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V Saudek

Publications and source records attributed to V Saudek.

33 records · Page 2Linked to original sources
PubMed1990

Sequence-specific 1H NMR assignment and secondary structure of neuropeptide Y in aqueous solution.

Sequence-specific assignment of the 1H NMR spectrum of the 36 amino acid polypeptide porcine neuropeptide Y (pNPY) at pH 3.1 is reported. It was achieved by use of standard two-dimensional techniques and by a combination of the sequential and main-chain-directed assignment strategies. The secondary structure was derived from inspection of the nuclear Overhauser spectra, slow hydrogen-deuterium exchange effects, chemical shifts of main-chain HA resonances, and coupling constants. These studies indicate that the C-terminal segment (residues 11-36) folds into an amphiphilic alpha-helix; the N-terminal segment, containing three prolines in both cis and trans conformations, assumes no regular structure. CD studies of pNPY at pH 3.1 and 7.4 show an increase in ordered structure at neutral pH. The difference spectrum, however, is typical of an alpha-helix and suggests a stabilization of residues 11-36, possibly via Maxfield-Scheraga pair interactions involving side-chain residues. This is supported by a comparison of the one-dimensional 1H NMR spectra of pNPY at pH 3.1 and 7.4, where no remarkable differences are observed.

Amino Acid Sequence↗
PubMed1990

Solution structure of the DNA-binding domain of the yeast transcriptional activator protein GCN4.

The solution structure of an active synthetic peptide containing both the leucine zipper and the adjacent basic domain of the yeast transcription factor GCN4 (residues 220-280) was determined by NMR. The two domains show structurally distinct behaviours. In the absence of DNA, the basic domain is, although very flexible, structured and fluctuating around a helical conformation. The leucine zipper region forms a long, uninterrupted helix. From a suitable set of NMR distances the three-dimensional structure of the leucine zipper monomeric sub-domain was calculated by distance geometry algorithms. The structure of the symmetrical parallel dimer was obtained by model building using the NMR information. A smaller peptide with the sequence of the isolated basic region (residues 1-35 of the 61 residue peptide) was also synthesized. Circular dichroism studies showed 30-40% helicity. A flexible helix spans the region between residues 8 and 21. The comparison of our results with suggested models is discussed in detail.

Algorithms↗
PubMed1989

1H-NMR study of endothelin, sequence-specific assignment of the spectrum and a solution structure.

The solution conformation of the recently discovered bi-cyclic, 21 amino acid vasoconstrictor peptide, Endothelin I, has been examined by 1H-NMR in deuterated dimethyl sulphoxide. A full sequential assignment has been achieved. In addition, 19 long range NOEs were detected which were employed as distance constraints in molecular dynamics calculations to yield a possible solution structure for this new peptide.

Amino Acid Sequence↗
PubMed1989

Mobility of secondary structure units of horse-muscle acylphosphatase. Relation to antigenicity.

The antigenic properties of acylphosphatase are compared with its various sequential characteristics (hydrophobicity, chemical shift of the main-chain 1H-NMR resonances, numbers and intensities of the nuclear Overhauser enhancements, hydrogen-deuterium exchange and sequential arrangement of the secondary structure units). The discussion is based on the complete sequential assignment of the 1H-NMR spectrum and the knowledge of the three-dimensional fold of the protein obtained by NMR spectroscopy from distance geometry calculations. Regions with very different degrees of mobility can be distinguished. It is found that all major antigenic sites are located in the most mobile surface loops.

Acid Anhydride Hydrolases↗
PubMed1989

The sequence-specific assignment of the 1H-NMR spectrum of an enzyme, horse-muscle acylphosphatase.

A complete range of two-dimensional NMR experiments was used for the assignment of the 1H-NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long-range patterns characteristics for secondary structure elements. This result had also provided the identification of the main-chain alpha and amide proton resonances. Several of the completely assigned spin systems were then identified as being part of the secondary structure units which led, after analysis of the primary amino acid sequence, to unambiguous sequence-specific assignments. The identification and assignment of the remaining side-chain resonances was then completed and are reported here. These results provide a complete data base for the three-dimensional structure determination of this enzyme in solution.

Acid Anhydride Hydrolases↗
PubMed1989

Identification and description of beta-structure in horse muscle acylphosphatase by nuclear magnetic resonance spectroscopy.

Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed subsequently and a list produced of nuclear magnetic resonance (n.m.r.) constraints derived from the spectra. The beta-structure consists of a beta-sheet with four antiparallel chains, one attached parallel chain, three tight turns and a beta-bulge. The conformation of the beta-sheet was determined by distance geometry calculation using the n.m.r. constraints (174 intraresidual, 107 sequential and 226 long-range distances, 32 torsion angles, phi, and 28 hydrogen bonds) as input. Observation of some interactions between the sheet and previously identified alpha-helical regions made it possible to give an outline of the three-dimensional structure of the enzyme.

Acid Anhydride Hydrolases↗
PubMed1989

Identification and description of alpha-helical regions in horse muscle acylphosphatase by 1H nuclear magnetic resonance spectroscopy.

It has been proposed that combination of intraresidue, sequential and longer range nuclear Overhauser enhancements occurring in 1H nuclear magnetic resonance spectra of protein chains folded in a helix show a regular characteristic pattern. As a test case the spectra of horse muscle acylphosphatase were searched for this pattern together with other typical signs of a helical conformation (i.e. chemical shift, coupling constants and slow 2H-H exchange). Two amino acid sequences complying with these requirements were found. Just a few amino acid spin system assignments were then sufficient to locate the two segments within the primary structure (residues 22 to 35 and 55 to 66), thus providing the sequential assignment. The assignment of the side-chains was completed and a list of all nuclear magnetic resonance constraints within the two segments (126 intra- and 180 interresidue distances, 21 torsion angles phi and 19 hydrogen bonds) was produced. Distance geometry calculation shows that each segment forms an alpha-helix. The mutual orientation of the two helices was established subsequently.

Acid Anhydride Hydrolases↗
PubMed1988

Secondary structure of acylphosphatase from rabbit skeletal muscle. A nuclear magnetic resonance study.

Sequence-specific assignment of 1H nuclear magnetic resonance spectra of acylphosphatase (EC 3.6.1.7) isolated from rabbit skeletal muscle have made it possible to identify short distance constraints from nuclear Overhauser enhancement spectra, to evaluate spin-spin coupling constants of many backbone amide hydrogens and to assess their slow exchange with deuterons in 2H2O solution. Analysis of these data show that the major regular secondary structure of the enzyme consists of five extended beta-strands, four of which are arranged in an antiparallel beta-sheet, while the fifth is attached parallel. A helix consisting of 11 residues has also been identified. Consideration of additional distance constraints between sequentially remote residues has allowed us to give an outline of the overall fold of the protein.

Acid Anhydride Hydrolases↗
PubMed1987

Chromotest estimation of SOS functions as a screening method for antitumor platinum complexes.

The chromotest which measures the induction of SOS functions in an Escherichia coli strain bearing the lacZ gene under the sfiA gene control was evaluated as a screening method for platinum complexes with antitumor activity. Four types of complexes were used: (a) those with two nitrogen ligands in cis position and chlorine or carboxylic acid as anionic ligand; (b) four nitrogen atoms surrounding platinum; (c) complexes involving sulphur and amino group of methionine; (d) complexes of tetravalent platinum. The trans-diaminodichloro complex was used as negative control. Groups (b) and (c) were inactive in the chromotest which correlated well with their absence of antitumor activity. Antitumor activity and a positive chromotest also correlated in group (d). In group (a), which includes complexes with well established antitumor activity, the chromotest separated complexes with fast and slow rate of hydrolysis. The results fitted well in the mutation induction test.

Alkaline Phosphatase↗
PubMed1985

The reaction of Pt-antitumor drugs with selected nucleophiles. I. The reaction of cis-[Pt(NH3)2Cl2] with glycine.

Various Pt(II)-glycine coordination compounds were characterized by 1H and 13C NMR spectroscopy, some of them also by electrophoretic and chromatographic behavior. The results were applied to the analysis of the reaction mixtures of cis-[Pt(NH3)2Cl2] and glycine obtained under various conditions. Cis-[Pt(NH3)2Cl2] reacts with glycine to give cis-diammine-(glycine-N,O)-Pt(II) and cis-diammine-bis(glycine-N-)Pt(II). Their ratio depends primarily on the pH of the reaction medium. Conformation of these compounds is discussed based on the observed Pt-C and Pt-H NMR coupling constants.

Antineoplastic Agents↗
PubMed1985

The reaction of Pt-antitumor drugs with selected nucleophiles. II. Preparation and characterization of coordination compounds of Pt(II) and L-histidine.

Various His-Pt(II) coordination compounds were prepared by reaction of K2PtCl4 or cis-[Pt(NH3)2Cl2](cis-DDP) with His and analyzed by 1H and 13C NMR spectroscopy, electrophoresis, and ion-exchange chromatography. His may be coordinated to Pt by the imidazol iminogroup and/or the alpha-aminogroup; the carboxygroup remains always free. Both bidentate as well as monodentate ligands were identified. Cis-DDP reacts with His to give a mixture of compounds where all these possibilities are present: cis-diamine-(histidine-N,N-)Pt(II) and three different types of cis-diammine-bis(histidine). HCl trans cleavage of compounds with bidentate His ligands leads to a mixture of two compounds having His ligated to Pt by an amino or imin group. The methods applied are suitable for analyzing reactions of His with cis-DDP under model conditions similar to physiological conditions.

Antineoplastic Agents↗
PubMed1982

A high-molecular mass derivative of trypsin-kallikrein inhibitor for potential medical use. II. Study of inhibitory activity.

Three derivatives of poly[alpha-N beta-(2-hydroxyethyl)-DL-asparagine, beta-N alpha-(2-hydroxyethyl)-DL-asparagine] were used as soluble carrier polymers for binding the trypsin-kallikrein inhibitor (TKI). In addition to the parent polymer an anionic derivative with 19.2 mol of carboxyl groups/100 mol of residues and a hydrophobized derivative with 18.0 mol of butyl groups/100 mol of residues were also used. All carriers contained 6.6 mol of 2-(4-aminobenzamido)ethyl residues/100 mol which were used for the binding after diazotation. All derivatives retained a high inhibitory activity toward trypsin and chymotrypsin whereas the effect on kallikrein was substantially reduced. The kinetic constants were estimated under the simplifying assumption, that classical kinetic equations for the enzyme-inhibitor interaction may be applied. The equilibrium dissociation constants (5 X 10(-9) mol/l) and association rate constants (1 X 10(5) l X mol-1 X s-1) with respect to trypsin are unaffected by the two chemical modifications of the carrier. The increase in the equilibrium dissociation constant (compared to native TKI) is probably a result of the decreasing strength of the enzyme-inhibitor complex rather than a manifestation of reduced rate of diffusion of the enzyme to the active center of the polymer-bound inhibitor.

Chymotrypsin↗
PubMed1979

Enzyme immobilization techniques on poly(glycidyl methacrylate-co-ethylene dimethacrylate) carrier with penicillin amidase as model.

Two types of bead-form macroporous carriers based on glycidyl methacrylate with ethylene dimethacrylate copolymers were used for the immobilization of penicillin amidase either directly or after chemical modification. Direct binding through oxirane groups, which is equally efficient at pH 4.2 and 7, is relatively slow and brings about an activity loss at low enzyme concentrations. The most efficient immobilization was achieved on glutaraldehyde-activated amino carrier, irrespective of whether the amino groups were formed by ammonia or 1,6-diaminohexane treatment of the original oxirane carrier. Hydrazine treatment gave lower immobilization yields. The same is true of the azide method independent of the length of the spacer. Most enzyme activity was preserved by coupling the carbodiimide-activated enzyme to the carrier with alkyl or arylamino groups at the end of a longer substituent. Immobilization on diazo-modified carrier gave average results. Rapid immobilization by a lysine-modified phosgene-treated carrier resulted in an activity loss. It is suggested that multipoint and very tight attachment of the enzyme molecule to the matrix decreased the activity. The immobilized activity is quite stable in solution and very stable upon lyophilization with sucrose.

Amidohydrolases↗