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V Rubio

Publications and source records attributed to V Rubio.

At least 91 records · Page 5Linked to original sources
PubMed1988

Carbamoyl-phosphate synthetase I. Kinetics of binding and dissociation of acetylglutamate and of activation and deactivation.

The dissociation of the cofactor, acetylglutamate, from the enzyme-cofactor complex formed by carbamoyl-phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO-3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s-1) is considerably slower than the rate of enzyme turnover (approximately equal to 6 s-1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5-2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme-cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor.

Adenosine Triphosphate↗
PubMed1987

Inactivation of mitochondrial carbamoyl phosphate synthetase induced by ascorbate, oxygen, and Fe3+ in the presence of acetylglutamate: protection by ATP and HCO3- and lack of inactivation of ornithine transcarbamylase.

Of the two mitochondrial enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) was and ornithine transcarbamylase (OTC) was not inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation [R. L. Levine, (1983) J. Biol. Chem. 258, 11828-11833]. The susceptibility of OTC was not increased by its substrates, products, or inhibitors, whereas that of CPS was markedly increased by acetylglutamate (its allosteric activator) when ATP was absent. Thus, acetylglutamate binds in the absence of ATP and exposes to oxidation essential groups of the enzyme. We estimate for this binding a KD value of 1.6 mM, which greatly exceeds the KD values (less than 10 microM) determined in the presence of ATP and bicarbonate. ATP, and even more, mixtures of ATP and bicarbonate protected CPS from inactivation. Acetylglutamate exposes the site for the ATP molecule that yields Pi, and it appears that ATP protects by binding at this site. Experiments of limited proteolysis with elastase suggest that oxidation prevents this binding of ATP and show that it accelerates cleavage of CPS by the protease, thus supporting the idea that oxidation may precede proteolysis. Trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerably faster than the native enzyme. Our results also support the idea that oxidative inactivation is site specific and requires sites on the enzyme for Me2+ and, possibly, for a nucleotide.

Adenosine Triphosphate↗
PubMed1987

Limited proteolysis reveals low-affinity binding of N-acetyl-L-glutamate to rat-liver carbamoyl-phosphate synthetase (ammonia).

Carbamoyl-phosphate synthetase was inactivated by elastase with first-order kinetics, and N-acetyl-L-glutamate speeded inactivation. From the dependence of the t1/2 value for inactivation on the concentration of acetylglutamate we estimate a Kd value for binding of the activator of 0.365 mM, which is approximately 600 times greater than in the presence of ATP, HCO3-, K+ and Mg2+. K+ and Mg2+ are not required for binding with low affinity, and in the absence of ATP they do not appear to increase the affinity for acetylglutamate. In the presence of acetylglutamate, mixtures of ATP, K+ and Mg2+ protect the enzyme from inactivation. ADP or AdoPP[NH]P partly replaced ATP in protecting the enzyme and thus binding of the nucleotide without further reaction is enough for protection. Two partial activities of the enzyme were inactivated by elastase to the same extent as the overall reaction, and thus elastase affects some property of the enzyme which is essential for catalysis. With other proteinases tested, inactivation was also accelerated by acetylglutamate and was slowed by mixtures of ATP, K+, Mg2+ and acetylglutamate, suggesting that changes in the accessibility of susceptible bonds are responsible for the changes in the degree of inactivation. It is concluded that elastase attacks at or close to the binding sites for ATP, and that exposure of the binding site for the ATP molecule that yields Pi (ATPA) upon binding of acetylglutamate causes the acceleration of the proteolytic inactivation.

Animals↗
PubMed1987

Transformation in Penicillium chrysogenum.

An auxotrophic mutant of Penicillium chrysogenum with a DNA rearrangement that affects the trpC region has been transformed to the Trp+ phenotype by using a plasmid that contains the trifunctional wild-type gene. A frequency of 40-80 transformants per microgram of input DNA was usually achieved. A low frequency of plasmid integration at the recipient mutated trpC gene was detected; however, most of the transformants integrated the plasmid DNA elsewhere into the genome. Some of the transformants contain multiple rearranged copies of the vector integrated in a tandem fashion.

DNA, Fungal↗
PubMed1986

Structure of a Cephalosporium acremonium mtDNA replicator.

We have investigated the ARS (autonomously replicating sequence) activity of a 1.94 kb mitochondrial DNA fragment of Cephalosporium acremonium and found that several subfragments of this piece of mtDNA conferred the ARS phenotype. The nucleotide sequence of the fragment shows: (i) a high A + T content (72.5%); (ii) a perfect consensus ARS sequence (ATTTATATTTA) in the subfragment with the highest ARS activity; (iii) a large number of ARS consensus-related sequences in the other subfragments, even in one lacking ARS activity; (iv) several potential hairpin structures. One of them contains the perfect consensus ARS sequence.

Acremonium↗
PubMed1986

Molecular cloning and characterization of the trpC gene from Penicillium chrysogenum.

We cloned the Penicillium chrysogenum trpC gene from a genomic library by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. The gene encodes a 2.7 kb poly(A)+ RNA. We localized the gene by sequence analysis in a 2.9 kb DNA insert found in the smallest plasmid selected from the library. Sequence data strongly suggest that the organization of the gene is similar to that described in other Ascomycetes. We found that a DNA fragment which codes only for the carboxy-terminal portion of the polypeptide is sufficient for complementation of the E. coli trpC9830 mutation.

Anthranilate Synthase↗
PubMed1986

A new case of arginase deficiency in a Spanish male.

A new case of arginase deficiency is reported in a male newborn from Spain. In contrast with the majority of the earlier cases, this infant showed severe protein intolerance of early onset. The diagnosis was based on the assay of the urea cycle enzymes in a postmortem liver sample. Levels of erythrocyte arginase were also determined in the parents and in a sister of the patient, and were consistent with heterozygosity. From a study of the pedigree it appears that arginase deficiency in this family presents a dramatic course.

Amino Acid Metabolism, Inborn Errors↗
PubMed1986

High-performance liquid chromatographic assay of argininosuccinate: its application in argininosuccinic aciduria and in normal man.

Argininosuccinate (ASA) is determined in a few minutes with little manipulation by reversed phase high-performance liquid chromatography (HPLC) using O-phthaldialdehyde. The two cyclic anhydrides of ASA are not formed during analysis but, if present, can be determined simultaneously. As little as 1 nl urine from a patient with argininosuccinic aciduria was sufficient for analysis; the ASA/creatinine ratio was 50.8 mmol g-1 and daily excretion was 5-7g ASA. We found small amounts of the two anhydrides in the patient's urine and we give factors to estimate, from their peaks, the corresponding amount of ASA. Urine from normal children showed a small acid-labile (at 100 degrees C) peak at the ASA position, which we tentatively assign to genuine ASA. From this peak less than 2mg ASA day-1 were excreted in our controls. Procedures for collection and storage of samples and the potential of this method for heterozygote detection are discussed.

Arginine↗
PubMed1986

ATPase activity of biotin carboxylase provides evidence for initial activation of HCO3- by ATP in the carboxylation of biotin.

When we incubated biotin carboxylase from Escherichia coli with ATP in absence of biotin we observed HCO3- -dependent ATP hydrolysis, which was activated by 10% ethanol in the same proportion as the activity of D-biotin carboxylation assayed in the presence of biotin. The two activities exhibited identical heat stability and were protected equally by glycerol; both required Mg2+ and K+ and showed similar dependency on the concentration of ATP. Biotin assay excluded potential contamination by traces of biotin as a cause of the observed ATP hydrolysis, and this was confirmed by the findings that carboxybiotin did not accumulate and that avidin was uninhibitory. Therefore we concluded that this HCO3- -dependent ATPase was genuinely a partial activity of biotin carboxylase. This partial activity supports a sequential mechanism for enzymatic carboxylation of biotin in which HCO3- is activated by ATP in a first step. It is consistent with the initial formation of the carbonic-phosphoric anhydride (HOCO2PO3(2-)), and it does not agree with models where biotin is phosphorylated by ATP prior to reaction with HCO3-. It appears that enzymes that use HCO3- for carboxylation, including biotin-dependent carboxylases, phosphoenolpyruvate carboxylase, and carbamoyl phosphate synthetase, activate HCO3- by a common mechanism involving the initial formation of the carbonic-phosphoric anhydride.

Adenosine Triphosphatases↗
PubMed1986

An efficient method for the introduction of viral DNA into Brevibacterium lactofermentum protoplasts.

A method for the introduction of a bacteriophage DNA into Brevibacterium lactofermentum protoplasts is described. Frequencies of 10(5) infective centres per micrograms DNA were easily achieved, the relationship between the number of infective centres and the amount of DNA being linear up to 5 micrograms DNA per assay. This method can be used to introduce foreign DNA into these bacteria.

Bacteriophages↗
PubMed1985

Determination of N-acetyl-L-glutamate using high-performance liquid chromatography.

Acetylglutamate in HClO4 tissue extracts is first separated from glutamate by ion exchange. It is then deacylated with aminoacylase, and the resulting glutamate, after adsorption to and elution from an AG 50 column, is quantitated by a fast-HPLC method using o-phthaldialdehyde precolumn derivatization, separation in a C18 reverse-phase column, and fluorescence detection. A linear response is obtained up to 2 nmol, the detection limit is 5 pmol, and the method is suitable for assay in 1 mg liver tissue and thus for needle biopsies. When samples were analyzed by this procedure and by earlier procedures based upon detection of glutamate with glutamate dehydrogenase or upon activation of carbamoyl phosphate synthetase the results were similar. The method, which is highly specific, compares favorably in sensitivity, precision, and accuracy with all other published procedures. Using this assay, no acetylglutamate has been found in chicken liver and rat kidney.

Animals↗
PubMed1983

Inactivation of carbamoyl phosphate synthetase (ammonia) by elastase as a probe to investigate binding of the substrates.

Rat liver carbamoyl phosphate synthetase I is inactivated by elastase. Addition of ATP, Mg2+, K+ and N-acetyl-L-glutamate (the physiological allosteric activator) protects entirely, whereas acetylglutamate alone speeds inactivation. We have exploited these properties to investigate binding of these ligands. Acetylglutamate binds with low affinity (KD 0.25 mM) in the absence of other ligands, and with higher affinity (KD much less than 0.1 mM) when ATP, Mg2+ and K+ are present. The apparent KD for ATP in the presence of acetylglutamate is intermediate between the KD values for the two ATP binding sites present in the enzyme; thus, binding of ATP to both sites is involved in protecting the synthetase. The data also indicate binding of MgATP and Mg2+ in the absence of acetylglutamate. The results provide further evidence for conformational changes associated with allosteric activation of the enzyme.

Animals↗
PubMed1983

Binding of N-acetyl-L-glutamate to rat liver carbamoyl phosphate synthetase (ammonia).

The binding of N-acetyl-L-glutamate, the physiological allosteric activator, to rat liver carbamoyl-phosphate synthetase (ammonia) was studied by techniques of rate of dialysis and of ultracentrifugation in the Airfuge. There is one binding site for acetylglutamate per enzyme monomer (Mr 165 000). K+, Mg2+ (free) and ATP were required to demonstrate binding. The concentrations of ATP required indicate that binding of ATPA (the ATP molecule that yields Pi) is needed. HCO-3 was not essential, but it enhanced binding of acetylglutamate. Glycerol also favored binding. Plots of Kd values versus the reciprocal of free Mg2+ and ATP concentrations are linear and indicate that ATPA, K+ and Mg2+ bind before acetylglutamate. In the presence of these ligands and HCO-3, ammonia increased drastically the Kd value for acetylglutamate, whereas in absence of HCO-3 ammonia had little effect. This suggests that acetylglutamate dissociates with the products and explains the higher Km for acetylglutamate in the synthetase (overall) reaction than in the ATPase (partial) reaction. In the absence of ATP acetylglutamate was bound with high affinity if ADP and carbamoyl phosphate were present. ADP or carbamoyl phosphate alone did not promote substantial binding. Binding of acetylglutamate at low concentration was slow; it was accelerated at higher concentrations of the activator. Exchange of bound acetylglutamate with acetylglutamate in solution was fast. A scheme proposed earlier for allosteric activation of the enzyme [Rubio, V., Britton, H. G. and Grisolia, S. (1983) Eur. J. Biochem. (in preparation)] is refined to incorporate the new information. Binding of ATPA, K+ and Mg2+ and formation of 'active CO2' (the central complex) are greatly favored by acetylglutamate.

Animals↗
PubMed1983

Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor.

Rat liver carbamoyl-phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. Km values for ATP, Mg2+ and K+ are greatly increased, the Km for HCO-3 is not changed much, and the Km for NH+4 is markedly reduced. Vmax for the synthetase reaction is less than 20% of that of the acetylglutamate-activated enzyme whereas Vmax for the ATPase activity is greater than 40% of that with acetylglutamate. Pulse-chase experiments with H14CO-3 show formation of less "active CO2" (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd = 20-30 microM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATPA (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 microM acetylglutamate the lag is much longer, but at 10 mM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time. From these findings we propose an allosteric model for activation of carbamoyl-phosphate synthetase in which there are two active states, R and R . AcGlu. Binding of ATPA is associated with the conversion of T to R. R . AcGlu differs from R in that transfer to carbamate of the gamma-phosphoryl group of ATPB appears to be facilitated.

Adenosine Triphosphatases↗