One-stage vs. chromogenic assays in haemophilia A.

Haemophilia A severity is closely correlated to the factor VIII (FVIII) activity, which can be measured in different ways. The original one-stage clotting assay is still the most widely used. The two-stage coagulation assay eliminated many of the drawbacks of the one-stage assay and was further developed into the chromogenic assay, a two-staged test with purified coagulation factors in the first stage, and a FXa-specific chromogenic substrate in the second stage. In many patients with mild or moderate haemophilia A, there is a discrepancy between the one-stage and the two-stage assays. If only the one-stage assay is used, some patients will have normal FVIII levels and not be diagnosed as having haemophilia or be considered to have a milder bleeding risk than is the case. Other patients who have normal FVIII activity will be diagnosed as haemophilia A. All haemophilia treatment centre laboratories should have access to both one-stage and chromogenic FVIII:C assays. Appropriate standards should be employed to enable accurate FVIII:C measurement. Different assays to measure inhibitor activity to infused FVIII have been developed since 1959. Inhibitor results based on the one-stage or chromogenic FVIII:C assays are well correlated, but the one-stage assay may be influenced by nonspecific inhibition.

In silico derived small molecules bind the filovirus VP35 protein and inhibit its polymerase cofactor activity.

The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein-protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID-NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35-NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins.

[Antioxidant and prooxidant action of nitric oxide donors and metabolites].

It has been shown that various nitric oxide donors and metabolites have similar effects on lipid peroxidation in rat myocardium homogenate. The formation of malondialdehyde, a secondary product of lipid peroxidation, was inhibited in a dose-dependent manner by PAPA/NONO (a synthetic nitric oxide donor), S-nitrosoglutathione, nitrite, and nitroxyl anion. The inhibition of lipid peroxidation was provided most efficiently by the administration of dinitrosyl-iron complexes with dextran and PAPA/NONO. S-nitrosoglutathione also inhibited the destruction of coenzymes Q9 and Q10 during free radical oxidation of myocardium homogenate. Low-molecular-weight dinitrosyl iron complexes with cysteine also promoted lipid peroxidation, which is probably due to iron release during the destruction dinitrosyl iron complexes. It is likely that the antioxidant action of nitric oxide derivatives is related to the reduction of ferry forms of hemoproteins and interaction of nitric oxide with lipid radicals.

Effect of an inhibitor of nitric oxide production on Cu-Zn/SOD and its cofactors in diabetic rats.

The aim of this study was to investigate the effect of an inhibitor of nitric oxide production, N(omega)-nitro-L-arginine methyl esther (L-NAME) on Cu-Zn/SOD (superoxide dismutase) enzyme activity and copper and zinc concentrations in diabetes-induced rats. The control group consisted of 12 male albino Sprague-Dawley rats, 10-12 wk of age and weighing 300 g. Twenty-six albino Sprague-Dawley rats, 10-12 wk of age and weighing 315 g, constituted the experimental group. The experimental group was divided into two groups. The first group (n=12) constituted streptozotocininduced (55 mg/kg, intraperitoneally) diabetic rats and the second group (n=14) was administered L-NAME (1 mg/kg/d) after streptozotocin induction. For determination of Cu-Zn/SOD activity, spectrophotometry was used. Zinc and copper concentrations were determined by atomic absorption spectrophotometry. Results showed that Cu-Zn/SOD activity was increased significantly in both experimental groups compared to controls, and the increase in the second group was higher than in the first group (p<0.01, p<0.01, p<0.05). Plazma zinc concentration was increased in the second group when compared with controls (p<0.05). Plasma copper was decreased significantly in the second group compared to controls and the first group (p<0.001, p<0.001). Red cell copper concentration was decreased significantly in the first group compared to controls (p<0.05). This study showed that L-NAME administration has ensured an additive effect on the antioxidant defense system, which was proved by the increase in Cu-Zn/SOD activity. This increase might have a protective effect against tissue damage in the acute period, with corresponding changes in zinc and copper concentrations.

Global effects of the energetics of coenzyme binding: NADPH controls the protein interaction properties of human cytochrome P450 reductase.

The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'-ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K(d)), the enthalpy (DeltaH(B)) and entropy (TDeltaS(B)) of binding, and the heat capacity change (DeltaC(p)). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K(d2)(')(,5)(')(-ADP) = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (DeltaC(p)) values for 2',5'-ADP (-210 cal mol(-)(1) K(-)(1)), NADP(+) (-230 cal mol(-)(1) K(-)(1)), and H(4)NADP (-220 cal mol(-)(1) K(-)(1)) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TDeltaS(B) = -13400 +/- 200 cal mol(-)(1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TDeltaS(B) = -11200 +/- 300 cal mol(-)(1), 35 degrees C) indicate that the number of accessible conformational substates of the protein increases upon 2',5'-ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez, A., Paine, M., Wolf, C. R., Scrutton, N. S., and Roberts, G. C. K. (2002) Biochemistry 41, 4626-4637]. A combination of calorimetry and stopped-flow spectrophotometry kinetics experiments showed that this linkage between coenzyme binding energetics and diffusional domain motion impinges directly on the molecular recognition of cytochrome c by CPR. Single-turnover reduction of cytochrome c by CPR (k(max) = 15 s(-)(1), K(d) = 37 microM) is critically coupled to coenzyme binding through ligand-induced motions that enable the FMN-binding domain to overcome a kinetically unproductive conformation. This is remarkable since the FMN-binding domain is not directly involved in coenzyme binding, the NADP(H) binding site being fully contained in the FAD-binding domain. Sequential rapid mixing measurements indicate that harnessing of coenzyme binding energy to the formation of a kinetically productive CPR-cytochrome c complex is a highly synchronized event. The inferred half-time for the decay of this productive conformation (tau(50)) is 330 +/- 70 ms only. Previously proposed structural and kinetic models are discussed in light of these findings.

Cyanide as a copper and quinone-directed inhibitor of amine oxidases from pea seedlings ( Pisum sativum) and Arthrobacter globiformis: evidence for both copper coordination and cyanohydrin derivatization of the quinone cofactor.

The interactions of cyanide with two copper-containing amine oxidases (CuAOs) from pea seedlings (PSAO) and the soil bacterium Arthrobacter globiformis (AGAO) have been investigated by spectroscopic and kinetic techniques. Previously, we rationalized the effects of azide and cyanide for several CuAOs in terms of copper coordination by these exogenous ligands and their effects on the internal redox equilibrium TPQ(amr)-Cu(II) right harpoon over left harpoon TPQ(sq)-Cu(I). The mechanism of cyanide inhibition was proposed to occur through complexation to Cu(I), thereby directly competing with O(2) for reoxidation of TPQ. Although cyanide readily and reversibly reacts with quinones, no direct spectroscopic evidence for cyanohydrin derivatization of TPQ has been previously documented for CuAOs. This work describes the first direct spectroscopic evidence, using both model and enzyme systems, for cyanohydrin derivatization of TPQ. K(d) values for Cu(II)-CN(-) and Cu(I)-CN(-), as well as the K(i) for cyanide inhibition versus substrate amine, are reported for PSAO and AGAO. In spite of cyanohydrin derivatization of the TPQ cofactor in these enzymes, the uncompetitive inhibition of amine oxidation is determined to arise almost exclusively through CN(-) complexation of Cu(I).

The role of the redox protein thioredoxin in cell growth and cancer.

The thioredoxins are ubiquitous proteins containing a conserved -Trp-Cys-Gly-Pro-Cys-Lys- redox catalytic site. Mammalian thioredoxin family members include thioredoxin-1 (Trx1), mitochondrial thioredoxin-2 (Trx2), and a larger thioredoxin-like protein, p32TrxL. Thioredoxin is reduced by NADPH and thioredoxin reductase and, in turn reduces oxidized cysteine groups on proteins. When thioredoxin levels are elevated there is increased cell growth and resistance to the normal mechanism of programmed cell death. An increase in thioredoxin levels seen in many human primary cancers compared to normal tissue appears to contribute to increased cancer cell growth and resistance to chemotherapy. Mechanisms by which thioredoxin increases cell growth include an increased supply of reducing equivalents for DNA synthesis, activation of transcription factors that regulate cell growth, and an increase in the sensitivity of cells to other cytokines and growth factors. The mechanisms for the inhibition of apoptosis by thioredoxin are just now being elucidated. Because of its role in stimulating cancer cell growth and as an inhibitor of apoptosis, thioredoxin offers a target for the development of drugs to treat and prevent cancer.

The N-terminus of the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for both nucleotide binding and heterotropic effects.

X-ray crystallographic studies indicate that the N-terminal region of the regulatory chain in Escherichia coli aspartate transcarbamoylase resides close to the effector binding site. The proximity of the N-terminal region to the binding site suggests it may be important for nucleotide binding and, therefore, the heterotropic mechanism. The N-terminal region of the structure is not well-defined since the electron density in this region is weak, indicating a flexible and mobile region. Furthermore, alanine scanning mutagenesis of residues 2-7 indicated that the N-terminal region may be involved in nucleotide binding and the heterotropic mechanism, especially, UTP recognition [Dembowski, N., and Kantrowitz, E. R. (1994) Protein Eng. 7, 673-679]. In order to investigate further the role of the N-terminal region in the heterotropic mechanism, the first 10 N-terminal residues of the regulatory chain were deleted using site-specific mutagenesis. This mutant enzyme was compared to the wild-type enzyme, and both solubility and functional differences were observed. The mutant enzyme forms an insoluble aggregate which can be solubilized by the addition of nucleotides, such as CTP, suggesting that the exposed nucleotide binding site is involved in aggregate formation. Kinetic analyses of the mutant enzyme showed a lower maximal velocity and slightly lower aspartate affinity. Apparent binding constants determined for CTP, ATP, UTP, and CTP in the presence of UTP suggest the heterotropic response is also altered. This study suggests that the N-terminal region of the regulatory subunit is important for controlling nucleotide binding, creating the high-affinity and low-affinity effector binding sites, and coupling the binding sites within the regulatory dimer.

The catalysis of redox cycling by pyrroloquinoline quinone (PQQ), PQQ derivatives, and isomers and the specificity of inhibitors.

Pyrroloquinoline quinone (PQQ) is a widely distributed redox-active cofactor and essential nutrient. For its detection in protein-free ultrafiltrates or dialysates, a highly sensitive amplification assay was developed on the basis of PQQ's ability to catalyze redox cycling at pH 10 in the presence of excess glycine, oxygen, and nitro blue tetrazolium. Herein, we examine the propensities of PQQ, PQQ triester, and its various isomers, and certain PQQ triester derivatives, to catalyze glycine-fueled redox cycling and show that PQQ is the most capable of catalyzing redox cycling. Furthermore, PQQ has a unique pattern of inhibition induced by a series of PQQ antagonists of different potencies. The data indicate that putative PQQ from a biological sample, separated by HPLC and detected by the glycine-fueled redox-cycling assay, can be further identified as PQQ based on the profile of inhibition it displays with the antagonists such as those employed in this study. The methodology presented here should facilitate the specific detection of PQQ in biological samples.

The 2.46 A resolution structure of the pancreatic lipase-colipase complex inhibited by a C11 alkyl phosphonate.

Pancreatic lipase belongs to the serine esterase family and can therefore be inhibited by classical serine reagents such as diisopropyl fluoride or E600. In an attempt to further characterize the active site and catalytic mechanism, we synthesized a C11 alkyl phosphonate compound. This compound is an effective inhibitor of pancreatic lipase. The crystal structure of the pancreatic lipase-colipase complex inhibited by this compound was determined at a resolution of 2.46 A and refined to a final R-factor of 18.3%. As was observed in the case of the structure of the ternary pancreatic lipase-colipase-phospholipid complex, the binding of the ligand induces rearrangements of two surface loops in comparison with the closed structure of the enzyme (van Tilbeurgh et al., 1993b). The inhibitor, which could be clearly observed in the active site, was covalently bound to the active site serine Ser152. A racemic mixture of the inhibitor was used in the crystallization, and there exists evidence that both enantiomers are bound at the active site. The C11 alkyl chain of the first enantiomer fits into a hydrophobic groove and is though to thus mimic the interaction between the leaving fatty acid of a triglyceride substrate and the protein. The alkyl chain of the second enantiomer also has an elongated conformation and interacts with hydrophobic patches on the surface of the open amphipathic lid. This may indicate the location of a second alkyl chain of a triglyceride substrate. Some of the detergent molecules, needed for the crystallization, were also observed in the crystal. Some of them were located at the entrance of the active site, bound to the hydrophobic part of the lid. On the basis of this crystallographic study, a hypothesis about the binding mode of real substrates and the organization of the active site is proposed.

An inducible NADP(+)-dependent D-phenylserine dehydrogenase from Pseudomonas syringae NK-15: purification and biochemical characterization.

An inducible NADP(+)-dependent D-phenylserine dehydrogenase [EC 1.1.1.-], which catalyzes the oxidation of the hydroxyl group of D-threo-beta-phenylserine, was purified to homogeneity from a crude extract of Pseudomonas syringae NK-15 isolated from soil. The enzyme consisted of two subunits identical in molecular weight (about 31,000). In addition to D-threo-beta-phenylserine, it utilized D-threo-beta-thienylserine, D-threo-beta-hydroxynorvaline, and D-threonine as substrates but was inert towards other isomers of beta-phenylserine and threonine. It showed maximal activity at pH 10.4 for the oxidation of D-threo-beta-phenylserine, and it required NADP+ as a natural coenzyme. NAD+ showed a slight coenzyme activity. The enzyme was inhibited by p-chloromercuribenzoate, HgCl2, and monoiodoacetate but not by the organic acids such as tartronate. The Michaelis constants for D-threo-beta-phenylserine and NADP+ were 0.44 mM and 29 microM, respectively. The N-terminal 27 amino acids sequence was determined. It suggested that the NADP(+)-binding site was located in the N-terminal region of the enzyme.

Interaction of glyceraldehyde-3-phosphate dehydrogenase with AMP as studied by means of a spin-labeled analog.

The binding of a spin-labeled AMP analog to tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle is described. The spin label, perdeuterated and 15N-substituted 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, was attached to C-8 of AMP (C8-SL-AMP). Up to 8 equivalents of C8-SL-AMP bind per enzyme tetramer, i.e., 2 per monomer. Combining sites are the adenine subsite of the coenzyme-binding domain and the phosphate site. Glyceraldehyde 3-phosphate causes a conformational change in the enzyme that brings C8-SL-AMP molecules bound to adjacent R-axis-related subunits closer to one another by 0.2-0.3 nm and allows for spin-spin interaction between the nitroxide radicals. Similar, but less pronounced structural changes take place upon lowering the pH from 8 to 7. Addition of a single equivalent of NAD+ to a complex of the enzyme with 7.6 equivalents of C8-SL-AMP leads to the release of almost 4 C8-SL-AMP molecules. This supports our previous findings that binding of just one NAD+ molecule induces conformational changes in all four subunits.

Changes in serum copper and zinc during treatment with anticancer drugs interfering with pyridoxal phosphate.

Hexamethylmelamine, pentamethylmelamine and procarbazine are anticancer drugs known to interfere with pyridoxal phosphate. This paper presents results on copper and zinc serum levels during the treatment with each of these drugs used as single agents. Six NZW rabbits weighing 2.7-4.5 kg were used in these experiments. Hexamethylmelamine and procarbazine were administered by gastric gavage and pentamethylmelamine by intravenous route at the daily doses of 100 mg, 30 mg and 50 mg/kg of body weight respectively for up to four days. Blood samples were collected in metal free tubes at fasting state before and during the treatment. Student's paired t-test was used for statistical analysis. The pretreatment serum copper concentration significantly (p = 0.05) increased and conversely the serum zinc concentration significantly (p = 0.05) decreased during each drug treatment. Consequently the copper/zinc ration significantly increased from 0.32, 0.33 and 0.27 to 1.16, 0.63 and 1.13 for hexamethylmelamine, pentamethylmelamine and procarbazine respectively. These results indicate, that daily administration of three anticancer drugs interfering with pyridoxal phosphate causes changes in serum copper and zinc levels with inversed relationship between both changes.

Inhibition of coenzyme activation of aspartate aminotransferase.

Forty compounds were surveyed for their effect on the activation of pig heart apoaspartate aminotransferase by pyridoxamine 5'-phosphate. Most of the nucleotides, sugar phosphates, coenzymes, phospholipid precursors and inorganic oxyanions tested were found to be inhibitory. With few exceptions, the only requirement for a substance to be inhibitory is the presence of a di- or polyanionic moiety analogous to the 5'-phosphate group of the cofactor. In spite of the lack of overall structural similarity to pyridoxamine 5'-phosphate, inorganic pyrophospate and apparently other inhibitors are characterized by dissociation constants comparable in magnitude to that previously reported for the natural cofactor. The physiological significance of the inhibition of coenzyme activation of apoaspartate aminotransferase by these common biological compounds is not known.