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R Schinzel

Publications and source records attributed to R Schinzel.

At least 55 records · Page 3Linked to original sources
PubMed1998

Growth dependence of alpha-glucan phosphorylase activity in Thermus thermophilus.

Glycogen from the thermophilic eubacterium Thermus thermophilus has been characterized by enzymatic, chemical and spectroscopic analysis. With an average chain length of seven glucose units, the glycogen from T. thermophilus is one of the most highly branched glycogens known. In contrast to other bacterial species, in T. thermophilus, accumulation of glycogen appears not be affected by low nitrogen concentration. For the first time, alpha-glucan phosphorylase activity and glycogen content were measured throughout the growth cycle of T. thermophilus in order to gain insight into glycogen metabolism. In contrast to the situation that prevails in Escherichia coli, additional carbon sources had no effect on alpha-glucan phosphorylase activity in T. thermophilus. Maximal activity of the thermophilic enzyme was found in the early logarithmic phase of growth, suggesting a function of the alpha-glucan phosphorylase in T. thermophilus as an outgrowth-specific enzyme.

Animals↗
PubMed1998

Advanced glycated albumin impairs protein degradation in the kidney proximal tubules cell line LLC-PK1.

Advanced glycation end-products (AGEs) are assumed to play a major role in the genesis of diabetic nephropathy and other diabetic complications. We studied the potential effect of AGEs on protein turnover and lysosomal proteinase activities in LLC-PK1 cells, a pig kidney proximal tubules cell line. Advanced glycated bovine serum albumin (AGE-BSA) was used as a model of AGEs and its action was compared to that of nonglycated BSA. AGE-BSA but not BSA (50 micromol/l) induced a significant increase in cell volume (BSA: 4870.6 +/- 74.8 fl, AGE-BSA: 5718.0 +/- 20.7 fl, p<0.01). Protein degradation rate was decreased by 13.8% after 48 hrs. incubation with AGE-BSA (p<0.01) while protein synthesis increased by 19,1%, (p<0.01). After incubation with AGE-BSA but not BSA activities of lysosomal cathepsins (B, L+B and H) decreased in a time- and dose-dependent fashion. This decline was neither caused by a shift in lysosomal pH outside the optimal range for cathepsins, nor by a direct inhibitory effect of AGEs modified proteins or peptides but most probably by inhibition of cathepsin B expression as measured by RT-PCR. It is supposed that impaired protease activities participated in decreased protein breakdown and cell enlargement. For the first time our data provide the evidence that AGEs induce hypertrophy of LLC-PK1 cells due to decreased protein breakdown resulting from reduced lysosomal proteinase activities with a concomitant stimulation of protein synthesis.

Animals↗
PubMed1997

Efficient downstream processing of maltodextrin phosphorylase from Escherichia coli and stabilization of the enzyme by immobilization onto hydroxyapatite.

Downstream processing by biospecific chromatography of maltodextrin phosphorylase from Escherichia coli, overexpressed in E. coli, was substantially improved by a novel approach using ceramic hydroxyapatite. Wild-type and a less active mutant enzyme were purified from crude bacterial cell extracts in one efficient separation step that yielded phosphorylase in purity > 95% in at least 90% recoveries. At pH 6.9 and 25 degrees C, wild-type and mutant phosphorylases eluted from the hydroxyapatite column at a phosphate concentration of 0.4 M whereas calcium ions failed to displace the enzymes. The dynamic capacity for phosphorylase binding in the presence of bulk proteins was approximately 3 mg enzyme ml-1 matrix. The interaction of E. coli phosphorylase with hydroxyapatite seems to be mediated by surface amino groups, so that the bound enzyme retained almost full catalytic activity. Compared to the soluble enzyme, immobilization onto hydroxyapatite resulted in a more than 30-fold stabilization of wild-type phosphorylase against thermal and proteolytic inactivation and thus could improve the operational stability of phosphorylase during conversion of polysaccharide to glucose 1-phosphate.

Chromatography↗
PubMed1997

Advanced glycation endproducts stimulate the MAP-kinase pathway in tubulus cell line LLC-PK1.

Advanced glycation endproducts (AGEs) are suggested to play an important role in diabetic nephropathy. They induce specific cellular responses such as the release of cytokines in different cell lines. The effect of AGEs on signal transduction pathways was investigated in the renal tubulus cell line LLC-PK1. Using a serine-phosphate-specific antibody AGE-induced cellular responses associated with phosphorylation/dephosphorylation events were demonstrated. In particular, the p42MAP kinase and its downstream target, the AP-1 complex, are shown to be activated by AGE-BSA but not by BSA. In contrast, only partial phosphorylation is observed for the p70S6-kinase. Thus, AGEs appear to induce specific signal transduction pathways.

Animals↗
PubMed1997

Influence of advanced glycation end-products and AGE-inhibitors on nucleation-dependent polymerization of beta-amyloid peptide.

Nucleation-dependent polymerization of beta-amyloid peptide, the major component of plaques in patients with Alzheimer's disease, is significantly accelerated by crosslinking through Advanced Glycation End-products (AGEs) in vitro. During the polymerization process, both nucleus formation and aggregate growth are accelerated by AGE-mediated crosslinking. Formation of the AGE-crosslinked amyloid peptide aggregates could be attenuated by the AGE-inhibitors Tenilsetam, aminoguanidine and carnosine. These experimental data, and clinical studies, reporting a marked improvement in cognition and memory in Alzheimer's disease patients after Tenilsetam treatment, suggest that AGEs might play an important role in the etiology or progression of the disease. Thus AGE-inhibitors may generally become a promising drug class for the treatment of Alzheimer's disease.

Alzheimer Disease↗
PubMed1997

The crystal structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase.

In animals, glycogen phosphorylase (GP) exists in an inactive (T state) and an active (R state) equilibrium that can be altered by allosteric effectors or covalent modification. In Escherichia coli, the activity of maltodextrin phosphorylase (MalP) is controlled by induction at the level of gene expression, and the enzyme exhibits no regulatory properties. We report the crystal structure of E. coli maltodextrin phosphorylase refined to 2.4 A resolution. The molecule consists of a dimer with 796 amino acids per monomer, with 46% sequence identity to the mammalian enzyme. The overall structure of MalP shows a similar fold to GP and the catalytic sites are highly conserved. However, the relative orientation of the two subunits in E. coli MalP is different from both the T and R state GP structures, and there are significant changes at the subunit-subunit interfaces. The sequence changes result in loss of each of the control sites present in rabbit muscle GP. As a result of the changes at the subunit interface, the 280s loop, which in T state GP acts as a gate to control access to the catalytic site, is held in an open conformation in MalP. The open access to the conserved catalytic site provides an explanation for the activity without control in this basic archetype of a phosphorylase.

Allosteric Regulation↗
PubMed1997

Advanced glycation endproducts in ageing and Alzheimer's disease.

Accumulation of advanced glycation endproducts (AGE) in the brain is a feature of ageing and degeneration, especially in Alzheimer's disease (AD). Increased AGE levels explain many of the neuropathological and biochemical features of AD such as extensive protein crosslinking (beta-amyloid and MAP-tau), oxidative stress and neuronal cell death. Oxidative stress and AGEs initiate a positive feedback loop, where normal age-related changes develop into a pathophysiological cascade. Combined intervention using antioxidants, metal chelators, anti-inflammatory drugs and AGE-inhibitors may be a promising neuroprotective strategy.

Aging↗
PubMed1997

Oligosaccharide substrate binding in Escherichia coli maltodextrin phosphorylase.

The crystal structure of E. coli maltodextrin phosphorylase co-crystallized with an oligosaccharide has been solved at 3.0 A resolution, providing the first structure of an oligosaccharide bound at the catalytic site of an alpha-glucan phosphorylase. An induced fit mechanism brings together two domains across the catalytic site tunnel. A stacking interaction between the glucosyl residue and the aromatic group of a tyrosine residue at a sub-site remote (8 A) from the catalytic site provides a key element in substrate recognition; mutation of this residue to Ala decreases the Kcat/Km by 10(4). Extrapolation of the results to substrate binding across the site of attack by phosphorolysis indicates a likely alteration in the glycosidic torsion angles from their preferred values, an alteration that appears to be important for the catalytic mechanism.

Crystallization↗
PubMed1997

Determination of advanced glycation end products in serum by fluorescence spectroscopy and competitive ELISA.

Recent studies suggest that advanced glycation endproducts play an important role in cardiovascular complications of ageing, diabetes and end-stage renal failure. Since highly elevated levels of advanced glycation endproducts are present in serum of patients on maintenance haemodialysis, an accurate and rapid assay for their determination would be useful. This would be particularly valuable for monitoring the removal of advanced glycation endproducts by novel dialysis membranes, as well as the effect of new drugs for the inhibition of their formation. Measurement of advanced glycation endproducts in serum was performed by two competitive ELISAs, using a monoclonal antibody directed against imidazolone, an advanced glycation endproduct formed by the reaction of arginine with 3-deoxyglucosone, and a polyclonal antibody directed against keyhole limpet haemocyanin-advanced glycation endproduct, as well as by quantitative fluorescence spectroscopy. Each of the assays showed significant differences between the controls and the maintenance haemodialysis patients. Advanced glycation endproduct levels determined by each of the ELISAs correlated with total and protein-bound fluorescence, but not with each other, suggesting a variable distribution of advanced glycation endproducts on serum proteins among the maintenance haemodialysis patients.

Aged↗
PubMed1996

Purification and characterisation of an alpha-glucan phosphorylase from the thermophilic bacterium Thermus thermophilus.

An alpha-glucan phosphorylase has been purified 4500-fold from the thermophilic bacteria Thermus thermophilus. In contrast to other bacterial phosphorylases the thermophilic enzyme seems neither to be inducible by maltose nor repressed by glucose. T. thermophilus phosphorylase shares major properties with known mesophilic phosphorylases such as pyridoxal 5'-phosphate content (1 M pyridoxal-P/M subunit), subunit molecular mass (about 90 kDa) and inhibitor constants. The optimum temperature of T. thermophilus phosphorylase was observed at 70 degrees C in the pH range 5.5-6.5. While at 25 degrees C the subunit composition of the thermophilic enzyme is an octameric form, the preferential form at the optimum temperature of 70 degrees C seems to be a dimer. Most remarkably, in the direction of synthesis and degradation the limiting size of the oligosaccharide substrate is shorter by one glucose residue than the minimum size of substrate degraded by other alpha-glucan phosphorylases. Maltotetraose and glycogen are degraded with rates similar to that observed with maltoheptaose (Vmax = 18 U/mg). Correspondingly, maltotriose functions as primer in the synthesis direction. Differences in fluorescence and absorption spectra of the cofactor and the failure of arsenate acting as a substrate indicate that the active site structure of T. thermophilus phosphorylase differs from that of known alpha-glucan phosphorylases.

Chromatography, Gel↗
PubMed1996

Mutational analysis of the oligosaccharide recognition site at the active site of Escherichia coli maltodextrin phosphorylase.

A mutagenesis approach was applied to identify specific amino acid residues that are tentatively involved in binding of the oligosaccharide substrate at the active site of Escherichia coli maltodextrin phosphorylase. From ten residues located within a proposed channel connecting the enzyme surface with the active site, nine displayed significant effects on the reaction with oligosaccharide substrates when exchanged by mutagenesis. While several mutant enzymes (N258A/D259A/N260A, N307A, E350A, and Y578F) exhibited moderate decreases in apparent binding (about 4-17-fold), two mutations, H536L and E67A, weakened apparent binding of oligosaccharide substrates by 2 orders of magnitude. Two further mutant enzymes (T346G and H310A) displayed a 10-fold increase in the apparent Km of the oligosaccharide in the degradation reaction, while binding in the synthesis direction seemed less affected, indicating partially differential binding modes of oligosaccharides in synthesis and degradation. Quite uniquely, the H310A mutant enzyme exhibits a more than 100-fold-lowered Ki for gluconolactone, indicating the existence of an inhibitor binding site similar to that expected for a carbonium ion-like transition state. This is further confirmed by the finding that glucose, which does not inhibit wild-type enzyme, became an inhibitor of the H310A mutant enzyme (Ki = 20 mM). Since mutation of D308 did reduce kcat about 10-100-fold while Km values remained unchanged, a participation of this residue in transition state binding is probable. The insight into substrate recognition derived from this mutagenesis study corroborates a binding model where maltopentaose fits into the phosphorylase b structure in a distorted form.

Amino Acid Sequence↗
PubMed1996

[New hypothesis on etiopathogenesis of Alzheimer syndrome. Advanced glycation end products (AGEs)].

Despite intense efforts, it has not yet been possible to clarify the etiopathogenesis of Alzheimer's dementia. There are, however, hypotheses which focus on certain aspects of this type of dementia, characterized by particular neuropathological alterations and clinical correlates. Recently, evidence has accumulated that advanced glycation endproducts (AGEs) could play an important role in the etiology of the Alzheimer's syndrome. AGEs are generated by an irreversible reaction through the non-enzymatic, long-term glycosylation of proteins. They are strongly resistant to proteolytic processes and induce protein crosslinking. They could thus inhibit the physiological functions of many proteins. Moreover, it is suggested that they contribute to the transformation of the soluble form of beta-amyloid into its unsoluble version. AGEs are also demonstrable in neurofibrillary tangles (NFTs). A further mechanism by which AGEs might be pathogenic is via their induction of oxidative stress. AGEs probably exert their pathological effects not only directly because of their chemical properties, but also by indirect receptor-mediated mechanisms. Further investigation of AGE-mediated mechanisms should reveal their role in the etiopathogenesis of the Alzheimer's syndrome and, finally, lead to the development of new pharmacological strategies aimed at inhibiting protein cross-linking.

Aged↗
PubMed1996

Advanced glycation endproducts-associated parameters in the peripheral blood of patients with Alzheimer's disease.

Advanced glycation endproducts (AGEs), structural components of beta-amyloid plaques and neurofibrillary tangels, have been implicated in the pathogenesis of Alzheimer's disease. AGE levels, measured by fluorescence, and their precursor molecules such as glucose and its Amadori product, fructosylamine, were measured to examine the question whether the reported increased level of AGEs in the brain is reflected in an increase in AGE-associated parameters in peripheral blood. Lactoferrin, proposed to play an important role in the interaction of AGEs with their receptors, was determined by ELISA. All AGE-associated parameters showed trends to lower values in patients with Alzheimer's disease compared with non-demented controls. Albumin and total iron were not significantly different between the groups. In contrast to diabetes and renal failure, where high levels of AGEs and their precursors are present in tissue as well as in peripheral blood, elevated CNS AGE levels in patients with Alzheimer's disease are manifested without detectable peripheral changes.

Aged↗
PubMed1996

Activation of E350A mutant maltodextrin phosphorylase by exogenously added acetate.

Site-directed mutagenesis of E350 to alanine in Escherichia coli maltodextrin phosphorylase reduced both enzyme activity (100-fold) and apparent binding of the oligosaccharide substrate (10-fold), suggesting a participation of this residue in binding of the substrate in the ground and transition states. The E350A mutant enzyme was found to be activated up to 20-fold by exogenous acetate ions which substitute for the deleted side chain. In contrast, apparent binding was not affected by acetate ions, indicating a dual role for the carboxylic group of this residue in catalysis and binding. Formate also appears to activate the E350A mutant enzyme, but this effect is obscured by the strong inhibitory effect of formate on the wild-type enzyme. For propionate ions, a weak 2-fold activation was noticed, while other compounds like trifluoroacetate and acetamide had no effects on the catalytic properties of either the E350A mutant enzyme or wild-type enzyme. If E350 was substituted by a glutamine, no activation was observed upon the addition of acetate ions. However, a weak activation by formate was found, confirming that activation by acetate is caused by specific binding at the mutated site.

Acetic Acid↗
PubMed1995

Photometric microtiter assay of inorganic phosphate in the presence of acid-labile organic phosphates.

A simple and rapid colorimetric microassay for inorganic phosphate in the mild acid pH range has enabled us to perform extensive enzyme kinetic studies even in the presence of high concentrations of acid-labile substrates. The assay is performed in a 96-well microtiter plate using 30-microliters samples containing between 0.5 and 100 nmol Pi. Compared to existing microassays, color formation is linear over a much wider range of phosphate concentrations and reaction conditions. Using a computer-assisted microtiter plate reader, the data can be directly transferred to kinetic analysis software. The above assay was used to determine simultaneously up to 12 rates of the Escherichia coli maltodextrin phosphorylase-catalyzed reaction in the direction of glycogen synthesis under conditions which include screening of substrates and inhibitors at variable substrate concentrations and pH on a single 96-well microtiter plate.

Acids↗
PubMed1995

A study of binary complexes of Escherichia coli maltodextrin phosphorylase: alpha-D-glucose 1-methylenephosphonate as a probe of pyridoxal 5'-phosphate-substrate interactions.

The glucose 1-phosphate (Glc-1-P) analog alpha-D-glucose 1-methylenephosphonate (Glc-1-MeP) inhibits competitively Escherichia coli maltodextrin phosphorylases against Glc-1-P (Ki = 0.20 mM) but also Pi (Ki = 0.36 mM). Exchange of the active site residue Glu637 to Asp by site-directed mutagenesis abolishes inhibition only in the synthesis direction (S-mode), while the degradative direction (P-mode) was not affected. Structural and conformational differences of the S-mode versus P-mode were also revealed by 31P-NMR spectroscopy by comparing chemical shifts of the cofactor pyridoxal-P in binary complexes formed either in the presence of Glc-1-MeP or of arsenate. In contrast the apparent pK of pyridoxal-P in both binary complexes was closely similar. Again, the total chemical shift of pyridoxal-P in the synthesis mode respectively degradative mode was differently affected in the binary complexes of the Glu637Asp mutant enzyme. This supports the contention that differential binding of the substrates in the synthesis or the degradative mode changes the arrangement and mutual interactions of cofactor phosphate and substrate phosphates.

Aspartic Acid↗