Functional groups in pig kidney aldehyde reductase

1981 ◽  
pp. 601-610
Author(s):  
T G Flynn ◽  
D Ferguson ◽  
W S Davidson
Keyword(s):  
Functional Groups ◽  
Aldehyde Reductase ◽  
Pig Kidney

Identification of Functional Groups of Pig Kidney Alkaline Phosphatase by Specific Inhibitors

1975 ◽  
Vol 30 (11-12) ◽  
pp. 829-831 ◽  
Author(s):  
Jan Ahlers
Keyword(s):  
Alkaline Phosphatase ◽  
Functional Groups ◽  
Pig Kidney ◽  
Kidney Alkaline Phosphatase ◽  
Specific Inhibitors ◽  
Regulatory Sites

Abstract Inactivation studies with 17 group-specific inhibitors showed that amino, hystidyl and tyrosyl residues probably are components of the active and/or regulatory sites of pig kidney alkaline phosphatase.

scholarly journals Some properties of pig kidney-cortex aldehyde reductase

1980 ◽  
Vol 191 (2) ◽  
pp. 619-626 ◽  
Author(s):  
F F Morpeth ◽  
F M Dickinson
Keyword(s):  
Active Site ◽  
Fluorescence Spectra ◽  
Dissociation Constants ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Kidney Cortex ◽  
Aldehyde Reductase ◽  
Pig Kidney ◽  
Binary Complexes ◽  
Spectrophotometric Titrations

Aldehyde reductase was purified from pig kidney cortex to homogeneity by a new procedure. The molecular weight of the enzyme was estimated by sedimentation equilibrium to be 43 700 and by gel electrophoresis in the presence of sodium dodecyl sulphate to be 41 700. The enzyme is clearly a monomer. The enzyme preparation contained no significant quantities of zinc, manganese or copper and had no essential histidine or thiol groups. Changes in the absorption and fluorescence spectra of NADPH were observed on formation of the enzyme-NADPH complexes. Large changes in the fluorescence spectra were also observed in the presence of sodium barbitone or Warfarin. These changes were used as the basis of active-site titrations, which showed that the enzyme had one active site per molecule. The dissociation constants of NADPH and NADP+ from binary complexes with the enzyme were estimated in spectrophotometric titrations.

scholarly journals The mechanism of hydrolysis of β-glycerophosphate by kidney alkaline phosphatase

1975 ◽  
Vol 149 (3) ◽  
pp. 535-546 ◽  
Author(s):  
J Ahlers
Keyword(s):  
Alkaline Phosphatase ◽  
Functional Groups ◽  
Functional Group ◽  
Substrate Binding ◽  
Activation Mechanism ◽  
Active Centre ◽  
Pig Kidney ◽  
Substrate Conversion ◽  
Mechanism Of Hydrolysis ◽  
Ph Range

1. To identify the functional groups that are involved in the conversion of β-glycerophosphate by alkaline phosphatase (EC 3.1.3.1) from pig kidney, the kinetics of alkaline phosphatase were investigated in the pH range 6.6-10.3 at substrate concentrations of 3 μM-30 mM. From the plots of log ṼH+ against pH and log ṼH+/KH+m against pH one functional group with pK = 7.0 and two functional groups with pK = 9.1 were identified. These groups are involved in substrate binding. Another group with pK = 8.8 was found, which in its unprotonated form catalyses substrate conversion. 2. GSH inhibits the alkaline phosphatase reversibly and non-competitively by attacking the bound Zn(II). 3. The influence of the H+ concentration on the activation by Mg2+ ions of alkaline pig kidney phosphate was investigated between pH 8.4 and 10.0. The binding of substrate and activating Mg2+ ions occurs independently at all pH values between 8.4 and 10.0. The activation mechanism is not affected by the H+ concentration. The Mg2+ ions are bound by a functional group with a pK of 10.15. 4. A scheme is proposed for the reaction between enzyme, substrate, Mg2+ and H+ and the overall rate equation is derived. 5. The mechanism of substrate binding and splitting by the functional groups of the active centre is discussed on the basis of a model. Mg2+ seems to play a role as an autosteric effector.

scholarly journals Kinetic studies of the mechanism of pig kidney aldehyde reductase

1981 ◽  
Vol 193 (2) ◽  
pp. 485-492 ◽  
Author(s):  
F F Morpeth ◽  
F M Dickinson
Keyword(s):  
Initial Rate ◽  
Kinetic Studies ◽  
Random Order ◽  
Alcohol Oxidation ◽  
Aldehyde Reductase ◽  
Pig Kidney ◽  
Binary Complexes ◽  
Inhibition Studies ◽  
Kinetics Of ◽  
Aldehyde Reduction

Initial-rate measurements were made of the oxidations of pyridine-3-methanol and glycerol by NADP+ and of the reduction of the corresponding aldehydes by NADPH catalysed by pig kidney aldehyde reductase. In addition, a brief survey of the specificity of the enzyme towards aldehyde substrates and its sensitivity to the inhibitors ethacrynic acid, sodium barbitone and warfarin was made. The detailed kinetic work indicates a compulsory mechanism for aldehyde reduction, with NADPH binding before aldehyde. For alcohol oxidation, however, it is necessary to postulate the formation of kinetically significant amounts of binary complexes of the type enzyme-alcohol to explain the results. Thus, for alcohol oxidation random-order addition of substrates may occur. Inhibition studies of the kinetics of aldehyde reduction in the presence of the corresponding alcohol product provide further evidence for the existence of enzyme-alcohol complexes. Finally, detailed kinetic studies were made of the inhibition of pyridine-3-aldehyde reduction by sodium barbitone. The mechanism of the inhibition is discussed.

scholarly journals Purification and characterization of an enzymically active cleavage product of pig kidney aldehyde reductase

1983 ◽  
Vol 209 (3) ◽  
pp. 597-607 ◽  
Author(s):  
J A Cromlish ◽  
T G Flynn
Keyword(s):  
Specific Activity ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cleavage Product ◽  
Aldehyde Reductase ◽  
Pig Kidney ◽  
Reductase Inhibitors ◽  
N Terminus ◽  
Minor Form

During the purification of pig kidney aldehyde reductase by an established procedure [Flynn, Cromlish & Davidson (1982) Methods Enzymol. 89, 501-506] a second enzyme with aldehyde reductase activity may be purified. When the procedure was performed in the presence of 5 mM-EDTA, only traces of the second reductase, pig kidney aldehyde reductase (minor form), were present. By the criterion of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, pig kidney aldehyde reductase (minor form) had Mr 35 000, in comparison with Mr 40 200 found for pig kidney aldehyde reductase. Amino acid analysis of both enzymes and tryptic-peptide-map comparisons indicated differences in primary structure. The N-terminus of pig kidney aldehyde reductase (minor form) had the sequence Lys-Val-Leu, in contrast with the blocked (acetylated) N-terminus of pig kidney aldehyde reductase. The C-terminal sequence of both enzymes was the same. Both reductases were immunologically identical by double immunodiffusion and rocket immunoelectrophoresis. Pig kidney aldehyde reductase (minor form) had 50% of the specific activity of pig kidney aldehyde reductase when tested with a variety of aldehyde substrates. Michaelis constants of both enzymes for these substrates and for NADPH were similar, but values for kcat. and kcat./Km indicated that catalytically pig kidney aldehyde reductase was the more efficient enzyme. Typical aldehyde reductase inhibitors, such as phenobarbital and sodium valproate, had the same effect on both enzymes. It was concluded that pig kidney aldehyde reductase (minor form) is an enzymically active cleavage product of pig kidney aldehyde reductase which is formed when the latter is purified in the absence of the metalloproteinase inhibitor EDTA.

Studies on the active site of pig kidney aldehyde reductase

1981 ◽  
Vol 9 (4) ◽  
pp. 273-275 ◽  
Author(s):  
T. GEOFFREY FLYNN ◽  
CHRISTINE GALLERNEAULT ◽  
DAVID FERGUSON ◽  
JAMES A. CROMLISH ◽  
WILLIAM S. DAVIDSON
Keyword(s):  
Active Site ◽  
Aldehyde Reductase ◽  
Pig Kidney
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