Molecular cloning,expression and characterization of Pekin duck interferon-λ

Interferons (IFNs) are the first line of defense against viral infections in vertebrates. Type III interferon (IFN-λ) is recognized for its key role in innate immunity of tissues of epithelial origin. Here we describe the identification of the Pekin duck IFN-λ ortholog (duIFN-λ). The predicted duIFN-λ protein has an amino acid identity of 63%, 38%, 37% and 33% with chicken IFN-λ and human IFN-λ3, IFN-λ2 and IFN-λ1, respectively. The duck genome contains a single IFN-λ gene that is comprised of five exons and four introns. Recombinant duIFN-λ up-regulated OASL and Mx-1 mRNA in primary duck hepatocytes. Our observations suggest evolutionary conservation of genomic organization and structural features implicated in receptor binding and antiviral activity. The identification and expression of duIFN-λ will facilitate further study of the role of type III IFN in antiviral defense and inflammatory responses of the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.     

Backbone resonance assignment of Alt a 1, a unique β-barrel protein and the major allergen of Alternaria alternata

Alt a 1 is the major allergen of the fungus Alternaria alternata and can be found in the cell wall of its spores. It is a cysteine linked homodimeric protein with a unique β-barrel fold as recently revealed by X-ray crystallography. Despite the elucidation of its structure, its biological function remains unknown. For Alternaria-sensitized patients, contact leads to respiratory allergy and in severe cases to asthma-related death. Here we report the sequence-specific Alt a 1 backbone ¹H, ¹⁵N and ¹³C chemical shift assignment.     

Kinetic method for differentiating mechanisms for ligand exchange reactions: application to test for substrate channeling in glycolysis.

We have derived analytical expressions for the kinetics of the two mechanisms involved in ligand substitution reactions. These mechanisms are (i) a dissociative mechanism in which the leaving ligand is first dissociated prior to the binding of the incoming ligand and (ii) an associative mechanism where a ternary complex is formed between the incoming ligand and the complex containing the leaving ligand. The equations obtained provide the theoretical basis for differentiating these two mechanisms on the basis of their kinetic patterns of the displacement reactions. Analysis of these equations shows that an associative mechanism can only generate an increasing kinetic pattern for the observed pseudo-first-ordered rate constants as a function of increasing concentration of the incoming ligand and plateaus, in most cases, at a value higher than the off-rate constant of the leaving ligand. However, a dissociative mechanism can generate either an increasing or a decreasing (kapp decreases with increasing concentrations of the incoming ligand) kinetic pattern, depending on the magnitudes of the individual rate constants involved, and, in either case, it will plateau at kapp equal to the koff of the leaving ligand. Therefore, the decreasing kinetic pattern is a hallmark for a dissociative mechanism. This general method was used to settle the dispute of whether NADH is transferred directly via the enzyme-enzyme complex between glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27).(ABSTRACT TRUNCATED AT 250 WORDS)     

The cooperative binding of fructose-1,6-bisphosphate to yeast pyruvate kinase.

The cooperative binding of the allosteric activator fructose-1,6-bisphosphate [Fru(1,6)P2] to yeast pyruvate kinase was investigated by equilibrium dialysis and fluorescence quench titration. The results show that yeast pyruvate kinase binds four molecules of Fru(1,6)P2 per tetramer and the observed fluorescence quench follows the binding of the ligand and not the cooperative T to R state transition. Additionally it is shown that the binding of Fru(1,6)P2 to yeast pyruvate kinase is compatible with the model of cooperativity that has been proposed and incorporates an intermediate state, R', with properties between those of the T and R states.     

The resolution of some steps of the reactions of lactate dehydrogenase with its substrates

1. The reaction of pig heart lactate dehydrogenase (EC 1.1.1.27) with NAD(+) and lactate to form pyruvate and NADH was followed by rapid spectrophotometric methods. The distinct spectrum of enzyme-bound NADH permits the measurement of the rate of dissociation of this compound. 2. The reduction of the first mole equivalent of NAD(+) per mole of enzyme sites can also be observed, and is much more rapid than the steady-state rate of NADH production. 3. At pH8 the dissociation of the enzyme-NADH complex is rate-determining for the steady-state oxidation of lactate. At lower pH some other step after the interconversion of the ternary complex and before the dissociation of NADH is rate-determining. Other evidence for a compulsory-order mechanism is provided.