THE INFLUENCE OF ETHANOL ON THE CONVERSION OF PREGNENOLONE TO PROGESTERONE BY HUMAN PLACENTAL MICROSOMES

1974 ◽  
Vol 76 (1) ◽  
pp. 178-188 ◽  
Author(s):  
H. Lübbert ◽  
K. Pollow ◽  
R. Wagner ◽  
J. Hammerstein
Keyword(s):  
Kinetic Parameters ◽  
Enzyme Preparation ◽  
Ethanol Concentration ◽  
Hydroxysteroid Dehydrogenase ◽  
Product Formation ◽  
Linear Increase ◽  
Maximal Velocity ◽  
Microsomal Enzyme ◽  
Temperature Optima ◽  
Substrate Concentrations

ABSTRACT The effects of ethanol on kinetic parameters of placental Δ5-3β-hydroxysteroid dehydrogenase were studied. In the presence of high pregnenolone concentrations (50 μm, [S] > Km) the microsomal enzyme preparation exhibited an almost linear increase in activity as the ethanol concentration in the medium was raised from 2.5 to 15 % (v/v). At lower substrate concentrations ([S] << Km) ethanol caused inhibition. Other effects of ethanol were: linearity of product formation with time was prolonged; the maximal velocity was markedly increased; the Km for pregnenolone slightly decreased with increasing ethanol concentrations (2.5 to 10 %, v/v) whereas the Km for NAD remained the same. The pH and temperature optima of the reaction were unaffected by ethanol. Other organic solvents caused similar effects.

Estimation of kinetic parameters of androgen-synthesizing enzyme activities in superfused Leydig cells from rat testes: Difference between endogenous and exogenous substrates

1984 ◽  
Vol 4 (6) ◽  
pp. 483-488 ◽  
Author(s):  
Nikolaus Kühn-Velten ◽  
Joachim Wolff ◽  
Wolfgang Staib
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Active Site ◽  
Enzyme Activities ◽  
Substrate Concentration ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Endogenous Substrate ◽  
Catalyzed Reactions ◽  
Actual Substrate

Kinetic parameters of 3β-hydroxysteroid dehydrogenase/isomerase, steroid-17α-monooxygenase, and steroid-17,20-lyase activities were estimated under steady-state conditions. Purified Leydig cells from rat testes were superfused with pregnenolone, progesterone, or 17α-hydroxyprogesterone. The Km values for both the monooxygenase- and the lyase-catalyzed reactions were by factors of five to ten higher if analyzed with the exogenously added substrate (0.98 and 0.65 μM, respectively) than if calculated from endogenous substrate derived from a precursor (0.10 and 0.13 μM, respectively). This discrepancy may be explained by different substrate partition between the intra- and extraceIJular spaces and by different substrate concentration at the active site of the respective enzyme, depending on whether the actual substrate is of exogenous or endogenous source.

scholarly journals Human Deoxycytidine Kinase Is a Valuable Biocatalyst for the Synthesis of Nucleotide Analogues

Catalysts ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 997 ◽  
Author(s):  
Katja F. Hellendahl ◽  
Sarah Kamel ◽  
Albane Wetterwald ◽  
Peter Neubauer ◽  
Anke Wagner
Keyword(s):  
Substrate Concentration ◽  
Food Additives ◽  
Product Yield ◽  
Building Blocks ◽  
Product Formation ◽  
Industrial Applications ◽  
Modified Nucleosides ◽  
Deoxycytidine Kinase ◽  
Wide Range ◽  
Substrate Concentrations

Natural ribonucleoside-5’-monophosphates are building blocks for nucleic acids which are used for a number of purposes, including food additives. Their analogues, additionally, are used in pharmaceutical applications. Fludarabine-5´-monophosphate, for example, is effective in treating hematological malignancies. To date, ribonucleoside-5’-monophosphates are mainly produced by chemical synthesis, but the inherent drawbacks of this approach have led to the development of enzymatic synthesis routes. In this study, we evaluated the potential of human deoxycytidine kinase (HsdCK) as suitable biocatalyst for the synthesis of natural and modified ribonucleoside-5’-monophosphates from their corresponding nucleosides. Human dCK was heterologously expressed in E. coli and immobilized onto Nickel-nitrilotriacetic acid (Ni-NTA) superflow. A screening of the substrate spectrum of soluble and immobilized biocatalyst revealed that HsdCK accepts a wide range of natural and modified nucleosides, except for thymidine and uridine derivatives. Upon optimization of the reaction conditions, HsdCK was used for the synthesis of fludarabine-5´-monophosphate using increasing substrate concentrations. While the soluble biocatalyst revealed highest product formation with the lowest substrate concentration of 0.3 mM, the product yield increased with increasing substrate concentrations in the presence of the immobilized HsdCK. Hence, the application of immobilized HsdCK is advantageous upon using high substrate concentration which is relevant in industrial applications.

scholarly journals Kinetics of Cl-dependent K fluxes in hyposmotically swollen low K sheep erythrocytes.

1991 ◽  
Vol 97 (2) ◽  
pp. 173-193 ◽  
Author(s):  
E Delpire ◽  
P K Lauf
Keyword(s):  
Red Blood Cells ◽  
Kinetic Study ◽  
Kinetic Parameters ◽  
Blood Cells ◽  
Red Cells ◽  
Maximal Velocity ◽  
Temperature Dependent ◽  
Low K ◽  
Kinetics Of ◽  
External K

A detailed kinetic study of K:Cl cotransport in hyposmotically swollen low K sheep red blood cells was carried out to characterize the nature of the outwardly poised carrier. The kinetic parameters were determined from the rate of K efflux and influx under zero-K-trans conditions in red cells with cellular K altered by the nystatin method and with different extracellular K or Rb concentrations. Although apparent affinities for efflux and influx were quite similar, the maximal velocity for K efflux was approximately two times greater than for influx. Furthermore, at thermodynamic equilibrium (i.e., when the ion product of K and Cl within the cell was equal to that outside) a temperature-dependent net K efflux was observed, approaching zero only when the external product reached approximately two times the internal product. The binding order of the ions to the transporter was asymmetric, being ordered outside (Cl binding first, followed by K) and random inside. K efflux but not influx was trans-inhibited by KCl. Trans inhibition of K efflux was used to verify the order of binding outside: trans inhibition by external Cl occurred in the absence of external K, but not vice versa. Thus K:Cl cotransport is kinetically asymmetric in hyposmotically swollen low K sheep red cells.

scholarly journals Substrate Specificity of Human Cytochrome P450 (CYP) 2C Subfamily and Effect of Azole Antifungal Agents on CYP2C8

2016 ◽  
Vol 19 (4) ◽  
pp. 423 ◽  
Author(s):  
Toshiro Niwa ◽  
Yurie Imagawa
Keyword(s):  
Cytochrome P450 ◽  
Antifungal Agents ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
Human Cytochrome ◽  
Km Value ◽  
Metabolic Activities ◽  
Substrate Concentrations ◽  
Hepatic Cytochrome ◽  
Demethylation Activity

PURPOSE: The metabolic activities of aminopyrine N-demethylation and tolbutamide methylhydroxylation by the human hepatic cytochrome P450 (P450 or CYP) 2C subfamily were compared and the effects of azole antifungal agent on the drug-metabolizing activity of CYP2C8 were investigated. METHODS: Aminopyrine N-demethylation and tolbutamide methylhydroxylation by CYP2C8, CYP2C9, and CYP2C19 were determined by the previous reported methods. The effects of five azole antifungal agents, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole, on the aminopyrine N-demethylation activity by CYP2C8 were investigated. RESULTS: With regard to aminopyrine N-demethylation, CYP2C19 had the lowest Michaelis constant (Km) and CYP2C8 had the highest maximal velocity (Vmax) among the CYP2C subfamily members. The Vmax/Km values for CYP2C8 were the highest, followed by CYP2C19. For tolbutamide methylhydroxylation, the Km and Vmax for CYP2C19 were three and six times higher than the corresponding values for CYP2C9, and the Vmax/Km value for CYP2C19 was twice that for CYP2C9, whereas hydroxylated tolbutamide formed by CYP2C8 was not detected. Fluconazole, itraconazole, and voriconazole at a concentration of 2 or 10 µM neither inhibited nor stimulated CYP2C8-mediated aminopyrine N-demethylation activity at substrate concentrations around the Km (5 mM). However, ketoconazole and miconazole noncompetitively inhibited CYP2C8-mediated aminopyrine N-demethylation with the inhibitory constant values of 1.98 and 0.86 µM, respectively. CONCLUSION: These results suggest that ketoconazole and miconazole might inhibit CYP2C8 clinically. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Purification and Properties of the Soluble 17β-Hydroxysteroid Dehydrogenase of Rabbit Uterus

1979 ◽  
Vol 34 (9-10) ◽  
pp. 726-737 ◽  
Author(s):  
Kunhard Pollow ◽  
Walter Eiger ◽  
Herrmann Heßlinger ◽  
Barbara Pollow
Keyword(s):  
Enzyme Activity ◽  
Dehydrogenase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Hydroxysteroid Dehydrogenase ◽  
Maximal Velocity ◽  
Ammonium Sulfate Precipitation ◽  
Ph Optimum ◽  
Mobility Data ◽  
Purification And Properties

Abstract 17 β-Hydroxysteroid dehydrogenase activity towards estradiol-17 β has been demonstrated in the 105,000 X g supernatant of rabbit uterus. Hydroxylapatite chromatography of the enzyme activity isolated by ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromato­graphy yielded a single 17 β-hydroxysteroid dehydrogenase activity. Further purification of the enzyme preparation by isoelectric focusing resulted in multiple peaks of activity. The molecular weight or the enzyme, calculated from mobility data on Sephadex gel, is approximately 64,000. Some properties of partially purified 17 β-hydroxysteroid dehydrogenase activity have been studied. Estradiol-17 β reacts at a faster rate than testosterone. The Km for estradiol is 4.16X 10-5 mol/1 for the NAD-linked enzyme activity and 4.37 X 10-5 mol/1 when NADP as cofactor was used. The ratio of the maximal velocity for NADP to that for NAD was 1.42. The pH-optimum for estradiol appears between 9.5 and 10.5 and for estrone between 5.5 and 6.5. The enzyme appears to be of the sulfhydryl type.

Regulation of Ca2+/calmodulin-dependent protein kinase II catalysis by N-methyl-D-aspartate receptor subunit 2B

2009 ◽  
Vol 419 (1) ◽  
pp. 123-136 ◽  
Author(s):  
Kurup K. Pradeep ◽  
John Cheriyan ◽  
Sudarsana Devi Suma Priya ◽  
Raveendran Rajeevkumar ◽  
Madhavan Mayadevi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinetic Parameters ◽  
Glutathione Transferase ◽  
Long Term Potentiation ◽  
Binding Motif ◽  
Sequence Motif ◽  
Maximal Velocity ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein

Binding of CaMKII (Ca2+/calmodulin-dependent protein kinase II) to the NR2B subunit of the NMDAR (N-methyl-D-aspartate-type glutamate receptor) in the PSD (postsynaptic density) is essential for the induction of long-term potentiation. In this study, we show that binding of NR2B to the T-site (Thr286-autophosphorylation site binding pocket) of CaMKII regulates its catalysis as reflected in the kinetic parameters. The apparent S0.5 (substrate concentration at half maximal velocity) and Vmax values for ATP were lower for phosphorylation of a GST (glutathione transferase)-fusion of NR2B(1271-1311) (with the phosphorylation site Ser1303) when compared with phosphorylation of the analogous sequence motif from NR2A. The co-operative behaviour exhibited by the CaMKII holoenzyme towards ATP for phosphorylation of GST–NR2A was significantly altered by the interaction with GST–NR2B. Disrupting the T-site-mediated binding by mutagenesis of either NR2B or CaMKII abolished the modulation of CaMKII activity by NR2B. The active site residue of α-CaMKII, Glu96, participates in effecting the modulation. The CaMKII-binding motif of the Drosophila voltage-gated potassium channel Eag interacted with the T-site of CaMKII with lower affinity and caused catalytic modulation to a lesser extent. The kinetic parameters of ATP for the Thr286-autophosphorylation reaction of CaMKII were also altered by NR2B in a similar manner. Interestingly, the NR2B sequence motif caused increased sensitivity of CaMKII activity to ATP, and saturation by lower concentrations of ATP, which, in effect, resulted in a constant level of activity of CaMKII over a broad range of ATP concentrations. Our findings indicate that CaMKII at the PSD may be regulated by bound NR2B in a manner that supports synaptic memories.

STUDIES ON 17β-HYDROXYSTEROID DEHYDROGENASE IN HUMAN ENDOMETRIUM AND ENDOMETRIAL CARCINOMA

1975 ◽  
Vol 80 (2) ◽  
pp. 355-364 ◽  
Author(s):  
K. Pollow ◽  
H. Lübbert ◽  
B. Pollow
Keyword(s):  
Endometrial Carcinoma ◽  
Ammonium Sulphate ◽  
Isoelectric Focusing ◽  
Hydroxysteroid Dehydrogenase ◽  
Maximal Velocity ◽  
Optimum Temperature ◽  
Ammonium Sulphate Precipitation ◽  
Optimum Ph ◽  
Triton X ◽  
Secretory Endometrium

ABSTRACT Microsomal 17β-hydroxysteroid dehydrogenase obtained from the human secretory endometrium (17β-HSD) was solubilized with triton X-100. A 4-fold purification was achieved by ammonium sulphate precipitation and isoelectric focusing. In the presence of glycerol the partially purified enzyme was stable at 4°C for at least 48 h. Using crude microsomes, the conversion of oestradiol to oestrone was linear with time and with the concentration of protein. The optimum temperature was approximately 40°C and the optimum pH 9.4. For the reduction of oestrone the optimum pH was 6.5. With NAD, oestradiol was oxidized approximately three times more rapidly than with NADP. Km-values for oestradiol were nearly the same in endometrial carcinoma and in proliferative and secretory endometrium (i. e. approximately 3 × 10−6 m). The maximal velocity was highest in secretory endometrium. Testosterone and androstenedione could also serve as substrates but they were interconverted more slowly than oestradiol and oestrone. Sulphhydryl groups were shown to be essential for catalysis.

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