scholarly journals Multiplicity of UDP-glucuronosyltransferases in fish. Purification and characterization of a phenol UDP-glucuronosyltransferase from the liver of a marine teleost, Pleuronectes platessa

1992 ◽  
Vol 284 (2) ◽  
pp. 417-423 ◽  
Author(s):  
D J Clarke ◽  
S G George ◽  
B Burchell
Keyword(s):  
Mammalian Species ◽  
Liver Microsomes ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Apparent Homogeneity ◽  
Molecular Masses ◽  
A Subunit ◽  
Udp Glucuronosyltransferase ◽  
High Degree ◽  
Sepharose 4B

The aim of this work was to determine if a non-mammalian species had multiple UDP-glucuronosyltransferase (UDPGT) isoforms. At least six highly purified UDPGT isoenzymes were partially resolved by anion-exchange chromatography and UDP-hexanolamine-Sepharose 4B affinity chromatography from liver microsomes of a fish, the plaice. Q-Sepharose FF, chromatofocusing and affinity-chromatographic procedures were employed to separate and purify the phenol UDPGT isoform to apparent homogeneity. The purified enzyme conjugated 1-naphthol, but not bilirubin or steroids, and displayed a pI of 7.0 and a subunit molecular mass of 55 kDa. Bilirubin and testosterone UDPGT activities were more labile and, although purified over 200-fold, these preparations also contained the phenol UDPGT and had multiple polypeptides with molecular masses of 52-57 kDa. Antisera to rat bilirubin/phenol UDPGT and testosterone/phenol UDPGT isoforms cross-reacted strongly with the partially purified plaice UDPGT isoforms of molecular masses 52, 53 and 57 kDa and less strongly with phenol UDPGT 54 kDa and 56 kDa isoforms. Fish and mammalian UDPGTs therefore apparently possess a high degree of evolutionary conservation.

Aniline hydroxylation and 7-ethoxycoumarin O-deethylation activities in solubilized and partially resolved liver cytochromes P-450 from ethanol-fed rats

1984 ◽  
Vol 62 (3) ◽  
pp. 266-271 ◽  
Author(s):  
V. Plourde ◽  
C. Hétu ◽  
J.-G. Joly
Keyword(s):  
Liver Microsomes ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Female Rats ◽  
Ethanol Administration ◽  
Molar Activity ◽  
Acidic Fraction ◽  
Chronic Ethanol Administration ◽  
And Control ◽  
Cytochrome P 450

Chronic ethanol administration in female rats enhances the apparent molar activity of liver microsomes for aniline hydroxylation and 7-ethoxycoumarin O-deethylation. Microsomal cytochromes P-450 from ethanol-fed and control rats have been solubilized and partially resolved in six fractions by anion-exchange chromatography. Induction of aniline hydroxylase activity by ethanol was associated with marked increases in the turnover numbers of the more basic cytochrome P-450 containing fractions in a reconstituted aniline hydroxylation system. Cytochrome P-450, exhibiting by far the highest 7-ethoxycoumarin O-deethylase activity, was eluted in a relatively acidic fraction; its turnover number with 7-ethoxycoumarin after ethanol consumption, however, did not differ significantly from that of the corresponding fraction from control microsomes. These observations suggest that induction of liver microsomal mixed function oxidases by ethanol may reflect the contribution of more than one cytochrome P-450 isozyme.

scholarly journals The Modified β-Ketoadipate Pathway in Rhodococcus rhodochrous N75: Enzymology of 3-Methylmuconolactone Metabolism

1998 ◽  
Vol 180 (24) ◽  
pp. 6668-6673 ◽  
Author(s):  
Chang-Jun Cha ◽  
Ronald B. Cain ◽  
Neil C. Bruce
Keyword(s):  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Sole Source ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Rhodococcus Rhodochrous ◽  
Natural Substrate ◽  
Ph Optimum ◽  
A Subunit ◽  
Hydrolysis Of

ABSTRACT Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified β-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native M r of 128,000, and a subunitM r of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.

scholarly journals Purification and characterization of an α2-macroglobulin-like proteinase inhibitor from plasma of the crayfish Pacifastacus leniusculus

1988 ◽  
Vol 255 (3) ◽  
pp. 801-806 ◽  
Author(s):  
H G Hergenhahn ◽  
M Hall ◽  
K Söderhäll
Keyword(s):  
Proteinase Inhibitor ◽  
Hydrophobic Interaction Chromatography ◽  
Acid Precipitation ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Pacifastacus Leniusculus ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Α2 Macroglobulin

An alpha 2-macroglobulin (alpha 2M)-like proteinase inhibitor from plasma of the crayfish Pacifastacus leniusculus was purified to apparent homogeneity by acid precipitation, hydrophobic interaction chromatography, affinity chromatography on concanavalin A-Sepharose and anion-exchange chromatography. The subunit Mr is about 190,000. Pore-size-limit electrophoresis proved the native protein to be a dimer. The purified protein resembled vertebrate alpha 2 Ms in that it protected trypsin from inhibition by soyabean trypsin inhibitor, and in its sensitivity to methylamine treatment. Methylamine also prevented the protein from being autolytically cleaved into Mr 60,000 and 140,000 fragments when subjected to heat treatment. The amino acid composition showed similarities with both human alpha 2 M and an alpha 2 M-like protein from the arthropod Limulus polyphemus. These data indicate that this Pacifastacus alpha 2M-like protein (P alpha 2M) may be a distantly related homologue of vertebrate alpha 2Ms.

Partial Purification and Some Properties of Brassica napus Lipase

1994 ◽  
Vol 49 (5-6) ◽  
pp. 293-301 ◽  
Author(s):  
Cornelia Fuchs ◽  
Gerd Hansen
Keyword(s):  
Brassica Napus ◽  
Size Exclusion Chromatography ◽  
Lipase Activity ◽  
Sodium Deoxycholate ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Partial Purification ◽  
Exclusion Chromatography ◽  
Molecular Masses

Abstract Lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from rape (Brassica napus cv. Ceres) was isolated from cotyledons of dark-grown seedlings. The enzyme was partially purified by poly­ ethylene glycol precipitation. Delipidation of the lipase with n-hexane was required prior to further purification by anion exchange chromatography and size exclusion chromatography. A purification factor of 337 was ultimately achieved and the purification process was moni­tored by SDS-PAGE. Here, at least two protein bands with molecular masses of 62 and 64 kD a respectively were found in the active fraction obtained by size exclusion chromatography. Sodium deoxycholate was found to stimulate the lipase activity, but appeared to cause aggregation of the enzyme. It was not possible to estimate the isoelectric point of the dialyzed rape lipase due to the high molecular mass of the aggregates. Two simple methods to detect lipase activity directly on polyacrylamide gel were applied. No esterase activity was found by using p-nitrophenyl acetate as substrate.

scholarly journals 4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2

1991 ◽  
Vol 274 (3) ◽  
pp. 833-842 ◽  
Author(s):  
H H Locher ◽  
T Leisinger ◽  
A M Cook
Keyword(s):  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Comamonas Testosteroni ◽  
Visible Absorption ◽  
Mononuclear Iron ◽  
Purification And Properties ◽  
A Subunit ◽  
Protein Components

Cell-free extracts of Comamonas testosteroni T-2 grown in toluene-p-sulphonate/salts medium catalyse the conversion of p-sulphobenzoate (PSB) into protocatechuate and sulphite by an NADH-requiring and Fe2(+)-activated dioxygenase. Anion-exchange chromatography of extracts yielded red (A) and yellow (B) protein fractions, both of which were necessary for dioxygenative activity. Further purification of each fraction by hydrophobic interaction chromatography and gel filtration led to two homogeneous protein components (A and B), which together converted 1 mol each of PSB, O2 and NADH into 1 mol each of protocatechuate, sulphite and, presumably, NAD+. The system was named 4-sulphobenzoate 3,4-dioxygenase (PSB dioxygenase system). Monomeric component B (Mr 36,000) was determined to be a reductase that contained 1 mol of FMN and about 2 mol each of iron and inorganic sulphur per mol. This component transferred electrons from NADH to the oxygenase component (A) or to, e.g., cytochrome c. Homodimeric component A (subunit Mr 50,000) of the PSB dioxygenase system contained one [2Fe-2S] centre per subunit and its u.v.-visible-absorption spectrum corresponded to a Rieske-type iron-sulphur centre. The requirement for activation by iron was interpreted as partial loss of mononuclear iron during purification of component A. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. The PSB dioxygenase system displayed a narrow substrate range: none of 18 sulphonated or non-sulphonated analogues of PSB showed significant substrate-dependent O2 uptake. The physical properties of the PSB dioxygenase system resemble those of other bacterial multi-component dioxygenase, especially phthalate dioxygenase. However, it differs from most characterized systems in its overall reaction; the product is a vicinal diphenol, and not a dihydrodiol.

scholarly journals Molecular cloning, expression and characterization of a ubiquitin conjugation enzyme (E217kB) highly expressed in rat testis

1995 ◽  
Vol 305 (1) ◽  
pp. 125-132 ◽  
Author(s):  
S S Wing ◽  
P Jain
Keyword(s):  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Histone H2a ◽  
Repair Gene ◽  
Ubiquitin System ◽  
Dna Repair Gene ◽  
Ubiquitin Conjugation ◽  
Molecular Masses ◽  
Ubiquitin Conjugating Enzymes

Ubiquitin-conjugating enzymes (E2s) play a key role in ubiquitin-mediated proteolysis by catalysing the conjugation of ubiquitin to protein substrates. We have previously reported the cDNA cloning of a 14 kDa conjugating enzyme [E2(14)k; Wing, Dumas and Banville (1992) J. Biol. Chem. 267, 6495-6501] that efficiently supported ubiquitination and protein degradation in reticulocyte extracts. Surprisingly, the structure of this E2 was markedly more similar to the Saccharomyces cerevisiae DNA repair gene RAD6, than to the S. cerevisiae UBC4/UBC5 genes which are required for the degradation of short-lived proteins and support much of the ubiquitination of yeast proteins. This suggested that mammalian homologues of UBC4/UBC5 remained to be identified. Using oligonucleotides derived from the S. cerevisiae UBC4 sequence as primers in a PCR reaction with rat muscle cDNA as a template, a 390 bp DNA fragment was amplified which predicted an amino acid sequence that was 83% identical to yeast UBC4. Screening a rat testes cDNA library identified a family of cDNAs which predicted two very similar proteins with basic pIs and molecular masses of approx. 16,700 Da. Isoform 2E was expressed in Escherichia coli and purified to homogeneity. It supported ubiquitination to reticulocyte and testis proteins more rapidly in vitro and produced larger conjugates than E2(14)k. Examination of RNA from different tissues indicated that this type of E2 was expressed in a broad spectrum of tissues but at particularly high levels in the testis. Fractionation of a testis extract by anion-exchange chromatography identified several putative ubiquitin protein ligase activities with which this E2 could interact in promoting conjugation of ubiquitin to proteins. One of these activities supported conjugation of ubiquitin to histone H2A, a substrate degraded in the ubiquitin system by a non-N-end rule mechanism. This paper reports the first cloning of a apparent mammalian homologue of S. cerevisiae UBC4/UBC5. Its high expression in testis and ability to efficiently support conjugation to testis proteins suggest that this family of E2s may play a role in the proteolysis that occurs during spermatogenesis.

N-Acetyl-ß-ᴅ-hexosaminidases of the Brine Shrimp Artemia: Partial Purification and Characterization

1991 ◽  
Vol 46 (9-10) ◽  
pp. 781-788 ◽  
Author(s):  
Klaus-Dieter Spindler ◽  
Brigitte Funke-Höpfner
Keyword(s):  
Brine Shrimp ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Strong Inhibitor ◽  
Molecular Masses ◽  
Dose Dependent ◽  
Enzyme I

Abstract N-Acetyl-β-ᴅ-hexosaminidases (EC 3.2.1.52) from Artemia nauplii were isolated and char­acterized. Three different enzymes I, II1 and II2 were separated according to their behaviour on anion exchange chromatography and gel filtration columns. Their apparent molecular masses were 83,000 ± 7000, 110,000 ± 10,000 and 56,000 ± 5000 Da with corresponding S-values of 8.6, 11.9 and 7.9. All three enzymes also differ in their apparent pH-optima (5.1, 4.5 and 6.1) and they all bind to concanavalin A. The three enzymes have about the same affinities (app. Km between 0.16 and 0.72 mmol/1) for the three substrates (p-nitrophenyl-N-acetyl-β-ᴅ-glucosamine or p-nitrophenyl-N-acetyl-β-ᴅ-galactosamine and N ,N′-diacetyl-chitobiose) and are therefore N-acetyl-β-ᴅ-hexosaminidases. In contrast, the three enzymes behave quite differently, both in terms of their inhibitor constants and the type of inhibition. The substrates inhibit both enzymes II1 and II2 but not enzyme I. On the other hand, N-acetyl-β-ᴅ-galactosamine inhibits enzyme I in a non-competi­tive way but not enzymes II1 and II2. All three enzymes are inhibited by the end product N-acetyl-β-ᴅ-glucosamine, enzyme I in a competitive manner, both enzymes II1 and II2 in a non-competitive way. 2-Acetamido-2-deoxy-ᴅ-galactonolactone is a strong inhibitor for en­zyme I (Ki = 13 μtmol/l) with much lower affinities towards enzymes II1 and II2 (Ki = 0.63 and 1.03 mmol/l). All three enzymes are inhibited in a dose-dependent way and completely reversi­ble by α-methyl-mannoside.

Carbohydrate Analysis of Bradyrhizobial (NC 92) Lipopolysaccharides by High Performance-Anion Exchange Chromatography with Pulsed Amperometric Detection

1999 ◽  
Vol 19 (3) ◽  
pp. 219-225 ◽  
Author(s):  
Hasi Das ◽  
Vani Jayaraman ◽  
Indranil Bhattacharya
Keyword(s):  
Anion Exchange ◽  
High Performance ◽  
Gel Filtration ◽  
Amperometric Detection ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Composition Analysis ◽  
Pulsed Amperometric Detection ◽  
Acid Hydrolysate ◽  
Sepharose 4B

Composition analysis of monosaccharides of Sepharose 4B purified NC 92 LPS and the polysaccharides fractions from Sephadex G-50 chromatography was performed by high performance anion exchange chromatography using pulsed amperometric detection. Rhamnose, mannose, galactose and glucose are present in a substantial amount in the purified LPS (Pk I). High molecular weight purified polysaccharides (PS I) obtained after sephadex gel filtration of the purified LPS (Pk I) acid hydrolysate showed an increase in glucose:galactose ratio. This indicates the presence of the peanut root lectin (PRA II) specific sugar in higher proportion on the O-antigen part of the LPS molecule, which may aid in the critical recognition reaction.

scholarly journals NADP-linked malic enzyme. Purification from maize leaves, Mr and subunit composition

1988 ◽  
Vol 254 (1) ◽  
pp. 229-233 ◽  
Author(s):  
M S Thorniley ◽  
K Dalziel
Keyword(s):  
Malic Enzyme ◽  
Enzyme Purification ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Exchange Chromatography ◽  
Subunit Composition ◽  
Sedimentation Equilibrium ◽  
Apparent Homogeneity ◽  
Maize Leaves ◽  
A Subunit

1. The isolation of NADP-linked malic enzyme (EC 1.1.1.40) from maize leaves is described, together with studies of its Mr and subunit composition. 2. The enzyme was purified to apparent homogeneity by affinity chromatography on N6-aminohexyl-2′,5′-bisphosphoadenosine-agarose, gel filtration with Sephadex G-100 and ion-exchange chromatography on DEAE-Sephadex A-50. A purification of 140-fold with a 30% yield was obtained. 3. A detailed study of the Mr by several methods revealed the existence of different Mr forms in solution. 4. In the presence of dithiothreitol the enzyme appears to be present in triethanolamine buffer, pH 7.5, as a tetramer with a subunit Mr of 60,000 and an S20,w of 10.75 S. 5. In phosphate buffer, pH 7.0, it seems to be a dimer of Mr 120,000 with an S20,w of 7.95 S. 6. In the absence of dithiothreitol, lower-Mr forms were detected by sedimentation-equilibrium and sedimentation-velocity studies in triethanolamine buffer. 7. Results from gel filtration gave Mr values of about 340,000 in both buffers.

Isolation and Purification of an Anticoagulant from Schizymenia dubyi by Fermentation

2007 ◽  
Vol 13 (5) ◽  
pp. 355-359 ◽  
Author(s):  
P.M. Ekanayake ◽  
C. Nikapitiya ◽  
M. De Zoysa ◽  
Y.J. Jeon ◽  
J. Lee
Keyword(s):  
Gel Electrophoresis ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Thrombin Time ◽  
Agarose Gel Electrophoresis ◽  
Fermentation Time ◽  
Preliminary Screening ◽  
Isolation And Purification ◽  
Time Period ◽  
Sepharose 4B

Natural fermentation is a method of extracting anticoagulant compounds from red algae Schizymenia dubyi. Preliminary screening was carried out on the basis of the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) assays. The optimal fermentation time period for the highest APTT, PT, and TT values was found to be 8 weeks. Purification of the fermented sample by DEAE-anion exchange chromatography followed by Sepharose-4B chromatography resulted in a single polysaccharide, which was reconfirmed by a single spot on agarose gel electrophoresis. The purified sample had >1000s APTT activity at 130 μg/mL. The molecular weight estimated by polyaccrylamide gel electrophoresis was >500,000 Da. This is the first report indicating the presence of the anticoagulant compound in S. dubyi.

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