scholarly journals Purification and properties of adenosine 5′-phosphosulphate sulphotransferase from Euglena

1991 ◽  
Vol 274 (2) ◽  
pp. 355-360 ◽  
Author(s):  
J Li ◽  
J A Schiff
Keyword(s):  
Isoelectric Focusing ◽  
Euglena Gracilis ◽  
Deae Cellulose ◽  
Reactive Dye ◽  
Multiple Forms ◽  
Freezing And Thawing ◽  
Disulphide Bonds ◽  
Purification And Properties ◽  
Sds Page ◽  
High Concentrations

Adenosine 5′-phosphosulphate sulphotransferase (APSST) was extracted from Euglena gracilis Klebs var. bacillaris mutant W10BSmL by freezing and thawing and was purified about 10,000-fold (to homogeneity) with 10.5% recovery by (NH4)2SO4 precipitation, Sephadex G-100 chromatography, Reactive Blue-agarose, Reactive Dye-agarose, DEAE-cellulose, preparative isoelectric focusing and non-inactivating SDS/PAGE. The active APSST, with a molecular mass of 102 kDa and multiple forms from pI 5.0 to 5.5, is a tetramer held together by covalent (probably disulphide) bonds. An apparent Km of the purified enzyme for adenosine 5′-phosphosulphate (APS) of 0.1 microM is obtained when dithiothreitol is used as the thiol. The enzyme is stimulated by Mg2+, Ca2+ or Ba2+, and uses almost any thiol; dithiothreitol and dithioerythritol give the highest activity. In the absence of APS, the enzyme is inactivated (and is rendered monomeric) by thiols but is protected from thiol inactivation by AMP, adenosine 5′-phosphoramidate (APA) or adenosine 5′-monosulphate (AMS), which also inhibit APSST activity somewhat. The enzyme resists inactivation by SDS in the absence of thiols; SDS stimulates APSST activity at low concentration, but high concentrations are inhibitory.

scholarly journals Purification and properties of l-3-glycerophosphate dehydrogenase from pig brain mitochondria

1980 ◽  
Vol 192 (1) ◽  
pp. 9-18 ◽  
Author(s):  
I R Cottingham ◽  
C I Ragan
Keyword(s):  
Isoelectric Focusing ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Brain Mitochondria ◽  
Exchange Chromatography ◽  
Glycerophosphate Dehydrogenase ◽  
Pig Brain ◽  
Purification And Properties ◽  
High Concentrations ◽  
Triton X

L-3-Glycerophosphate dehydrogenase (EC 1.1.99.5) was purified from pig brain mitochondria by extraction with deoxycholate, ion-exchange chromatography and (NH4)2SO4 fractionation in cholate, and preparative isoelectric focusing in Triton X-100. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single subunit of mol.wt. 75 000. The enzyme contains non-covalently bound FAD and low concentrations of iron and acid labile sulphide. No substrate reducible e.p.r. signals were detected. The conditions of purification, particularly the isoelectric focusing step, lead to considerable loss of FAD and possibly iron-sulphur centres. It is therefore not possible to decide with certainty whether the enzyme is a flavoprotein or a ferroflavoprotein. The enzyme catalyses the oxidation of L-3-glycerophosphate by a variety of electron acceptors, including ubiquinone analogues. A number if compounds known to inhibit ubiquinone oxidoreduction by other enzymes of the respiratory chain failed to inhibit L-3-glycerophosphate dehydrogenase, except at very high concentrations.

scholarly journals Deoxyribonucleic acid polymerases of Euglena gracilis. Purification and properties of two distinct deoxyribonucleic acid polymerases of high molecular weight

1975 ◽  
Vol 151 (2) ◽  
pp. 227-238 ◽  
Author(s):  
A G McLennan ◽  
H M Keir
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Euglena Gracilis ◽  
Deoxyribonucleic Acid ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Optimum Conditions ◽  
Electrophoretic Mobilities ◽  
Substrate Activation ◽  
Purification And Properties

Two DNA polymerases of high molecular weight, pol A (mol.wt. 190 000) and pol B (mol.wt. 240 ooo), have been purified 6300-fold and 1600-fold respectively from an extramitochondrial supernatant of a bleached strain of Euglena gracilis. They have very similar requirements when assayed with an ‘activated’-DNA primer-template [the optimum conditions of pH and ionic (K+ and Mn2+) composition being 7.2, 25 mM and 0.2 mM respectively]. 0.2 mM-Mn2+ was about 1.5-2-fold as effective as 2 mM-Mg2+, owing to substrate activation by deoxyribonucleoside 5′-triphosphates in the presence of Mn2+. Km values for the triphosphates in the absence of activation were about 10(-6)M with Mn2+ and 8 × 10(-6) M with Mg2+ for both enzymes. They were inhibited to the same extent by N-ethylmaleimide, novobiocin and o-phenanthroline, but differed in their chromatographic behaviour on DEAE-cellulose and in their electrophoretic mobilities on polyacrylamide gel. No evidence was found for the existence in these cells of a DNA polymerase of low molecular weight, but there were indications that a third enzyme of high molecular weight might exist.

The purification and properties of an amino acid arylamidase from bovine milk

1969 ◽  
Vol 47 (2) ◽  
pp. 173-178 ◽  
Author(s):  
A. Mellors
Keyword(s):  
Amino Acid ◽  
Milk Fat ◽  
Specific Activity ◽  
Bovine Milk ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Divalent Metal Ions ◽  
Purification And Properties ◽  
Milk Fat Globule ◽  
High Concentrations

An amino acid arylamidase is present in bovine milk and is associated with the "microsomes" of the milk-fat globule membrane. It has been purified by DEAE-cellulose chromatography of a 0.1 M NaCl extrast of milk microsomes. The specific activity of the purified arylamidase was increased 12 700-fold over that of the milk. Three peaks of arylamidase activity could be recognized after the chromatography. One form was apparently bound to casein. The major peak of arylamidase activity hydrolyzes lysyl-, alanyl-, valyl-, and arginyl-β-naphthylamides at similar rates, with little activity against glycyl- and histidyl-β-naphthylamides. The arylamidase requires the restoration of sulfhydryl groups by dithiothreitol for maximum activity. It is inhibited by EDTA and some divalent metal ions, and only calcium ions restore the EDTA-inactivated enzyme. The optimum pH for the hydrolysis of lysyl-β-naphthylamide is pH 7.7, and high concentrations of this substrate are inhibitory.

scholarly journals The N-acetylhexosaminidase components of the ram testis and epididymis

1973 ◽  
Vol 133 (3) ◽  
pp. 593-599 ◽  
Author(s):  
Sarah Bullock ◽  
Bryan Winchester
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Isoelectric Focusing ◽  
Catalytic Properties ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Multiple Forms ◽  
The Individual

Three and four N-acetylhexosaminidase components, from ram testis and epididymis respectively, have been separated by ion-exchange chromatography on DEAE-cellulose. Although they all have the same molecular weight (approx. 140000) and very similar catalytic properties towards the synthetic substrates, 4-methylumbelliferyl N-acetyl-β-glucosaminide and N-acetyl-β-galactosaminide, isoelectric focusing of the individual components showed that each had a distinct pI value. Isoelectric focusing has also been used to demonstrate the occurrence of multiple forms in ejaculated ram semen.

scholarly journals Purification of multiple forms of the soluble 17α-hydroxy steroid dehydrogenase or rabbit liver.

1975 ◽  
Vol 147 (3) ◽  
pp. 457-461 ◽  
Author(s):  
S Hasnain ◽  
D G Williamson
Keyword(s):  
Substrate Specificity ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Rabbit Liver ◽  
Multiple Forms ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

Eight distinct forms of the soluble 17alpha-hydroxy steroid dehydrogenase of rabbit liver were resolved by DEAE-cellulose chromatography and isoelectric focusing. Five of these enzymes were homogeneous as judged by polyacrylamide-gel electrophoresis. Substrate-specificity studies carried out with oestradiol-17alpha and oestradiol-17alpha 3-glucuronide revealed a variation in activity toward these substrates among the different purified enzyme forms. Three forms of the 17alpha-hydroxy steroid dehydrogenase exhibited equal activity toward both oestrogen substrates, whereas three forms of the enzyme displayed a greater activity toward the glucuronide derivative of oestradiol-17alpha. One enzyme in particular is essentially specific for oestradiol-17alpha 3-glucuronide, its activity toward oestradiol-17alpha being only one-thirtieth that observed with the 3-glucuronide derivative.

scholarly journals Purification and properties of a phenol sulphotransferase from Euglena using l-tyrosine as substrate

1994 ◽  
Vol 298 (1) ◽  
pp. 45-50 ◽  
Author(s):  
T Saidha ◽  
J A Schiff
Keyword(s):  
Euglena Gracilis ◽  
Deae Cellulose ◽  
Enzyme Reaction ◽  
Amino Group ◽  
Purification Procedure ◽  
Inhibition Kinetics ◽  
Tyrosine Derivatives ◽  
Purification And Properties ◽  
Membrane Bound ◽  
Tyrosine D

A purification procedure based on (NH4)2SO4 precipitation, and chromatography on Affi-Gel Blue, DEAE-cellulose, hydroxyapatite and Bio-Gel P-60 yields a stable 6400-fold-purified active monomeric phenol (tyrosine) sulphotransferase of 26 kDa from W10BSmL, an aplastidic mutant of Euglena gracilis var. bacillaris. The apparent Km for adenosine 3′-phosphate 5′-phosphosulphate (PAPS) is 15 microM (60 microM tyrosine as substrate); adenosine 5′-phosphosulphate is inactive. L-Tyrosine gave the lowest apparent Km (33 microM) (with PAPS at 30 microM), but tyrosine esters, tyrosinamide, L-p-hydroxyphenylglycine and a number of tyrosine dipeptides were also active, with higher Km values. Nitrophenols (m- and p-) and chlorophenols (o-, m- and p-) were active, with higher Km values than for tyrosine. D-Tyrosine was inactive as a substrate, as was D-p-hydroxyphenylglycine and a number of other tyrosine derivatives lacking the carboxy carbonyl or the amino group, or having extra ring substituents or the hydroxy group in the wrong position. Adenosine 3′,5′-bisphosphate and tyrosine O4-sulphate, products of the enzyme reaction with PAPS and tyrosine as substrates, showed competitive (Ki = 20 microM) and uncompetitive (Ki = 500 microM) inhibition kinetics respectively. This appears to be the first phenol sulphotransferase to accept tyrosine as substrate. This membrane-bound enzyme may be involved in tyrosine transport as well as detoxification.

scholarly journals Purification and properties of hyaluronidase from human liver. Differences from and similarities to the testicular enzyme

1982 ◽  
Vol 205 (1) ◽  
pp. 69-74 ◽  
Author(s):  
E W Gold
Keyword(s):  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Human Umbilical Cord ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Significant Difference ◽  
High Concentrations ◽  
Different Response

Human liver hyaluronidase was purified to homogeneity by (NH4)2SO4 fractionation, chromatography on hydroxyapatite and DEAE-cellulose, and preparative disc polyacrylamide-gel electrophoresis. The enzyme had a pH optimum of 3.8-4.0, a molecular weight (determined by gel filtration) of 76000, and a Km of 0.05 mg/ml for purified human umbilical-cord hyaluronic acid. It generally resembled hyaluronidases studied in other tissues which are believed to be lysosomal, but shared a number of characteristics with a partially purified bovine testicular hyaluronidase. Neither enzyme exhibited inhibition by high concentrations of substrate, but both were competitively inhibited by dermatan sulphate and keratan sulphate. Both enzymes exhibited increased activity in the presence of albumin, probably owing to an increased susceptibility of substrate to enzyme action. The liver enzyme was inhibited by NaCl, but the testicular enzyme exhibited an increase in activity in the presence of the salt which was similar to the effect observed with albumin. The different response toward Cl- ion appeared to be the most significant difference between the two enzymes.

scholarly journals Purification and properties of urease from bovine rumen

1977 ◽  
Vol 163 (3) ◽  
pp. 495-501 ◽  
Author(s):  
S Mahadevan ◽  
F D Sauer ◽  
J D Erfle
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Maximum Velocity ◽  
Maximum Activity ◽  
Rumen Bacteria ◽  
Multiple Forms ◽  
Bovine Rumen ◽  
Molecular Weights ◽  
Purification And Properties ◽  
High Concentrations

Urease (urea amidohydrolase, EC 3.5.1.5) was extracted from the mixed rumen bacterial fraction of bovine rumen contents and purified 60-fold by (NH4)2SO4 precipitation, calcium phosphate-gel adsorption and chromatography on hydroxyapatite. The purified enzyme had maximum activity at pH 8.0. The molecular weight was estimated to be 120000-130000. The Km for urea was 8.3 X 10(-4) M+/-1.7 X 10(-4) M. The maximum velocity was 3.2+/-0.25 mmol of urea hydrolysed/h per mg of protein. The enzyme was stabilized by 50 mM-dithiothreitol. The enzyme was not inhibited by high concentrations of EDTA or phosphate but was inhibited by Mn2+, Mg2+, Ba2+, Hg2+, Cu2+, Zn2+, Cd2+, Ni2+ and Co2+. p-Chloromercuribenzenesulfphonate and N-ethylmaleimide inhibited the enzyme almost completely at 0.1 mM. Hydroxyurea and acetohydroxamate reversibly inhibited the enzyme. Polyacrylamide-gel electrophoresis showed that the mixed rumen bacteria produce ureases which have identical molecular weights and electrophoretic mobility. No multiple forms of urease were detected.

scholarly journals Rabbit β-glucuronidase. Purification and properties, and the existence of multiple forms

1974 ◽  
Vol 138 (3) ◽  
pp. 395-405 ◽  
Author(s):  
Roger T. Dean
Keyword(s):  
Molecular Weight ◽  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Multiple Forms ◽  
Hydroxyapatite Gel

1. β-Glucuronidase (EC 3.2.1.31) was purified from rabbit liver by a procedure involving autolysis, (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration, sedimentation in a sucrose gradient, and isoelectric focusing. 2. Electron microscopy revealed ferritin as the major contaminant in later stages of purification and also showed aggregates of enzyme molecules. Particular attention was paid to the removal of ferritin. 3. The purified enzyme was homogeneous in polyacrylamide-gel electrophoresis both in non-dissociating conditions and in the presence of sodium dodecyl sulphate, and in Ouchterlony gel diffusion and immunoelectrophoresis against polyspecific antisera. 4. Sedimentation in sucrose gradients gave a molecular weight of 300000, whereas gel filtration indicated 440000. 5. Subunits of 75000 molecular weight were observed in gel electrophoresis in the presence of sodium dodecyl sulphate and in gel filtration in the presence of urea. 6. The Km value for p-nitrophenyl β-d-glucuronide was 0.6mm, and the enzyme was extremely sensitive to lactone inhibitors. It was also inhibited by Hg2+ ions. 7. Multiple forms were observed in the pure enzyme by isoelectric focusing, with pI values of 4.5–5.8. Subunits showed similar heterogeneity. The origin of the multiple forms was investigated in detail, and the possibility of artifact generation largely excluded. Some of the forms of lowest pI disappeared after neuraminidase digestion. The nature of the residual heterogeneity remains to be elucidated.

Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

1990 ◽  
Vol 48 (1) ◽  
pp. 282-283
Author(s):  
José L. Carrascosa ◽  
José M. Valpuesta ◽  
Hisao Fujisawa
Keyword(s):  
Dna Sequences ◽  
Expression Vector ◽  
Three Dimensional ◽  
Deae Cellulose ◽  
Dna Packaging ◽  
Dimensional Structure ◽  
Two Dimensional ◽  
Freezing And Thawing ◽  
Three Dimensional Structure ◽  
Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

Sign in / Sign up

Export Citation Format

Share Document