scholarly journals A comparison of nitrophenyl esters and lactones as substrates of cytosolic aldehyde dehydrogenase

1996 ◽  
Vol 316 (1) ◽  
pp. 225-232 ◽  
Author(s):  
Trevor M. KITSON ◽  
Kathryn E. KITSON
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Visible Region ◽  
Stopped Flow ◽  
Site Model ◽  
Rate Determining Step ◽  
Steady State Rate ◽  
State Rate ◽  
Reporter Group ◽  
Hydrolysis Of

1. p-Nitrophenyl (PNP) acetate and propionate show a burst of p-nitrophenoxide release when their hydrolysis is catalysed by sheep liver cytosolic aldehyde dehydrogenase. This is not seen in the presence of NAD+ or NADH, implying a change in rate-determining step. 2. 6-Nitrodihydrocoumarin (6-NDC) shows no burst of absorbance in the visible region. We propose that the pKa of the transient ‘reporter group’ produced during the hydrolysis of this lactone is high (approx. 10) and that the incipient covalently linked p-nitrophenoxide moiety is protonated immediately on formation. The small burst seen in the hydrolysis of 5-nitro-2-coumaranone (5-NC) suggests that the pKa of its reporter group is about 8.5. 3. NADH markedly enhances the steady-state rate with the lactones. 5-NC shows a large rapid burst of colour development in the presence of NADH; this implies that NADH decreases the pKa of the reporter group to 7–7.5. 4. In the presence of NAD+, 5-NC and 6-NDC give an unusual ‘negative burst’ in the stopped-flow traces. We propose that, under these circumstances, acylation of the enzyme is extremely fast and that the first event seen in the stopped-flow traces is protonation of the reporter group. NAD+ also greatly increases the steady-state rate. 5. With the lactones in the presence of NADH, the kcat value (nearly 6 s-1), a measure of the deacylation rate, is compatible with the single-site model for dehydrogenase and esterase activities.

scholarly journals Effect of pyrophosphate ions and alkaline pH on the kinetics of propionaldehyde oxidation by sheep liver cytosolic aldehyde dehydrogenase

1991 ◽  
Vol 273 (3) ◽  
pp. 691-693 ◽  
Author(s):  
J P Hill ◽  
P D Buckley ◽  
L F Blackwell ◽  
R L Motion
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Alkaline Ph ◽  
Activation Effect ◽  
Ph Values ◽  
Sheep Liver ◽  
Steady State Rate ◽  
State Rate ◽  
Rate Limiting ◽  
Kinetics Of

Pyrophosphate ions activate the steady-state rate of oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase at alkaline pH values. The steps in the mechanism governing the release of NADH from terminal enzyme. NADH complexes have been shown to be rate-limiting at pH 7.6 [MacGibbon, Buckley & Blackwell (1977) Biochem J. 165, 455-462]. These steps are shown to be also rate-limiting at more alkaline pH values, and it is through an acceleration of these steps that pyrophosphate ions exert their activation effect.

scholarly journals Further studies of the action of disulfiram and 2,2′-dithiodipyridine on the dehydrogenase and esterase activities of sheep liver cytoplasmic aldehyde dehydrogenase

1982 ◽  
Vol 203 (3) ◽  
pp. 743-754 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Chloral Hydrate ◽  
Enzyme Molecule ◽  
Catalysed Reaction ◽  
Dehydrogenase Reaction ◽  
Steady State Rate ◽  
State Rate ◽  
Usual Site ◽  
Esterase Activities

1. Pre-modification of cytoplasmic aldehyde dehydrogenase by disulfiram results in the same extent of inactivation when the enzyme is subsequently assayed as a dehydrogenase or as an esterase. 2. 4-Nitrophenyl acetate protects the enzyme against inactivation by disulfiram, particularly well in the absence of NAD+. Some protection is also provided by chloral hydrate and indol-3-ylacetaldehyde (in the absence of NAD+). 3. When disulfiram is prevented from reacting at its usual site by the presence of 4-nitrophenyl acetate, it reacts elsewhere on the enzyme molecule without causing inactivation. 4. Enzyme in the presence of aldehyde and NAD+ is not at all protected against disulfiram. It is proposed that, under these circumstances, disulfiram reacts with the enzyme-NADH complex formed in the enzyme-catalysed reaction. 5. Modification by disulfiram results in a decrease in the amplitude of the burst of NADH formation during the dehydrogenase reaction, as well as a decrease in the steady-state rate. 6. 2,2′-Dithiodipyridine reacts with the enzyme both in the absence and presence of NAD+. Under the former circumstances the activity of the enzyme is little affected, but when the reaction is conducted in the presence of NAD+ the enzyme is activated by approximately 2-fold and is then relatively insensitive to the inactivatory effect of disulfiram. 7. Enzyme activated by 2,2′-dithiodipyridine loses most of its activity when stored over a period of a few days at 4 degrees C, or within 30 min when treated with sodium diethyldithiocarbamate. 8. Points for and against the proposal that the disulfiram-sensitive groups are catalytically essential are discussed.

A New Method for Determining Individual Rate Constants for Specific Non-chromophoric Substrates of Proteolytic Enzymes

1975 ◽  
Vol 53 (4) ◽  
pp. 564-571
Author(s):  
Lewis J. Brubacher
Keyword(s):  
Steady State ◽  
Rate Constants ◽  
Proteolytic Enzymes ◽  
Enzyme Mechanism ◽  
Steady State Kinetics ◽  
Steady State Rate ◽  
State Rate ◽  
Individual Rate ◽  
Kinetics Of ◽  
Hydrolysis Of

Equations are developed for the pre-steady state kinetics of the proteolytic enzyme-catalyzed hydrolysis of a substrate A in the presence of a monitoring substrate (or covalent inhibitor) S of known properties. A two-intermediate acyl–enzyme mechanism is assumed in which the first intermediate is in instantaneous equilibrium with enzyme and substrate. The appearance of the first product of substrate S is characterized by two relaxation rate constants. From these constants it is possible to determine the dissociation constant and the acylation and deacylation rate constants of substrate A. Criteria are also developed for using the steady state rate parameters of A to establish conditions for which the slower relaxation process is equivalent to the deacylation rate constant of A. The technique of premixing enzyme with substrate A has certain advantages in this approach.

scholarly journals Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase

1978 ◽  
Vol 171 (3) ◽  
pp. 533-538 ◽  
Author(s):  
A K H MacGibbon ◽  
S J Haylock ◽  
P D Buckley ◽  
L F Blackwell
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Sheep Liver ◽  
Rate Limiting Step ◽  
Dehydrogenase Reaction ◽  
Steady State Rate ◽  
Michaelis Constants ◽  
State Rate ◽  
Liver Aldehyde Dehydrogenase

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.

scholarly journals The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step

1989 ◽  
Vol 259 (3) ◽  
pp. 709-713 ◽  
Author(s):  
E Rigney ◽  
T J Mantle ◽  
F M Dickinson
Keyword(s):  
Steady State ◽  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Protein Fluorescence ◽  
Biliverdin Reductase ◽  
Rate Determining Step ◽  
Steady State Rate ◽  
State Rate ◽  
Kinetics Of

When the production of bilirubin by biliverdin reductase was monitored at 460 nm by stopped-flow spectrophotometry a ‘burst’ was observed with a first-order rate constant at pH 8 of 20 s-1. The steady-state rate was established on completion of the ‘burst’. When the reaction was monitored at 401 nm there was no observed steady-state rate, but a diminished pre-steady-state ‘burst’ reaction was still seen with a rate constant of 22 s-1. We argue that the rate-limiting reaction is the dissociation of bilirubin from an enzyme.NADP+.bilirubin complex. With NADPH as the cofactor the hydride-transfer step was shown to exhibit pH-dependence associated with an ionizing group with a pK of 7.2. The kinetics of NADPH binding to the enzyme at pH 7.0 were measured by monitoring the quenching of protein fluorescence on binding the coenzyme.

Corticosterone secretion in rats as a function of ACTH input and adrenal blood flow

1965 ◽  
Vol 209 (4) ◽  
pp. 811-814 ◽  
Author(s):  
John C. Porter ◽  
M. S. Klaiber
Keyword(s):  
Blood Flow ◽  
Steady State ◽  
Steady Increase ◽  
Constant Input ◽  
Corticosterone Secretion ◽  
Steady State Rate ◽  
State Rate

The rate of secretion of corticosterone from the left adrenal of rats receiving a constant input of ACTH was determined for different flows of blood through the adrenal during the 2- to 3-hr interval following hypophysectomy. Two hours after hypophysectomy the secretion of corticosterone was low in all groups regardless of flow. An input of 0.26 mU ACTH/min caused a steady increase in secretion for 30–40 min before a steady-state rate was attained. The average steady-state rate of secretion was 1.1, 2.4, 3.5, 6.2, 7.2, 6.2, and 6.2 µg/5 min for flows of 0.005, 0.012, 0.023, 0.034, 0.039, 0.051, and 0.058 ml/min, respectively. Under the conditions of these experiments where the input of ACTH was 0.26 mU/min the secretion of corticosterone increased significantly with time of input of ACTH and with flow of blood through the adrenal.

scholarly journals The Natural Rate of Interest and the Optimal Rate of Interest in the Steady State

2021 ◽  
pp. 17-41
Author(s):  
Carl Christian von Weizsäcker ◽  
Hagen M. Krämer
Keyword(s):  
Steady State ◽  
Public Debt ◽  
Fundamental Equation ◽  
Optimal Rate ◽  
Natural Rate ◽  
Real Rate ◽  
Capital Theory ◽  
Steady State Rate ◽  
Natural Rate Of Interest ◽  
State Rate

AbstractThe “natural rate of interest” is the hypothetical, risk-free real rate of interest that would obtain in a closed economy, if net public debt were zero. It is considerably less than the optimal steady-state rate of interest, which is equal to the system’s growth rate. This holds for a very general “meta-model.” The fundamental equation of capital theory holds on the optimal steady-state path: T = Z − D, where T is the overall economic period of production, Z is the representative private “waiting period” of consumers and D is the public debt ratio. Prosperity is at least 30% lower at the natural rate of interest than at the optimal rate.

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