Abstract: 1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (H2NADH) has been investigated as a reduced coenzyme analog in the reaction between trans-4-N,N-dimethylaminocinnamaldehyde (I) (lambdamax 398 nm, epsilonmax 3.15 X 10-4 M-minus 1 cm-minus 1) and the horse liver alcohol dehydrogenase-NADH complex. These equilibrium binding and temperature-jump kinetic studies establish the following. (i) Substitution of H2NADH for NADH limits reaction to the reversible formation of a new chromophoric species, lambdamax 468 nm, epsilonmax 5.8 x 10-4 M-minus 1 cm-minus 1. This chromophore is demonstrated to be structurally analogous to the transient intermediate formed during the reaction of I with the enzyme-NADH complex [Dunn, M. F., and Hutchison, J. S. (1973), Biochemistry 12, 4882]. (ii) The process of intermediate formation with the enzyme-NADH complex is independent of pH over the range 6.13-10.54. Although studies were limited to the pH range 5.98-8.72, a similar pH independence appears to hold for the H2NADH system. (iii) Within the ternary complex, I is bound within van der Waal's contact distance of the coenzyme nicotinamide ring. (iv) Formation of the transient intermediate does not involve covalent modification of coenzyme. Based on these findings, we conclude that zinc ion has a Lewis acid function in facilitating the chemical activation of the aldehyde carbonyl for reduction, and that reduced coenzyme plays a noncovalent effector role in this substrate activating step.

Abstract: The pregnane X receptor (PXR) is a ligand-activated transcription factor and member of the nuclear receptor superfamily. Activation of PXR represents an important mechanism for the induction of cytochrome P450 3A (CYP3A) enzymes that can convert acetaminophen (APAP) to its toxic intermediate metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Therefore, it was hypothesized that activation of PXR plays a major role in APAP-induced hepatotoxicity. Pretreatment with the PXR activator, pregnenolone 16alpha-carbonitrile (PCN), markedly enhanced APAP-induced hepatic injury, as revealed by increased serum ALT levels and hepatic centrilobular necrosis, in wild-type but not in PXR-null mice. Further analysis showed that following PCN treatment, PXR-null mice had lower CYP3A11 expression, decreased NAPQI formation, and increased maintenance of hepatic glutathione content compared to wild-type mice. Thus, these results suggest that PXR plays a critical role in APAP-induced hepatic toxicity, probably by inducing CYP3A11 expression and hence increasing bioactivation.

Abstract: The Federation Equestre Internationale has permitted the use of altrenogest in mares for the control of oestrus. However, altrenogest is also suspicious to misuse in competition horses for its potential anabolic effects and suppression of typical male behaviour, and thus is a controlled drug. To investigate the pharmacokinetics of altrenogest in horses we conducted an elimination study. Five oral doses of 44 mug/kg altrenogest were administered to 10 horses at a dose interval of 24 h. Following administration blood and urine samples were collected at appropriate intervals. Altrenogest concentrations were measured by liquid chromatography-tandem mass spectrometry. The plasma levels of altrenogest reached maximal concentrations of 23-75 ng/mL. Baseline values were achieved within 3 days after the final administration. Urine peak concentrations of total altrenogest ranged from 823 to 3895 ng/mL. Twelve days after the final administration concentrations were below the limit of detection (ca 2 ng/mL).

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 effect of His-heme misligation on folding has been investigated for a triple mutant of yeast iso-2 cytochrome c (N26H,H33N,H39K iso-2). The variant contains a single misligating His residue at position 26, a location at which His residues are found in several cytochrome c homologues, including horse, tuna, and yeast iso-1. The amplitude for fast phase folding exhibits a strong initial pH dependence. For GdnHCl unfolded protein at an initial pH<5, the observed refolding at final pH 6 is dominated by a fast phase (tau(2f)=20 ms, alpha(2f)=90 %) that represents folding in the absence of misligation. For unfolded protein at initial pH 6, folding at final pH 6 occurs in a fast phase of reduced amplitude (alpha(2f) approximately 20 %) but the same rate (tau(2f)=20 ms), and in two slower phases (tau(m)=6-8 seconds, alpha(m) approximately 45 %; and tau(1b)=16-20 seconds, alpha(1b) approximately 35 %). Double jump experiments show that the initial pH dependence of the folding amplitudes results from a slow pH-dependent equilibrium between fast and slow folding species present in the unfolded protein. The slow equilibrium arises from coupling of the His protonation equilibrium to His-heme misligation and proline isomerization. Specifically, Pro25 is predominantly in trans in the unligated low-pH unfolded protein, but is constrained in a non-native cis isomerization state by His26-heme misligation near neutral pH. Refolding from the misligated unfolded form proceeds slowly due to the large energetic barrier required for proline isomerization and displacement of the misligated His26-heme ligand.