Biomedical subjects

Judit Ovádi

Publications and source records attributed to Judit Ovádi.

6 recordsLinked to original sources

PubMed2003

Glucose conversion by multiple pathways in brain extract: theoretical and experimental analysis.

Experimental and model studies were performed to characterize the flux of glucose metabolism and the sharing of glucose-6-phosphate (Glu6P) by the upper parts of glycolytic and pentosephosphate pathways in the brain extract. A mathematical model based upon the kinetic equations of the individual enzymes was evaluated to fit the experimental data. Glucose is converted to glucose-6-phosphate by hexokinase that controls almost exclusively the glucose metabolism. Experiments showed that this crossroad-metabolite was shared between glycolysis and pentosephosphate pathway in the brain extract in a ratio of 1.5:1. This ratio was favorable to the pentosephosphate pathway by the addition of high excess of exogenous glucose-6-phosphate dehydrogenase, standardly used for the activity assay of hexokinase, but still a significant part (17+/-3%) of the common intermediate was converted into the direction of glycolysis. Stimulation of glucose-6-phosphate formation via moderate (30-50%) increase of hexokinase activity by adding exogenous hexokinase or tubulin resulted in the slight increase of the relative flux into direction of glycolysis. The model correctly described all of these observations. However, when the activity of hexokinase was doubled with exogenous enzyme, significantly less glucose-6-phosphate was converted into direction of glycolysis than predicted. This discrepancy shows that the system did not behave in this case as an ideal one, which could be due to the formation of distinct pools for the intermediate.

Animals↗

PubMed2002

A simple method for the determination of dissociation constants by displacement ELISA.

Previously, we have described a linearization procedure for the determination of dissociation constants of antigen-antibody interactions using data from enzyme-linked immunosorbent assays (ELISA) [J. Immunol. Methods 143 (1991) 119]. Here, we present a new linearization procedure-based partly on the former one-to evaluate quantitatively the data of displacement ELISAs. It renders possible the determination of dissociation constants of complexes formed between immobilised antigens and the displacing molecules without knowing the explicit concentration of the antigen and the dissociation constant and stoichiometry of the immunocomplex. In addition, determination of the concentration of specific antibody in the serum is not necessary since the equations use only the ratio of serum concentration and apparent dissociation constant of the immunocomplex (both of which can be expressed as serum dilutions). The method has been applied to determine the strength of immobilised hexokinase-tubulin interactions (K(d)=1.2 microM) from a displacement ELISA using antihexokinase serum. The applicability and limitations of the procedure are discussed on the basis of both the experimental and theoretical data.

Antibodies↗

PubMed2002

Brain-specific p25 protein binds to tubulin and microtubules and induces aberrant microtubule assemblies at substoichiometric concentrations.

Previously, we have demonstrated the presence of a protein factor [tubulin polymerization perturbing protein (TPPP)] in brain and neuroblastoma cell but not in muscle extract that uniquely influences the microtubule assembly. Here we describe a procedure for isolation of this protein from the cytosolic fraction of bovine brain and present evidence that this protein is a target of both tubulin and microtubules in vitro. The crucial step of the purification is the cationic exchange chromatography; the bound TPPP is eluted at high salt concentrations, indicating the basic character of the protein. By IDA-nanoLC-MS analysis of the peptides extracted from the gel-digested purified TPPP, we show the presence of a single protein in the purified fraction that corresponds to p25, a brain-specific protein the function of which has not been identified. Circular dichroism data have revealed that, on one hand, the alpha-helix content of p25 is very low (4%) with respect to the predicted values (30-43%), and its binding to tubulin induces remarkable alteration in the secondary structure of the protein(s). As shown by turbidimetry, pelleting experiments, and electron microscopy, p25 binds to paclitaxel-stabilized microtubules and bundles them. p25 induces formation of unusual (mainly double-walled) microtubules from tubulin in the absence of paclitaxel. The amount of aberrant tubules formed depends on the p25 concentration, and the process occurs at substoichiometric concentrations. Our in vitro data suggest that p25 could act as a unique MAP in vivo.

Animals↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMedDate not supplied

On the origin of intracellular compartmentation and organized metabolic systems.

The history of the development of the ideas and research of organized metabolic systems during last three decades is shortly reviewed. The cell cytoplasm is crowded with solutes, soluble macromolecules such as enzymes, nucleic acids, structural proteins and membranes. The high protein density within the large compartments of the cells predominantly determines the major characteristics of cellular environment such as viscosity, diffusion and inhomogeneity. The fact that the solvent viscosity of cytoplasm is not substantially different from the water is explained by intracellular structural heterogeneity: the intrinsic macromolecular density is relatively low within the interstitial voids in the cell because many soluble enzymes are apparently integral parts of the insoluble cytomatrix and are not distributed homogeneously. The molecular crowding and sieving restrict the mobility of very large solutes, binding severely restrict the mobility of smaller solutes. One of consequence of molecular crowding and hindered diffusion is the need to compartmentalize metabolic pathway to overcome diffusive barriers. Although the movement of small molecules is slowed down in the cytoplasm, the metabolism can successfully proceed and even be facilitated by metabolite channeling which directly transfers the intermediate from one enzyme to an adjacent enzyme without the need of free aqueous-phase diffusion. The enhanced probability for intermediates to be transferred from one active site to the other by sequential enzymes requires stable or transient interactions of the relevant enzymes, which associate physically in non-dissociable, static multienzyme complexes--metabolones, particles containing enzymes of a part or whole metabolic systems. Therefore, within the living cell the metabolism depends on the structural organization of enzymes forming microcompartments. Since cells contain many compartments and microenvironments, the measurement of the concentration of metabolites in whole cells or tissues gives an average cellular concentration and not that which is actually sensed by the active site of a specific enzyme. Thus, the microcompartmentation could provide a mechanism which can control metabolic pathways. Independently and in parallel to the developments described above, the ideas of compartmentation came into existence from the necessity to explain important physiological phenomena, in particular in heart research and in cardiac electrophysiology. These phenomena demonstrated the physiological importance of the biophysical and biochemical mechanisms described in this review.

Cell Compartmentation↗

PubMedDate not supplied

Functional aspects of cellular microcompartmentation in the development of neurodegeneration: mutation induced aberrant protein-protein associations.

Research in the last 10 years has revealed that the development of neurodegeneration is a multistep process during which one or few specific mutant protein species of altered conformation initiate aberrant protein-protein interactions resulting in aggregates forming plaques. This review focuses on the heteroassociations of the mutant proteins with subcellular structures, such as cytoskeleton, cell membranes or with glycolytic enzymes, which may be crucial in the initiation of neurodegeneration such as in Huntington's disease or Alzheimer's disease. Triosephosphate isomerase enzymopathy is a unique glycolytic enzyme deficiency coupled with neurodegeneration. We present data on the mutation induced misfolding process, which likely plays a crucial role in the enhanced associations of the enzyme with the truncated fragment of the isomerase, with the red cell membrane or with the microtubular network. On the basis of our recent clinical and experimental results obtained with two compound heterozygote Hungarian brothers it became obvious that the mutations alone are not sufficient to explain the development of the neurological sympthomes. This underscores the fact that the mutations alone are not enough for the development of the clinical phenotype of a disease.

Animals↗