scholarly journals Purification and physicochemical characterization of a human cytotoxic factor produced by a human haemic cell line

1981 ◽  
Vol 194 (3) ◽  
pp. 847-856 ◽  
Author(s):  
H Neumann ◽  
A Karpas
Keyword(s):  
Molecular Weight ◽  
Cell Line ◽  
Concanavalin A ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Cytotoxic Factor ◽  
Factor I ◽  
Factor Ii

A cytotoxic factor, produced by a human lymphoblastoid cell line [Karpas (1977) Br. J. Cancer 35, 152–160; Karpas (1977) Br. J. Cancer 36, 437–445], was purified both from the cell extracts and from the culture medium containing the cell lysate, by using ammonium sulphate precipitation, DEAE-cellulose chromatography, gel filtration and affinity chromatography on concanavalin A–Sepharose and on [3H]amino-ethanol–glass beads. Two factors, Factor I and Factor II, were separated by DEAE-cellulose chromatography. Factor I was eluted from this column at 30 mM-aminoethanol/HCl buffer, pH 8.0, whereas Factor II was bound strongly to DEAE-cellulose and was eluted only at 325 mM-aminoethanol/HCl buffer, pH 8.0. The purified Factor I migrated as a single band on polyacrylamide-gel electrophoresis. Its isoelectric point, pI, was 8.0 +/- 0.3. Its sedimentation coefficient, S20,w, was 3.5 +/- 0.1 S and its apparent molecular weight, Mr, was 65 000 +/- 1000 as determined by sedimentation-velocity and sedimentation-equilibrium measurements. A linear relationship between molecular weight and concentration was found in equilibrium runs, suggesting a non-spherical shape of the molecule. Factor I is not a glycoprotein, inasmuch as it does not bind to concanavalin A–Sepharose. It consists of two subunits (Mr 32 000 +/- 4000), migrating on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis as a single band. Factor II had pI 6.0 +/- 0.4 and Mr 75 000 +/- 3000. Factors I and II are thus different proteins.

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.

Fractionation of Chromatin Components

1973 ◽  
Vol 51 (5) ◽  
pp. 709-720 ◽  
Author(s):  
John J. Monahan ◽  
Ross H. Hall
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Urea Solution ◽  
Equilibrium Density ◽  
Tissue Culture Cell ◽  
Low Molecular Weight ◽  
Nonhistone Proteins

A general method for isolation and fractionation of chromatin into its four major components, DNA, RNA, histories, and nonhistone proteins, is described. The procedure avoids the use of strongly acidic or alkaline conditions, or the use of ionic detergents or phenol. As few as 14 × 106 cells can be used. The procedure is reasonably rapid and has been used successfully with a number of tissue culture cell lines. The chromatin components are dissociated in a 3 M NaCl – 5 M urea solution containing 2-mercaptoethanol and EDTA. The DNA and high molecular weight RNA are collected by high-speed centrifugation and DNA is separated from the RNA by means of Cs2SO4 equilibrium density centrifugation. The histones, nonhistone proteins, and low molecular weight RNA's are fractionated using DEAE-cellulose column chromatography and polyacrylamide gel electrophoresis. A small amount (< 1%) of protein is present in the DNA and RNA fractions. At least 11 low molecular weight RNA subfractions can be detected by means of polyacrylamide gel electrophoresis.

scholarly journals Purification and characterization of mouse α-lactalbumin and preparation of its antibody

1980 ◽  
Vol 185 (1) ◽  
pp. 227-237 ◽  
Author(s):  
Y Nagamatsu ◽  
T Oka
Keyword(s):  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Major Species ◽  
Alpha Lactalbumin

alpha-Lactalbumin was purified to apparent homogeneity from mouse milk by combined use of gel filtration, chromatography on DEAE-cellulose and hydroxyapatite, and concanavalin A-Sepharose affinity chromatography. Mouse alpha-lactalbumin exists in several species with different charges and in two molecular-size forms. The smaller form, which constituted over 90% of total alpha-lactalbumin, included two major and two minor species, each of which showed different electrophoretic mobility on polyacrylamide-gel electrophoresis, but gave the same single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in two different buffer systems and over the range 10-15% acrylamide concentrations. The molecular weight was estimated as 14 100. The two major species of the smaller form had the same amino acid composition and contained no significant amount of carbohydrate. The larger form of alpha-lactalbumin, consisting of two species with different charges, was present in a small amount (less than 10%) in the milk and was isolated by its ability to interact with concanavalin A-Sepharose. Each of the two species also gave the same single band of apparent mol.w.t 18 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, this value may be anomalous, since this larger form appears to be glycosylated, and glycoproteins can behave anomalously on sodium dodecyl sulphate/polyacrylamide gels by binding less sodium dodecyl sulphate. All species of mouse alpha-lactalbumin from milk were active in the lactose synthase reaction and showed identical immunological properties, as determined by the mono-specific antibody prepared against the small major species. The presence of both the larger and the smaller forms, each in a percentage concentration similar to that found in milk, was also demonstrated in alpha-lactalbumin induced by hormones in organ cultureof pregnant-mouse mammary gland.

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.

Purification and Characterization of Ginsenoside-Hydrolyzing β-Glucosidase from Wheat Bran

2013 ◽  
Vol 641-642 ◽  
pp. 906-909
Author(s):  
Chun Zhi Zhang ◽  
Ming Chen ◽  
Hai Chen Guo ◽  
Guo Ren Zu ◽  
Li Chen
Keyword(s):  
Molecular Weight ◽  
Wheat Bran ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Purification And Characterization ◽  
Enzyme Solution ◽  
Crude Enzyme Solution

The ginsenoside-hydrolyzing β-glucosidase that can converse the major ginsenosides into the minor ginsenosides was isolated from wheat bran, and the enzyme was purified and characterized. The crude enzyme solution extracted from wheat bran could hydrolyse the protopanaxadiol-type ginsenosides such as Rb1, Rc, Rd and Rg3, but could not hydrolyse the protopanaxatriol-type ginsenosides such as Re and Rg2. The enzyme fractionated on the DEAE-Cellulose DE-52 column was purified to one spot in SDS polyacrylamide gel electrophoresis, and the molecular weight of enzyme in the fraction 34, 47, and 61 was approximately 62 kDa, 62 kDa, and 68 kDa, respectively.

Purification Of Factors IX And X From Clinical Concentrate

1981 ◽  
Author(s):  
G C Russell ◽  
G Kemble ◽  
E G D Tuddenham
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Factor Ix ◽  
Factor X ◽  
Clinical Factor

Human factors IX and X have been purified to homogeneity from clinical factor IX concentrate that had been rejected for therapeutic use due to particulate contamination. (It was necessary to start with this material since in the UK, plasma is not commercially available). The procedure involved barium citrate adsorption followed by ammonium sulphate elution, DEAE- cellulose chromatography, gel filtration on Sephacryl S-200 and affinity chromatography on heparin sepharose gel. The preparation of factor IX at this stage showed a single band on SDS-polyacrylamide gel electrophoresis, of molecular weight 58,000. No change in molecular weight was observed in the presence of 2-mercaptoethanol. A further affinity chromatography column - poly (homoarginine) Sepharose or dextran sulphate sepharose - was necessary to obtain homogeneous factor X. The preparation obtained showed a single band on SDS-polyacrylamide gel electrophoresis of molecular weight 67,000. In the presence of 2-mercaptoethanol, two bands were obtained of molecular weights 49000 and 17000 representing the heavy and light chains respectively of factor X. The purified coagulation proteins contained no activated species detectable by nonactivated partial thromboplastin time or by chromogenic substrate (S2222) assay. Prothrombin protein Sand protein C are by-products of this purification procedure.

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

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