The assay and partial characterization of 3β-hydroxysteroid sulfotransferase of the hamster epididymis

M. Bouthillier; G. Bleau; A. Chapdelaine; K. D. Roberts

doi:10.1139/o85-010

The assay and partial characterization of 3β-hydroxysteroid sulfotransferase of the hamster epididymis

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o85-010 ◽

1985 ◽

Vol 63 (1) ◽

pp. 71-76 ◽

Cited By ~ 5

Author(s):

M. Bouthillier ◽

G. Bleau ◽

A. Chapdelaine ◽

K. D. Roberts

Keyword(s):

Organic Phase ◽

Enzyme Preparation ◽

Enzymatic Reaction ◽

Partial Characterization ◽

Optimal Conditions ◽

Steroid Nucleus ◽

Optimum Ph ◽

High Concentrations

Using a partially purified enzyme preparation obtained from hamster epididymis, a simple assay has been developed to measure the sulfurylation of dehydroisoandrosterone (DHA) and desmosterol in the presence of 3′-phosphoadenosine 5′-phospho[35S]sulfate ([35S]PAPS). After stopping the enzymatic reaction with methanol and KCl, the 35S-labelled steroid sulfates are readily extracted into an organic phase. Optimal conditions for the sulfurylation of the two steroids were compared; optimum pH is 8.7 for DHA and 9.8 for desmosterol. Sulfoconjugation of desmosterol increases with magnesium concentrations up to 6 mM, while 40 mM concentrations of the divalent ion are required for the optimal sulfurylation of DHA. Maximum sulfurylation of these steroids requires the presence of 15 mM cysteine. Michaelis–Menten kinetics are observed with DHA which has an apparent Km of 32 μM, while desmosterol inhibits sulfotransferase activity at high concentrations. Saturation of the enzyme with PAPS results in an allosteric behaviour. Only the 3β-hydroxyl function of the steroid nucleus appears to be an appropriate sulfate acceptor for the epididymal hydroxysteroid sulfotransferase.

Download Full-text

Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.5b01665 ◽

2015 ◽

Vol 63 (23) ◽

pp. 5682-5693 ◽

Cited By ~ 67

Author(s):

Michael Merz ◽

Thomas Eisele ◽

Pieter Berends ◽

Daniel Appel ◽

Swen Rabe ◽

...

Keyword(s):

Enzyme Preparation ◽

Partial Characterization ◽

Industrial Relevance

Download Full-text

Multiple forms of glycosidases in an enzyme preparation from Aspergillus niger: Partial characterization of a β-apiosidase

Enzyme and Microbial Technology ◽

10.1016/s0141-0229(96)00221-9 ◽

1997 ◽

Vol 21 (1) ◽

pp. 39-44 ◽

Cited By ~ 16

Author(s):

Ziya Günata ◽

Isabelle Dugelay ◽

M.J. Vallier ◽

J.C. Sapis ◽

C. Bayonove

Keyword(s):

Aspergillus Niger ◽

Enzyme Preparation ◽

Partial Characterization ◽

Multiple Forms

Download Full-text

Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.55604 ◽

2020 ◽

Vol 25 (2) ◽

pp. 127

Author(s):

Kezia Abib Yerah Tjandra ◽

Kartika Sari Dewi ◽

Asrul Muhamad Fuad ◽

Trisanti Anindyawati

Keyword(s):

Pichia Pastoris ◽

Trichoderma Reesei ◽

Specific Activity ◽

Catalytic Efficiency ◽

Endoglucanase Activity ◽

Gap Promoter ◽

Sds Page ◽

Optimum Ph ◽

High Concentrations

Trichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively.

Download Full-text

Brain arylamidase. Purification and characterization of the soluble bovine enzyme

Biochemical Journal ◽

10.1042/bj1120335 ◽

1969 ◽

Vol 112 (3) ◽

pp. 335-342 ◽

Cited By ~ 27

Author(s):

A. S. Brecher ◽

J. B. Suszkiw

Keyword(s):

Soluble Fraction ◽

Substrate Inhibition ◽

Gel Filtration ◽

Bovine Brain ◽

Purification And Characterization ◽

Sephadex Column ◽

Optimum Ph ◽

Ammonium Sulphate Fractionation ◽

High Concentrations

1. An enzyme acting on aminoacyl-β-naphthylamides has been isolated from the soluble fraction of bovine brain and purified 205-fold by means of ammonium sulphate fractionation, hydroxyapatite adsorption and DEAE-Sephadex column chromatography. 2. Arylamidase requires thiol groups for retention of its activity, is heat-labile and is susceptible to freezing. p-Chloromercuribenzoate and N-ethylmaleimide inactivate the enzyme rapidly. 3. Metal ions are not required for its activity, but stimulation by Mn2+ and Mg2+ and inactivation by Co2+ and Zn2+ are observed. 4. Optimum pH7·5 in phosphate buffer was exhibited for all substrates tested except l-leucyl-β-naphthylamide, for which optimum pH is 6·5. 5. Km values for a number of substrates have been obtained and substrate inhibition at high concentrations was demonstrated. 6. The molecular weight is approx. 70000 as determined by Sephadex-gel filtration.

Download Full-text

Isolation and partial characterization of a peptidase with trypsin-like activity from bovine adenohypophysis

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19892276 ◽

1989 ◽

Vol 54 (8) ◽

pp. 2276-2286

Author(s):

Tsezengijn Dash ◽

Tomislav Barth ◽

Jiřina Slaninová ◽

Jana Barthová ◽

Hana P. Mašková ◽

...

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Partial Characterization ◽

Chromogenic Substrate ◽

Fluorogenic Substrate ◽

Basic Amino Acids ◽

Optimum Ph ◽

Reproducible Method ◽

Hydrolysis Of

A reproducible method has been developed for the isolation of the adenohypophyseal enzyme with a trypsin-like activity. The enzyme is able to hydrolyze Nα-benzoyl-L-arginine-p-nitroanilide, a fluorogenic substrate CBzl-Arg-Arg-β-naphthyl amide and some peptides with one or two accumulated basic amino acids in the chain. The optimum pH for hydrolysis of the chromogenic substrate was within the range 6.0-7.0 (Km = 0.66 mmol l-1), in the case of the fluorogenic substrate the range was between 7.0 and 7.5 (Km = 1.2 μmol l-1). The enzyme is activated by cysteine and dithiothreitol and inhibited by SH-poisons. The molecular weight of the enzyme, determined by means of two independent methods, was approximately 25 kDA.

Download Full-text

Purification and Characterization of a New Thermostable, Haloalkaline, Solvent Stable, and Detergent Compatible Serine Protease fromGeobacillus toebiiStrain LBT 77

BioMed Research International ◽

10.1155/2016/9178962 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 17

Author(s):

Wajdi Thebti ◽

Yosra Riahi ◽

Omrane Belhadj

Keyword(s):

Serine Protease ◽

Hot Spring ◽

Deae Cellulose ◽

Ammonium Sulfate Precipitation ◽

Purification And Characterization ◽

Optimum Ph ◽

High Concentrations ◽

First Time ◽

Solvent Stable

A new thermostable, haloalkaline, solvent stable SDS-induced serine protease was purified and characterized from a thermophilicGeobacillus toebiiLBT 77 newly isolated from a Tunisian hot spring. This study reveals the potential of the protease fromGeobacillus toebiiLBT 77 as an additive to detergent with spectacular proprieties described for the first time. The protease was purified to homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography. It was a monomeric enzyme with molecular weight of 30 kDa. The optimum pH, temperature, and NaCl for maximum protease activity were 13.0, 95°C, and 30%, respectively. Activity was stimulated by Ca2+, Mg2+, DTNB,β-mercaptoethanol, and SDS. The protease was extremely stable even at pH 13.25, 90°C, and 30% NaCl and in the presence of hydrophilic, hydrophobic solvents at high concentrations. The high compatibility with ionic, nonionic, and commercial detergents confirms the utility as an additive to cleaning products. Kinetic and thermodynamic characterization of protease revealedKm=1 mg mL−1, Vmax=217.5 U mL−1,Kcat/Km=99 mg mL−1 S−1,Ea=51.5 kJ mol−1, andΔG⁎=56.5 kJ mol−1.

Download Full-text

Partial characterization of a soluble ATPase from pea cotyledon mitochondria

Canadian Journal of Biochemistry ◽

10.1139/o77-120 ◽

1977 ◽

Vol 55 (8) ◽

pp. 812-818 ◽

Cited By ~ 11

Author(s):

Charles Grubmeyer ◽

Ian Duncan ◽

Mary Spencer

Keyword(s):

Partial Characterization ◽

Mitochondrial Enzyme ◽

Mammalian Mitochondria ◽

High Concentrations

A partially purified soluble ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pea cotyledon mitochondria was characterized. Inhibition patterns with azide, NaF, and cold, and a stimulation by 2,4-dinitrophenol were typical of F1-ATPases from mammalian mitochondria. The enzyme hydrolysed GTP, ITP, and ATP, but not CTP, UTP, ADP, or IDP. ATPase and ITPase activities were strongly inhibited by ADP and to a lesser extent by IDP. Distinctive properties of the pea mitochondrial enzyme were activation by high concentrations of CaCl2 and stimulation by NaCl.

Download Full-text

Computer Simulation of Amperometric Biosensor Response to Mixtures of Compounds

Nonlinear Analysis Modelling and Control ◽

10.15388/na.2002.7.2.15190 ◽

2002 ◽

Vol 7 (2) ◽

pp. 3-14 ◽

Cited By ~ 3

Author(s):

R. Baronas ◽

J. Christensen ◽

F. Ivanauskas ◽

J. Kulys

Keyword(s):

Computer Simulation ◽

Enzymatic Reaction ◽

Diffusion Equations ◽

Response Data ◽

Finite Difference Technique ◽

Stationary Diffusion ◽

Biosensor Array ◽

Menten Kinetic ◽

Biosensor Response

A mathematical model of amperometric biosensors has been developed. The model bases on non-stationary diffusion equations containing a non-linear term related to Michaelis-Menten kinetic of the enzymatic reaction. The model describes the biosensor response to mixtures of multiple compounds in two regimes of analysis: batch and flow injection. Using computer simulation, large amount of biosensor response data were synthesised for calibration of a biosensor array to be used for characterization of wastewater. The computer simulation was carried out using the finite difference technique.

Download Full-text