scholarly journals Effect of different compounds on 1-aspartamido-β-N-acetylglucosamine amidohydrolase from human liver

1978 ◽  
Vol 171 (3) ◽  
pp. 799-802 ◽  
Author(s):  
B Dugal
Keyword(s):  
Enzyme Activity ◽  
Human Liver ◽  
Gel Filtration

The effect of varous compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was studied. N-Acetylcysteine inhibited the nezyme non-competitively (Ki 3.2 mM), whereas 3-hydroxybutanone inhibited competitively (Ki 4.1 mM). Methionine, isoleucine and cystathionine apparently enhanced the enzyme activity. The enzyme had a mol. wt. of 63000 as determined by gel filtration. The present studies differentiate between the aspartylglucosylaminase from human liver and that obtained from various other sources.

Stimulation of β-(l → 6)-glucanase production by oxidized pustulan

1972 ◽  
Vol 18 (10) ◽  
pp. 1543-1550 ◽  
Author(s):  
Robert G. Brown
Keyword(s):  
Enzyme Activity ◽  
Isoelectric Focusing ◽  
Gel Filtration ◽  
Tween 80 ◽  
Sole Source ◽  
Cellulase Production ◽  
Low Degree ◽  
Degree Of Oxidation ◽  
High Degree ◽  
Stimulation Of

A strain of Penicillium lilacinum, isolated from soil, produced pustulanase, β-(1 → 3)-glucanase, (EC. 3.2.1.6) and cellulase (EC.3.2.1.4) when cultivated on a medium containing pustulan as the sole source of carbon. If pustulan was replaced by ketopustulan, the production of pustulanase was stimulated about 10-fold although the amount of stimulation was dependent on the degree of oxidation of pustulan. β-(1 → 3)-Glucanase production was stimulated slightly by ketopustulan; however, the degree of oxidation did not affect significantly the yield of this enzyme. Cellulase production was either unaffected by the oxidized polymer, or at higher degrees of oxidation, decreased. Tween 80 stimulated the production of the three enzymes in media containing ketopustulan with a low degree of oxidation but was inhibitory to pustulanase and cellulase production in media containing ketopustulan with a high degree of oxidation. A combination of gel filtration and isoelectric focusing revealed that each enzyme activity was attributable to at least two proteins.

scholarly journals Characterization of the binding of plasminogen activators to plasma membranes from human liver

1992 ◽  
Vol 287 (3) ◽  
pp. 911-915 ◽  
Author(s):  
G Nguyen ◽  
S J Self ◽  
C Camani ◽  
E K O Kruithof
Keyword(s):  
Human Liver ◽  
Plasma Membranes ◽  
Gel Filtration ◽  
Mannose Receptor ◽  
Hepatoma Cells ◽  
Tissue Type ◽  
High Affinity ◽  
Liver Membranes ◽  
Membrane Bound ◽  
Pai 1

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.

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 Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

1992 ◽  
Vol 288 (2) ◽  
pp. 475-482 ◽  
Author(s):  
I Ishii-Karakasa ◽  
H Iwase ◽  
K Hotta ◽  
Y Tanaka ◽  
S Omura
Keyword(s):  
Enzyme Activity ◽  
Culture Medium ◽  
Gel Filtration ◽  
Partial Purification ◽  
Gastric Mucus ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Optimum Ph ◽  
New Type

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

scholarly journals Analysis of the forms of acetylcholinesterase from adult mouse brain

1975 ◽  
Vol 147 (2) ◽  
pp. 205-214 ◽  
Author(s):  
E D Adamson ◽  
S E Ayers ◽  
Z A Deussen ◽  
C F Graham
Keyword(s):  
Enzyme Activity ◽  
Mouse Brain ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Acetylcholinesterase Activity ◽  
Total Activity ◽  
Polyacrylamide Gel ◽  
Soluble Extract ◽  
Molecular Sizes

The solubilization of 80% of the acetylcholinesterase activity of mouse brain was performed by repeated 2h incubations of homogenates at 37 degrees C in an aqueous medium. Analysis of the soluble extract by gel filtration on Sephadex G-200 showed that up to 80% of the enzyme activity was eluted in a peak which was estimated to consist of molecules of about 74000mol.wt. This peak was called the monomer form of the enzyme. After 3 days at 4 degrees C, the soluble extract was re-analysed and was eluted from the column in four peaks of about 74000, 155000, 360000 and 720000 mol.wt. Since the total activity of the enzyme in these peaks was the same as that in the predominantly monomer elution profile of fresh enzyme, we concluded that the monomer had aggregated, possibly into dimers, tetramers and octomers. Extracts of the enzyme were analysed by polyacrylamide-gel electrophoresis and the resulting multiple bands of enzyme activity on gels were shown to separate according to their molecular sizes, that is by molecular sieving. All these forms had similar susceptibilities to the inhibitors eserine, tetra-isopropyl pyrophosphoramide and compound BW 284c51 [1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide]. Thus the forms of the enzyme in mouse brain which can be detected by gel filtration and polyacrylamide-gel electrophoresis may all be related to a single low-molecular-weight form which aggregates during storage. This supports similar suggestions made for the enzyme in other locations.

Purification of p-hydroxyphenylpyruvate hydroxylase from rat liver—requirements for cofactors

1976 ◽  
Vol 54 (5) ◽  
pp. 423-431 ◽  
Author(s):  
Kun-Tsan Lin ◽  
John C. Crawhall
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Gel Filtration ◽  
Protein Band ◽  
Final Concentration ◽  
Assay Method ◽  
Enzyme Extract ◽  
Crude Enzyme Extract ◽  
Single Protein

Theenzyme p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27)from rat liver was studied with the assay method which measures the release of 14CO2 from p-hydroxyphenyl [carboxyl-,14C]pyruvate. Extensive dialysis of the crude enzyme extract against Tris buffer or purification involving ammonium sulfate, gel filtration, and ion exchange results in loss of enzyme activity that can be reactivated by Fe2+, dichlorophenolindophenol, and various other agents. The effect of these activators depends critically on their final concentration in the assay media.A 70-fold purification of the enzyme fraction yielded a preparation which behaved as a single protein band in Sephadex G-150. It had an isoelectric point at 5.85 and molecular weight of 63 000. The enzyme obtained appears to be different in some respects from those described by other workers from the liver of dog, human, chicken, and frog.

A three step purification procedure for human liver ferritin

1980 ◽  
Vol 58 (6) ◽  
pp. 494-498 ◽  
Author(s):  
M. Pagé ◽  
J. Lagueux ◽  
C. Gauthier
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Purification Procedure ◽  
Human Liver Ferritin ◽  
Normal Human Liver ◽  
Normal Human ◽  
Liver Ferritin

We describe a method for the purification of normal human liver ferritin by ultrafiltration, gel filtration on Sephacryl S-300, and affinity chromatography on DEAE-Affi Gel Blue. The purity of the ferritin obtained was verified by immunoelectrophoresis, Ouchterlony immunodiffusion, polyacrylamide gel electrophoresis, and electrofocusing. This rapid method yields 32% of the original ferritin.

Inhibitory effect of six herbal extracts on CYP2C8 enzyme activity in human liver microsomes

Xenobiotica ◽  
2014 ◽  
Vol 45 (5) ◽  
pp. 406-412 ◽  
Author(s):  
Ahmed A. Albassam ◽  
Mohamed-Eslam F. Mohamed ◽  
Reginald F. Frye
Keyword(s):  
Enzyme Activity ◽  
Human Liver ◽  
Liver Microsomes ◽  
Inhibitory Effect ◽  
Human Liver Microsomes ◽  
Herbal Extracts

scholarly journals Measurement of 1-aspartamido-β-N-acetylglucosamine amidohydrolase activity in human tissues

1977 ◽  
Vol 163 (1) ◽  
pp. 9-14 ◽  
Author(s):  
B Dugal
Keyword(s):  
Enzyme Activity ◽  
Incubation Time ◽  
Human Liver ◽  
Enzyme System ◽  
Total Concentration ◽  
Reaction Times ◽  
Human Tissues ◽  
Human Organs ◽  
Crude Homogenate ◽  
Enzyme Fraction

The activity of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was measured in normal and diseased human liver, brain and kidney. Organs from patients with aspartylglucosaminuria show very little activity. Crude homogenates of human organs show a reaction catalysed by a complex enzyme system. With homogenate, the formation of product was linear with time up to about 6 h. Reaction times longer than 6-7h resulted in a decrease in the total concentration of product. This phenomenon was not found with the partially purified enzyme fraction. Linearity of the enzyme activity with different protein concentrations was found, independent of the incubation time. Longer incubation of the crude homogenate resulted in the utilization of the product, N-acetylglucosamine. This phenomenon was not observed with the partially purified enzyme fraction. This amidase from human organs differs from that obtained from other sources and apparently represents a rather complex enzyme system.

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