Thallium activation and inhibition of yeast aldehyde dehydrogenase

The activity, stability and spectroscopic properties of yeast K+ -activated aldehyde dehydrogenase were measured at various times after removal from, and after returning to a solution containing K+. Enzyme activity is rapidly lost on removal of most of the K+ and rapidly regained if K+ is replaced immediately. These activity changes are slower than likely rates of K+ dissociation and association. These rapid changes in concentration result in altered enzyme stability with enzyme in K+ the more stable. U.v. difference spectra are produced whenever enzyme in an activating environment (K+ or Tl+) is compared with enzyme in a non-activating environment (Tris+ or Li+). These spectral changes occur within 10s. The saturation characteristics with K+ are hyperbolic for all three phenomena of activation, stabilization and spectral change, with estimated apparent dissociation constants (Ks) for K+ of 7.5 mM, 5.5 mM and 6 mM respectively. Continued incubation of enzyme in the absence of K+ results in the accumulation of an enzyme form that re-activates only slowly on replacing K+. Stability characteristics in various concentrations of K+ over equivalent time scales are consistent with the existence of additional conformations. Spectroscopic evidence also indicates such additional slow conformation changes. Results have been interpreted in terms of two separate conformation transitions induced or stabilized by K+.

Section: Cation-induced Spectral Changesmentioning

confidence: 99%

“…Here a difference spectrum can still be seen, though much suppressed. This reflects the higher affinity of enzyme for Tl+ (Bostian et al, 1975); that is, there is more Vol. 183 active enzyme in the TlV cell.…”

Section: Cation-induced Spectral Changesmentioning

confidence: 99%

Kinetic and spectroscopic evidence of cation-induced conformation changes in yeast K+-activated aldehyde dehydrogenase

Betts¹,

Poole²,

Springham³

et al. 1979

“…1800 spectrophotometer, or for experiments involving benzaldehyde as substrate, by monitoring NADH production fluorimetrically in a double monochromator spectrofluorimeter. The standard spectrophotometric assay described in detail elsewhere (Bostian et al, 1975;Bostian & Betts, 1978) varied here only in the type of aldehyde or coenzyme and concentrations of these substrates and of K+. In monitoring NADH fluorescence, assays were performed in a total reaction mixture volume of 0.5 ml in quartz Spectrosil fluorescence cells (5mm x 5mm x 40 mm).…”

Section: Methodsmentioning

confidence: 99%

“…Aldehyde dehydrogenase concentration was determined by enzyme activity calculated from a specific activity of 34 units/mg under standard assay conditions (Bostian et al, 1975;Bostian & Betts, 1978); 1 enzyme unit transforms 1,umol of substrate/min. The molarity of enzyme active sites was based on four active sites per molecule of mol.wt.…”

Section: Methodsmentioning

confidence: 99%

Kinetics and reaction mechanism of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae

Bostian¹,

Betts²

1978

Data from steady-state kinetic analysis of yeast K+-activated aldehyde dehydrogenase are consistent with a ternary complex mechanism. Evidence from alternative substrate analysis and product-inhibition studies supports an ordered sequence of substrate binding in which NAD+ is the leading substrate. A preincubation requirement for NAD+ for maximum activity is also consistent with the importance of a binary enzyme-NAD+ complex. Dissociation constant for enzyme-NAD+ complex determined kinetically is in reasonable agreement with that determined by direct binding. The order of substrate addition proposed here differs from that proposed for a yeast aldehyde dehydrogenase previously reported. Different methods of purification produced an enzyme that showed similar kinetic characteristics to those reported here.

“…pyruvate kinase (Kayne & Reuben, 1970;Reuben & Kayne, 1971), Na+ + K+-activated ATPase (Grisham et al, 1974), the ionophore lasalocid, X-537A (Briggs et al, 1980), and gramicidin (Turner et al, 1982;Hinton et al, 1982). Such studies are possible because Tl + can often replace K + ions (Britten & Blank, 1968;Inturisi, 1969a,b;Robinson, 1970;Kayne, 1971;Bostian et al, 1975Bostian et al, , 1982. In addition, T1+ has been suggested as a fluorescence probe (Cornelius et al, 1974;Manners et al, 1971).…”

mentioning

confidence: 99%

Arylthallium(III) reagents for protein modification. Inhibition of lactate dehydrogenase from various sources by o-carboxyphenylthallium(III) bistrifluoroacetate

Bunni¹,

Douglas²

1984

The use of organothallium compounds for protein/macromolecule modification and as probes for n.m.r. and fluorescence is introduced. Lactate dehydrogenase from a number of species was rapidly and specifically inhibited by o-carboxyphenylthallium(III) bistrifluoroacetate and p-methylphenylthallium(III) bistrifluoroacetate. Inhibition of rabbit muscle lactate dehydrogenase by o-carboxyphenylthallium(III) bistrifluoroacetate was time-dependent and not reversible by gel filtration. A small degree of re-activation was possible by incubation with dithiothreitol. The time course of the inactivation kinetics showed two phases, only the first, and faster, of which was efficiently prevented by the presence of cofactor, NADH. Inhibition rates depended on the structure of the thallium reagent, its concentration and the temperature. No significant inhibition was found by thallous acetate or thallic trifluoroacetate. Saturation kinetics were observed for the inhibition by o-carboxyphenylthallium(III) bistrifluoroacetate of the pig heart enzyme. The possibilities of various cross-linking activities of these reagents are addressed. Mechanisms of the inhibition are discussed.