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At least 397 records · Page 22Linked to original sources
PubMed2006

Two new phosphoglycerate kinase mutations associated with chronic haemolytic anaemia and neurological dysfunction in two patients from Spain.

We report two previously undescribed mutations of the phosphoglycerate kinase gene (PGK1) leading to enzyme deficiency. In both cases, the patients were of Spanish origin and they exhibited a severe life-long chronic haemolytic anaemia associated with progressive neurological impairment. Sequence analysis of the first patient's entire PGK1 gene found a novel missense mutation (140T > A). This mutation caused an amino acid change of Ile to Asn at 46th position from the NH(2)-terminal serine residue (Ile46Asn), which has been called PGK-Barcelona based on the place of origin of the patient. In the second patient, a G to A transversion was discovered at nucleotide 958 (958G > A). This caused a Ser319Asn amino acid substitution. Since this mutation had not been previously described, the provisional name of PGK-Murcia was given to this deficient enzyme. The crystal structure of porcine PGK was used as a molecular model to investigate how these mutations may affect enzyme structure and function. In both cases, the mutations did not modify any of the PGK binding sites for ATP or 3PG, so their consequence is related to a loss of enzyme stability rather than a decrease of enzyme catalytic function.

Anemia, Hemolytic↗
PubMed1986

Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings.

3-Hydroxy-3-methylglutaryl-CoA reductase (NADPH) was solubilized with polyoxyethylene ether (Brij) W-1 from a heavy-membrane fraction, sedimented at 16000 X g from a cell-free homogenate of four-day-old, dark-grown radish seedlings (Raphanus sativus L.). Approximately 350-fold purification of the solubilized enzyme activity was achieved by (NH4)2SO4 precipitation followed by column chromatography on DEAE-Sephadex A-50, blue-dextran-agarose and HMG-CoA-hexane-agarose. The presence of detergent, which was required at all times to maintain activity, did not interfere with the chromatographic procedures used. Sucrose density centrifugation suggested an apparent molecular mass of 180 kDa with subunits of 45 kDa (polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate). The enzyme was stable at 67.5 degrees C for 30 min in the presence of glycerol, dithioerythritol and detergent. Studies of enzyme stability and activation indicate that the enzyme is a hydrophobic protein with free thiol groups that are essential for full activity. The activation energy was estimated to be 92 kJ (Arrhenius plot). Antibodies raised against rat liver and yeast hydroxymethylglutaryl-CoA (HMG-CoA) reductase failed to bind or inactivate the radish enzyme. When both HMG-CoA and NADPH concentrations were varied, intersecting patterns were obtained with double-reciprocal plots. The apparent Km values determined in this way are 1.5 microM [(S)-HMG-CoA], and 27 microM (NADPH). Concentrations of NADPH greater than 150 microM caused substrate inhibition at low HMG-CoA concentrations resulting in deviations from linearity in secondary plots. Analysis of these data and the product inhibition pattern suggest a sequential mechanism for the reduction of HMG-CoA to mevalonic acid with HMG-CoA being the first substrate binding to the enzyme, followed by NADPH.

Animals↗
PubMedDate not supplied

[Interrelation between thrombin structure and its stability].

Temperature inactivation of human thrombin has been studied when finding out the mechanism of this enzyme stabilization by amino acids. Effect of a number of amino acids on thrombin in the conditions (pH) of the highest activity of proteinase has been investigated. It is established that most amino acids are characterized to more or less extent by the protective action, when hampering the temperature inactivation of the enzyme. The correspondence was mainly found between the stabilizing effect of amino acids and thrombin specificity. Thrombin is stabilized by L-arginine and DL-lysine more intensively than by other amino acids. A stabilizing effect of L-glutamic acid was shown in contrast to the action of the latter on trypsin that was obviously connected with the original structure of the active centre of thrombin, that is the availability of anionic binding centre which includes Lys68, Arg72, Arg77. High thrombin stabilization by such amino acids as phenylalanine, DL-serine, DL-methonine was an exception. It was established that amino acids stabilize thrombin with formation of a compound with the reactive centre of its molecule, like the compounds enzyme-substrate. The macrostructure stability probably depends, to a considerable extent, on the state of the enzyme reactive centre: thrombin molecules, which contain a free reactive centre, are more labile than those which reactive centre is bound to the reagent of more or less specific character. The inhibition of the autolysis process may be another manifestation of thrombin stabilization by amino acids.

Amino Acids↗
PubMed1999

Role of the C-terminal helix 9 in the stability and ligandin function of class alpha glutathione transferase A1-1.

Helix 9 at the C-terminus of class alpha glutathione transferase (GST) polypeptides is a unique structural feature in the GST superfamily. It plays an important structural role in the catalytic cycle. Its contribution toward protein stability/folding as well as the binding of nonsubstrate ligands was investigated by protein engineering, conformational stability, enzyme activity, and ligand-binding methods. The helix9 sequence displays an unfavorable propensity toward helix formation, but tertiary interactions between the amphipathic helix and the GST seem to contribute sufficient stability to populate the helix on the surface of the protein. The helix's stability is enhanced further by the binding of ligands at the active site. The order of ligand-induced stabilization increases from H-site occupation, to G-site occupation, to the simultaneous occupation of H- and G-sites. Ligand-induced stabilization of helix9 reduces solvent accessible hydrophobic surface by facilitating firmer packing at the hydrophobic interface between helix and GST. This stabilized form exhibits enhanced affinity for the binding of nonsubstrate ligands to ligandin sites (i.e., noncatalytic binding sites). Although helix9 contributes very little toward the global stability of hGSTA1-1, its conformational dynamics have significant implications for the protein's equilibrium unfolding/refolding pathway and unfolding kinetics. Considering the high concentration of reduced glutathione in human cells (about 10 mM), the physiological form of hGSTA1-1 is most likely the thiol-complexed protein with a stabilized helix9. The C-terminus region (including helix9) of the class alpha polypeptide appears not to have been optimized for stability but rather for catalytic and ligandin function.

Acrylamide↗
PubMed2001

N-terminal portion acts as an initiator of the inactivation of pepsin at neutral pH.

Porcine pepsin, an aspartic protease, is unstable at neutral pHs where it rapidly loses activity, however, its zymogen, pepsinogen, is stable at neutral pHs. The difference between the two is the presence of the prosegment in pepsinogen. In this study, possible factors responsible for instability were investigated and included: (i) the distribution of positively charged residues on the surface, (ii) an insertion of a peptide in the C-terminal domain and (iii) the dissociation of the N-terminal fragment of pepsin. Mutations to change the number and the distribution of positive charges on the surface had a minor effect on stability. No effect on stability was observed for the deletion of a peptide from the C-terminal domain. However, mutations on the N-terminal fragment had a major impact on stability. At pH 7.0, the N-fragment mutant was inactivated 5.8 times slower than the wild-type. The introduction of a disulfide bond between the N-terminal fragment and the enzyme body prevented the enzyme from denaturing. The above results showed that the inactivation of pepsin was initiated by the dissociation of the N-fragment and that the sequence of this portion was a major determinant for enzyme stability. Through this study, we have created porcine pepsin with increased pH stability at neutral pHs.

Amino Acid Sequence↗
PubMed2002

Extremely high stability of African oil palm tree peroxidase.

A detailed kinetic study on thermal inactivation of African oil palm tree peroxidase (AOPTP) at different pHs has been carried out. The enzyme does not undergo inactivation over a broad range from pH 2 to 12 at ambient temperature. Complete inactivation of AOPTP is observed only at 70 degrees C and extremal pHs like <3.0 and >12.0, whereas under neutral conditions, its activity shows no changes. The study of AOPTP inactivation kinetics in the presence of dithiothreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) showed that calcium ions, disulfide bonds and the interaction between apo-AOPTP and heme are important structural elements responsible for the enzyme stability. The guanidium hydrochloride (GdHCl)-induced inactivation of AOPTP indicated that the hydrogen-bonding network plays also a significant role in stabilizing the active structure of the enzyme. AOPTP is stable toward hydrogen peroxide treatment, especially under neutral conditions. The comparison of AOPTP stability to that of other peroxidases shows that AOPTP is the most stable peroxidase reported so far.

Dithiothreitol↗
PubMedDate not supplied

[Choice of the initial enzyme correlation in immobilizing polyenzyme systems].

An optimal, in respect to productivity (activity X stability), enzyme ratio for immobilization of multienzyme systems was calculated by using the kinetic parameters (KM and Vmax), data on the stability and yield of each enzyme during immobilization. The experimental data, obtained during combined immobilization of invertase, mutarotase and glucose oxidase, illustrate the theoretical propositions.

Animals↗
PubMedDate not supplied

Mechanism for high stability of liposomal glucose oxidase to inhibitor hydrogen peroxide produced in prolonged glucose oxidation.

Glucose oxidase (GO) was encapsulated in the liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) to increase the enzyme stability through its decreased inhibition because of hydrogen peroxide (H(2)O(2)) produced in the glucose oxidation. The GO-containing liposomes (GOLs) were completely free of the inhibition even in the complete conversion of 10 mM glucose at 25 degrees C because the H(2)O(2) concentration was kept negligibly low both outside and inside liposomes throughout the reaction. It was interestingly revealed that the H(2)O(2) produced was decomposed not only by a slight amount of catalase originally contained in the commercially available GO but also by the lipid membranes of GOL. As compared to the GOL-catalyzed reaction, the free GO-catalyzed reaction more highly accumulated H(2)O(2) because of the more rapid glucose conversion despite containing free catalase, leading to the completely inhibited GO before reaching a sufficient glucose conversion. This suggested that only the liposomal catalase could continue to catalyze the H(2)O(2) decomposition. The effect of the glucose oxidation rate, i.e., the H(2)O(2) production rate on the liposomal GO inhibition, was also examined employing the various GOLs with different permeabilities to glucose present in their external phase. It was concluded that the liposomal GO free of the inhibition could be obtained when the GOL-catalyzed H(2)O(2) formation rate was limited by such a suitable lipid bilayer as POPC membrane so that the rate was well-balanced with the sum of the above two H(2)O(2) decomposition rates. The highly stable GOL obtained in the present paper was shown to be a useful biocatalyst for the prolonged glucose oxidation.

Enzyme Stability↗
PubMed2004

Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability.

The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5-25 degrees C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t(1/2) value of 16 min for thermal inactivation during incubation at 40 degrees C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly(244) was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly(244)-->Pro substitution into the enzyme. Stability studies showed that the t(1/2) value for thermal inactivation of the mutant during incubation at 40 degrees C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k(cat)/K(m) value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.

Amino Acid Sequence↗
PubMed1994

Proline-protein interactions: protection of structural and functional integrity of M4 lactate dehydrogenase.

A well defined labile isozyme, rabbit muscle M4-lactate dehydrogenase was denatured under freeze-thaw, heat and GuHCl treatment in the presence and absence of proline, and the corresponding structural changes of the enzyme were monitored through fluorescence and CD spectral studies. The data reveal that proline confers protection to the structural integrity of the enzyme, thereby protecting its activity. This was attributed to its property of forming hydrophilic colloids in aqueous media with a hydrophobic backbone interacting with protein. Unlike other osmolytes, proline is proposed to act on the enzyme stability not only by inducing preferential hydration of proteins but also through the interactions of its multimeric hydrophobic backbone with the solvent-accessible hydrophobic regions of the enzyme.

Animals↗
PubMed1989

Comparison of the thermolability and hydrophobic properties of high- and low-molecular-weight forms of rabbit liver arginyl-tRNA synthetase.

Two preparations with arginyl-tRNA synthetase activity have been obtained from rabbit liver post-microsomal fraction: a) a high-molecular-weight containing the multienzyme aminoacyl-tRNA synthetase complex and b) a low-molecular-weight preparation containing free enzymes. Thermal inactivation of arginyl-tRNA synthetase in both preparations has been compared in a solution which was successively supplemented with tRNA, reduced glutathione, L-ascorbic acid, ZnCl2 and Triton X 100. Moreover, hydrophobic properties of both enzyme preparations have been compared. It was found that the complexed arginyl-tRNA synthetase is more stable than the free enzyme. A role of hydrophobic interactions in the maintenance of the complexed enzyme stability is suggested.

Amino Acyl-tRNA Synthetases↗
PubMed1999

Bioenergetic and structural consequences of allotopic expression of subunit 8 of yeast mitochondrial ATP synthase. The hydrophobic character of residues 23 and 24 is essential for maximal activity and structural stability of the enzyme complex.

Subunit 8 (Y8), a mitochondrially encoded subunit of the F0 sector of the F1F0-ATP synthase is essential for oxidative phosphorylation. We have previously introduced the technique of allotopic expression to study the structure/function of Y8, whereby an artificial Y8 gene is expressed in the nucleus of cells lacking a functional mitochondrial Y8, thus generating assembly of a functional F1F0-ATPase complex. In this paper we show that when a gene encoding an essentially unmodified version of Y8 is allotopically expressed, ATP synthesis and hydrolysis rates, as well as efficiency of oxidative phosphorylation, were similar to those of the parental wild-type strain in which Y8 is naturally expressed in mitochondria. We then tested the requirement for the hydrophobicity of the central domain (residues 14-32), which possibly represents a transmembrane stem, by introducing adjacent negative charges at different positions of Y8. One of the variants thus generated, which carries the double substitution Leu23-->Asp, Leu24-->Asp, when expressed in a strain lacking endogenous Y8, gave rise to cells which grew very slowly by oxidative phosphorylation. Measurement of bioenergetic parameters showed two major defects in these cells relative to control cells allotopically expressing unmodified Y8. First, the activity of the F1F0-ATP synthase was significantly decreased. ATP synthesis and state 3 of respiration were reduced by approximately 30-40%. ATP hydrolysis was reduced by approximately 30% and was almost insensitive to the F0 inhibitor oligomycin. Second, the physical coupling between the two sectors of the enzyme, as well as the stability of the F1 sector itself, were affected as shown by decreased recovery of F0 sector [8, 9, b, oligomycin sensitivity-conferring protein (OSCP), d, h and f] and F1 sector (alpha, gamma, delta) subunits in immunoprecipitates of ATP synthase. This study indicates that Y8 not only performs an important role in the structure of the mitochondrial complex but also in its activity. We conclude that the hydrophobic character of amino acids 23 and 24 in the middle of the putative transmembrane stem of Y8 is essential for coupling proton transport through F0 to ATP synthesis on F1.

Adenosine Triphosphate↗
PubMed2006

Purification and characterization of a nitrilase from Aspergillus niger K10.

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg(-1)) at 45 degrees C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of D: -sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.

Amino Acid Sequence↗
PubMed2005

ATP-independent activation of natriuretic peptide receptors.

Natriuretic peptide receptor A (NPR-A) is an essential cardiovascular regulator that is stimulated by atrial natriuretic peptide and B-type natriuretic peptide, whereas natriuretic peptide receptor B (NPR-B) stimulates long bone growth in a C-type natriuretic peptide-dependent manner. Many reports indicate that ATP is essential for NPR-A and NPR-B activation. Current models suggest that natriuretic peptide binding to receptor extracellular domains causes ATP binding to intracellular kinase homology domains, which derepresses adjacent catalytic domains. Here, we report 100-fold activations of natriuretic peptide receptors in the absence of ATP. The addition of a nonhydrolyzable ATP analog had no effect at early time periods (measured in seconds) but increased cGMP production about 2-fold after longer incubations (measured in minutes), consistent with a stabilization, not activation, mechanism. These data indicate that ATP does not activate natriuretic peptide receptors as has been repeatedly reported. Instead, ATP increases activity primarily by maintaining proper receptor phosphorylation status but also serves a previously unappreciated enzyme stabilizing function.

Adenosine Triphosphate↗
PubMed1986

NAD+-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1. Stabilization against temperature and urea induced inactivation.

Chemical modification of the NAD+-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1 results in considerable enzyme stabilization towards urea and temperature induced inactivation. The stabilizing effect was shown to originate from the suppression of hydrogenase tetramer dissociation. The magnitudes of the stabilizing effects (5-fold and more) were in agreement with the values predicted on the basis of the enzyme thermoinactivation mechanism postulated earlier. Hydrophobic interactions are considered to be critical for the stability of the enzyme quaternary structure. Various methods of hydrogenase immobilization were tested. The enzyme was immobilized with a high retention of activity on aminated silochrom via its carboxylic groups.

Acrolein↗
PubMed1990

Dependence on Ca2+ of lipoprotein lipase stability.

Macrophages continuously secrete lipoprotein lipase (LPL) into the culture medium. When LPL was collected from thioglycollate-elicited peritoneal macrophages (Tg-Mø) or J774.1 cells over a 4 h period in Ca2+ and Mg2(+)-free Dulbecco's modified Eagle's medium (d-DMEM) the activity in the collection medium was reduced by 40-62% and 23%, respectively, as compared to that expressed in full medium (DMEM). Ca2+ supplementation during the collection period in d-DMEM augmented LPL activity in the medium; about 1 mM Ca2+ was required for attainment of activity comparable to that expressed in DMEM. Addition of Ca2+ during the assay did not enhance LPL activity collected into d-DMEM. Addition of EGTA to the assay mixture reduced LPL activity by 34-60% and when present in the collection medium, EGTA led to a reduction in enzyme activity greater than 90%. A 4 h incubation of Tg-Mø in 3 mM EGTA led to an almost complete loss of intracellular Ca2+ (measured by efflux of 45Ca2+ from preloaded cells), yet there was no change in the overall synthesis and secretion of proteins and in the phagocytic capability of the cells. LPL activity in the enzyme collection medium after its removal from cell monolayers was stable at least up to 4 h at 0 degrees C and at 23 degrees C. Activity was progressively lost with increased temperatures: up to 40% loss at 37 degrees C in 4 h. Addition of EGTA to the above medium led to an enhanced rate of irreversible enzyme inactivation: 76-86% loss of activity in 4 h at 37 degrees C. No inactivation was observed at 0 degrees C and at 23 degrees C in the presence of EGTA. The results indicate a critical role for Ca2+ in enzyme stabilization.

Animals↗
PubMedDate not supplied

Stabilization of the restriction enzyme EcoRI dried with trehalose and other selected glass-forming solutes.

The stabilization of the restriction enzyme EcoRI by its incorporation into aqueous glass-forming carbohydrate or polymer solutions, followed by vacuum-drying to low moisture, has been studied. Glass-forming solutes included trehalose, sucrose, lactose, maltose, raffinose, maltodextrin DE 10, and poly(vinylpyrrolidone) (molecular weight 40,000, PVP). Among the solutes examined, trehalose and sucrose protected the enzyme most effectively during storage at 37 and 45 degrees C. The restriction enzyme dried with trehalose or sucrose maintained its activity without detectable loss for at least 20 days at 37 degrees C and 12 days at 45 degrees C. In contrast, the activity of the enzyme dried with maltodextrin or PVP was reduced during vacuum desiccation and also it decreased remarkably during storage at the same temperatures. Stored (37/45 degrees C) vacuum-dried trehalose and sucrose systems were either a dense paste or a very viscous syrup, and this indicated that they were not glassy. Moreover, no relationship was found between the glass transition temperatures (Tg) of the pure added solute and enzyme protection during storage, since, e.g., sucrose which has significantly lower Tg values protected the enzyme much better than either maltose, lactose, maltodextrin, or PVP. The trisaccharide raffinose offered good protection of enzyme activity, and its role as a novel excipient matrix for labile enzyme stabilization deserves further investigation. The stability of enzyme EcoRI was rapidly lost when the vacuum-dried trehalose and sucrose systems were humidified to 58% relative humidity and stored at 45 degrees C, and this was attributed to disaccharide crystallization.

Deoxyribonuclease EcoRI↗
PubMed2005

An improved purification procedure for cyclosporin synthetase.

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation-->gel filtration-->hydrophobic interaction chromatography-->anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24-29 degrees C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

Chromatography↗