Search PubMed⌕ Search

Biomedical subjects

I Simon

Publications and source records attributed to I Simon.

At least 73 records · Page 4Linked to original sources
PubMed1995

Interaction of guanosine nucleotides and their analogs with elongation factor Tu from Thermus thermophilus.

Transient kinetic experiments on the interaction of nucleotide-free EF-Tu from Thermus thermophilus with nucleotides using intrinsic protein fluorescence, extrinsic nucleotide fluorescence and fluorescence resonance energy transfer show that nucleotide binding is in general at least a two-step process. The first step is a weak initial binding, which is followed by a relatively slow isomerization of the protein-nucleotide complex in which changes of both intrinsic and extrinsic fluorescence, as well as energy transfer, occur. The values obtained for the equilibrium and kinetic constants confirm the earlier observation that EF-Tu has a higher affinity for GDP than GTP. This is mainly due to a lower dissociation rate constant for GDP, in combination with a somewhat higher effective association rate constant. Modifications of the triphosphate moiety of GTP are quite well tolerated by EF-Tu, with GTP gamma S displaying the same affinity as GTP and with GppNHp and GppCH2p being only ca. 2-3-fold less strongly bound. Caged GTP is bound about 6-fold more weakly than GTP. These results suggest that the binding of GppNHp and GppCH2p is likely to be similar to that of GTP. The photolytic protecting group of caged GTP (or the loss of one of the negative charges on the gamma-phosphate group) appears to interfere to a certain extent with the interaction with the protein, but the affinity is high enough to permit generation of 1:1 complexes for dynamic structural studies. Discrimination between GDP and ADP is dramatic, with a difference of 6 orders of magnitude in affinity.

Adenosine Diphosphate↗
PubMed1995

Substrate and product structural requirements for binding of nucleotides to H-ras p21: the mechanism of discrimination between guanosine and adenosine nucleotides.

The interaction of the protein product of the H-ras oncogene with a series of nucleoside di- and triphosphates has been examined to investigate the tolerance of the active site to departures from the GTP or GDP structures. Nucleotides which bind relatively strongly could be used as competitors of GDP in a simple filter binding assay to give semiquantitave estimates of their affinities. For more weakly binding nucleotides or to obtain quantitative data, a transient kinetic method was used which was based on determination of the association and dissociation rate constants. The results obtained indicate that substantial modification of the sugar or phosphate structure is tolerated with little or moderate loss of affinity, but that large losses in affinity occur on modification of the base structure. In particular, replacing the guanine by an adenine residue leads to a dramatic loss of affinity. Thus, discrimination against ATP and ADP is very high (relative affinities of ATP and GTP 1:10(7)). This is due not only to loss of positive (stabilizing) interactions, but especially to the introduction of negative ones.

Acid Anhydride Hydrolases↗
PubMed1995

Independence divergence-generated binary trees of amino acids.

The discovery of the relationship between amino acids is important in terms of the replacement ability, as used in protein engineering homology studies, and gaining a better understanding of the roles which various properties of the residues play in the creation of a unique, stable, 3-D protein structure. Amino acid sequences of proteins edited by evolution are anything but random. The measure of nonrandomness, i.e. the level of editing, can be characterized by an independence divergence value. This parameter is used to generate binary tree relationships between amino acids. The relationships of residues presented in this paper are based on protein building features and not on the physico-chemical characteristics of amino acids. This approach is not biased by the tautology present in all sequence similarity-based relationship studies. The roles which various physico-chemical characteristics play in the determination of the relationships between amino acids are also discussed.

Amino Acid Sequence↗
PubMed1995

Enhancement of glucose disposal in patients with type 2 diabetes by alpha-lipoic acid.

Insulin resistance of skeletal muscle glucose uptake is a prominent feature of Type II diabetes (NIDDM); therefore pharmacological interventions should aim to improve insulin sensitivity. Alpha-lipoic acid (CAS 62-46-4, thioctic acid, ALA), a natural occurring compound frequently used for treatment of diabetic polyneuropathy, enhances glucose utilization in various experimental models. To see whether this compound also augments insulin mediated glucose disposal in NIDDM, 13 patients received either ALA (1000 mg/Thioctacid/500 ml NaCl, n = 7) or vehicle only (500 ml NaCl, n = 6) during a glucose-clamp study. Both groups were comparable in age, body-mass index and duration of diabetes and had a similar degree of insulin resistance at baseline. Acute parenteral administration of ALA resulted in a significant increase of insulin-stimulated glucose disposal; metabolic clearance rate (MCR) for glucose rose by about 50% (3.76 ml/kg/min = pre vs. 5.82 ml/kg/min = post, p < 0.05), whereas the control group did not show any significant change (3.57 ml/kg/min = pre vs. 3.91 ml/kg/min = post). This is the first clinical study to show that alpha-lipoic acid increases insulin stimulated glucose disposal in NIDDM. The mode of action of ALA and its potential use as an antihyperglycemic agent require further investigation.

Aged↗
PubMed1994

New alignment strategy for transmembrane proteins.

In this paper an algorithm which locates helical transmembrane segments is described. It is shown that given the location of transmembrane helices of a protein, corresponding helices in another membrane related protein can be pinpointed. The method seems to be extremely insensitive to sequence identity but highly sensitive to the property of a sequence to assume transmembrane helical structure. As an example, using the present method, a sequence alignment between bacteriorhodopsin and human rhodopsin is carried out and it provides a good starting point for homology modeling of this G-protein coupled receptor. It is difficult to obtain this particular alignment using the traditional methods because of poor sequence homology. There are indications that hint at the broader range of applicability of the presented method.

Algorithms↗
PubMed1994

Allelic inactivation regulates olfactory receptor gene expression.

We suggest a model in which a hierarchy of controls is exerted on the family of odorant receptor genes to assure that a sensory neuron expresses a single receptor from a family of 1000 genes. We propose that a cis-regulatory element directs the stochastic expression of only one gene from a large array of linked receptor genes. Moreover, only one allelic array encoding multiple receptor genes is active in an individual neuron. We demonstrate that in a neuron expressing a given receptor, expression derives exclusively from one allele. In addition, we observe that alleles encoding the odorant receptors are replicated asynchronously, a phenomenon consistently associated with allelic inactivation. This model, involving inactivation of one allelic array and cis control of the active array, provides a mechanism such that individual neurons express one or a small number of receptors.

Alleles↗
PubMed1994

Chaos game representation of protein structures.

Chaos game representation (CGR) was proposed recently to visualize nucleotide sequences as one of the first applications of this technique in the field of biochemistry. In this paper we would like to demonstrate that representations similar to CGR can be generalized and applied for visualizing and analyzing protein databases. Examples of applications will be presented for investigating regularities, and motifs in the primary structure of proteins, and for analyzing possible structural attachments on the super-secondary structure level of proteins. A further application will be presented for testing structure prediction methods using CGR.

Computer Simulation↗
PubMed1994

Different sequence environments of amino acid residues involved and not involved in long-range interactions in proteins.

No method has yet been available to decode information, hidden in the protein primary structure, on long-range interactions of amino acids. Even a limited amount of information on long-range interactions could help in conformational energy calculations of protein structures and could lead to a better understanding of how the primary structure of proteins determines their conformation. The sequence environments of amino-acid residues were compared from the viewpoint of their participation in long-range interactions. By using the simplest definition, residues were considered as partners in a long-range interaction if they were at least 20 residues apart in the sequence and their C alpha distance was less than 7 A. In spite of this rather crude definition, an analysis of 88 unrelated proteins has shown that the sequence environments (10 residues on each side) of those amino acids which are involved in long-range interactions and of those which are not are significantly different according to the criteria of mathematical statistics. Moreover, in many cases the differences are so pronounced that the involvement of a given amino acid in long-range interactions can be predicted from its sequence environment.

Amino Acid Sequence↗
PubMed1993

Allele-specific replication timing of imprinted gene regions.

Several lines of evidence suggest that the paternal and maternal genomes may have different expression patterns in the developing organism and this has been confirmed by the identification of endogenous genes that are parentally imprinted in the mouse. Little is known about the precise mechanisms involved in the process, but structural differences between the two alleles must somehow provide cis-acting signals for directing parental-specific transcription. Cell-cycle replication time is one parameter that has been shown to be associated with both tissue-specific gene expression and the allele-specific transcription patterns of the X chromosomes in female cells. For this reason we have examined the replication timing patterns for the chromosomal regions containing the imprinted genes Igf2, Igf2r, H19 and Snrpn in the mouse. At all of these sites, and their corresponding positions in the human genome, the two homologous alleles replicate asynchronously and it is always the paternal allele that is early-replicating. Thus imprinted genes appear to be embedded in large DNA domains with differential replication patterns, which may provide a structural imprint for parental identity.

Alleles↗
PubMed1992

Different sequence environments of cysteines and half cystines in proteins. Application to predict disulfide forming residues.

Protein sequences are often derived by translating genetic information, rather than by classical protein sequencing. At the DNA level cysteines and half cystines are indistinguishable. Here we show that the sequential environments of 'free' cysteine and half cystine are different. A possible origin of this difference is discussed and a simple method to predict cysteines and half cystines from the amino acid sequence is also presented.

Amino Acid Sequence↗
PubMed1991

Identification of the cytochrome P450 IIIA family as the enzymes involved in the N-demethylation of tamoxifen in human liver microsomes.

The antiestrogen tamoxifen (Tam or Nolvadex, ICI)-Z-1-[4-[2-(dimethylamino) ethoxy]phenyl]-1,2-diphenyl-1-butene is widely used in treatment of hormone-dependent breast cancer. The drug is extensively metabolized by cytochrome P450 dependent hepatic mixed function oxidase in man, yielding mainly the N-desmethyl metabolite (DMT). This study has been carried out to determine the P450 enzyme involved in the N-oxidative demethylation of Tam in microsomal samples from 25 human livers (23 adults, two children). This metabolic step was inhibited by carbon monoxide up to 75%. Tam was demethylated into DMT with an apparent Km of 98 +/- 10 microM; rates varied between 37 and 446 pmol/min/mg microsomal protein. These metabolic rates were strongly correlated with 6 beta-hydroxylation of testosterone (r = 0.83) and erythromycin N-demethylase (r = 0.75), both activities known to be associated with P450 IIIA enzyme. To further assess whether or not the Tam demethylation pathway is catalysed by the same P450, the inhibitory effect of TST on this reaction was determined. The competitive inhibition had an apparent Ki of 100 +/- 10 microM. Drugs such as erythromycin, cyclosporin, nifedipine and diltiazem were shown to inhibit in vitro the metabolism of tamoxifen. Furthermore the P450 IIIA content of liver microsomal samples, measured by Western blot technique using a monoclonal P450NF (nifedipine) antibody, was strongly correlated with DMT formation (r = 0.87). Tam N-demethylase activity was inhibited by more than 65% with polyclonal anti-human anti-P450NF. All these in vitro observations establish that a P450 enzyme of the IIIA sub-family is involved in the oxidative demethylation of tamoxifen in human liver.

Animals↗
PubMed1991

Calculation of protein conformation as an assembly of stable overlapping segments: application to bovine pancreatic trypsin inhibitor.

Conformations of bovine pancreatic trypsin inhibitor were calculated by assuming that the final structure as well as properly chosen overlapping segments thereof are simultaneously in low-energy (not necessarily the lowest-energy) conformational states. Therefore, the whole chain can be built up from building blocks whose conformations are determined primarily by short-range interactions. Our earlier buildup procedure was modified by taking account of a statistical analysis of known amino acid sequences that indicates that there is nonrandom pairing of amino acid residues in short segments along the chain, and by carrying out energy minimization on only these segments and on the whole chain [without minimizing the energies of intermediate-size segments (20-30 residues long)]. Results of this statistical analysis were used to determine the variable sizes of the overlapping oligopeptide building blocks used in the calculations; these varied from tripeptides to octapeptides, depending on the amino acid sequence. Successive stages of approximations were used to combine the low-energy conformations of these building blocks in order to keep the number of variables in the computations to a manageable size. The calculations led to a limited number of conformations of the protein (only two different groups, with very similar structure within each group), most residues of which were in the same conformational state as in the native structure.

Amino Acid Sequence↗