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.

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.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

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 Detection, purification and characterization of a human cancer-associated galactosyltransferase acceptor

1979 ◽  
Vol 178 (2) ◽  
pp. 279-287 ◽  
Author(s):  
D K Podolsky ◽  
M M Weiser
Keyword(s):  
Molecular Weight ◽  
Human Cancer ◽  
Deae Cellulose ◽  
Structural Data ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Purification And Characterization ◽  
Peptide Moiety ◽  
Cellulose Column

A low-molecular-weight acceptor of galactosyltransferase activity was detected in sera and effusions of patients with extensive maligant disease. This substance was purified to homogeneity from both human serum and effusion by using sequential charcoal/Celite and DEAE-cellulose column chromatography. The purified acceptor was shown to act as substrate for both purified normal and cancer-associated human galactosyltransferase (EC 2.4.1.22) isoenzymes, but had a higher affinity for the cancer-associated isoenzyme (Km = 20 microM) than for the normal isoenzyme (Km = 500 microM). The substrate was found to be a glycopeptide with mol.wt. approx. 3600 determined by polyacrylamide-gel chromatography. Carbohyydate analysis demonstrated only the presence of glucosamine and mannose. Amino acid analysis revealed that the peptide moiety consisted of eight different amino acids, including two residues of asparagine and one residue of serine, but no threonine. These structural data suggest that the acceptor is a fraction of an asparagine-glucosamine type of glycoprotein.

scholarly journals Partial purification and characterization of chick-embryo prolyl 3-hydroxylase

1979 ◽  
Vol 183 (2) ◽  
pp. 303-307 ◽  
Author(s):  
K Tryggvason ◽  
K Majamaa ◽  
J Risteli ◽  
K I Kivirikko
Keyword(s):  
Molecular Weight ◽  
Chick Embryo ◽  
Ethylene Glycol ◽  
Affinity Chromatography ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Chick Embryo Extract

Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.

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.

scholarly journals Purification and characterization of a low molecular weight xylanase from solid-state cultures of Aspergillus fumigatus Fresenius

1999 ◽  
Vol 30 (2) ◽  
pp. 114-119 ◽  
Author(s):  
Claudio Henrique Cerri e Silva ◽  
Jurgen Puls ◽  
Marcelo Valle de Sousa ◽  
Edivaldo Ximenes Ferreira Filho
Keyword(s):  
Molecular Weight ◽  
Solid State ◽  
Aspergillus Fumigatus ◽  
Gel Filtration ◽  
Low Molecular Weight ◽  
Transferase Activity ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Sds Page

A xylan-degrading enzyme (xylanase II) was purified to apparent homogeneity from solid-state cultures of Aspergillus fumigatus Fresenius. The molecular weight of xylanase II was found to be 19 and 8.5 kDa, as estimated by SDS-PAGE and gel filtration on FPLC, respectively. The purified enzyme was most active at 55 °C and pH 5.5. It was specific to xylan. The apparent Km and Vmax values on soluble and insoluble xylans from oat spelt and birchwood showed that xylanase II was most active on soluble birchwood xylan. Studies on hydrolysis products of various xylans and xylooligomers by xylanase II on HPLC showed that the enzyme released a range of products from xylobiose to xylohexaose, with a small amount of xylose from xylooligomers, and presented transferase activity.

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 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 Purification and Characterization of the Glucose Oxidase from Penicillium notatum

2018 ◽  
Vol 9 (01) ◽  
Author(s):  
Baydaa A. Hassan ◽  
Mohammed A. Jebor ◽  
Zahra M. Ali
Keyword(s):  
Molecular Weight ◽  
Glucose Oxidase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Penicillium Notatum ◽  
Oxidase Enzyme ◽  
Filtration Technique

This study aims to purification and characterization of the glucose oxidase enzyme from Penicillium notatum, the enzyme was purified by ammonium sulfate precipitation (60%), dialysis and gel filtration chromatography using sephadex G-200, A trial for the purification of glucose oxidase using gel filtration technique resulted in one type of glucose oxidase with specific activity of (62.382 U/mg) with (7.385 folds) purification. the purified glucose oxidase had a maximum activity at pH = 5.5, 45 °C, glucose oxidase was stable with pH values ranging between (5 – 6) and the enzyme was maintained the activity when it incubated into (25 -35) °C for 15 minutes, analyses of the glucose oxidase for molecular weight was carried out by PAGE and SDS-PAGE electrophoresis, which revealed 78 KDa, also molecular weight of the glucose oxidase was achieved by gel filtration technique and was found 87 KDa this means that enzyme consisting of only one subunit, the Km and Vmax value of glucoamylase (B) were (19.6 mM, 7.5 mM/min ) respectively using different concentration of glucose.

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