Abstract: Equilibrium constants for coenzyme binding to liver alcohol dehydrogenase have been determined over the pH range 10--12 by pH-jump stop-flow techniques. The binding of NADH or NAD+ requires the protonated form of an ionizing group (distinct from zinc-bound water) with a pKa of 10.4. Complex formation with NADH exhibits an additional dependence on the protonation state of an ionizing group with a pKa of 11.2. The binding of trans-N,N-dimethylaminocinnamaldehyde to the enzyme . NADH complex is prevented by ionization of the latter group. It is concluded from these results that the pKa-11.2-dependence of NADH binding most likely derives from ionization of the water molecule bound at the catalytic zinc ion of the enzyme subunit. The pKa value of 11.2 thus assigned to zinc-bound water in the enzyme . NADH complex appears to be typical for an aquo ligand in the inner-sphere ligand field provided by the zinc-binding amino acid residues in liver alcohol dehydrogenase. This means that the pKa of metal-bound water in zinc-containing enzymes can be assumed to correlate primarily with the number of negatively charged protein ligands coordinated by the active-site zinc ion.

Abstract: Chemical relaxation studies on the system horse liver alcohol dehydrogenase, nicotinamide adenine dinucleotide, and ethanol were conducted observing fluorescence changes between 400 and 500 nm. Temperature-jump experiments were performed at pH 6.5, 7.0, 8.0, and 9.0; concentration-jump experiments at pH 9.0. The reciprocal of the slowest relaxation time was found to be linearly dependent upon the enzyme concentration for relatively low enzyme concentrations, as predicted earlier. Use of the wide pH-range necessitated expression of the four apparent dissociation constants of the catalytic reaction cycle in terms of pH-independent constants. The system was described in terms of only one (or two) catalysis-linked protons not associated with the electron transfer. Protonic steps in a buffered system are in rapid equilibrium, too fast to be measured with the equipment available. Assuming only two of the four bimolecular reaction steps in the four-step cycle are fast compared to the remaining two, six cases may be considered with six expressions for the reciprocal of the slowest relaxation time. Comparison with the experimental data revealed that the bimolecular reaction steps governing the slowest relaxation time change with pH. Above the effective time resolution of the temperature-lump instrument with fluorescence detection (0.1 msec) only one other relaxation time was detectable and only at pH 9. This relaxation time, found to be independent of the concentration of all reactants within experimental error (r = 10 +/- 5 msec), is most likely due to an interconversion among ternary complexes.

Abstract: Several years ago, Theorell and Czerlinski conducted experiments on the system of horse liver alcohol dehydrogenase, reduced nicotinamide adenine dinucleotide and imidazole, using the first version of the temperature jump apparatus with detection of changes in fluorescence. These early experiments were repeated with improved instrumentation and confirmed the early experiments in general terms. However, the improved detection system allowed to measure a slight concentration dependence of the relaxation time of around 3 ms. Furthermore, the chemical relaxation time was smaller than the one determined earlier (by factor 2). The data were evaluated much more rigorously than before, allowing an appropriate interpretation of the results. The observed relaxation time is largely due to rate constants in an interconversion of ternary complexes, which are faster than three (of the four) dissociation rate constants, determined previously by Theorell and McKinley-McKee.1,2 This fact contributed to earlier difficulties of finding any concentration dependence. However, the binding of imidazole to the binary enzyme-coenzyme complex can be made to couple kinetically into the interconversion rate of the two ternary complexes. The observed signal derives largely from the ternary complex(es). A substantial fluorescence signal change is associated with the observed relaxation process, suggesting a relocation of the imidazole in reference to the nicotinamide moiety of the bound coenzyme. Nine models are considered with two types of coupling of pre-equilibria (none-all). Quantitative evaluations favor the model with two ternary complexes connected by an interconversion outside the four-step (bimolecular) cycle. The ternary complex outside the cycle has much higher fluorescence yield than the one inside. The interconversion equilibrium is near unity for imidazole. If it would be shifted very much to the side of the “dead-end” complex (as in isobutyramide?!), stimulating action could not take place.

Abstract: The length of test period of stationary performance testing (SPT) of stallions of the Dutch Warmblood riding horse population is evaluated. Heritability of successive judgements of traits and the phenotypic and genetic relations between successive judgements are estimated. Data from 1983-1988 are used, covering scores from 206 mostly 3-year-old stallions. Ten subjectively scored traits are considered: walk, trot, canter, riding ability, jumping ability, free jumping, cross country, character, stable behaviour, training report. Traits are successively scored at about 25, 50, 80 and 100 days in SPT. Missing scores are predicted on basis of the available scores using multiple partial regression coefficients. Validity of this method is checked in an independent data set for walk, trot and canter. The correlations between predicted and realized scores average 0.74, 0.77 and 0.79 when first, first and second, and first, second and third judgements are available, respectively. Variance and covariance components are estimated by restricted maximum likelihood (REML) procedures. Data from SPT are analysed using an animal model. Estimates of heritability are high and constant for gaits during the successive judgements. Except for stable behaviour estimates of heritability, the traits decrease slightly during the successive judgements. Estimates of the phenotypic and genetic correlations between successive judgements are high. It is concluded that length of SPT can be shortened and selection during SPT can be intensified.

Abstract: The objective of this study is to estimate several genetic parameters in the Dutch Warmblood riding horse population. The traits involved are performances in jumping and dressage competition. The following parameters are estimated: heritabilities for jumping and dressage; phenotypic and genetic correlations between jumping and dressage; and phenotypic and genetic correlations between performances at different ages. These parameters are estimated by restricted maximum likelihood (REML). Data are from 6899 horses with performances in jumping and 10 408 horses with performances in dressage competition. The horses are sired by 205 and 237 stallions for the two traits, respectively. The progeny range in age from 4 to 8 years old. The performance trait is a cumulatively derived score, that reflects the level of performance in competition. A square root transformation of the score is most appropriate to normalize the data. For estimation of phenotypic and genetic parameters the data is split into two data sets according to the age of the sires (offspring sired by older vs. younger stallions). For estimating correlations between performances at 4, 5 and 6 years of age, performances of the offspring out of previous years are linked to the data. The most unbiased estimates of heritability for jumping and dressage are from data derived from the youngest offspring sired by the younger stallions and are 0.20 and 0.10, respectively. Genetic correlation between jumping and dressage ranges from -0.27 to 0.10. The phenotypic correlation between these traits ranges from 0.15 to 0.26. Phenotypic and genetic correlations between performances at 4, 5 and 6 years average 0.95 and 0.75, respectively. These latter results have important implications for genetic evaluation of breeding candidates in the population.