Biomedical subjects

J Bozal

Publications and source records attributed to J Bozal.

At least 19 recordsLinked to original sources

PubMed1993

Inhibition of rat hepatic mitochondrial aldehyde dehydrogenase isozymes by repeated cyanamide administration: pharmacokinetic-pharmacodynamic relationships.

The inhibition of rat hepatic mitochondrial aldehyde dehydrogenase (ALDH) isozymes was studied in apparent steady-state conditions after repeated intra-peritoneal cyanamide administration. The low-Km mitochondrial ALDH isozyme was more susceptible to cyanamide-induced inhibition (DI50 = 0.104 mg kg-1) than the high-Km isozyme (DI50 = 8.52 mg kg-1), with almost complete inhibition occurring at 0.35 mg kg-1 total cyanamide administered for the low-Km isozyme. The relationships between plasma and liver cyanamide concentrations and the inhibition of high-Km ALDH were established by means of the sigmoid Imax model. The effect of dosing rate on the plasma concentration of cyanamide at apparent steady-state showed non-linearity, indicating that clearance or first-pass metabolism of cyanamide during its absorption after intraperitoneal administration did not remain constant throughout the range of doses studied.

Aldehyde Dehydrogenase↗

PubMed1989

Inactivation of low-Km rat liver mitochondrial aldehyde dehydrogenase by cyanamide in vitro. A catalase-mediated reaction.

The inactivation of the affinity chromatography purified low-Km rat liver mitochondrial aldehyde dehydrogenase (ALDH)--free of catalase activity--by the alcohol sensitizing agent cyanamide was studied in vitro. This ALDH-purified preparation was not susceptible to cyanamide inactivation at concentrations up to 2.5 mM. On the other hand, ALDH activity appears to be irreversibly inhibited when the incubation mixture contained ALDH, catalase, NAD+ and cyanamide. Influence of catalase, NAD+ and cyanamide concentrations in the incubation mixtures on the ALDH activity were also established. The time course of the concentration of cyanamide in an incubation mixture when ALDH activity was inhibited by cyanamide in the presence of catalase and NAD+, was evaluated by HPLC. No disappearance of cyanamide was observed for a period of time up to 24 hr. This result suggests that no metabolic conversion of cyanamide to an active inhibitory form takes place, as has been suggested recently.

Aldehyde Dehydrogenase↗

PubMed1989

Purification and characterization of chicken brain cytosolic aspartate aminotransferase.

Aspartate aminotransferase from the cytosolic fraction of chicken brain was isolated with acceptable yield and high degree of purity. The enzyme appeared in multiple molecular forms: alpha, beta, gamma, and delta (alpha predominates), as detected by polyacrylamide gel electrophoresis with specific staining. These different forms of the enzyme were separated by DEAE-Sephacel chromatography, and showed different isoelectric points and maximal velocities values, whereas their molecular weight, optimum pH and Michaelis constants were very similar. Generation process studies suggest that minors subforms of the enzyme could be raised from alpha form by a mechanism in which the oxidation of particular amino acid groups are involved.

Animals↗

PubMed1989

Cytosolic aspartate aminotransferases from different chicken tissues: purification and characterization of their multiple forms.

Cytosolic aspartate aminotransferases from chicken heart, liver, spleen, skeletal muscle and breast muscle differed in number of their molecular forms, detected by polyacrylamide gel electrophoresis and specific staining. The number of molecular forms varied from tissue to tissue but the electrophoretic mobilities of a given form in all tissues were analogous. Within a single tissue most of the enzyme activity was present as the lowest-running band (alpha form) and the rest was distributed in minor bands termed (B,tau, alpha and epsilon forms). We report a method for the purification of cytosolic aspartate aminotransferases from various chicken tissues. The procedure can be carried out in one week and allows the obtention of isolated molecular forms of the enzyme, independently of the tissue under study. Separation of multiple forms was also achieved by chromatofocusing. The isoelectric points determined by this method for a given form in all five tissues were analogous and differed from those of the molecular forms of the enzyme from other origins. An Mr of 100,000 was obtained for all molecular forms of the five chicken tissues studied.

Animals↗

PubMed1988

Purification and partial characterization of brain adenosine deaminase: inhibition by purine compounds and by drugs.

Rat brain adenosine deaminase (E.C. 3.5.4.4.) was purified 667-fold from the supernatant fraction by the following techniques: heat treatment (60 degrees C), fractionation with ammonium sulfate, column chromatography on DEAE-Sepharose, and preparative gel electrophoresis. The purified enzyme was homogeneous by the criterion of polyacrylamide disc gel electrophoresis and isoelectric focusing. Amino acid composition is given. The isoelectric point of the enzyme (5.2) was determined by isoelectric focusing on agarose. The apparent molecular weight was estimated to be 39,000 (Stokes Radius [Rs] = 27.3 A) using a calibrated Sephacryl S-300 column. The study of the influence of the temperature on the initial reaction rates allowed calculation of Ea (8.9 Kcal/mole) and delta H (5.0 Kcal/mole) values. The variation of V and Km with pH suggests the existence of a sulfhydryl group and an imidazole group in the enzyme-substrate complex. The enzyme had a Km (adenosine) of 4.5 X 10(-5) M and was inhibited by inosine, guanosine, adenine, and hypoxanthine but not by other intermediates of purine metabolism. None of the inhibitors were active as substrates. The enzyme was also inhibited by dimethyl sulfoxide and ethanol. Inhibition by ethanol can account partially for the CNS depressant effects of levels 3 and 4 of alcohol intoxication. A number of drugs having therapeutic uses such as sedative, anxiolytic, analgesic, and relaxant are modulators of the enzyme. Among these, lidoflazine, phenylbutazone, and chlordiazepoxide are the most potent as inhibitors (Ki 30, 54, and 83 microM, respectively), whereas medazepam is the most potent as activator (Ka 0.32 mM). Thus, it is concluded that some drugs that inhibit adenosine uptake also modulate adenosine deaminase activity. Besides, since the enzyme is located extracellularly [Franco et al, 1986], these drugs can modulate the physiological effects exerted by extracellular adenosine.

Adenosine Deaminase↗

PubMed1988

Isolation and characterization of bovine brain myelin distribution of 5'-nucleotidase.

Myelin was isolated from bovine brain by several published procedures and modifications of these procedures. High activity of the myelin marker (2',3'-cyclic nucleotide 3'-phosphohydrolase) and low activity of contaminants markers in white matter homogenates in respect to cerebral cortex showed the white matter to be better than the cerebral cortex or the whole brain for myelin isolation. A procedure is described for the preparation of purified myelin from bovine white matter which yielded a content of protein (40%), myelin marker (51%), and 5'-nucleotidase (25%) in purified myelin higher than by any used method. Acetylcholinesterase or succinate dehydrogenase was lower than 7% of its activity in the white matter homogenate, and monoamine oxidase and NADPH:cytochrome c reductase were not recovered in myelin fraction. Morphologically, myelin fraction was shown to mainly consist of multilamellar membranes of different sizes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of myelin fraction showed a characteristic protein pattern of myelin. When our procedure was applied to frozen white matter, lower protein (32%) and myelin marker (34%) and similar 5'-nucleotidase activity (24%) were recovered in myelin, increasing its recovery in denser fractions of white matter.

5'-Nucleotidase↗

PubMed1988

Localization of 5'-nucleotidase in bovine brain myelin fraction and myelin subfractions.

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and 5'-nucleotidase (5'N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5'N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5'N activity maxima suggest that myelin may be a predominant locus of 5'N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5'N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5'N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (LUbrol WX) on 5'N activity were studied, and the results also suggest the intrinsic nature of 5'N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5'N activity, but not for the solubilization of this enzyme.

5'-Nucleotidase↗

PubMed1988

Generation process of cytosolic aspartate aminotransferase molecular forms by several treatments.

Alpha-, beta-, and gamma-forms of chicken liver cytosolic aspartate aminotransferase generate variants on storage (4 degrees C, 25 days). The variants developed from each isolated form appeared as evenly spaced bands with increasing anodic mobilities after polyacrylamide gel electrophoresis (PAGE), pH 8.8, and specific staining. Their mobilities coincided with those of the more negatively charged forms present in fresh tissue. Development of faster-running variants on storage was avoided by addition of thiol reagents to the freshly isolated forms. In their presence, beta- and gamma-forms were partially transformed into one and two variants with lower anodic mobilities analogous to those of native alpha- and beta-forms. Short pH and heat treatments did not modify the electrophoretic patterns of the alpha-, beta-, and gamma-forms, but the incubation with 5 mM L-ascorbic acid (37 degrees C, 7 h) produced more anodic active bands. The formation of these variants was inhibited by the presence, in the incubation mixture, of superoxide dismutase and catalase. The kinetic parameters of the forms submitted to the different treatments were similar to those of the freshly isolated subforms. The results obtained suggest that minor subforms of the enzyme could be generated in vivo by a mechanism in which the oxidation of particular amino acid groups is involved.

Animals↗

PubMed1988

Purification of malate dehydrogenase from chicken liver mitochondria. Existence of a small quantity of cytosolic isoenzyme.

1. A new purification method for chicken liver mitochondrial malate dehydrogenase is described. The application of affinity chromatography through 5'AMP-Sepharose and Blue-Sepharose permits to obtain homogeneous preparations, with good yields (47%), in a short time (48 hr). 2. The 5'AMP-Sepharose chromatography reveals the presence of two malate dehydrogenase species in the mitochondrial extracts. 3. A comparative study of these forms point out the cytosolic nature of the minority form and suggests that its presence could be due to a slight interaction of the cytosolic malate dehydrogenase with mitochondrial membranes.

Animals↗

PubMed1988

An improved purification method for cytosolic malate dehydrogenase from several sources.

A new purification method for cytosolic malate dehydrogenases from several sources has been developed. The procedure, employing chromatographies on 5'AMP-Sepharose, DEAE-Sephacel and Blue-Sepharose, allows for a rapid isolation of the enzyme (approximately 40 hours), in large quantities, with good yields (45-54%). The specific activity of final preparations were around 1300 I.U./mg and were judged homogeneous by polyacrylamide gradient gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size exclusion chromatography and isoelectric focusing.

Animals↗

PubMed1988

Simultaneous purification and characterization of aspartate aminotransferase isoenzymes from chicken liver.

Cytosolic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) were purified to homogeneity from chicken liver, without previous fractionation of the subcellular components. The procedure includes initial heat treatment and ammonium sulfate fractionation. The two isoenzymes can then be separated by a DEAE-Sepharose chromatography using a linear gradient of L-aspartate (reaction substrate). The separated fractions can be further purified by a parallel step with HA-Ultrogel prior to octyl-Sepharose (c-AAT) and CM-Sepharose (m-AAT) chromatographies. Michaelis constants, pI values, inhibition by adipate and subforms generation with time were studied for both isoenzymes.

Animals↗

PubMed1987

Microheterogeneity of the malate dehydrogenase from several sources.

Different homogeneously purified cytosolic malate dehydrogenases gave, on isoelectric focusing, several active bands. The phenomenon could not be assigned to differences in their molecular weights or to alterations in the enzyme preparations during the purification procedure. Resolution of the multiple malate dehydrogenase active bands was achieved by chromatofocusing. The aged isolated subforms always yielded the original electrofocusing pattern. This fact suggests that conformational isomerism is a likely explanation for the charge heterogeneity of the enzymes studied.

Animals↗

PubMed1987

Intramitochondrial location and some characteristics of chicken liver aspartate aminotransferase.

Chicken liver mitochondrial aspartate aminotransferase was found to be located in the intermembrane space and bound to the inner mitochondrial membrane. Purification of two mitochondrial fractions containing aspartate aminotransferase activity was performed. Both fractions showed similar chromatographic behaviour and identical isoelectric point and molecular weight values. There were no significant differences in the general kinetic mechanism, Km values, substrates inhibition and effect of various anions on the activity of mitochondrial aspartate aminotransferase purified from both fractions.

Animals↗

PubMed1987

A kinetic method for quantification of aspartate aminotransferase isoenzymes.

A spectrophotometric assay is proposed to determine the levels of aspartate aminotransferase (AAT) isoenzymes from chicken liver by a steady-state kinetic method which depends on the differential inhibition of these isoenzyme forms by high concentrations of substrate 2-oxoglutarate at pH 6.2. The use of a standard curve permits the determination of the percentage of chicken liver c-AAT and m-AAT isoenzymes. This method yields results in good correlation with those achieved by different extent adipate inhibition and by differential centrifugation.

Animals↗

PubMed1987

Lactate dehydrogenase activity in the mitochondrial fraction of chicken liver: enzyme binding and kinetic behavior of soluble and bound enzyme.

Chicken liver crude mitochondrial fraction showed lactate dehydrogenase activity (6.5% of cytoplasmic enzyme). Most of the mitochondrial lactate dehydrogenase was solubilized by sonication of the mitochondrial fraction in 0.15 M NaCl, pH 6. Total extracted lactate deshydrogenase activity was 3-fold higher than the initial pellet activity. Different isoenzymatic compositions were observed for cytosoluble and mitochondrial extracted lactate dehydrogenase. The pI, values of the 5 lactate dehydrogenase isoenzymes were found to be independent of their origin. The cytosoluble lactate dehydrogenase and the separated H4,H3M and H2M2 isoenzymes were able to bind to the chicken liver mitochondrial fraction in 5 mM sodium phosphate buffered medium, and could be solubilized afterwards with 0.15 M NaCl, pH 6. The enzyme bound to the mitochondrial fraction was less active than the soluble one. Particle saturation by the bound enzyme occurred with all mitochondrial fractions assayed. According to the Langmuir isotherm, the non-sonicated mitochondrial fractions contain a single type of binding sites for lactate dehydrogenase; in contrast, the sonicated mitochondrial fraction should contain different binding sites. Chicken liver crude or sonicated active mitochondrial fractions showed a hyperbolic behavior with respect to NADH and a non-hyperbolic one with respect to pyruvate. This mechanism is different from the bi-bi compulsory order mechanism of the soluble enzyme. With hydroxypyruvate as the substrate, the active mitochondrial fraction fit a sequential mechanism but lost the rapid-equilibrium characteristics of the soluble enzyme.

Animals↗

PubMed1987

Chicken liver purine nucleoside phosphorylase activity dependency of the redox state of sulfhydryl groups.

1. Double reciprocal plots (1/v vs 1/S) for nucleoside substrates of chicken liver purine nucleoside phosphorylase were non linear at high inosine or deoxyinosine concentrations (greater than 0.1 mM). The appearance of downward curvatures may be correlated with the oxidation of sulfhydryl groups of the enzyme. 2. 5,5'-Dithiobis-(2-nitrobenzoic acid) reacts with four sulfhydryl groups in the native enzyme, but upon denaturation with sodium dodecylsulfate six sulfhydryl groups react with this reagent. 3. Inosine, ribose-1-phosphate, hypoxanthine and orthophosphate partially protect sulfhydryl groups from the reaction with Ellman's reagent. 4. Inhibition of purine nucleoside phosphorylase by p-chloromercuribenzoate and 5,5'-dithiobis-(2-nitrobenzoic acid) follows a second order reaction kinetics.

Animals↗

PubMed1987

Comparative analysis of the malate dehydrogenases isolated from the cytosolic fraction of several tissues of guinea-pig Cavia porcellus.

The specific activities of the malate dehydrogenase and lactate dehydrogenase present in the soluble fraction of several guinea-pig tissues are reported. The electrophoretic patterns showed always two forms (A and B) with malate dehydrogenase activity and the five isoenzymes of lactate dehydrogenase. Chromatography of the different soluble fractions through 5' AMP-Sepharose allowed both molecular forms of malate dehydrogenase to be separated and obtained free from lactate dehydrogenase. Comparative studies of the two forms of malate dehydrogenase evidenced that the A and B forms exhibited cytosolic and mitochondrial characteristics, respectively.

Animals↗

PubMed1987

Distribution of adenosine deaminase in some rat tissues. Inhibition by ethanol and dimethyl sulfoxide.

The level of adenosine deaminase in various rat tissues has been tested. The enzyme activity of cytosolic fractions decreased in the following order: lung greater than spleen greater than small intestine greater than stomach greater than kidney greater than heart greater than liver greater than skeletal muscle greater than forebrain greater than cerebellum. The enzyme had identical patterns from tissue to tissue with respect to Km, V, and Ki values for ethanol and for dimethyl sulfoxide, with respect to electrophoretic behaviour and to inhibition by antibodies anti-rat brain adenosine deaminase.

Adenosine Deaminase↗