scholarly journals Purification and properties of four species of lysyl oxidase from bovine aorta

1979 ◽  
Vol 177 (1) ◽  
pp. 203-214 ◽  
Author(s):  
Herbert M. Kagan ◽  
Kathleen A. Sullivan ◽  
Theodore A. Olsson ◽  
Anne L. Cronlund
Keyword(s):  
Metal Ion ◽  
Lysyl Oxidase ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Active Species ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Apparent Homogeneity ◽  
Multiple Species

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49–60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000–33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of α-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu2+, and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.

scholarly journals Isolation of palmitoyl-CoA hydrolases from human blood platelets

1981 ◽  
Vol 199 (3) ◽  
pp. 639-647 ◽  
Author(s):  
R K Berge ◽  
L E Hagen ◽  
M Farstad
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Cytosol Fraction ◽  
Molecular Weights ◽  
Apparent Homogeneity ◽  
Coa Hydrolase ◽  
Human Blood Platelets

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].

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 Characterization of an Inducible Chlorophenol O-Methyltransferase from Trichoderma longibrachiatum Involved in the Formation of Chloroanisoles and Determination of Its Role in Cork Taint of Wines

2003 ◽  
Vol 69 (9) ◽  
pp. 5089-5095 ◽  
Author(s):  
Juan-José R. Coque ◽  
María Luisa Álvarez-Rodríguez ◽  
Germán Larriba
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Ph Optimum ◽  
Trichoderma Longibrachiatum ◽  
Molecular Weights ◽  
Apparent Homogeneity ◽  
Halogenated Phenols ◽  
Cork Taint

ABSTRACT A novel S-adenosyl-l-methionine (SAM)-dependent methyltransferase catalyzing the O methylation of several chlorophenols and other halogenated phenols was purified 220-fold to apparent homogeneity from mycelia of Trichoderma longibrachiatum CECT 20431. The enzyme could be identified in partially purified protein preparations by direct photolabeling with [methyl-3H]SAM, and this reaction was prevented by previous incubation with S-adenosylhomocysteine. Gel filtration indicated that the M r was 112,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme was composed of two subunits with molecular weights of approximately 52,500. The enzyme had a pH optimum between 8.2 and 8.5 and an optimum temperature of 28°C, with a pI of 4.9. The Km values for 2,4,6-trichlorophenol and SAM were 135.9 ± 12.8 and 284.1 ± 35.1 μM, respectively. S-Adenosylhomocysteine acted as a competitive inhibitor, with a Ki of 378.9 ± 45.4 μM. The methyltransferase was also strongly inhibited by low concentrations of several metal ions, such as Cu2+, Hg2+, Zn2+, and Ag+, and to a lesser extent by p-chloromercuribenzoic acid, but it was not significantly affected by several thiols or other thiol reagents. The methyltransferase was specifically induced by several chlorophenols, especially if they contained three or more chlorine atoms in their structures. Substrate specificity studies showed that the activity was also specific for halogenated phenols containing fluoro, chloro, or bromo substituents, whereas other hydroxylated compounds, such as hydroxylated benzoic acids, hydroxybenzaldehydes, phenol, 2-metoxyphenol, and dihydroxybenzene, were not methylated.

scholarly journals Tamm–Horsfall urinary glycoprotein. The subunit structure

1970 ◽  
Vol 120 (2) ◽  
pp. 425-432 ◽  
Author(s):  
A. P. Fletcher ◽  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Disulphide Bonds ◽  
Terminal Amino ◽  
Urinary Glycoprotein

1. Subunit molecular weights of 76000–82000 were obtained for native and alkylated Tamm–Horsfall glycoprotein by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. 2. A further estimate of the subunit molecular weight of 79000±4000 was obtained by disc gel electrophoresis in sodium dodecyl sulphate. 3. A minimum value of the chemical molecular weight of 79000±6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 4. Similar values were obtained for the subunit molecular weight of Tamm–Horsfall glycoprotein from patients with cystic fibrosis. 5. On ultracentrifugation both in 1.0% sodium dodecyl sulphate and in 70% formic acid, Tamm–Horsfall glycoprotein sedimented as a single component, slightly faster than serum albumin. 6. On reduction of the disulphide bonds the same subunit molecular weight was obtained, which suggested that these bonds are intrachain.

scholarly journals Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain

1973 ◽  
Vol 135 (2) ◽  
pp. 367-373 ◽  
Author(s):  
C.-C. Liu ◽  
C.-H. Chung ◽  
M.-L. Lee
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Mammalian Brain ◽  
Acid Activation ◽  
High Concentration ◽  
Molecular Weights ◽  
Fractionation Column ◽  
Quaternary Structures

l-Tryptophan-activating enzyme [l-tryptophan–tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different Km and Vmax. values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg2+, ATP, in any combination.

scholarly journals Partial characterization of lysyl oxidase from several human tissues

1985 ◽  
Vol 230 (3) ◽  
pp. 639-643 ◽  
Author(s):  
H Kuivaniemi
Keyword(s):  
Human Skin ◽  
Oxidase Activity ◽  
Lysyl Oxidase ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Active Species ◽  
Skin Fibroblasts ◽  
Human Tissues ◽  
Human Skin Fibroblasts ◽  
Tissue Samples

Lysyl oxidase activity was assayed in urea extracts of a number of human tissues, proving to be highest in skin. Antibodies to human placental lysyl oxidase completely inhibited the activity of crude lysyl oxidase from all the human tissues studied, with no significant differences in the amounts of antiserum required for 50% inhibition. By contrast, marked differences were found in this value between skin tissue samples from different species. The Mr of lysyl oxidase in crude extracts of human skin and in the medium of cultured human skin fibroblasts was 30 000 by gel filtration, no active species with a higher Mr being detectable. Four forms of lysyl oxidase activity were seen in DEAE-cellulose chromatography of urea extract from human skin, all having Mr 30 000. Antibodies to human placental lysyl oxidase stained a 30 000-Mr protein in urea extracts of all the human tissues studied and in the medium of cultured human skin fibroblasts when examined by immunoblotting after sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis, but they also stained high-Mr material. The findings suggest that there are no immunologically distinct lysyl oxidase isoenzymes in the various human tissues and that the true Mr of lysyl oxidase in crude urea extracts is 30 000.

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

scholarly journals Purification and properties of α-mannosidase II from Golgi-like membranes of baculovirus-infected Spodoptera frugiperda (IPLB-SF-21AE) cells

1997 ◽  
Vol 324 (3) ◽  
pp. 951-956 ◽  
Author(s):  
Jianxin REN ◽  
Francis J. CASTELLINO ◽  
Roger K. BRETTHAUER
Keyword(s):  
Spodoptera Frugiperda ◽  
Reaction Pathway ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Mannose Residue ◽  
Lepidopteran Insect ◽  
Reducing Conditions ◽  
Apparent Homogeneity ◽  
Purification And Properties

An α-mannosidase II-like activity was identified in baculovirus-infected Spodoptera frugiperda (IPLB-SF21-AE) cells. The enzyme responsible was purified from Golgi-type membranes to apparent homogeneity by using a combination of steps including DEAE-cellulose, hydroxyapatite, concanavalin A–Sepharose and gel filtration chromatography. The molecular mass of this purified protein was approx. 120 kDa by SDS/PAGE under reducing conditions and approx. 240 kDa under non-reducing conditions, indicating that the enzyme is a disulphide-linked dimer. Substrates demonstrated to undergo hydrolysis with this enzyme were GlcNAc-Man5-GlcNAc-GlcNAc (non-reduced and reduced) and p-nitrophenyl α-d-mannopyranoside. The oligosaccharide substrate was converted into GlcNAc-Man3-GlcNAc-GlcNAc through an intermediate GlcNAc-Man4-GlcNAc-GlcNAc. Treatment of the isolated intermediate oligosaccharide with endoglycosidase H resulted in its conversion into GlcNAc-Man4-GlcNAc. This indicated that it contained the α-1,3-linked mannose residue on the α-1,6-linked mannose arm and showed that the α-1,6-linked mannose residue on the α-1,6-linked mannose arm had been preferentially hydrolysed by the mannosidase. The oligosaccharide lacking the β-1,2-linked GlcNAc residue on the α-1,3-linked mannose arm (Man5-GlcNAc-GlcNAc) was not hydrolysed in the presence of the enzyme. Metal ions were not required for enzymic activity on any of the substrates, but Cu2+ was strongly inhibitory. The activity of the enzyme was inhibited at low concentrations of swainsonine, but much higher concentrations of 1-deoxymannojirimycin were required to achieve inhibition. All of these properties are characteristic of mannosidase II enzymes from other eukaryotic tissues. The presence of mannosidase II in lepidopteran insect cells would allow entry of N-linked glycoproteins into the complex processing reaction pathway or into the terminal Man3-GlcNAc-GlcNAc pathway.

scholarly journals α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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