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: Horse liver alcohol dehydrogenase has been claimed to exhibit presteady-state “half-of-the-sites” reactivity with aromatic substrates under some circumstances. To clarify the role of half-of-the-sites reactivity in liver alcohol dehydrogenase the direct sampling of the alcohol product formed immediately after initiation of the reaction was studied using a rapid sampling device and [4α-3H]NADH. Liver alcohol dehydrogenase which contained a very low mole-ratio of [4α-3H]NADH bound to one subunit of the dimer was rapidly mixed with excess 4-(2'-imidazolylazo)benzaldehyde substrate and nonradioactive NADH to initiate the reaction, which was allowed to proceed for a short time before it was quenched. If strong HClO4 quench was used isolation of total free and bound azoalcohol product was possible. If NaOH quench was used isolation only of the azoalcohol product released by the enzyme was possible since most enzyme-bound azoalcohol was reversed back to azoaldehyde by the base. The pH-jump reversal reaction also was characterized spectroscopically by stopped flow technique. Nearly fullsites reactivity was observed for reaction in either direction. Furthermore (4α-3H]NADH bound firstly to one subunit in the dimer reacted essentially identically to NADH bound secondly to the other subunit. Thus, half-of-the-sites reactivity was not observed in these experiments nor did they give any indication of liver alcohol dehydrogenase active site nonequivalence induced by coenzyme binding or reaction.

Abstract: Twenty-six yearling horses were tested on a serial reversal learning discrimination combining spatial and brightness cues. An original discrimination of rewarded or nonrewarded stimuli was made followed by 20 daily reversals for position/brightness discrimination. Learning criteria were defined as 11 out of 12 correct, with the last eight responses all correct. Each horse was allowed 30 trials per discrimination to achieve criteria. Mean errors (ME) and mean trials (MT) required to achieve criteria were computed for each horse. A relative learning ability index (LAI) was calculated by the formula 1000/MT/ME. A daily emotionality score, based on a scale of one (tranquil) to six (very excitable) was assigned each horse each day after testing and a mean computed for each horse. A single subjective trainability score, based on a scale of one (difficult to train) to six (easy to train) was obtained for each horse from an independent trainer. Linear regression analyses for all 26 horses revealed a reduction in MT and ME (P<.01) over the 21-day test period indicating evidence of learning to learn. Differences (P<.05) were evident between sexes for MT and ME. Significant correlations between trainability scores and learning ability indices MT, ME, and LAI were evident for colts and geldings but not for fillies. Pooled data showed significant correlations between ME and trainability. There was a negative correlation (P<.05) between emotionality and trainability scores for all 26 horses, although the filly group did not exhibit significant correlation between these parameters.