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Biomedical subjects

V Rubio

Publications and source records attributed to V Rubio.

At least 109 records · Page 6Linked to original sources
PubMed1983

Nitrogen metabolism in the sheep fetus. Observations on the liver and placenta.

Ammonium ions added in large quantity disappear rapidly from the reservoir of the sheep placenta perfused in situ through the umbilical vessels. Ammonium ions are removed from the reservoir of perfused sheep fetal livers of 108-141 days of conceptual age at a rate of at least 1 mumol/min/g liver. The majority appears as urea. There is little or no change in glutamine concentration. Hepatic carbamoylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinase and arginase are present, even at 97 days of conceptual age, in adequate amounts to account for the observed urea production. With the exception of arginase, all levels rise with fetal age. The levels in the maternal liver are comparable with those at 106 days of conceptual age. Arginase is high in the younger fetuses, falls progressively with fetal age and is very low in the mother. It is concluded that (a) the perfused placenta is permeable to ammonia and the placenta may be able to clear ammonia from the fetal circulation at a rate comparable with that of fetal liver; (b) the fetal liver converts ammonia to urea at a rate comparable with the urea production of the fetus; (c) there is virtually no glutamine production by the fetal liver; (d) adequate amounts of the enzymes of urea synthesis are present even in the immature fetal liver to account for the total urea production of the fetus, and (e) the anomalously low arginase level in the maternal liver may conserve maternal arginine, and the high levels in the younger fetuses may be related to fetal polyamine production from maternally derived arginine.

Ammonia↗
PubMed1981

Carbamoyl phosphate synthetase I of human liver. Purification, some properties and immunological cross-reactivity with the rat liver enzyme.

The purification of mitochondrial carbamoyl phosphate synthetase I (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) from small samples of human liver is described. The enzyme is composed of a single polypeptide of Mr 160 000 +/- 500 as shown by SDS-polyacrylamide gel electrophoresis in the presence of reducing agents. The synthetase migrates in polyacrylamide gradient gels in the absence of detergents at a rate corresponding to a Mr of 165 000. Estimates of the molecular weight of the native enzyme by gel filtration and density gradient centrifugation yield a value of 178 000. The results indicate that the enzyme exists predominantly as monomeres. Amino acids composition, isoelectric point, stability, Km values and the ability to catalyze partial reactions have been measured and compared with known properties of carbamoyl phosphate synthetases from other sources. From the available data a high degree of evolutionary conservation of the ammonia-dependent synthetase is suggested. This is also supported by the demonstration of extensive immunological cross-reactivity between the human and rat enzymes.

Amino Acids↗
PubMed1981

Mechanism of activation of bicarbonate ion by mitochondrial carbamoyl-phosphate synthetase: formation of enzyme-bound adenosine diphosphate from the adenosine triphosphate that yields inorganic phosphate.

The mechanism of the reaction catalyzed by rat liver mitochondrial carbamoyl-phosphate synthetase has been studied by using [beta-18O2]ATP and HC18O-3, monitoring the isotopic composition of adenosine triphosphate (ATP) and inorganic phosphate (Pi) by high-resolution 31P NMR spectroscopy. In the presence of both HCO3- and acetylglutamate, the enzyme catalyzes the exchange of oxygen atoms between the beta, gamma bridging and the beta nonbridging positions of ATP. Addition of NH3 stops the exchange, Pi released by the ATPase activity of the enzyme in the absence of NH3 contains one oxygen atom from HC18O3- but there is no incorporation of 18O into ATP. There is no significant incorporation of [14C]ADP or 32Pi into ATP. It is concluded that in the enzyme-ATPA.HCO30.ATPB complex formed in the presence of ATP and HCO3- there is reversible transfer of the gamma-PO3 group of ATPA (the molecule that yields Pi) to HCO3- without dissociation of products. The beta-PO3 of the enzyme-bound ADP that is formed can rotate. Virtually all of the complex appears to be in the form in which ATPA is cleaved, but in the absence of NH3, ATP is reconstituted and dissociates from the complex on at least 75% of the occasions. On the remainder, the carbonyl phosphate is cleaved in an irreversible process that yields Pi and a low-energy form of carbonic acid (probably HCO3-). NH3 reacts rapidly and irreversibly with the complex, and at saturation the rate (greater than 10 times the rate of Pi release in the absence of NH3) is sufficient to prevent dissociation of ATPA. In the absence of HCO3- an enzyme-ATPA.ATPB complex is formed, but cleavage of the bond between beta, gamma bridging oxygen and P gamma of ATPA does not occur.

Adenosine Diphosphate↗
PubMed1981

Human carbamoylphosphate synthetase I.

Carbamoylphosphate synthetase (ammonia) isolated from human liver has been characterized. It is composed of monomers (constituted by a single polypeptide of 160,000 +/- 5,000 Mr) that can associate into dimers, although in the native state the monomer predominates. Physical and kinetic properties and amino acid composition are very similar to those found for the rat liver enzyme. There is extensive immunological cross-reactivity between the enzymes from both species. These results are discussed in the light of a possible regulatory role of carbamoylphosphate synthetase. The implications of these findings on a number of pathological states are also discussed.

Amino Acid Metabolism, Inborn Errors↗
PubMed1979

Mechanism of carbamoyl-phosphate synthetase. Properties of the two binding sites for ATP.

Carbamoyl phosphate synthethase I synthesizes carbamoyl phosphate from ammonia, HCO3- and two molecules of ATP, one of which, ATPA, yields Pi while the other, ATPB, yields the phosphoryl group of carbamoyl phosphate. Pulse-chase experiments with [gamma-32P]ATP without added HCO3- demonstrate separate binding sites for ATPA and ATPB. Bound ATPA dissociates readily from its site (t1/2 approximately 1--2 s) and the Kd is 0.2--0.7 mM. For the ATPB binding site the t1/2 for dissociation is 5--12 s and the Kd approximately 10 mM. Kd for ATPA seems to increase with enzyme concentration whereas Kd for ATPB does not change. HClO4 releases the ATP unchanged from the enzyme . ATPB and enzyme . ATPB . ATPA complexes. In the presence of HCO3-, ATP and N-acetylglutamate, an enzyme . ATPB . HCO3- . ATPA complex is formed. Its formation by the addition of HCO3- to the enzyme . ATPB . ATPA complex appears to involve an initial bimolecular addition reaction followed by an isomerization. Treatment with HClO4 releases Pi from ATPA but ATPB is released unchanged. Spontaneous hydrolysis of ATPA is responsible for the ATPase activity of the enzyme. Thus, a covalent bond may form between HCO3- and ATPA. However, ATPA can dissociate rapidly (t1/2 less than 10 s). The Kd for ATPA is approximately 0.2 mM. ATPB appears unable to dissociate from the enzyme . ATPB . HCO3- . ATPA complex since the t1/2 for dissociation of ATPB from the enzyme is lengthened about five times in the presence of 19 mM HCO3- and at 1 mM ATP. ATPA may also hydrolyse in this complex and be replaced by another molecule of ATP in the absence of exchange of ATPB. However, the ATPA binding site must be occupied to prevent ATPB release. ATPB may be bound in a pocket which becomes inaccessible to the solution when HCO3- and ATPA also bind. In contrast, HCO3- does not inhibit the binding of ATPB to the enzyme. Various intermediate steps in the formation of the enzyme . ATPb . HCO3- . ATPA complex are discussed. Additional evidence is presented that the ATPB binding site is only periodically accessible to ATP in solution and that ATPB in the steady-state reaction binds when the products leave. Since greater than 1.3 mol ATPB and greater than 1.8 mol ATPA bind/mol enzyme dimer, the enzyme monomer may be an active species.

Adenosine Triphosphate↗
PubMed1979

Mechanism of carbamoyl-phosphate synthetase. Binding of ATP by the rat-liver mitochondrial enzyme.

This paper demonstrates, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its gamma-phosphoryl group to carbamoyl phosphate. This bound APTB can react with NH3, HCO-3 and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N-acetylglutamate, but not NH3 or HCO-3, are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10--30 microns ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer. Binding of ATPB follows a slow exponential time course (t1/2 8--16 s, 22 degrees C), independent of ATP concentration and little affected by NH3, NCO-3 or by incubation of the enzyme with unlabelled ATP prior to the pulse of [gamma-32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO-3 when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation. Pulse-chase experiments in the absence of NH3 and HCO-3 (less than 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO-3 in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 micron) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine(Ap5A) fails to inhibit the enzyme significantly. A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321--329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent to conformational change postulated by others from steady-state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO-3-dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.

Adenosine Triphosphate↗
PubMed1977

Mechanism of mitochondrial carbamoyl-phosphate synthetase: synthesis and properties of active CO2, precursor of carbamoyl phosphate.

This paper demonstrates the formation of "active CO2" (CO2-P), a precursor of carbamoyl phosphate (CP), with frog liver carbamoyl-phosphate synthetase. Absence of ammonia is essential for the demonstration by pulse incubation with H14CO3- of CO2-P. Adenosine triphosphate (ATP) and acetylglutamate are required for the synthesis of CO2-P, which is highly unstable in aqueous solutions (t1/2 = 0.75 s at 24 degrees C at neutral pH). In the absence of ammonia, CO2-P attains rapidly a steady-state level, which depends on the concentration of ATP and HCO3-. The "apparent KM'S" are approximately equal to those found for the adenosine triphosphate (ATPase) activity of the enzyme. The maximum level of CO2-P is limited by the amount of enzyme, and approximates 4 mol of intermediate/mol of enzyme. The unprotonated form of ammonia seems to be the species reacting with CO2-P to produce CP. The reaction of CO2-P and NH3 is very fast (rate constant kn = 8 x 10(4) M-1 S-1) and does not consume free ATP. Therefore, the 2 mol of ATP necessary for CP synthesis binds or reacts with the enzyme and/or CO2 prior to reaction with NH3. The reaction of CO2-P with NH3 also takes place in acetone under conditions at which the enzyme is not active, suggesting little or no assistance from enzyme catalysis or that a part of the catalytic site is "frozen" by the solvent in the active conformation. In the light of these and other findings, a new scheme is proposed for the mechanism of frog liver carbamoyl-phosphate synthetase and some considerations are made on the chemical nature of the intermediate and on the possible evolutionary significance of the reaction of CO2-P with NH3 in acetone.

Acetone↗
PubMed1977

Evidence pointing to the main role of lysosomes in mitochondrial proteolysis at neutral pH.

Recombination experiments using radioactive mitochondria and mitoplasts, and nonradioactive lysosomes or digitonin-soluble fraction of mitochondria, show equal rates of proteolysis and of inactivation of carbamyl phosphate synthetase; the amount of lysosomal protein was equal in both cases on the basis of N-acetyl-beta-glucosaminidase activity. Therefore, lysosomes seem to be responsible for all the proteolytic activity exhibited by the digitonin soluble fraction of mitochondrial preparations. Since this fraction contains ca. 90% of the proteolytic activity present in mitochondrial preparations, most of the proteolysis can be attributed to lysosomal contamination. These findings and stability characteristics "in vitro" and "in vivo" of some matrix enzymes are presented and discussed in relation to protein turnover.

Acetylglucosaminidase↗