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C Valcarce

Publications and source records attributed to C Valcarce.

16 recordsLinked to original sources
PubMed1999

The epidermal growth factor precursor. A calcium-binding, beta-hydroxyasparagine containing modular protein present on the surface of platelets.

Various human body fluids and secretions contain a soluble form of the epidermal growth factor (EGF) precursor. The EGF precursor molecule contains eight EGF modules in addition to EGF itself. Using monoclonal antibodies specific for the EGF modules 7 and 8, we have purified the soluble form of the EGF precursor from human urine to homogeneity. The protein was shown to have a molecular mass of about 160 kDa and the N-terminal sequence SAPNHWSXPE. EGF modules 2, 7 and 8 of the precursor have the consensus sequence for post-translational beta-hydroxylation of Asp/Asn residues. We identified the presence of erythro-beta-hydroxy-aspartic acid (Hya) in acid hydrolysates of the EGF precursor (2.4 M.M protein-1). As the DNA sequence encodes Asn in the corresponding position, the Hya represents erythro-beta-hydroxyasparagine (Hyn). The Hyn-containing modules have a consensus calcium-binding motif immediately N-terminal of the first Cys residue. The synthetic EGF module 2 (residues 356-395) of the EGF precursor was found to bind calcium with low affinity, Kd approximately 3.5 mM, i.e. similar to the affinity of other isolated calcium-binding EGF modules. EGF module 7, when part of the intact protein, was found to bind Ca2+ with a Kd approximately 0.2 microM, i.e. approximately 10(4)-fold higher than that of isolated EGF modules presumably due to the influence of neighboring modules. We have detected EGF precursor in platelet-rich plasma and demonstrated it to be associated to platelets. The platelets were found to have 30-160 EGF molecules each.

Amino Acid Sequence↗
PubMed1994

Mammalian adaptation to extrauterine environment: mitochondrial functional impairment caused by prematurity.

In this paper we report that, compared with term rat neonates, both mitochondrial content and function are diminished in liver of preterm neonates (delivered 24 h before full term) compromising cellular energy provision in the postnatal period. In addition, there is a parallel reduction in the content of mRNAs encoding mitochondrial proteins in preterm rats. Also, efficient oxidative phosphorylation is not attained in these pups until 3 h after birth. Although isolated liver mitochondria from preterm neonates show a two-fold increase in F1-ATPase beta-subunit and cytochrome c oxidase activity 1 h after birth, the abnormal coupling efficiency between respiration and oxidative phosphorylation (ADP/O ratio) is due to maintenance of high H(+)-leakage values in the inner mitochondrial membrane. Postnatal reduction of the H+ leak occurs concomitantly with an increase in intra-mitochondrial adenine nucleotide concentration. Accumulation of adenine nucleotides in preterm and term liver mitochondria parallels the postnatal increase in total liver adenine nucleotides. Delayed postnatal induction of adenine biosynthesis most likely accounts for the lower adenine nucleotide pool in the liver of preterm neonates. The delayed postnatal accumulation of adenine nucleotides in mitochondria is thus responsible for the impairment in oxidative phosphorylation displayed by organelles of the preterm liver.

Adaptation, Physiological↗
PubMed1994

Calcium-dependent interaction between gamma-carboxyglutamic acid-containing and N-terminal epidermal growth factor-like modules in factor X.

The N-terminal epidermal growth factor (EGF)-like module in factor X binds a single Ca2+ with low affinity (Kd = 2.2 mM). When it is linked to the gamma-carboxyglutamic acid (Gla)-containing module, however, the affinity increases approximately 20-fold (Kd = 120 microM), indicating an interaction between the two modules and making the site in the N-terminal EGF-like module essentially saturated at physiological Ca2+ concentrations. We have now used the thioredoxin system to probe Ca(2+)-induced conformational changes and interaction between modules in the light chain of factor X. Thioredoxin, in conjunction with thioredoxin reductase and NADPH, allows direct measurements of the rate and extent of disulfide bond reduction. Most disulfide bonds accessible to the reducing agent were found to be located in the light chain of the protein. Moreover, those disulfide bonds that were resistant to reduction by thioredoxin in the presence of Ca2+, but were readily reduced in the absence of the metal ion, were located in the N-terminal EGF-like module and in the Gla module, whereas disulfide bonds in the C-terminal EGF-like module appeared to be equally accessible whether Ca2+ was present or not. Comparison of the rate of disulfide bond reduction in the isolated modules with that in mixtures of modules indicated that a Ca(2+)-dependent interaction occurred between the Gla and the N-terminal EGF-like module. This interaction was mediated by the C-terminal alpha-helical part of the Gla module. The affinity between the modules was low and could not be determined accurately owing to competing equilibria, presumably Ca(2+)-dependent aggregation of the isolated Gla module. By comparing the rates of disulfide bond reduction in Gla module-containing fragments before and after decarboxylation of Gla, we could demonstrate that Ca2+ binding to sites in the Gla module as well as to the single site in the EGF-like module contribute to the interaction between the two modules.

1-Carboxyglutamic Acid↗
PubMed1993

Calcium affinity of the NH2-terminal epidermal growth factor-like module of factor X. Effect of the gamma-carboxyglutamic acid-containing module.

The NH2-terminal epidermal growth factor (EGF)-like module of vitamin K-dependent coagulation factors IX and X and protein C each has one calcium binding site. This module (residues 45-86) from factor X has been isolated previously and found to bind calcium with a Kd of 2.2 mM at physiological pH and ionic strength. We have now demonstrated that it binds calcium with a Kd of 120 microM in a fragment that consists of the Gla module and the NH2-terminal EGF-like module. The presence of the Gla module (residues 1-44) increases the calcium affinity of the site in the EGF-like module approximately 20-fold, thus making it essentially saturated in vivo. Decarboxylation of the Gla residues to Glu has no significant effect on the calcium affinity of the EGF-like module. A proteolytic fragment of factor X (residues 29-86) and a synthetic peptide (residues 34-86), folded to a native conformation, were used to demonstrate that the contribution of the Gla module to the calcium affinity of the site in the EGF-like module is mediated by its 17 COOH-terminal residues, 12 of which form an alpha-helix in the intact Gla module. In the NMR structure of the NH2-terminal EGF-like module in factor X, five calcium ligating groups have been identified (Selander-Sunnerhagen, M., Ullner, M., Persson, E., Teleman, O., Stenflo, J., and Drakenberg, T. (1992) J. Biol. Chem. 267, 19642-19649). As calcium usually requires seven to eight oxygen ligands, there is reason to believe that the Gla module contributes ligands, or negative charge, to increase the calcium affinity. Our findings suggest that the calcium affinity of EGF-like modules in other proteins may also be influenced by neighboring modules.

1-Carboxyglutamic Acid↗
PubMed1991

Interaction of adenine nucleotides with the adenine nucleotide translocase regulates the developmental changes in proton conductance of the inner mitochondrial membrane.

2-h-old neonatal liver mitochondria, when depleted of adenine nucleotides, showed an 'ohmic' current-voltage relationship and a higher passive proton permeability of the membrane, resembling fetal mitochondrial behaviors for the proton conductance. Incubation of fetal mitochondria with ATP, GDP or carboxyatractyloside promoted a significant reduction in the passive proton permeability of the membrane and the appearance of the characteristic biphasic behavior for the proton conductance. It is concluded that the postnatal increase in intramitochondrial adenine nucleotide concentration promotes, by the interaction of the nucleotides with the adenine nucleotide translocase, the reduction in the passive proton permeability of the mitochondrial membrane, allowing efficient energy conservation in the neonatal liver.

Adenine Nucleotides↗
PubMed1991

The gamma-carboxyglutamic acid and epidermal growth factor-like domains of factor X. Effect of isolated domains on prothrombin activation and endothelial cell binding of factor X.

Factor Xa is the enzymatically active constituent of the prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin. We have isolated fragments, from tryptic digests of factor X, that consists of the gamma-carboxyglutamic acid (Gla) region linked to one or two epidermal growth factor (EGF)-like domains. Calcium ion binding measurements indicated that these fragments have a native conformation. The factor X-GlaEGF fragments inhibit factor Xa-induced blood clotting in a manner suggesting that they compete with factor Xa for phospholipid binding sites. The same conclusion was reached when thrombin generation was studied in a system of purified components (factor Xa, factor Va, prothrombin, phospholipid, and Ca2+). There was no evidence for a strong interaction between the EGF-like domains of factor Xa and factor Va in either system. However, experiments in the purified system without phospholipid indicated a direct, albeit weak, interaction between the Gla region of factor Xa and factor Va and between the COOH-terminal EGF-like domain of factor Xa and factor Va. Using domain-specific Fab fragments, we have confirmed that the conformation of the serine protease region alters dramatically upon activation of factor X. Furthermore, we have demonstrated that the conformation of the Gla region is affected by the activation, whereas the EGF-like domains appear to be unaltered. The association constant for factor X binding to endothelial cells was two orders of magnitude lower than that for binding of factor IX to these cells. Binding of the Gla and GlaEGF fragments suggested Gla-mediated binding to phospholipid rather than binding to a specific receptor.

1-Carboxyglutamic Acid↗
PubMed1990

Immunological detection of the mitochondrial F1-ATPase alpha subunit in the matrix of rat liver peroxisomes. A protein involved in organelle biogenesis?

Rat liver peroxisomes contain in their matrix the alpha-subunit of the mitochondrial F1-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No beta-subunit of the mitochondrial F1-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature alpha-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the alpha-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.

Animals↗
PubMed1990

Rapid postnatal developmental changes in the passive proton permeability of the inner membrane in rat liver mitochondria.

Titration of mitochondrial respiration against the membrane potential with the inhibitor malonate has been carried out during the perinatal period in isolated rat liver mitochondria. Neonatal and adult mitochondria exhibited the characteristic "nonohmic" behavior for the proton conductance (CmH+). In contrast, fetal mitochondria exhibited an "anomalous" "ohmic" behavior for CmH+. The calculated passive proton permeability of the membrane undergoes a profound reduction during the first postnatal hour. The results reported demonstrate that the hypothesis [Pollak, J.K. & Sutton, R. (1980) Trends Biochem. Sci. 5, 23-27] of the existence of a "leaky" mitochondria in the fetal rat liver, and of its sudden neonatal change towards a state of higher energy conservation of the proton electrochemical gradient, is correct.

Animals↗
PubMed1988

Postnatal development of rat liver mitochondrial functions. The roles of protein synthesis and of adenine nucleotides.

It has been proposed that the acquisition of efficient energy-transducing mitochondria after birth is mediated by an ATP-dependent mechanism "that causes the rapid maturation of mitochondria without requiring either transcription or translation" (Pollak, J. K., and Sutton, R. (1980) Trends Biochem. Sci. 5, 23-27). Investigation of developmental changes in rat liver mitochondria during the first 6 postnatal h showed that fetal mitochondria had low State 4, State 3, and uncoupled rates of respiration, inefficient coupling between respiration and phosphorylation, and low membrane potentials and proton electrochemical gradients under State 4 conditions. In contrast, hepatic mitochondria from 1-h-old neonates showed increased respiratory control and ADP/O ratios and adult proton electrochemical gradient and membrane potential values. In parallel with these changes, mitochondria became enriched in adenine nucleotides and underwent a 50% reduction in matrix volume. During the first postnatal hour, an increase in mitochondrial succinic dehydrogenase, cytochrome c oxidase, and F1-ATPase activities takes place in the neonatal liver concurrent with a preferential postnatal increase in the in vivo rates of protein synthesis for mitochondrial proteins. In particular, the amount of F1-ATPase increased from 109 +/- 9 to 206 +/- 5 ng/microgram of mitochondrial protein in the first hour of postnatal life. Inhibitors of cytosolic protein synthesis present during the first 2 h of life blocked the postnatal increase in respiratory control and ADP/O ratios, succinic dehydrogenase activity, and F1-ATPase content; but they had no effect on the increase in adenine nucleotide concentrations and mitochondrial volume contraction. This indicates that the acquisition of an efficient coupling between respiration and phosphorylation is dependent on de novo protein synthesis and cannot be brought about by the postnatal increase in adenine nucleotides. The increase of State 4 and uncoupled rates of respiration during the first 2 postnatal h was resistant to protein synthesis inhibitors. We suggest that the postnatal increase in these parameters is due to the reduction of mitochondrial volume occurring during that time, which, in turn, may be triggered by the concurrent enrichment in adenine nucleotides.

Adenine Nucleotides↗
PubMed1986

Alanine and lactate as gluconeogenic substrates during late gestation.

Rates of alanine incorporation into glucose by isolated liver cells of fed rats are 5-fold higher than those observed when lactate was used as substrate. The rates of gluconeogenesis from alanine and lactate in isolated liver cells of fed pregnant rats increase 50 and 200-400%, respectively, over virgins during the last 3 days of gestation. The results support the existence of an increase in the alanine-glucose cycle in the late pregnancy as an important homeostatic pathway in the supply of glucose to the growing fetus.

Alanine↗
PubMed1985

Increased gluconeogenesis in the rat at term gestation.

In this study the contribution of maternal gluconeogenesis to the glucose homeostasis of the maternal-fetal unit has been studied in fed term pregnant rats. We have measured the activity of two gluconeogenic enzymes, the rates of lactate turnover and the rates of gluconeogenesis from lactate in fed term pregnant rats. A decrease in plasma glucose and liver glycogen concentrations, and an increase of plasma lactate and alanine concentrations were observed in fed 22-day pregnant rats compared to virgin controls. Also, liver and kidney phosphoenolpyruvate carboxykinase activities and liver lactate dehydrogenase and hexose bisphosphatase activities significantly increased in fed term pregnant rats compared to virgin rats. The lactate turnover rate and the rate of gluconeogenesis in vivo from L-[U14C] Lactate increased four- and two-fold respectively in fed pregnant rats compared to fed virgins.

Alanine↗
PubMed1983

Postnatal hypoglycaemia and gluconeogenesis in the newborn rat. Delayed onset of gluconeogenesis in prematurely delivered newborns.

The concentrations of glucose and lactate in the blood and of liver glycogen, and the phosphoenolpyruvate carboxykinase activity in liver and kidney of term and preterm newborn rats, were studied during the first 6 h post partum. Rates of lactate turnover and gluconeogenesis in vivo from [U-14C]lactate at 3 h and 6 h post partum were also quantified. The development of the prolonged postnatal hypoglycaemia observed after birth in the premature newborn rat is associated with lower rates of glucose production through glycogenolysis and gluconeogenesis; liver glycogenolysis was the main contributing factor to the glucose available during the neonatal period studied in both groups. Delayed induction of liver phosphoenolpyruvate carboxykinase activity was observed in premature newborn rats. Renal phosphoenolpyruvate carboxykinase activity increased 72% from birth in preterm newborns, but only a 25% increase was found in term newborns during the same experimental period. The gluconeogenesis in vivo from [U-14C]lactate paralleled the appearance of cytosolic phosphoenolpyruvate carboxykinase activity in the liver of both groups of newborns. Blood lactate concentrations remained higher in preterm than in term newborns. The postnatal utilization of lactate via the gluconeogenic pathway in either group of newborns was always less than 20% of the total lactate used. The results presented are discussed in relation to the development of postnatal hypoglycaemia and gluconeogenesis in the premature newborn rat.

Animals↗