Biomedical subjects

J L Rendón

Publications and source records attributed to J L Rendón.

18 recordsLinked to original sources

PubMed2001

Unfolding kinetics of glutathione reductase from cyanobacterium Spirulina maxima.

The kinetics of the irreversible unfolding of glutathione reductase (NAD[P]H:GSSG oxidoreductase, EC 1.6.4.2.) from cyanobacterium Spirulina maxima was studied at pH 7.0 and room temperature. Denaturation was induced by guanidinium chloride and the changes in enzyme activity, aggregation state, and tertiary structure were monitored. No full reactivation of enzyme was obtained, even after very short incubation times in the presence of denaturant. Reactivation plots were complex, showing biphasic kinetics. A very fast early event in the denaturation pathway was the dissociation of tetrameric protein into reactivatable native-like dimers, followed by its conversion into a nonreactivatable intermediary, also dimeric. In the final step of the unfolding pathway the latter was dissociated into denatured monomers. Fluorescence measurements revealed that denaturation of S. maxima glutathione reductase is a slow process. Release of the prostethic group FAD was previous to the unfolding of the enzyme. No aggregated species were detected in the unfolding pathway, dismissing the aggregation of denatured polypeptide chains as the origin of irreversibility. Instead, the transition between the two dimeric intermediates is proposed as the cause of irreversibility in the denaturation of S. maxima glutathione reductase. A value of 106.6 +/- 3 kJ mol(-1) was obtained for the activation free energy of unfolding in the absence of denaturant. No evidence for the native monomer in the unfolding pathway was obtained which suggests that the dimeric nature of glutathione reductase is essential for the maintenance of the native subunit conformation.

Bacterial Proteins↗

PubMed2000

Aggregation, dissociation and unfolding of glucose dehydrogenase during urea denaturation.

The effect of urea on glucose dehydrogenase from Bacillus megaterium has been studied by following changes in enzymatic activity, conformation and state of aggregation. It was found that the denaturation process involves several transitions. At very low urea concentrations (below 0.5 M), where the enzyme is fully active and tetrameric, there is a conformational change as monitored by an increase in intensity of the tryptophan fluorescence and a maximum exposure of organized hydrophobic surfaces as reported by the fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5.5'-disulfonic acid. At slightly higher urea concentrations (0.75-2 M), a major conformational transition occurs, as monitored by circular dichroism and fluorescence measurements, in which the enzyme activity is completely lost and is concomitant with the formation of interacting intermediates that lead to a highly aggregated state. Increasing urea concentrations cause a complete dissociation to lead first a partially and eventually the complete unfolded monomer. These phenomena are fully reversible by dilution of denaturant. It is concluded that after urea denaturation, the folding/assembly pathway of glucose dehydrogenase occurs with the formation of intermediate species in which transient higher aggregates appear to be involved.

Anilino Naphthalenesulfonates↗

PubMed1999

An alternative model for the transmembrane segments of the yeast H+-ATPase.

An alternative topological model for the yeast plasma membrane H(+)-ATPase from K. lactis was deduced by joint prediction, using 11 algorithms for the prediction of transmembrane segments complemented with hydrophobic moment analysis. Similarly to the model currently used in the literature, this alternative model contains 10 transmembrane segments, four in the N-half and six in the C-half of the protein. However, the distribution of the membrane-associated segments on the C-half of the enzyme differs in both models. Nine of the 10 transmembrane segments are highly hydrophobic with low hydrophobic moments, and are probably involved in structural roles. The fifth transmembrane segment is, on the other hand, less hydrophobic, with the highest hydrophobic moment, suggesting that this segment might have a dynamic role in the coupling of the hydrolysis of ATP with the translocation of protons across the membrane. The alignment of the Ca(2+)-ATPase, the Na(+)/K(+)-ATPase and the H(+)-ATPase sequences showed that these proteins have the same topology in the N-half, but important differences were found at the C-half of the enzymes. In contrast with the mammalian ATPases, the fifth transmembrane segment in the H(+)-ATPase appears early in the sequence, giving rise to a shorter cytoplasmic central loop. This alternative model will be useful in the designing of site-directed mutagenesis experiments and contains information for the fitting of the amino acid sequence into the transmembrane region of the three-dimensional model of the ATPase.

Algorithms↗

PubMed1999

Presence of two enzymes, different from the F1F0-ATPase, hydrolyzing nucleotides in human term placental mitochondria.

The hydrolysis of ATP, ADP or GTP was characterized in mitochondria and submitochondrial particles since a tightly-bound ATPase associated with the inner mitochondrial membrane from the human placenta has been described. Submitochondrial particles, which are basically inner membranes, were used to define the location of this enzyme. Mitochondria treated with trypsin and specific inhibitors were also used. The oxygen consumption stimulated by ATP or ADP was 100% inhibited in intact mitochondria by low concentrations of oligomycin (0.5 microgram/mg) or venturicidine (0.1 microgram/mg), while the hydrolysis of ATP or ADP was insensitive to higher concentrations of these inhibitors but it was inhibited by vanadate. Oligomycin or venturicidine showed a different inhibition pattern in intact mitochondria in relation to the hydrolysis of ATP, ADP or GTP. When submitochondrial particles were isolated from mitochondria incubated with oligomycin or venturicidine, no further inhibition of the nucleotide hydrolysis was observed, contrasting with the partial inhibition observed in the control. By incubating the placental mitochondria with trypsin, a large fraction of the hydrolysis of nucleotides was eliminated. In submitochondrial particles obtained from mitochondria treated with trypsin or trypsin plus oligomycin, the hydrolysis of ATP was 100% sensitive to oligomycin at low concentrations, resembling the oxygen consumption; however, this preparation still showed some ADP hydrolysis. Native gel electrophoresis showed two bands hydrolyzing ADP, suggesting at least two enzymes involved in the hydrolysis of nucleotides, besides the F1F0-ATPase. It is concluded that human placental mitochondria possesses ADPase and ATP-diphosphohydrolase activities (247).

Adenosine Triphosphate↗

PubMed1997

Thermal denaturation of glutathione reductase from cyanobacterium Spirulina maxima.

The thermal unfolding of glutathione reductase (NAD[P]H:GSSG oxidoreductase EC 1.6.4.2.) from cyanobacterium Spirulina maxima was monitored by differential scanning calorimetry and circular dichroism at neutral pH. Covalent cross-linking of enzyme at different temperatures revealed dimer as the species undergoing the thermal transition. A single endotherm was observed, but its thermodynamic parameters showed dependence on the scan rate. In the transition zone, aggregation of the dimeric species was observed. Analysis of the enzyme heated at 80 degrees C revealed that the resultant species retained a high content of secondary structure. The addition of low concentrations of guanidinium hydrochloride resulted in a full cooperative thermal transition. A model in which the dimeric protein undergoes a partial unfolding in a kinetically controlled fashion is proposed, such that the experimental value of delta H(cal) results from the simultaneous occurrence of endothermic and exothermic events.

Bacterial Proteins↗

PubMed1996

Human placental estradiol 17 beta-dehydrogenase: structural and catalytic changes during urea denaturation.

The denaturation behavior of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) in urea was studied by following changes in enzyme activity, conformation and oligomeric state. Results showed that the native --> unfolded transition follows a complex pattern, in which changes in both secondary and tertiary structure are simultaneous with changes in the aggregation state of enzyme. At relatively low urea (< 3 M), a major conformational transition, as monitored by CD and fluorescence measurements, is concomitant with an expanded state of the enzyme that coincides with its inactivation and the formation of polymeric species. Protein structural changes were also monitored by using the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid. The combined data suggest the existence of a molten globule state of dimeric enzyme promoted by low urea concentrations. Dilution of urea at this stage results in a full recovery of the enzymatic activity as well as of the native dimeric structure. Between 3 and 5 M urea estradiol 17 beta-dehydrogenase exists as a mixture of high molecular mass species which may be resolved by electrophoresis. In this range of urea concentration, only minor conformational changes were detected, although inactivation becomes to be irreversible. Above 5 M urea a second conformational transition takes place. Electrophoretic analysis of cross-linked samples revealed this stage results in the complete dissociation of enzyme toward unfolded monomer. It is concluded that the inactivation and unfolding of estradiol 17 beta-dehydrogenase during denaturation by urea occurs with the formation of intermediate species with different stability in which a molten globule-like state appears to be involved. The irreversibility of the process above urea 3 M is explained as the inability of aggregated enzyme to dissociate into native dimers.

Anilino Naphthalenesulfonates↗

PubMed1996

Detergent solubilization of 3 beta-hydroxysteroid dehydrogenase from dog pancreas.

Functional 3 beta-hydroxysteroid dehydrogenase coupled with isomerase (3 beta-HSD) was extracted from dog pancreatic mitochondria by treatment with the zwitterionic detergent CHAPSO. Increasing concentrations of this detergent led to a progressive and simultaneous solubilization of the pregnene (C-21) and androstene (C-19) dehydrogenase activities. Optimal solubilization of both C-21 and C-19 3 beta-HSD activities was achieved at a detergent/protein ratio of 0.6 (w/w). One hundred thirty percent of the initial particulate enzyme activities were recovered in the 105,000 g supernatant fluid with a 2.5-fold increase in the enzymatic specific activities. The C-21/C-19 activity ratios were 1.3 for mitochondria and 1.39 for the solubilized preparation. The apparent Km values for steroid substrates were unchanged after solubilization. Treatment of the mitochondrial suspension with sodium deoxycholate, CTAB, Lubrol XW, Brij 58, Emulgen 913 and Triton X-100 markedly decreased the 3 beta-HSD activities as a function of the detergent concentration and failed in to achieve solubilization.

3-Hydroxysteroid Dehydrogenases↗

PubMed1995

Denaturing behavior of glutathione reductase from cyanobacterium Spirulina maxima in guanidine hydrochloride.

The influence of guanidine hydrochloride (Gdn-HCl) on glutathione reductase from Spirulina maxima has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, thiol groups accessibility, and gel filtration chromatography. It was found that the denaturation process involves several intermediate states. At low, Gdn-HCl concentrations (Cm = 0.4 M), reductase activity was fully lost. However, below 3 M Gdn-HCl, this inhibition was freely reversible upon removal of the denaturing agent. Gel filtration experiments revealed that this reversible inhibition was not due to dissociation of the tetrameric enzyme. Structural studies strongly suggest that the conformation of this intermediate state is similar to that of native enzyme. A model in which a local region of the polypeptide chain assumes an extended conformation (D. T. Haynie, and E. Freire, Proteins 16,115-140) is proposed for the reversibly inactivated enzyme. Between 3 and 4 M Gdn-HCl (Cm = 3.5), the enzyme activity was irreversibly lost, this inhibition being concomitant with the loss of ellipticity, changes in both wavelength and intensity at the maximum of fluorescence emission, and dissociation of the enzyme into unfolded monomers; these results reveal that gross changes in the protein conformation occur under these conditions. At 4 M Gdn-HCl an equilibrium exists between the denatured forms of dimer and monomer, which is completely shifted toward the unfolded monomers at 5 M Gdn-HCl. Irreversibility in the Gdn-HCl-induced denaturation of S. maxima glutathione reductase was not due to aggregation of the unfolded enzyme.

Chromatography, Gel↗

PubMed1995

Human placental 17 beta-hydroxysteroid dehydrogenase: secondary structure and circular dichroism demonstration of conformational changes upon NADP binding.

The secondary structure of human placental 17 beta-hydroxysteroid dehydrogenase in the absence and presence of NADP has been studied by circular dichroism spectroscopy. The conformational analysis of the NADP-containing enzyme shows that is an alpha/beta protein with 60% of regular secondary structure (38% of alpha helix, 22% of beta-strand structures), 20% of beta-turn and 20% of non-repetitive structure. These results were in good agreement with the information obtained using statistical and homology methods based on amino-acid sequence. On the other hand, 25% alpha-helix, 55% beta-strand, and 20% non-repetitive structure were estimated by circular dichroism for the cofactor-free enzyme. Addition of varying concentrations of NADP to the cofactor free enzyme is accompanied by circular dichroism spectral changes. From the variation in the magnitude of the positive band at 193 nm with increasing NADP concentration, a dissociation constant of 34 nM was obtained.

17-Hydroxysteroid Dehydrogenases↗

PubMed1993

Effect of inorganic phosphate on the self-associating properties of glutathione reductase from Spirulina maxima.

In the presence of millimolar concentrations of inorganic phosphate, native Spirulina maxima glutathione reductase (NAD[P]H:GSSG oxidoreductase EC 1.6.4.2.) changes its aggregation state. The oligomeric structure of the enzyme was notably dependent upon phosphate molarity, ranging from a dimer-tetramer equilibrium at relatively low phosphate concentrations into a tetramer-octamer equilibrium at moderate or high phosphate concentrations. In spite of the changes in quaternary structure, the tetramer remains as the most stable and abundant species. Sodium chloride solutions were not able to produce a similar effect, thus discarding an unspecific ionic strength effect.

Cyanobacteria↗

PubMed1992

Purification and characterization of glutathione reductase from Rhodospirillum rubrum.

Glutathione reductase (NAD(P)H:GSSG oxidoreductase EC 1.6.4.2.) was purified 1160-fold to homogeneity from the nonsulfurous purple bacteria Rhodospirillum rubrum (wild type). Specific activity of the pure preparation was 102 U/mg. The enzyme displayed a typical flavoprotein absorption spectrum with maxima at 274,365, and 459 nm and an absorbance ratio A280/A459 of 7.6. The amino acid analysis revealed an unusually high content of glycine and arginine residues. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) showed a total of two free thiol groups per subunit, one of which is made accessible only under denaturing conditions. An isoelectric point of 5.2 was found for the native enzyme. Km values, determined at pH 7.5, were 6.1 and 90 microM for NADPH and GSSG, respectively. NADH was about 2% as active as NADPH as an electron donor. The enzyme's second choice in disulfide substrate was the mixed disulfide of coenzyme A and glutathione, for which the specific activity and Km values were 5.1 U/mg and 3.4 mM, respectively. A native molecular weight of 118,000 was found, while denaturing electrophoresis gave a value of 54,400 per subunit, thus suggesting that R. rubrum glutathione reductase exists as a dimeric protein. Other physicochemical constants of the enzyme, such as Stokes radius (4.2 nm) and sedimentation coefficient (5.71 S), were also consistent with a particle of 110,000.

Amino Acids↗

PubMed1990

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in canine pancreas.

Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with steroid-delta 5-4-isomerase was demonstrated for the first time in the pancreas. The enzyme complex was assayed by measuring the conversion of pregnenolone to progesterone as well as of dehydroepiandrosterone to androstenedione and found to be localized primarily in the mitochondrial fraction of dog pancreas homogenates. The delta 5-3 beta-hydroxysteroid dehydrogenase used either NAD+ or NADP+ as co-substrates, although maximal activity was observed with NAD+. In phosphate buffer, pH 7.0 and 37 degrees C, the apparent Km values of the dehydrogenase were 6.54 +/- 0.7 microM for pregnenolone and 9.61 +/- 0.8 microM for NAD+. The apparent Vmax was determined as 0.82 +/- 0.02 nmol min-1 mg-1. Under the same conditions the Km values for dehydroepiandrosterone and NAD+ were 3.3 +/- 0.2 microM and 9.63 +/- 1.6 microM, respectively, and the apparent Vmax was 0.62 +/- 0.01 nmol min-1 mg-1.

3-Hydroxysteroid Dehydrogenases↗

PubMed1989

Dimer-tetramer equilibrium of glutathione reductase from the cyanobacterium Spirulina maxima.

Glutathione reductase [NAD(P)H:GSSG oxidoreductase; EC 1.6.4.2] from cyanobacterium Spirulina maxima exists as an equilibrium system between a dimer (S20,W = 5.96) and a tetramer (S20,W = 8.49) which has a very slow interconversion rate at neutral pH. Our results showed that the apparent dissociation constant (kd) was 4.61 X 10(-7) M. The proportion of both forms at pH 7.0 did not alter at either 4 or 25 degrees C. However, electrophoretic analysis at various pH values showed that at 25 degrees C a gradual transition takes place between oligomers with an apparent pKa of 7.55. When dimers aggregate to form tetramers, the reaction involves the uptake of eight protons (K = 1.58 X 10(-64) M9). At pH 7.7, the equilibrium shifts completely from dimers-tetramers to dimers when temperature is increased, which would suggest that the dissociation is an endothermic process. Thermodynamic parameters obtained from the temperature study show that the dissociation of glutathione reductase is characterized by positive entropy and enthalpy changes. Neither NADPH nor GSSG have any effect on the dimer-tetramer equilibrium. Measurements of reductase activity indicate that the tetramer is almost certainly active, whereas the dimer is either less active or inactive.

Cyanobacteria↗

PubMed1988

17 beta-hydroxysteroid dehydrogenase activity in canine pancreas.

The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively.

17-Hydroxysteroid Dehydrogenases↗

PubMed1986

Purification, properties, and oligomeric structure of glutathione reductase from the cyanobacterium Spirulina maxima.

Glutathione reductase [NAD(P)H:GSSG oxidoreductase EC 1.6.4.2] from cyanobacterium Spirulina maxima was purified 1300-fold to homogeneity by a simple three-step procedure involving ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and affinity chromatography on 2',5'-ADP-Sepharose 4B. Optimum pH was 7.0 and enzymatic activity was notably increased when the phosphate ion concentration was increased. The enzyme gave an absorption spectrum that was typical for a flavoprotein in that it had three peaks with maximal absorbance at 271, 370, and 460 nm and a E1%271 of 23.3 Km values were 120 +/- 12 microM and 3.5 +/- 0.9 microM for GSSG and NADPH, respectively. Mixed disulfide of CoA and GSH was also reduced by the enzyme under assay conditions, but the enzyme had a very low affinity (Km 3.3 mM) for this substrate. The enzyme was specific for NADPH. The isoelectric point of the native enzyme at 4 degrees C was 4.35 and the amino acid composition was very similar to that previously reported from other sources. The molecular weight of a subunit under denaturing conditions was 47,000 +/- 1200. Analyses of pure enzyme by a variety of techniques for molecular weight determination revealed that, at pH 7.0, the enzyme existed predominantly as a tetrameric species in equilibrium with a minor dimer fraction. Dissociation into dimers was achieved at alkaline pH (9.5) or in 6 M urea. However, the equilibrium at neutral pH was not altered by NADPH or by disulfide reducing reagents. The Mr and S20,w of the oligomeric enzyme were estimated to be 177,000 +/- 14,000 and 8.49 +/- 0.5; for the dimer, 99,800 +/- 7000 and 5.96 +/- 0.4, respectively. Low concentrations of urea increased the enzymatic activity, but this increase was not due to changes in the proportions of both forms.

Amino Acids↗

PubMed1985

A single step procedure for purification of estradiol 17 beta-dehydrogenase from human placenta.

A new and simple procedure for purification to homogeneity of the soluble estradiol 17 beta-dehydrogenase [E C.1.1.1.62] from human placenta has been developed. The purification is achieved in a single step by affinity-absorption of Cibachrome Blue F3G-A coupled to Sepharose and selective elution with NADP+. Homogeneous estradiol 17 beta-dehydrogenase has a specific activity of 7.2 units/mg. and has been purified 2400-fold with a 93% recovery.

17-Hydroxysteroid Dehydrogenases↗

PubMed1983

Glutathione reductase in evolution.

The disulfide reducing activities of GSSG-and CoASSG-reductases were measured on partially purified extracts from a variety of prokaryotes and eukaryotes. Glutathione-reductase was found in varying amounts in all eukaryotes and prokaryotes, used in this study, with the exception of the two strict anaerobes Clostridium tartarivorum and Desulfovibrio vulgaris, and the two primitive Archaebacteria Methanosarcina barkeri and Halobacterium halobium. CoASSG-reductase was found in some eukaryotes and prokaryotes, but showed no clear pattern of distribution other than its absence whenever GSSG-reductase was not present. The absence of GSSG-reductase activity in organisms lacking GSH, confirms that glutathione metabolism is not universal and suggests that this enzyme might be useful as a marker in classifying organisms. The data suggest that glutathione-reductase occurs as a result of the change from a reducing to a oxidizing atmosphere in the primitive Earth.

Aerobiosis↗

PubMedDate not supplied

A novel ATP-diphosphohydrolase from human term placental mitochondria.

This report describes an ATP-diphosphohydrolase activity associated with the inner membrane of human term placental mitochondria. An enriched fraction containing 30 per cent of the total protein and 80 per cent of the total ATP-diphosphohydrolase activity was obtained from submitochondrial particles. ATP-diphosphohydrolase activity was characterized in this fraction. The enzyme had a pH optimum of 8 and catalysed the hydrolysis of triphospho- and diphosphonucleosides other than ATP or ADP. Pyrophosphate was also hydrolysed, but AMP or other monoester phosphates were not. The activity of ATP-diphosphohydrolase was dependent on Mg(2 + ), Ca(2 + )or Mn(2 + )and the enzyme substrate was the cation-nucleotide complex. An excess of free cation produced inhibition.ATP-diphosphohydrolase activity was stimulated at micromolar concentrations of calcium or magnesium in the presence of La-PPi. Negative cooperativity kinetics was observed with all substrates tested. The V(max)ranged from 150 to 300nmol of Pi released/mg/min. The [S](0.5)for nucleotides was 1-10m m and 182m m for PPi. The enzyme was inhibited by orthovanadate, but not by l -phenylalanine, oligomycin, sodium azide, P(1),P(5)-di(adenosine-5')pentaphosphate or sodium fluoride.The experimental evidence showing absence of inhibition by sodium azide and sodium fluoride, hydrolysis of pyrophosphate but not of monoester phosphates, and negative cooperativity suggested that this enzyme was a novel ATP-diphosphohydrolase.

Apyrase↗