. Within the first postoperative day all extrinsic and intrinsic pathway factors became normal except factors IX, VII, and X, which recovered within the next 24 hr. Of interest are the changes in factor VIII, which reached a high concentration with an increase in its antigenic fraction during the study. However, coagulation inhibitors showed a different pattern. In fact, antithrombin III (AT-III) and protein C (PC) needed from 7 to 14 days to reach normal values. Total protein S (TPS) and free protein S (FPS) did not recover until day 7, whereas heparin cofactor II (HC-II) remained at subnormal levels throughout the study. Thrombin-antithrombin III complex (TAT) values were strikingly elevated in the immediate postoperative period. Fibrinolysis parameters showed plasminogen (PL) levels in the normal range until day 4. Antiplasmin (AP) followed a curve parallel to that of plasminogen but its levels were higher during this observation period. Similarly the initial elevation in plasminogen activator inhibitor 1 endothelial type (PAI-1) levels remained high until days 4 and 7. In summary, it can be concluded that during the postoperative phase after OLT a hypercoagulable state is developed as a result of diminished anticoagulant and fibrinolytic activity. This coagulation might be a nontechnical factor contributing to the thrombotic vascular complications of some liver recipients.

Adolescent↗
PubMed1992

Protein C, protein S and C4b-binding protein in neonatal severe infection and septic shock.

We have studied the behaviour of total protein S, free protein S, protein C and C4b-binding protein fifteen neonates with severe infections, eight with septic shock and in a group of ten healthy newborns. Protein C was decreased in shock and septic patients, but only the shock group showed significant differences compared to normal neonates. Total protein S was normal in both groups of patients, although free protein S had significantly lower values in shock and nonshock infants. C4b-binding protein was higher than normal in septic and shock patients compared to the control group. Decreased values of protein C and free protein S can be explained by the activation of coagulation and their subsequent consumption. On the other hand, the increased levels of C4b-binding protein can affect the distribution of protein S in plasma, producing a shift in protein S to the complexed inactive form. These findings can contribute to an increased risk of microthrombosis during neonatal sepsis.

Carrier Proteins↗
PubMed1991

Influence of anions on the activation of carbamoyl phosphate synthetase (ammonia) by acetylglutamate: implications for the activation of the enzyme in the mitochondria.

Rat liver carbamoyl phosphate synthetase is shown to be inhibited by anions competitively with acetylglutamate (the allosteric activator of the enzyme) with a potency decreasing in the order NO3- greater than SO4(2-) greater than Cl- approximately HCO3-. Inhibition by chloride accounts for most of the inhibition reported [Lund, P., and Wiggins, D. (1987) Biochem. J. 243, 273-276] in Tris buffer. Mes, acetate, and isethionate give little or no inhibition and phosphate inhibits noncompetitively. Plots of the KA value for acetylglutamate versus the concentration of chloride or nitrate are curved upward and binding assays demonstrate that the inhibitory anions displace acetylglutamate from the enzyme. Thus, the anions may compete with the carboxyls of acetylglutamate for positive charges at the binding site. Of the organic anions found in the mitochondrial matrix, alpha-ketoglutarate, malate, succinate, and citrate increase substantially the KA for acetylglutamate. Changes in the concentrations of ATP, HCO3-, NH4+, and Mg2+, and high concentrations of protein (60 mg/ml serum albumin) influence the KA value. Changes in the concentration of the enzyme have no effect. Under assay conditions approaching the ionic, buffer, and substrate concentrations expected to occur in the mitochondrial matrix, the KA value for acetylglutamate is 27 microM and the Vmax is decreased about 50%. These results indicate that physiological changes in the level of acetylglutamate significantly influence the degree of activation of carbamoyl phosphate synthetase in vivo.

Animals↗
PubMed1991

Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain.

The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [14C]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1991

N-acetyl-L-glutamate in brain: assay, levels, and regional and subcellular distribution.

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200-300 times lower than the levels of N-acetyl-L-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 micrograms/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-L-aspartyl-L-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.

Animals↗