scholarly journals Purification and characterization of human hepatic cysteine-conjugate β-lyase

1986 ◽  
Vol 235 (2) ◽  
pp. 569-575 ◽  
Author(s):  
H Tomisawa ◽  
S Ichihara ◽  
H Fukazawa ◽  
N Ichimoto ◽  
M Tateishi ◽  
...  
Keyword(s):  
Human Liver ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Elimination Reaction ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Beta Elimination ◽  
Alpha Beta ◽  
Hydroxyapatite Gel

Cysteine-conjugate beta-lyase (EC 4.4.1.13) was purified about 880-fold from human liver obtained post mortem. The purification procedure included (NH4)2SO4 precipitation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and chromatofocusing. The purified enzyme cleaves the C-S bond of several S-aryl-L-cysteines to yield equimolar amounts of thiols, pyruvic acid and ammonia via an alpha beta-elimination reaction. The Mr of the enzyme was estimated to be 88,000 by gel filtration. The enzyme is thermolabile, has a pH optimum of 8.5, and an apparent Km of 0.7 mM towards S-(p-bromophenyl)-L-cysteine. The enzyme requires pyridoxal 5′-phosphate as a cofactor, and hence the enzyme activity was completely abolished by hydroxylamine. No effect of EDTA or thiol-blocking reagents was observed on the activity of the enzyme.

Purification and Characterization of Two Proteins from Culture Filtrates of Mycobacterium tuberculosis H 37 Ra Strain

1970 ◽  
Vol 1 (2) ◽  
pp. 164-168
Author(s):  
Thomas M. Daniel ◽  
Lavenia E. Ferguson
Keyword(s):  
Molecular Weight ◽  
Mycobacterium Tuberculosis ◽  
Skin Testing ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Purification And Characterization ◽  
Culture Filtrates ◽  
Zone Electrophoresis

Two proteins have been purified from culture filtrates of Mycobacterium tuberculosis , H 37 Ra strain by a procedure combining gel filtration, diethylaminoethyl (DEAE)-cellulose chromatography, and zone electrophoresis. The two proteins are similar in molecular weight but differ slightly in charge. The faster migrating protein, designated a 1 , is not antigenic. The slower migrating protein, designated a 2 , is antigenic both with respect to antisera and as a skin-testing antigen.

scholarly journals Solubilization and characterization of pellet-associated human brain α-L-fucosidase activity

1985 ◽  
Vol 229 (3) ◽  
pp. 679-685 ◽  
Author(s):  
R L Hopfer ◽  
J A Alhadeff
Keyword(s):  
Human Brain ◽  
Isoelectric Focusing ◽  
Human Liver ◽  
Gel Filtration ◽  
Compelling Evidence ◽  
Supernatant Fluid ◽  
Ph Optimum ◽  
Triton X ◽  
Soluble Supernatant

The pellet-associated portion of human brain alpha-L-fucosidase (which represents approx. 20% of the homogenate activity) was solubilized with 0.5% (w/v) Triton X-100, characterized with regard to several properties and compared with the corresponding properties of the soluble supernatant-fluid enzyme in an attempt to find a second alpha-L-fucosidase in human brain. The solubilized and soluble alpha-L-fucosidase activities exhibited complete stability after storage at 2-4 degrees C for up to 29 days, comparable thermostability after preincubation at 50 degrees C, comparable apparent Km values (0.07-0.08 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside, comparable hydrophobicity, comparable isoelectric-focusing profiles (six major forms, with pI values between 4.5 and 5.8) and comparable immunoprecipitation curves (with the IgG fraction of antisera prepared against human liver alpha-L-fucosidase). Differences in three properties were found between solubilized and soluble alpha-L-fucosidase activities: the solubilized activity was less stable to storage at −20 degrees C, had a 0.5-pH-unit neutral shift in its pH optimum (6.0) and had smaller Mr forms after gel filtration on Sephadex G-200. The overall results indicate that the pellet-associated and soluble portions of human brain alpha-L-fucosidase are quite similar in most of their properties. Thus there is still no compelling evidence for the existence of a second mammalian alpha-L-fucosidase.

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

scholarly journals Purification and characterization of β-N-acetylhexosaminidase I2 from human liver

1986 ◽  
Vol 234 (1) ◽  
pp. 157-162 ◽  
Author(s):  
N N Dewji ◽  
D R De-Keyzer ◽  
J L Stirling
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Alpha Subunit ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.

Purification and characterization of exo-β-1,3-glucanase from a hatching supernatant of Strongylocentrotus intermedius

1983 ◽  
Vol 61 (1) ◽  
pp. 54-62 ◽  
Author(s):  
Kiyoshi Takeuchi
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Protein Band ◽  
Divalent Ions ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Single Protein

Exo-β-1,3-glucanase from the sea urchin embryos was purified 114-fold from the initial hatching supernatant by the following procedures: (a) gel filtration on Sephadex G-100; (b) hydrophobic chromatography on 4-phenylbutylamine-Sepharose (PBA-Sepharose); (c) two ion-exchange chromatographic steps on DEAE-cellulose; (d) gel filtration on Ultrogel AcA 34; (e) gel filtration on Sephadex G-100. The purified enzyme contained 2.2% carbohydrate and gave a single protein band corresponding to a molecular weight of 136 000 following electrophoresis on sodium dodecyl sulfate (SDS) – urea – polyacrylamide gel. Gel filtration on Ultrogel AcA 34 with a nondenaturing solvent gave a molecular weight of 130 000 ± 6000. The enzyme displayed an optimum pH at 5.0–5.5 and hydrolysed laminarin and PS(curdlan)-beads at the nonreducing ends, releasing glucose. Although activity of the purified enzyme was not affected by SDS, urea, some divalent ions, and 2-mercaptoethanol, both dithiothreitol and Hg2+ were markedly inhibitory.

scholarly journals Purification and characterization of a second form of acid lipase in human liver

1987 ◽  
Vol 248 (1) ◽  
pp. 139-144 ◽  
Author(s):  
E R Sjoberg ◽  
J D Hatton ◽  
J S O'Brien
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Polyacrylamide Gel ◽  
Acid Lipase ◽  
Purification And Characterization ◽  
Cellulose Acetate Electrophoresis

We describe here the purification and characterization of a form of acid lipase from human liver (designated ALII), which differed from the more abundant Mr-29000 form (ALI). ALII was solubilized from frozen human liver with Triton X-100 and purified 8500-fold by chromatography over concanavalin A-sepharose, CM-cellulose and finally h.p.l.c. over a Mono S column. ALII migrated as a single band on polyacrylamide-gel electrophoresis in both the presence and the absence of SDS. The Mr of ALII was estimated to be 58,500 by SDS/polyacrylamide-gel electrophoresis. Gel filtration on Sephacryl S-200 gave an apparent Mr of 69,000. 4-Methylumbelliferyl (4MU) palmitate, cholesterol oleate and triolein were substrates for ALII, with apparent Vmax values of 5000, 1100 and 2500 nmol/min per mg respectively and Km values of 1.0, 1.5 and 1.8 mM respectively. Cholesterol oleate and triolein were hydrolysed optimally by ALII at pH 4.5, whereas 4MU palmitate was hydrolysed optimally at pH 5.3. Antisera were raised against ALI and ALII and, on immunoblot analysis, no antigenic similarity was observed between ALI and ALII. Cellulose acetate electrophoresis followed by reaction with 4MU palmitate revealed two forms of lipase, corresponding to ALI and ALII. The two enzymes were also separated by hydrophobic chromatography. The activity of ALII was stimulated by several proteins and was partially inhibited by millimolar concentrations of NaCl, CaCl2 and MgSO4.

Purification and Characterization of Activation Fragments F1 and F2 from Human Prothrombin

1975 ◽  
Author(s):  
A. P. Ball ◽  
J. Fenton ◽  
D. L. Aronson ◽  
R. B. Franza ◽  
A. M. Young
Keyword(s):  
Gel Electrophoresis ◽  
Large Scale ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Scale Production ◽  
Purification And Characterization ◽  
Large Scale Production ◽  
Human Thrombin ◽  
A Chain

Considerable quantities of the non-thrombin portions of human prothrombin (II) have become available as a byproduct of the large-scale production of human thrombin (Biochim. Biophys. Acta 229, 26). Components not adsorbed on CG-50 are further purified by DEAE-cellulose chromatography and gel filtration, yielding the NH2-terminal fragment (F1) and the inner fragment (F2) which are homogeneous by SDS gel electrophoresis. SDS gel electrophoresis of reduced F1 indicates variable amounts of a two-chain derivative, F1’, with one chain migrating just ahead of F1 and one just ahead of the thrombin A-chain. The F1 → F1’ conversion is catalyzed by thrombin with the creation of a new MH2-terminal threonine. Ultracentrifugal patterns of human F1 and F2 closely resemble those of the bovine fragments. NH2-terminal residues were found to be alanine (± threonine) for F1 and serine for F2. Minor deviations from the reported amino acid compositions of bovine F1 and F2 were observed, primarily in the acidic residues. Other properties include:Immunization of rabbits with F1 gave a precipitating antibody to F1 which cross-reacts with II, but native F2 does not appear to be immunogenic. 3H-F1 is rapidly cleared from the blood of rabbits (T 1/2 20 min), with a major portion detectable in the urine.

PURIFICATION AND CHARACTERIZATION OF CAPRINE PROLACTIN

1974 ◽  
Vol 60 (2) ◽  
pp. 359-367 ◽  
Author(s):  
A. S. McNEILLY ◽  
P. ANDREWS
Keyword(s):  
Simple Procedure ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Product Yield ◽  
Pituitary Hormones ◽  
Major Product ◽  
Purification And Characterization ◽  
Pituitary Glands ◽  
Ovine Prolactin

SUMMARY Prolactin was isolated from frozen goat pituitary glands by a simple procedure involving gel filtration and chromatography on DEAE-cellulose. The major product (yield, 2·5 mg/g pituitary tissue) had high pigeon crop sac-stimulating activity (27 i.u./mg) and was free of growth hormone and other pituitary hormones. The molecular weight was similar to that of ovine prolactin. Caprine prolactin was immunologically indistinguishable from ovine prolactin in radioimmunoassays, in which ovine prolactin antiserum and either ovine or caprine prolactin labelled with 125I were used. The results indicate that caprine and ovine prolactin are closely related and that radioimmunoassay for ovine prolactin may be used to estimate caprine prolactin in serum.

scholarly journals The separation and characterization of the methylumbelliferyl β-galactosidases of human liver

1976 ◽  
Vol 157 (1) ◽  
pp. 189-195 ◽  
Author(s):  
P S Cheetham ◽  
N E Dance
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Neuraminidase Treatment ◽  
High Molecular Weight Component ◽  
Electrophoretic Procedure ◽  
Beta Galactosidase

1. A previously uncharacterized form of human liver acid beta-galactosidase (EC 3.2.1.23), possibly a dimer of molecular weight 160 000, was resolved by gel filtration. It has the same ability to hydrolyse GM1 ganglioside as the two other acid beta-galactosidase forms. 2. The low-molecular-weight forms of acid beta-galactosidase undergo salt-dependent aggregation. 3. The high-molecular-weight component may consist of the low-molecular-weight forms bound to membrane fragments. It can be converted completely into a mixture of these forms. 4. The neutral beta-galactosidase activity can be resolved into two forms by DEAE-cellulose chromatography. They differ in their response to Cl-ions. 5. A new nomenclature is suggested for the six beta-galactosidases so far found in human liver. 6. The enzymic constituents of the beta-galactosidase bands resolved by electrophoresis were re-examined. The A band contains three components. A two-dimensional electrophoretic procedure for resolving the A band is described. 7. The effect of neuraminidase treatment on the behaviour of beta-galactosidases in various separation systems is examined.

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.

Sign in / Sign up

Export Citation Format

Share Document