1. Sheep liver cytoplasmic aldehyde dehydrogenase showed little pH dependence of V or k,,,. Some buffer anion effects were noted.2. The oxidation of aldehydes at pH 7.6 was quantitative but irreversible. The initial velocity data indicated a sequential mechanism for the addition of substrates. Inhibition by NADH and the product analogue 2-bromo-2-phenylacetic acid, together with the known tight binding of NADH to the free enzyme, indicated an ordered mechanism with NAD+ as leading substrate.3 . Values for the rate of binding and dissociation of NAD+ were obtained from the steady-state data. The values obtained were virtually identical with those which could be calculated from the data for the horse liver cytoplasmic enzyme. Close similarities are in general apparent for the horse and sheep liver cytoplasmic enzymes and with other tissue aldehyde dehydrogenases.4. Apparent substrate activation was observed with high concentrations of both acetaldehyde and propionaldehyde, a limiting value of 0.25 spl being obtained for k,,,. No isotope effect was observed on V using [l-'Hlpropionaldehyde as substrate suggesting that NADH release might be rate-limiting in the steady-state.5. The implications of the non-linear steady-state behaviour are discussed.In recent years aldehyde dehydrogenases from various sources have been purified to homogeneity [1,2]. More recently the purification of sheep liver cytoplasmic and mitochondrial enzymes has been reported [3] and the two major isozymes of aldehyde dehydrogenase from horse liver, which also appear to be cytoplasmic and mitochondrial in origin, have been purified and characterised [4]. An enzyme from human liver has also been studied [ 5 ] and the kinetics of aldehyde oxidation investigated, although its cellular localization is unknown.All the kinetic investigations so far reported suggest sequential mechanisms but the order of substrate addition varies from one enzyme to another. Pig brain, horse liver and bovine liver enzymes have been reported to follow compulsory order mechanisms in which NAD+ binds before aldehyde [2,6-8], but evidence has also been presented for the yeast enzyme [9] which indicates an ordered mechanism in which aldehyde binds first.The human liver enzyme appears to be partially random in its addition of its substrates, although most of the reaction is considered to proceed by means of an initial binary NAD' . enzyme complex [5]. In view of the importance of these enzymes in an understanding of human alcohol metabolism we have undertaken a detailed kinetic study of sheep liver cytoplasmic aldehyde dehydrogenase in order to determine the steady-state parameters for the reaction. MATERIALS AND METHODS MaterialsNADH (grade 111) and NAD' (grade 111) were obtained from Sigma Chemical Co. (St Louis, Mo., U.S.A.). The NADH was obtained as preweighed vials and the concentration was checked by absorbance measurements at 340 nm assuming a molar absorption coefficient (8) of 6 . 2 2~ lo3 M-l cm-I [lo]. NADt was used without further purification since no detectable...

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The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.

A transient release of protons with an amplitude corresponding to one proton per active site has been observed for the oxidation of propionaldehyde, acetaldehyde, and benzaldehyde by sheep liver cytoplasmic aldehyde dehydrogenase at pH 7.6 with phenol red as indicator. At saturating substrate levels, the rate constants for the proton burst are in each case the same, and for acetaldehyde and propionaldehyde show the same dependence on the concentrations of the substrates, as the rate constants for the transient production of NADH reported previously [MacGibbon, A.K.H., Blackwell, L.F., & Buckley, P.D. (1977) Biochem. J. 167, 469-477]. Although, with propionaldehyde as a substrate, a full proton burst is also observed at pH 6.0, no proton burst is observed at pH 9.0. For 4-nitrobenzaldehyde, there is no burst in NADH production, but a burst in proton release is observed, showing that proton release precedes hydride transfer. No protons were released during the binding of the substrate analogues acetone and chloral hydrate nor on reaction of the enzyme with the inhibitor tetraethylthiuram disulfide (disulfiram). A model is proposed in which the rate-limiting step in the pre-steady-state phase of the reaction is a conformational change which occurs after the binding of aldehydes to the enzyme. As a result of the conformational change, the environment of a functional group on the enzyme, which initially has a pKa of about 8.5, is perturbed to give a final pKa value for the group of less than 5. Computer simulations were used to show that the model accurately reproduces all of the experimental data. The lack of observation of a second transient proton release, as required by the overall stoichiometry, argues that its release occurs in a slow step prior to NADH dissociation.

An in-vitro culture system was developed in which primary mouse follicles from 12-16-day-old mice grew to the preovulatory stage. The important determinants of growth in culture were the inclusion of stroma with the primary follicles, the age of the mouse, the presence of FSH and LH, the use of culture dishes with a hydrophobic membrane and the use of post-menopausal human serum to supply growth factors. During culture the pieces of ovarian tissue containing the primary follicles coalesced to form characteristic spherical clusters. The cultured follicles appeared to be normal as determined by the appearance and organization of the granulosa cells, the appearance of the antrum and the accompanying steroidogenesis, but the ova had not resumed meiosis. The results show that the growth of mouse follicles starting from the primary stage is critically dependent on adequate concentrations of FSH.