scholarly journals Isopentenyl pyrophosphate isomerase from liver

1968 ◽  
Vol 106 (4) ◽  
pp. 835-840 ◽  
Author(s):  
P. W. Holloway ◽  
G. Popják
Keyword(s):  
Ammonium Sulphate ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Pig Liver ◽  
Isopentenyl Pyrophosphate ◽  
Km Value ◽  
Ammonium Sulphate Fractionation ◽  
Dimethylallyl Pyrophosphate

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) was purified from extracts of pig liver by ammonium sulphate fractionation and by gel filtration. After about 20-fold purification the preparations were free of phosphatase and prenyltransferase (EC 2.5.1.1), the two enzymes that could have interfered with the assays. The isomerase has a distinct pH optimum at 6·0 and is activated by Mn2+ in preference to Mg2+. The Km value for isopentenyl pyrophosphate is 4×10−6m. The equilibrium of the reaction favours the formation of dimethylallyl pyrophosphate. The reversibility of the isomerase reaction was demonstrated directly by the formation of isopentenyl pyrophosphate from dimethylallyl pyrophosphate. It is suggested that two prenyl isomerases might exist, one involved in the synthesis of trans- and another in the synthesis of cis-polyprenyl substances.

scholarly journals Cathepsin D from pig myometrium. Characterization of the proteinase

1984 ◽  
Vol 219 (3) ◽  
pp. 899-904 ◽  
Author(s):  
R Barth ◽  
E G Afting
Keyword(s):  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Equilibrium Studies ◽  
Main Band ◽  
Km Value ◽  
Ph Range

The purification of cathepsin D from pig uterus by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose was described previously [Afting & Becker (1981) Biochem. J. 197, 519-522]. In this paper, chemical and physical properties of the proteinase are presented. The purified enzyme showed three bands on SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis, one main band corresponding to an Mr of 31 000 and two minor bands with Mr values of 43 000 and 15 000 respectively. Gel filtration on Bio-gel P-150 and sedimentation-diffusion equilibrium studies give an Mr for the main band of about 35 000. The pI of the enzyme was determined to be 7.2. Haemoglobin was the best substrate, with a Km value of 6.4 X 10(-6)M. It was hydrolysed with a pH optimum between 3.0 and 3.3 for a substrate concentration of 100 microM. The proteinase was stable over the pH range of 3.5-6.5. At pH 6 the enzyme showed stability up to a temperature of 50 degrees C; at pH 3 the activity was already decreased below 40 degrees C. Carbohydrate studies resulted in the staining of all three bands on an SDS/polyacrylamide gel by thymol/H2SO4. After treatment with endo-beta-N-acetylglucosaminidase H, all three bands were shifted to a region of lower Mr. Of various inhibitors tested, only pepstatin was strongly inhibiting, with a Ki of 2.1 X 10(-9)M.

scholarly journals The role of adenosine 3′:5′-cyclic monophosphate in the regulation of insulin release. Properties of islet-cell adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1972 ◽  
Vol 129 (4) ◽  
pp. 945-952 ◽  
Author(s):  
D. J. Sams ◽  
W. Montague
Keyword(s):  
Cyclic Amp ◽  
Insulin Release ◽  
Islets Of Langerhans ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Supernatant Fraction ◽  
Phosphodiesterase Activity ◽  
Ph Optimum ◽  
Cyclic Monophosphate ◽  
Km Value

1. An assay has been developed with sufficient sensitivity for determination of the adenosine 3′:5′-cyclic monophosphate diesterase activity in islets of Langerhans, and has been used to investigate the response of the enzyme to various agents which are known to affect insulin release. 2. The subcellular distribution of the enzyme in islets of Langerhans prepared from guinea-pig pancreas was investigated and over 70% of the activity present in the original homogenate was recovered in the supernatant fraction. 3. Gel filtration of the activity present in the supernatant fraction on Sephadex G-200 gave a single peak of activity with an apparent molecular weight of 200000. The phosphodiesterase activity in the peak fraction showed two apparent Km values for adenosine 3′:5′-cyclic monophosphate (cyclic AMP) of 3μm and 30μm, suggesting the presence of two activities. The pH optimum of the activity with the low Km value was 8.7. 4. Theophylline, caffeine, 3-isobutyl-1-methylxanthine (SC-2964), glibenclamide, tolbutamide, xylitol and leucine were inhibitors of the activity with the low Km value; imidazole and arginine stimulated the activity, and glucose and diazoxide were without significant effect. 5. It is suggested that the agents theophylline, caffeine, SC-2964, glibenclamide, tolbutamide, leucine and imidazole may alter the intracellular concentration of cyclic AMP in islets of Langerhans by affecting the cyclic AMP phosphodiesterase activity in islet cells and in this way may affect insulin release.

scholarly journals Purification and properties of a human seminal proteinase

1972 ◽  
Vol 126 (5) ◽  
pp. 1135-1140 ◽  
Author(s):  
Frank N. Syner ◽  
Kamran S. Moghissi
Keyword(s):  
Ethyl Ester ◽  
Seminal Plasma ◽  
Gel Filtration ◽  
Enzyme Assays ◽  
Human Seminal Plasma ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Ammonium Sulphate Fractionation ◽  
Substrate Concentrations

1. A method is described for the purification of a proteinase, present in human seminal plasma and previously shown to accelerate migration of spermatozoa through cervical mucus in vitro. A 25-fold purification was achieved in three steps, consisting of ammonium sulphate fractionation, chromatography on CM-cellulose and gel filtration. 2. The enzyme displays some properties similar to chymotrypsin: pH optimum 7.5–8.0; substrate preference of casein, haemoglobin and benzoyltyrosine ethyl ester but not benzoylarginine ethyl ester; mol.wt. 33000. However, it is unaffected by 1mm-di-isopropyl phosphofluoridate or 1mm metal cations, and in this respect differs from chymotrypsin. 3. The properties of the enzyme strongly resemble those of the ‘chymotrypsin-like’ enzyme discovered in seminal plasma by Lundquist et al. (1955). 4. The use of dimethyl-casein permitted the performance of enzyme assays at substrate concentrations five times higher (up to 50mg/ml) than could be achieved with ordinary casein (10mg/ml).

Large-scale isolation of fraction 1 leaf protein (18S) from lucerne (Medicago sativa L)

1976 ◽  
Vol 86 (3) ◽  
pp. 495-501 ◽  
Author(s):  
W. T. Jones ◽  
J. L. Mangan
Keyword(s):  
Molecular Weight ◽  
Medicago Sativa ◽  
Ammonium Sulphate ◽  
Large Scale ◽  
Gel Filtration ◽  
Low Molecular Weight ◽  
Fraction 1 Protein ◽  
Ammonium Sulphate Fractionation ◽  
Medicago Sativa L ◽  
Soluble State

SummaryFraction 1 protein (18S) can be isolated in large quantities (order 100 g) in a soluble state by heating lucerne juice, adjusted to pH 6·7 to 6·9, from a Pirie extractor to 63 °C for 10 min. Low speed oentrifugation (2500 g) removed coagulated chloroplast fragments and most of the heat-denatured Fraction 2 proteins. Fraction 1 (18S) protein (> 95% pure) was purified from low molecular weight materials (sugars, phenolics etc.) by ammonium sulphate fractionation and gel filtration on Sephadex G-75 gels.

scholarly journals Purification and Characterization of Peroxidase From Anthracnose Disease Infected Papaya (Carica papaya L.)

2014 ◽  
Vol 6 (2) ◽  
pp. 49-57 ◽  
Author(s):  
L Bari ◽  
P Hassan ◽  
N Absar ◽  
S Khatun ◽  
MI Hossain
Keyword(s):  
Ammonium Sulphate ◽  
Gel Filtration ◽  
Potassium Cyanide ◽  
Ammonium Sulphate Precipitation ◽  
Reducing Conditions ◽  
Sds Page ◽  
Peroxidase Enzyme ◽  
Km Value ◽  
Temperature Optima

Peroxidase enzyme was isolated and purified from the pulp of disease infected ripen papaya of local variety by 90% ammonium sulphate precipitation, chromatography on DEAEcellulose followed by hydrophobic chromatography on Phenyl Sepharose CL-4B and the purifications achieved was about 7.2 fold with 2.5% recovery. The purified enzyme was homogeneous as judged by polyacrylamide slab gel electrophoresis. The purified enzyme had a Mr of about 55,000 and 50 000 as determined by gel filtration on Sephadex G-100 and SDS-PAGE, respectively. The molecular mass of the enzyme was found to be very similar under both reducing and non-reducing conditions indicating that the enzyme contains no subunit. The enzyme has the following characteristics: pH optima at 6.0, temperature optima around 38°C, enzyme activity was found to be strongly inhibited in the presence of potassium cyanide and Fe+2 while the activity was found to be remarkably increased in the presence of ammonium sulphate. The Km value for the peroxidase obtained with pyrogallol as substrate was 0.027 mM. DOI: http://dx.doi.org/10.3329/bjmb.v6i2.17643 Bangladesh J Med Biochem 2013; 6(2): 49-57

scholarly journals Production and optimization of Aspergillus niger glucoamylase using amylopectin from guinea corn starch as the sole carbon source

2017 ◽  
Vol 52 (4) ◽  
pp. 263-272
Author(s):  
PC Okwuenu ◽  
AL Ezugwu ◽  
FC Chilaka
Keyword(s):  
Carbon Source ◽  
Ammonium Sulphate ◽  
Sole Carbon Source ◽  
Corn Starch ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Glucoamylase Activity ◽  
Ammonium Sulphate Precipitation ◽  
Specific Activities ◽  
Two Peaks

A Fourteen day experimental study was carried out to determine the day of highest glucoamylase activity using amylopectin from guinea corn starch as the sole carbon source. Two peaks of high activity were observed on the fifth and twelveth days, and were thus mass produced. Specific activities for crude enzymes were found to be 729.45 U/mg and 1046.82 U/mg for day five and twelve harvested enzymes respectively. Ammonium sulphate saturations, 70% and 20%, were found suitable to precipitate proteins with highest glucoamylase activity for day five and twelve harvested enzymes respectively. After ammonium sulphate precipitation and gel filtration, specific activities were found to be 65.98 U/mg and 180.52 U/mg respectively for day five harvested enzyme and 61.51 U/mg and 272.81 U/mg for day twelve harvested enzyme. The pH optimum for day five harvested enzyme were found to be 7.5, 7.5 and 6.0 using tiger nut, cassava and guinea corn starches as substrates respectively, also, the pH optimum for day twelve harvested enzyme were found to be 5.0, 8.5 and 7.0 using tiger nut, cassava and guinea corn starches as substrate, respectively. Optimum temperatures were found to be 50˚C and 45˚C for day five and twelve harvested enzymes, respectively. Km and Vmax, of day five harvested enzyme were found to be 770.75 mg/ml and 2500 μmol/min, 158.55 mg/ml and 500 μmol/min and 46.23 mg/ml and 454.53 μmol/min using cassava, guinea corn and tiger nut starches as substrate respectively. Km and Vmax of day twelve harvested enzyme were found to be 87.1 mg/ml and 384.61 μmol/min, 29.51 mg/ml and 243.90 μmol/min, and 2364 mg/ml and 2500 μmol/min, using cassava, guinea corn and tiger nut starches as substrate respectively.Bangladesh J. Sci. Ind. Res. 52(4), 263-272, 2017

scholarly journals Subunits From Reduced and S-Carboxymethylated Ribulose Diphosphate Carboxylase (Fraction I Protein)

1969 ◽  
Vol 22 (2) ◽  
pp. 463 ◽  
Author(s):  
KE Moon ◽  
EOP Thompson
Keyword(s):  
Ammonium Sulphate ◽  
Gel Filtration ◽  
Fraction I Protein ◽  
Ammonium Sulphate Fractionation ◽  
Fraction I ◽  
Ribulose Diphosphate Carboxylase

Fraction I protein isolated from spinach beet chloroplasts was purified by ammonium sulphate fractionation and Sephadex G�200 gel filtration. The isolated protein was reduced and S-carboxymethylated, and the dissociated protein resolved into two distinct subunits by gel filtration on Sephadex G-200 in an 8M urea buffer at pH 10�0.

scholarly journals Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

1974 ◽  
Vol 141 (1) ◽  
pp. 211-217 ◽  
Author(s):  
Hilary A. Rolton ◽  
Hamish M. Keir
Keyword(s):  
Molecular Weight ◽  
Ethylene Glycol ◽  
Substrate Concentration ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Kidney Cells ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Hyperbolic Curve ◽  
Km Value

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a Km value for dCMP of 0.91mm. dCTP activation required the presence of Mg2+ or Mn2+ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg2+ or Mn2+ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).

scholarly journals Pemurnian dan Karakterisasi Enzim Lipase dari Aspergillus oryzae pada Kopra Berjamur

2012 ◽  
Vol 14 (1) ◽  
pp. 26
Author(s):  
Seniwati Dali Dali ◽  
Abdul Rauf Patong ◽  
Muhammad Noor Jalaluddin ◽  
Pirman Andi Parenrengi
Keyword(s):  
Ammonium Sulphate ◽  
Aspergillus Oryzae ◽  
Enzyme Purification ◽  
Maximum Activity ◽  
Crude Enzyme ◽  
Lipase Catalysis ◽  
Lipase Enzyme ◽  
Km Value ◽  
Ammonium Sulphate Fractionation

An investigation on purification and characterization of lipase enzyme production Aspergillus oryzae from copra by fermentation of oliveoil has been carried out. This enzyme can be produced by fermenting olive oil in a medium containing Aspergillus oryzae. Crude enzyme isobtained by centrifuging the medium cultures containing Aspergillus oryzae at 3500 rpm for 30 minutes and than adding 0.2 M borat buffer(pH 8.2). Enzyme activity was determined from paranitrophenol as product of lipase catalysis of paranitrophenylbutirat (0.2 M) assubstrate measured by the Vorderwulbecke method. Prepurification process was by ammonium sulphate fractionation. Precipitation60-80% ammonium sulphate produced maximum activity of enzyme. Purification by Q sepharosa FF and sephadex G-75 collumchromatography produced four and three fractions with purifity of 12.85 and 20.25 times than crude enzyme respectively. Characterizationof this enzyme showed optimum condition at pH 8.2, temperature at 350C, the Km value at 0.046 M, and Vmaks is 1.926 μmol/menit and themolecular weight at 40.7 kDa.

scholarly journals Isolasi, Pemurnian dan Karakterisasi Fitase Bacillus subtilis dari Holiwood Gresik

2010 ◽  
Vol 15 (2) ◽  
pp. 113-119 ◽  
Author(s):  
Leny Yuanita ◽  
Aline Puspita ◽  
Suzana Surodjo ◽  
Sri Hidayati ◽  
Farid Al Amin ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Control Group ◽  
Optimum Temperature ◽  
Ph Optimum ◽  
Control Group Design ◽  
Group Design ◽  
Optimum Ph ◽  
Post Test ◽  
Km Value

The aim of the research were isolation, purification and characterization of Bacillus subtilis phytase from Holiwood Gresik. The research was done in two stages; the first include enzyme isolation, precipitation with amonium sulphate, dialysis, gel filtration chromatography, SDS-PAGE analysis, while second determining optimum pH, optimum temperature, the effect of pH and temperature to enzim stability, the values of KM and Vmax Bacillus subtilis phytase from Holiwood Gresik. The first stage research design were One Shot Case Study and Post Test Only Control Group Design, while the second stage were Post Test Only Control Group Design and Factorial Design. The data being analyzed by one-way and two-way Anova. The results of research showed that Bacillus subtilis phytase has the molecular mass of 36.5 kDa, optimum pH at 6.5–7.0, optimum temperature at 41° C and it was found to be stable for 30 minute incubation at pH 7 or 30° C with 2% or 3% lost of its activity respectively. KM value was 0.62 mM and VMax 0.393 mmol/ml/minute.

Sign in / Sign up

Export Citation Format

Share Document