scholarly journals ‘Affinity’ chromatography of steroid-transforming enzymes with a non-steroidal ligand

1979 ◽  
Vol 177 (2) ◽  
pp. 401-408
Author(s):  
A G Renwick ◽  
S M Chambers ◽  
P Willcox
Keyword(s):  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
Chicken Liver ◽  
Rabbit Muscle ◽  
Chromatographic Behaviour ◽  
Liver Preparation ◽  
The Mean ◽  
Hormonal Steroids ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

The chromatographic behaviour of an avian oestradiol-17 beta dehydrogenase, the 3(17) beta-hydroxy steroid dehydrogenase from Pseudomonas testosteroni and cortisone reductase from Streptomyces dehydrogenans was studied on columns of p-(phenoxypropoxy)aniline attached to CNBr-activated Sepharose. The ligand was effective in adsorbing the oestradiol dehydrogenase from a partially purified extract of chicken liver, and the cortisone reductase was perferentially retained when mixtures of the three dehydrogenases were applied to columns in 10mM-buffer. Under these conditions the 3(17)beta-hydroxy steroid dehydrogenase was eluted in the front, but was adsorbed in the presence of 3 M-KCl. beta-N-Acetylglucosaminidase present in the liver preparation was not retained by the ligand, whereas lactate dehydrogenase from rabbit muscle was adsorbed in a manner similar to the retention pattern found on affinity chromatography with !2′,5′-ADP–Sepharose. The mean overall purification of the oestradiol dehydrogenase was 13-fold, with a mean recovery of 53%. p-(Phenoxypropoxy)aniline offers promise for the purification of steroid-transforming enzymes where elution with substrate or cofactor is not wanted. It is also suggested that the ligand may be of service in the purification of receptors of hormonal steroids.

scholarly journals Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography

1975 ◽  
Vol 151 (3) ◽  
pp. 631-636 ◽  
Author(s):  
R I Brinkworth ◽  
C J Masters ◽  
D J Winzor
Keyword(s):  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
Equilibrium Constants ◽  
Mouse Tissue ◽  
Rabbit Muscle ◽  
Muscle Lactate ◽  
Sodium Phosphate Buffer ◽  
A Value ◽  
Series Of Experiments ◽  
Reduced Nicotinamide Adenine Dinucleotide

Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 × 104M−1 for the interaction of enzyme with NADH at 5°C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 × 105M−1, 3 × 105M−1, 4 × 105M−1, 7 × 105M−1 and 2 × 106M−1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase.

A metabolic stability determination of Tetrahydrothiazolopyridine derivative a selective 11β-hydroxy steroid dehydrogenase type 1 (11β-hsd1) inhibitor

2017 ◽  
Vol 8 (3) ◽  
Author(s):  
MURUGESH KANDASAMY ◽  
MUHAMMED SALIHIN ◽  
MALLIKARJUNA RAO PICHIKA ◽  
SLAVKO KOMARNYTSKY ◽  
THIRUMURUGAN RATHINASABAPATHY
Keyword(s):  
Metabolic Stability ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

scholarly journals Validation of Recombinant Chicken Liver Bile Acid Binding Protein as a Tool for Cholic Acid Hosting

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 645
Author(s):  
Giusy Tassone ◽  
Maurizio Orlandini ◽  
Massimo Olivucci ◽  
Cecilia Pozzi
Keyword(s):  
Bile Acid ◽  
High Resolution ◽  
Affinity Chromatography ◽  
Bile Acids ◽  
Drug Carriers ◽  
Chicken Liver ◽  
Purification Procedure ◽  
Acid Binding Protein ◽  
Bile Acid Binding ◽  
Exogenous Substances

Bile acids (BAs) are hydroxylated steroids derived from cholesterol that act at the intestinal level to facilitate the absorption of several nutrients and also play a role as signaling molecules. In the liver of various vertebrates, the trafficking of BAs is mediated by bile acid-binding proteins (L-BABPs). The ability to host hydrophobic or amphipathic molecules makes BABPs suitable for the distribution of a variety of physiological and exogenous substances. Thus, BABPs have been proposed as drug carriers, and more recently, they have also been employed to develop innovative nanotechnology and biotechnology systems. Here, we report an efficient protocol for the production, purification, and crystallization of chicken liver BABP (cL-BABP). By means of target expression as His6-tag cL-BABP, we obtained a large amount of pure and homogeneous proteins through a simple purification procedure relying on affinity chromatography. The recombinant cL-BABP showed a raised propensity to crystallize, allowing us to obtain its structure at high resolution and, in turn, assess the structural conservation of the recombinant cL-BABP with respect to the liver-extracted protein. The results support the use of recombinant cL-BABP for the development of drug carriers, nanotechnologies, and innovative synthetic photoswitch systems.

scholarly journals Kinetic Studies of Rabbit Muscle Lactate Dehydrogenase

1962 ◽  
Vol 237 (5) ◽  
pp. 1668-1675
Author(s):  
Virginia Zewe ◽  
Herbert J. Fromm
Keyword(s):  
Lactate Dehydrogenase ◽  
Kinetic Studies ◽  
Rabbit Muscle ◽  
Muscle Lactate

DIFFICULTIES IN THE DIAGNOSIS OF THE ADRENOGENITAL SYNDROME IN INFANCY

PEDIATRICS ◽  
1972 ◽  
Vol 49 (2) ◽  
pp. 198-205
Author(s):  
C. H. Shackleton ◽  
F. L. Mitchell ◽  
J. W. Farquhar
Keyword(s):  
Hydroxylase Deficiency ◽  
Adrenogenital Syndrome ◽  
Steroid Excretion ◽  
21 Hydroxylase Deficiency ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

Pregnanetriol was not excreted by an infant (7 days old) who was later shown to have a defect in steroid 21-hydroxylase. However, the excretion of this compound increased during the following days (1.2 mg on the thirteenth day of life). A high excretion of 3β-hydroxy-Δ steroids was the most noticeable abnormality in steroid excretion noted on the seventh day of life (e.g., 3β, 16α-dihydroxy-5-pregnen-20-one, 15 mg; 3β, 21-dihydroxy-5-pregnen-20-one, 1.4 mg and 3β, 16α-dihydroxy-5-androsten-17-one, 7.4 mg). This high 3β-hydroxy-Δ steroid excretion results in difficulties in distinguishing a defect in 3β-hydroxy steroid dehydrogenase from a 21-hydroxylase deficiency. At the age of 14 months the principal steroids excreted were those predominant in other cases of 21-hydroxylase deficiency, viz. pregnanetriol and 5β-pregnane-3α, 17α, 20α-triol-11-one (11-oxo-pregnanetriol).

Optimal α-Oxobutyrate Concentration for Assay of α-Hydroxybutyrate Dehydrogenase Activity in Normal and Pathological Sera at 30 °C

1974 ◽  
Vol 20 (4) ◽  
pp. 494-496 ◽  
Author(s):  
Leslie M Shaw ◽  
Jane Gray
Keyword(s):  
Myocardial Infarction ◽  
Liver Disease ◽  
Lactate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Substrate Concentration ◽  
Optimal Concentration ◽  
The Mean

Abstract Optimal concentration of substrate, α-oxobutyrate, for determination of α-hydroxybutyrate dehydrogenase (HBD) activity in normal and pathological sera at 30 °C (with a centrifugal analyzer) is 12 mmol/ liter. With this substrate concentration, HBD activity averaged 146% of that found with the suboptimal concentration, 3.3 mmol/liter, usually used. Furthermore, day-to-day precision was significantly better. HBD/LD ranges (LD, lactate dehydrogenase), determined for sera from patients with myocardial infarction and liver disease, did not overlap and were similar to those established by Elliot et al. [Clin. Sci. 23, 305 (1962)], who used a suboptimal α-oxobutyrate concentration of 3.3 mmol/liter at 25 °C. At 30 °C the mean HBD/LD ratios for sera from patients with myocardial infarction were most different from those for patients with liver disease when the optimal concentration of α-oxobutyrate was used.

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