scholarly journals Purification and characterization of the ferredoxin component of 25-hydroxycholecalciferol 1α-hydroxylase

1979 ◽  
Vol 177 (2) ◽  
pp. 673-678 ◽  
Author(s):  
J A Kulkoski ◽  
J G Ghazarian
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Molar Absorptivity ◽  
Polyacrylamide Gel ◽  
Antigenic Determinants ◽  
Paramagnetic Resonance ◽  
Nadph Cytochrome ◽  
25 Hydroxycholecalciferol

The chick kidney mitochondrial iron–sulphur protein (ferredoxin), a component of the NADPH–cytochrome P-450 reductase functional in the 1 alpha-hydroxylation of 25-hydroxycholecalciferol, was purified to homogeneity by chromatography on DEAE-cellulose, gel filtration on Sephadex G-100 and preparative electrophoresis on polyacrylamide gel. A novel NADPH–cytochrome c reductase assay utilizing crude renal NADPH–ferredoxin reductase was used for the detection of the ferredoxin. A mol. wt. of 53 000 was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by Sephadex G-100 gel filtration of the 125I-labelled ferredoxin. The ferredoxin has a sedimentation constant (S 20, w) of 2.66S, an A411/A280 of 0.4, and a molar absorptivity of 7300 cm-1 . M-1. The electron-paramagnetic-resonance spectrum after reduction with Methyl Viologen and dithionite was characteristic of ferredoxins with signals at g = 1.956 and 2.025. Two iron and two labile sulphur atoms per molecule of ferredoxin were released by acid. Ouchterlony immunodiffusion tests by using goat anti-(bovine adrenal ferredoxin) antiserum showed precipitin reactions with the bovine adrenal ferredoxin and the chick renal ferredoxin as antigens, suggesting that the renal ferredoxin shares antigenic determinants(s) with the natural adrenal antigen. Amino acid analysis showed that of the total number of residues per molecule of ferredoxin, glutamic acid and aspartic acid are the most abundant residues, comprising 17 and 15% respectively.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

scholarly journals The purification and molecular properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa

1981 ◽  
Vol 197 (2) ◽  
pp. 427-436 ◽  
Author(s):  
G A Nimmo ◽  
J R Coggins
Keyword(s):  
Molecular Weight ◽  
Neurospora Crassa ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Phosphate Synthase

Neurospora crassa contains three isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, which are inhibited by tyrosine, tryptophan and phenylalanine respectively, and it was estimated that the relative proportions of the total activity were 54%, 14% and 32% respectively. The tryptophan-sensitive isoenzyme was purified to homogeneity as judged by polyacrylamide-gel electrophoresis and ultracentrifugation. The tyrosine-sensitive and phenylalanine-sensitive isoenzymes were only partially purified. The three isoenzymes were completely separated from each other, however, and can be distinguished by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and Ultrogel AcA-34 and polyacrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the tryptophan-sensitive isoenzyme contained one type of subunit of molecular weight 52000. The molecular weight of the native enzyme was found to be 200000 by sedimentation-equilibrium centrifugation, indicating that the enzyme is a tetramer, and the results of cross-linking and gel-filtration studies were in agreement with this conclusion.

scholarly journals Purification and properties of arginase from human liver and erythrocytes

1978 ◽  
Vol 175 (2) ◽  
pp. 449-454 ◽  
Author(s):  
J Berüter ◽  
J P Colombo ◽  
C Bachmann
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Kinetic Properties ◽  
Purification Procedure

Arginase was isolated from human liver and erythrocytes. The purification procedure used acetone precipitation, heat-treatment, (NH4)2SO4 precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-200 in the presence of 2-mercaptoethanol. Both enzymes migrated to the anode at pH8.3 on polyacrylamide-gel electrophoresis. After incubation at pH8.0 and 37 degrees C the purified anionic liver arginase migrated to the cathode on polyacrylamide-gel electrophoresis. It is assumed that the multiple forms of the enzyme reported in the literature are partly artifacts of the purification procedure. The liver arginase showed a mol.wt. of 107000 determined by gel filtration and a sedimentation coefficient of 5.9S. Treatment of the liver enzyme with 0.25% sodium dodecyl sulphate at pH10 demonstrated an oligomeric structure of the enzyme with a mol.wt. of the subunit of 35000. The kinetic properties determined for the purified liver arginase showed an optimum pH of 9.3 and an optimal MnCl2 concentration of 2mM. The Km for L-arginine was 10.5 mM and for L-canavanine 50mM, and L-lysine exhibited a competitive type of inhibition with a Ki of 4.4mM. L-Homoarginine was not a substrate for liver arginase.

scholarly journals Purification and properties of S-adenosylmethionine: aldoxime O-methyltransferase from Pseudomonas sp. N.C.I.B. 11652

1985 ◽  
Vol 226 (1) ◽  
pp. 147-153 ◽  
Author(s):  
D B Harper ◽  
J T Kennedy
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Pseudomonas Sp ◽  
Ph Optimum

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.

scholarly journals Purification of uroporphyrinogen decarboxylase from human erythrocytes. Immunochemical evidence for a single protein with decarboxylase activity in human erythrocytes and liver

1983 ◽  
Vol 215 (1) ◽  
pp. 45-55 ◽  
Author(s):  
G H Elder ◽  
J A Tovey ◽  
D M Sheppard
Keyword(s):  
Gel Electrophoresis ◽  
Solid Phase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Human Erythrocytes ◽  
Decarboxylase Activity ◽  
Uroporphyrinogen Decarboxylase

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified 4419-fold to a specific activity of 58.3 nmol of coproporphyrinogen III formed/min per mg of protein (with pentacarboxyporphyrinogen III as substrate) from human erythrocytes by adsorption to DEAE-cellulose, (NH4)2SO4 fractionation, gel filtration, phenyl-Sepharose chromatography and polyacrylamide-gel electrophoresis. Progressive loss of activity towards uroporphyrinogens I and III occurred during purification. Experiments employing immunoprecipitation, immunoelectrophoresis and titration with solid-phase antibody indicated that all the uroporphyrinogen decarboxylase activity of human erythrocytes resides in one protein, and that the substrate specificity of this protein had changed during purification. The purified enzyme had a minimum mol.wt. of 39 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gel filtration gave a mol.wt. of 58 000 for the native enzyme. Isoelectric focusing showed a single band with a pI of 4.60. Reaction with N-ethylmaleimide abolished both catalytic activity and immunoreactivity. Incubation with substrates or porphyrins prevented inactivation by N-ethylmaleimide. An antiserum raised against purified erythrocyte enzyme precipitated more than 90% of the uroporphyrinogen decarboxylase activity from human liver. Quantitative immunoprecipitation and crossed immunoelectrophoresis showed that the erythrocyte and liver enzymes are very similar but not identical. The differences observed may reflect secondary modification of enzyme structure by proteolysis or oxidation of thiol groups, rather than a difference in primary structure.

scholarly journals Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fusarium solani

1977 ◽  
Vol 167 (3) ◽  
pp. 685-692 ◽  
Author(s):  
David B. Harper
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Fusarium Solani ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sole Source ◽  
Polyacrylamide Gel ◽  
Ph Optimum

1. A strain of the fungus Fusarium solani able to use benzonitrile as sole source of carbon and nitrogen was isolated by elective culture. 2. Respiration studies indicate that the nitrile, after degradation to benzoate, is catabolized via catechol or alternatively via p-hydroxybenzoate and 3,4-dihydroxybenzoate. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme mediating the conversion of benzonitrile into benzoate and ammonia. 4. The nitrilase enzyme was purified by DEAE-cellulose chromatography, (NH4)2SO4 precipitation and gel filtration on Sephadex G-200. The homogeneity of the purified enzyme preparation was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme showed a broad pH optimum between pH7.8 and 9.1 and a Km with benzonitrile as substrate of 0.039mm. The activation energy of the reaction deduced from an Arrhenius plot was 48.4kJ/mol. 6. The enzyme was susceptible to inhibition by thiol-specific reagents and certain heavy metal ions. 7. Gel filtration gave a value of 620000 for the molecular weight of the intact enzyme. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that the enzyme was composed of eight subunits of mol.wt. 76000. 8. Rates of enzymic attack on various substrates indicated that the nitrilase has a fairly broad specificity and that the fungus probably plays an important role in the biodegradation of certain nitrilic herbicides in the environment.

Photoactivated linkage of catechol O-methyltransferase and S-adenosyl-L-methionine

1984 ◽  
Vol 62 (10) ◽  
pp. 964-969 ◽  
Author(s):  
Peter H. Yu
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Ultraviolet Light ◽  
Paper Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Comt Inhibitors ◽  
Isoelectric Focussing

The formation of a stably linked complex of tritiated S-adenosyl-L-methionine (AdoMet) and catechol O-methyltransferase (COMT) has been achieved by irradiating the enzyme and ligand in Tris–HCl buffer (pH 7.5) with ultraviolet light at 254 nm. The reaction is specific as shown by a number of criteria. COMT inhibitors such as S-adenosylhomocysteine can block this photoactivated linkage. The [3H]AdoMet–COMT adduct has been shown to be a homogeneous protein by Sephadex gel filtration, sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and isoelectric focussing. After extensive proteolysis of the [3H]AdoMet–COMT adduct with pronase P, one major labelled product was released. This fragment could be separated by paper chromatography and was shown to be chromatographically identical to that released from the [3H]AdoMet – phenylethanolamine N-methyltransferase adduct.

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

scholarly journals Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein

1984 ◽  
Vol 220 (3) ◽  
pp. 811-818 ◽  
Author(s):  
M P Waalkes ◽  
S B Chernoff ◽  
C D Klaassen
Keyword(s):  
Metal Binding ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Or Groups

Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.

scholarly journals Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

1981 ◽  
Vol 195 (3) ◽  
pp. 545-560 ◽  
Author(s):  
Heinz Fankhauser ◽  
Jerome A. Schiff ◽  
Leonard J. Garber
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Low Ph ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Unknown Compound ◽  
Reactive Blue 2

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH4)2SO4 precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent Km for the last-mentioned compound is 0.82mm. The apparent Km for ammonia (assuming NH3 to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.

Sign in / Sign up

Export Citation Format

Share Document