Binding of Au(CN)2- and Pt(CN)4-2- to horse liver alcohol dehydrogenase. A 35C1NMR relaxation study.

The binding of Au(CN)2- and Pt(CN)4-2- ions to the coenzyme binding site of horse liver alcohol dehydrogenase (alcohol : NAD+ oxidoreductase EC 1.1.1.1) has been studied by 35C1 nuclear magnetic relaxation. Longitudinal relaxation rates were analyzed in terms of a simple model and binding constants for Au(CN)2-, Pt(CN)4-2- and C1- were estimated. From a comparison between transverse and longitudinal relaxation rates the correlation time and the quadrupole coupling constant of bound chloride ion were obtained. The quadrupole coupling constant estimated from a simple electrostatic model for chloride ion interacting with an arginine group agrees with the experimental value.

An EPR study of the blue copper center in horse liver alcohol dehydrogenase.

Active-site specifically reconstituted Cu2+ horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) shows optical and EPR spectra similar to those of native blue copper (Type 1) proteins. EPR spectra at different temperatures and frequencies reveal a heterogeneity of the copper center: a minor 'non-blue' species with axial line shape (g parallel = 2.16, g perpendicular = 2.04; A parallel = 100 x 10(-4) cm-1), which accounts for approximately 10% of the total copper and is not accessible to ligands and a major blue species with rhombic line shape (g1 = 2.21, g2 = 2.06, g3 = 2.03, A1 = 50 x 10(-4) cm-1, A2 = 30 x 10(-4) cm-1, A3 = 76 x 10(-4) cm-1), which is accessible to ligands and participates in redox reactions. The major blue species in cupric horse liver alcohol dehydrogenase is metastable, since it is reduced in a process markedly accelerated in the presence of oxygen or hydrogen peroxide. In addition, the reduction depends on the presence of exogenous metal ligands or coenzymes. Whereas the binary complex enzyme-NAD+ is even more susceptible to bleaching than the free enzyme, the cupric center is stable towards bleaching in the binary complex enzyme-NADH. In the discussion the redox properties and coordination chemistry of Cu2+ in horse liver alcohol dehydrogenase and copper proteins are compared.

Identification of tyrosine as a functional residue in the active site of Escherichia coli dUTPase.

dUTP nucleotidohydrolase (dUTPase, EC 3.6.1.23) from E. coli contains a total of six tyrosine residues per trimer. About half of them were found to be susceptible to acetylation with N-acetylimidazole or to nitration with tetranitromethane with concomitant loss of activity. Deacetylation with N-hydroxylamine leads to full reactivation. Inhibitory products of dUTP hydrolysis, i.e., dUMP and inorganic pyrophosphate together with the cofactor Mg2+ protect significantly against inactivation and chemical modification. In the Cu(2+)-dUTPase complex, charge transfer from Cu2+ to the tyrosinate anion was perturbed by the presence of the substrate dUTP. These results, together with the occurrence of one tyrosine residue in a strictly conserved sequence motif suggest the critical importance of this residue for the function of the enzyme.

Copper(II)-substituted horse liver alcohol dehydrogenase: structure of the minor species.

Oxygen treatment of horse liver alcohol dehydrogenase EE isozyme substituted with Cu(II) at the catalytic site leads to bleaching with concomitant reduction to Cu(I) of approximately 90% of total Cu(II). The Cu(II) of the remaining 'minor species' cannot be reduced nor does it interact with exogenous ligands, e.g. 2-mercaptoethanol, imidazole, pyrazole, or azide ions. The EPR spectrum is axial with a super-hyperfine splitting of 15.6 G indicating binding of one nitrogen atom to Cu(II). These data as well as the energies and intensities of the absorption and CD spectra suggest the Cu(II) ion of the minor species to be located in the catalytic site of HLADH in a position and geometry different from that of the major species.

The primary structure of two polypeptide chains from preparations of homeostatic thymus hormone (HTH alpha and HTH beta) entire-chain identities to two histones.

The primary structures of the 2 polypeptide chains (HTH alpha and HTH beta) of the homeostatic thymus hormone (HTH) were determined. The entire structures were found to be identical to those of histones H2A and H2B, respectively, without evidence for sub-types, proteolytic processings, or other peptide fragments. The results show that suggestions for new extranuclear and hormone-like histone functions apply to HTH preparations with intact protein chains of the H2 histones.

Designed inhibitors of horse liver alcohol dehydrogenase. Effect of active-site metal ion substitution.

3-(p-Butoxyphenyl)propionamide, -thioamide and -hydrazide and the formamide of p-butoxybenzylamine were tested as inhibitors of cadmium(II) and cobalt(II) active-site substituted alcohol dehydrogenase. The results agree with a direct coordination of these inhibitors except for the hydrazide to the active-site metal ion, in the enzyme-NADH-inhibitor complex. The hydrazide might be situated at some distance from the metal ion without a direct coordination bond.

Transient kinetics of ligand binding and role of the C-terminus in the dUTPase from equine infectious anemia virus.

Transient kinetics of the equine infectious anemia virus deoxyuridine 5'-triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. Rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a one-step mechanism for substrate binding. A C-terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. As a result, the activity of the dUTPase was completely quenched, but the rate constants and fluorescent signal of the truncated enzyme were affected only to a minor degree. We conclude that the flexible C-terminus is not a prerequisite for substrate binding, but indispensable for catalysis.

Mono- and binuclear Zn-beta-lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions.

The Bacteroides fragilis Zn-beta-lactamase is active with a mono- and a binuclear zinc site. The apoenzyme produced by removal of both Zn ions does not recover full activity upon readdition of Zn2+ in contrast to an active mono-Zn form prepared at pH 6.0. Differences in k(cat) values observed are substrate-dependent implying distinct mechanisms for the mono- and binuclear species. The substrate profile of a Zn,Cd hybrid obtained by selective exchange of one zinc ion is different from that of the Zn2 enzyme with a remarkable 15-fold increased activity with cefoxitin as substrate.

Kinetic and spectroscopic characterization of native and metal-substituted beta-lactamase from Aeromonas hydrophila AE036.

Two metal ion binding sites are conserved in metallo-beta-lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur-to-metal charge transfer bands are observed for all mono- and di-metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non-competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed.

The nicotinamide subsite of glyceraldehyde-3-phosphate dehydrogenase studied by site-directed mutagenesis.

Directed mutagenesis has been used to study the nicotinamide subsite of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Residue Asn313 is involved together with the carboxyamide moiety of the nicotinamide ring in a complex network of hydrogen bonding interactions which fix the position of the pyridinium ring of NAD to which hydride transfer occurs at the C-4 position in the catalytic reaction. The asparagine side-chain has been replaced by that of the Thr and Ala residues and results in mutants with very similar properties. Both mutants show much weaker binding of NAD and lower catalytic efficiency. The mutant Asn313----Thr still exhibits strict B-stereospecificity in hydride transfer and retains the property of negative co-operativity in NAD binding. These experiments strongly suggest that the mutant enzyme undergoes the apo----holo sub-unit structural transition associated with coenzyme binding but that the nicotinamide ring is no longer as rigidly held in its pocket as in the wild type enzyme. The results shed light on the details of the molecular interactions which are responsible for negative co-operativity in this enzyme.

Half-life of single-chain urokinase-type plasminogen activator (scu-PA) and two-chain urokinase-type plasminogen activator (tcu-PA) in patients with acute myocardial infarction.

The pharmacokinetics of urokinase (two-chain urokinase-type plasminogen activator, tcu-PA) and single-chain urokinase-type plasminogen activator (scu-PA) were studied in 20 patients with acute myocardial infarction (AMI). Ten consecutive patients received 2.5 million units tcu-PA by bolus injection within 5 min during the first 6 h after AMI (group I). Ten further consecutive patients received 250,000 U tcu-PA within 5 min, followed by 4.5 million U scu-PA by intravenous infusion over 40 min (group II). An enzyme immunoassay was developed for urokinase antigen determinations, and a fibrin plate assay for determinations of fibrinolytic activity was applied. Using a 3-compartment model, in group I 98% of urokinase antigen were cleared with a half-life of 60.8 min. After scu-PA, urokinase antigen was cleared with half-lives (area under the curve in parentheses) of 6.9 min (74.8%), 26.5 min (23.6%), and 329.7 min (2.2%). The half-disappearance times of fibrinolytic activity were 18 and 8 min in group I and II, respectively. A more pronounced decrease of plasminogen was observed after tcu-PA.

Separation and characterisation of bovine histone H1 subtypes by combined ion-exchange and reversed-phase chromatography and mass spectrometry.

In order to separate and identify histone H1 subtypes from calf thymus we used both electrospray mass spectrometry (ES-MS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) after a three-step chromatographic procedure consisting of reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC) and ion-exchange chromatography (IEC). Under the RP-HPLC conditions described, we obtained two baseline-separated H1-fractions which were characterised by MALDI-TOF-MS. The determined masses ranged from 22,850 to 22,590 for the first fraction and from 22,070 to 21,250 for the second fraction. Further, it was shown that the first fraction contained at least four and the second one at least five subtypes of the histone class H1. Four homogeneous pure H1 subtypes were obtained by a combination of IEC followed by SEC and RP-HPLC. The molecular masses of these four subtypes determined by ES-MS were 22,606, 22,761, 21,347 and 21,263. We obtained six additional molecular masses of histone H1 subtypes from three heterogeneous fractions, namely 22,066, 21,802, 20,586 and 19,817 by ES-MS and 22,800 and 22,675 by MALDI-TOF-MS. The retention times of these fractions and the molecular masses were in agreement with the data obtained from RP-HPLC fractions by MALDI-TOF-MS.

Kinetics and mechanisms of the recombination of Zn2+, Co2+, and Ni2+ with the metal-depleted catalytic site of horse liver alcohol dehydrogenase.

The kinetics of the recombination of the metal-depleted active site of horse liver alcohol dehydrogenase (LADH) with metal ions have been studied over a range of pH and temperature. The formation rates were determined optically, by activity measurements, or by using the pH change during metal incorporation with a pH-indicator as monitor. The binding of Zn2+, Co2+, and Ni2+ ions occurs in a two-step process. The first step is a fast equilibrium reaction, characterized by an equilibrium constant K1. The spectroscopic and catalytic properties of the native or metal-substituted protein are recovered in a slow, monomolecular process with the rate constant k2. The rate constants k2 5.2 X 10(-2) sec-1 (Zn2+), 1.1 X 10(-3) sec-1 (Co2+), and 2 X 10(-4) sec-1 (Ni2+). The rate constants increase with increasing pH. Using temperature dependence, the activation parameters for the reaction with Co2+ and Ni2+ were determined. Activation energies of 51 +/- 2.5 kJ/mol (0.033 M N-Tris-(hydroxymethyl)methyl-2-aminomethane sulfonic acid (TES), pH 6, 9) for Co2+ and 48.5 +/- 4 kJ/mol (0.033 M TES, pH 7, 2) for Ni2+ at 23 degrees C were found. The correspondent activation entropies are - 146 +/- 10 kJ/mol K for Co2+ and - 163 +/- 9 kJ/mol K for Ni2+. Two protons are released during the binding of Zn2+ to H4Zn(n)2 LADH in the pH range 6.8-8.1. The binding of coenzyme, either reduced or oxidized, prevents completely the incorporation of metal ions, suggesting that the metal ions enter the catalytic site via the coenzyme binding domain and not through the hydrophobic substrate channel.

Spectral evidence for three metal-linked ionization equilibria in the interaction of cobalt(II) horse liver alcohol dehydrogenase with coenzyme and substrate.

The visible absorption bands in the region 525-575 nm of the catalytic cobalt ion in cobalt(II) horse liver alcohol dehydrogenase show characteristic pH-dependent changes both in the free enzyme and its complexes with nicotinamide adenine dinucleotide (NAD+) and NAD+ plus ethanol or 2,2,2-trifluoroethanol. In the free enzyme, the change of the coordination environment has an apparent pK of about 9.4. In the binary complex with NAD+ the spectral changes are complex, indicating changes in the coordination sphere in a lower pH range with an estimated pK value of about 7.9. The ternary complexes enzyme X NAD+ X ethanol and enzyme X NAD+ X 2,2,2-trifluoroethanol exhibit very similar, characteristic spectral features; their apparent pK values are 6.3 and less than 4, respectively. We ascribe these pK values to the ionization of the alcohol bound in the ternary complexes. The results demonstrate that the catalytic cobalt ion is sensing changes of the ionization state of the protein when going from low pH forms to high pH forms both in the absence and presence of coenzyme and substrate/inhibitor.

Active site-specifically reconstituted nickel(II) horse liver alcohol dehydrogenase: optical spectra of binary and ternary complexes with coenzymes, coenzyme analogues, substrates, and inhibitors.

Insertion of nickel ions into the empty catalytic site of horse liver alcohol dehydrogenase yields an active enzyme with 65% metal substitution and about 12% intrinsic activity. The electronic absorption spectrum is characterized by bands at 357 nm (2900 M-1 cm-1), 407 nm (3500 M-1 cm-1), 505 nm (300 M-1 cm-1), 570 nm (approximately equal to 130 M-1 cm-1), and 680 nm (approximately equal to 80 M-1 cm-1). The absorption and CD spectra are similar to those of nickel(II) azurin and nickel(II) aspartate transcarbamoylase and prove coordination of the nickel(II) ions to sulfur in a distorted tetrahedral coordination geometry. Changes of the spectra upon ligand binding at the metal or conformation changes of the protein induced by coenzyme, or both, indicate alterations of the metal geometry. The chromophoric substrate trans-4-(N, N-dimethylamino)-cinnamaldehyde forms a ternary complex with Ni(II) liver alcohol dehydrogenase and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide-adenine-dinucleotide, stable between pH 6 and 10. The corresponding ternary complex with NADH is only stable at pH greater than 9.0. The spectral redshifts induced in the substrate are 11 nm larger than those found in the zinc enzyme. We suggest direct coordination of the substrate to the catalytic metal on which acts as a Lewis acid in both substrate coordination and catalysis.

Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions.

Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.

Expression in Escherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation.

Human alcohol dehydrogenase (ADH, beta beta isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing. The expression system was obtained by ligation of a cDNA fragment corresponding to the beta-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter. The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3% of the total amount of protein. Recombinant ADH was separated from E. coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis. The N-terminal sequence was identical to that of the authentic beta-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.

Histone H1 suppresses tumor growth of leukemia cells in vitro, ex vivo and in an animal model suggesting extracellular functions of histones.

Purified histone H1 exerts growth inhibition of leukemia cells independent of lineage, stage, and maturation. At 200 micrograms/ml, H1 proved cytotoxic in 19 of 21 of the tested leukemia-derived cell lines and for 11 of 16 of the fresh tumor samples from leukemia patients. In all cases, normal peripheral blood mononuclear cells and bone marrow cells remained unaffected. Multicellular spheroids from the Burkitt's lymphoma cell line IM-9 were growth arrested at 500 micrograms H1/ml. The clonogenic growth of the Burkitt's lymphoma cell line Daudi was arrested at 160 micrograms H1/ml. Synthetic H1-peptides as well as peptides and proteins with biochemical properties similar to H1 had no inhibitory growth effect at equimolar concentrations. Furthermore, 250 micrograms H1 injected into a Burkitt's lymphoma (Daudi), xenotransplanted into nude mice, arrested tumor growth. As shown by electron microscopy and flow cytometry, incubation of leukemia cells with H1 resulted in severe plasma membrane damage and ultimately cytolysis. This report characterizes a 33-kd protein that binds H1 and is responsible for the cell death via destruction of the cell membrane integrity. New extranuclear functions of histones are presented.

[Effect of histone on hematopoietic stem cells (CFU-S) in normal and irradiated organism]. / Vozdeistvie gistona na krovetvornye kletki-predshestvenniki (KOEs) normal'nogo i obluchennogo organizma.

Radiotherapeutic activity of histone fractions H1 and H2A/H2B were studied. It was demonstrated that both fractions are able to reduce the damaging effect of ionizing radiation on spleen colony forming unit (CFU-S) population. Histone preparations stimulated colony-forming activity of bone marrow cells exposed to dose of 0.5-3.0 Gy both in the case of incubation with preparations and intravenous or intraperitoneal administration into recipients of irradiated cells. The effect of histones and accessory thymocytes on CFU-S population is compared.