Bovine hepatic carboxylesterases chromatographic fractionation, gel filtration, and molecular weight estimation

1969 ◽  
Vol 47 (8) ◽  
pp. 799-805 ◽  
Author(s):  
D. J. Ecobichon
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bovine Liver ◽  
Weight Estimation ◽  
Molecular Weights ◽  
Starch Gel ◽  
Two Peaks ◽  
Second Peak

The carboxylesterase activity of bovine liver was fractionated by DEAE-cellulose chromatography into two peaks of activity. Electrophoresis in starch gel showed that the peak eluted first was composed of a group of five electrophoretically slow bands while the second peak was a single, rapidly migrating band. Gel filtration of the crude extract on Sephadex G-100 and G-200 yielded a single peak of esterase activity containing both the electrophoretically slow and fast bands. The determination of molecular weights by gel filtration on Sephadex G-200 and G-100 yielded estimates of 52 000 and 55 000, respectively. The molecular weight estimates of the DEAE-cellulose fractionated electrophoretically slow and fast bands on Sephadex G-100 were identical, namely 55 000.

Cytoplasmic Carboxylesterases of Human and Domestic Animal Liver: Aggregation, Dissociation and Molecular Weight Estimation

1972 ◽  
Vol 50 (1) ◽  
pp. 9-15 ◽  
Author(s):  
D. J. Ecobichon
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Gel Filtration ◽  
Domestic Animal ◽  
Starch Gel Electrophoresis ◽  
Weight Estimation ◽  
Animal Liver ◽  
Starch Gel ◽  
Species Specific ◽  
Two Peaks

The cytoplasmic carboxylesterases of bovine, ovine, equine and human liver were fractionated by starch gel electrophoresis and by gel filtration on Sephadex. While species-specific, heterogeneous bands were observed in starch gel, the esterases of the bovine, ovine and equine liver were eluted from Sephadex G-100 as single peaks of activity, each with a characteristic elution volume. Gel filtration of human liver extracts yielded two peaks of activity, one containing electrophoretically slow esterases, the other electrophoretically fast esterases. Extracted equine and human hepatic carboxylesterases aggregated readily on storage or concentration, forming larger units which could be dissociated by a combination of acidic pH and high salt concentration. Molecular weight estimates of the hepatic esterases by gel filtration on Sephadex G-100 and G-200 yielded values of 65 000 for ovine, 55 000 for bovine, 96 000 and 70 000 for equine variants and 180 000 and 65 000 for human variants. The observations suggested that the cytoplasmic enzymes in relatively crude hepatic extracts had a lower molecular weight than those in concentrated or partially purified preparations which formed stable dimers or trimers.

scholarly journals A chromatographic preparation of ox growth hormone

1966 ◽  
Vol 100 (3) ◽  
pp. 593-600 ◽  
Author(s):  
M Wallis ◽  
HBF Dixon
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Gel Electrophoresis ◽  
Anterior Pituitary ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Starch Gel Electrophoresis ◽  
Highly Active ◽  
Cross Linkage ◽  
Starch Gel

1. A method is described for the chromatographic preparation of ox growth hormone. It involves chromatography of an extract of anterior pituitary lobes on DEAE-cellulose, followed by rechromatography on a dextran gel of low cross-linkage (Sephadex G-100). 2. The product is highly active in growth-hormone assays, and is obtained in good yield. It was homogeneous by several criteria, but showed some heterogeneity on starch-gel electrophoresis. 3. The molecular weight of the hormone was estimated from its behaviour on gel-filtration columns under various conditions. Evidence that the hormone may dissociate into sub-units under some conditions is presented.

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.

IDENTIFICATION AND PURIFICATION OF A NEW PROTEIN FROM WATER-SOLUBLE EXTRACTS OF HUMAN ADRENAL GLAND

1979 ◽  
Vol 82 (3) ◽  
pp. 383-NP ◽  
Author(s):  
M. A. AL-AWQATI ◽  
Y. B. GORDON ◽  
T. CHARD
Keyword(s):  
Molecular Weight ◽  
Adrenal Gland ◽  
Gel Filtration ◽  
Water Soluble ◽  
Double Immunodiffusion ◽  
Molecular Weights ◽  
Physicochemical Methods ◽  
Two Peaks ◽  
Sepharose 4B ◽  
New Protein

An homogenate of human foetal adrenal gland was subjected to negative immunoabsorption by column chromatography using anti-whole human serum coupled to Sepharose 4B. Two peaks were eluted and used to immunize rabbits. The antisera produced were absorbed and tested for specificity by double immunodiffusion. Two antigens, which appeared to be specific to the adrenal gland, were identified having molecular weights of 25 000 and 65 000 as determined by gel filtration. The lower molecular weight antigen was isolated by physicochemical methods and found to be a protein. The amino acid composition is reported.

scholarly journals Plasmin Inhibitors in Human Urine

1977 ◽  
Author(s):  
H. Sumi ◽  
Y. Takada ◽  
A. Takada
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Molecular Form ◽  
Membrane Filter ◽  
Gel Filtration ◽  
Total Activity ◽  
Native Form ◽  
Molecular Weights ◽  
Two Peaks

By the gel filtration of urinary protein on Sephadex G-200, two peaks of activity to hydrolyze Nα-acetylglycyl-L-lysine methyl ester (AGLMe) were detected. One was the native form of urokinase, and the molecular weight was about 54,000. The other was of high molecular weight, and eluted in void fraction. The high molecular form was thought to be a complex of urokinase and urinary plasmin inhibitor (UPI). By using Arg-Sepharose, membrane filter (M.W. 10,000), and pevikon block electrophoresis, we could isolate four types of UPI from normal human urine. One UPI was positively charged at pH 8.6, and of high molecular weight. Other types were negatively charged, and the molecular weights by gel filtration on Sephadex G-200 were about 67,000 (UPI6.7), 45,000 (UPI4.5), and 22, 000 (UPI2.2), respectively. In acrylamide disc gel electrophoresis, UPI57 migrated to serum prealbumin fraction, and UPI45 and UPI? 2 were less anionic. Negative-charged UPIs could be adsorbed on trypsin-Sepharose, and were thought to be identical to urinary trypsin inhibitors. Purified UPIs showed strong inhibition on caseinolytic- and esterolytic-activities of plasmin, and the total activity was about 16 UPIU(inhibited 16 casein U of plasmin)/liter of urine.

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 The separation and characterization of marmoset kidney β-d-galactosidase and β-d-glucosidase

1977 ◽  
Vol 167 (3) ◽  
pp. 765-773 ◽  
Author(s):  
R J Pierce ◽  
R G Price
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Starch Gel Electrophoresis ◽  
Lysosomal Fraction ◽  
Glucosidase Activity ◽  
Starch Gel

beta-D-Galactosidase and beta-D-glucosidase activities were determined in homogenates of marmoset kidney by using the appropriate 4-methylumbelliferyl glycoside, beta-D-Galactosidase activity was separated into two main components by ion-exchange chromatography on DEAE-cellulose, starch-gel electrophoresis, isoelectric focusing and gel filtration on Sephadex G-200. One form designated A had a pI of 5.1, was loosely bound to DEAE-cellulose at pH7.0, remained near the origin on starch-gel electrophoresis at pH 7.0 and had an apparent molecular weight of 160000. The second beta-D-galactosidase component, designated B, was associated with the total beta-D-glucosidase activity, had a pI of 4.3, was firmly bound to DEAE-cellulose, migrated rapidly towards the anode on starch-gel electrophoresis and had an apparent molecular weight of 50000. The optimum pH values of beta-D-galactosidase A and B were 4.5 and 6.0 respectively. beta-D-Galactosidase A was activated by 0.1 M-NaC1 but the activity of the B form was inhibited by 1 M-NaC1 at pH 4.5. beta-D-galactosidase had a bimodal distribution, the A form being recovered in the lysosomal fraction whereas the B form was present in the soluble fraction, as was the major portion of the beta-D-glucosidase activity. The lysosomal and soluble forms were further characterized by DEAE-cellulose chromatography.

scholarly journals Mannosidosis in Angus cattle. The enzymic defect

1974 ◽  
Vol 137 (2) ◽  
pp. 363-371 ◽  
Author(s):  
N. C. Phillips ◽  
D. Robinson ◽  
B. G. Winchester ◽  
R. D. Jolly
Keyword(s):  
Gel Filtration ◽  
Residual Activity ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Starch Gel Electrophoresis ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Angus Cattle ◽  
Starch Gel ◽  
Component C

Normal calf α-mannosidase activity exists in at least three forms separable by chromatography on DEAE-cellulose and by starch-gel electrophoresis. Two components, A and B, have optimum activity between pH3.75 and 4.75, but component C has an optimum of pH6.6. Components A and B are virtually absent from the tissues of a calf with mannosidosis and the residual activity is due to component C. The acidic and neutral forms of α-mannosidase differ in their molecular weights and sensitivity to EDTA, Zn2+, Co2+ and Mn2+. An acidic α-mannosidase component (pH optimum 4.0) accounts for most of the activity in normal plasma but it is absent from the plasma of a calf with mannosidosis. Although the acidic α-mannosidase component is probably related to tissue components A and B, it can be distinguished from them by ion-exchange chromatography and gel filtration. The optimum pH of the low residual activity in the plasma from a calf with mannosidosis is pH5.5–5.75. The results support the hypothesis that Angus-cattle mannosidosis is a storage disease caused by a deficiency of lysosomal acidic α-mannosidase activity.

Determination Of Molecular Size Of VIII:C In Whole Plasma By Electron Irradiation

1981 ◽  
Author(s):  
J Harmon ◽  
G A Jamieson ◽  
G Rock
Keyword(s):  
Molecular Weight ◽  
Electron Irradiation ◽  
Gel Filtration ◽  
Molecular Size ◽  
Target Size ◽  
Platelet Poor Plasma ◽  
Molecular Weights ◽  
Normal Plasma ◽  
Biological Mixtures

Loss of activity during electron irradiation provides a means of determining the molecular size of specific molecules in complex biological mixtures. This technique has established a molecular weight of 200,000 for Factor VIII:C in concentrates prepared from citrated plasma in which calcium is sequestered by chelation (Aronson, et al. Thromb. Diath. Haemorrh. 8:270, 1962): this value is similar to that obtained by more conventional techniques. However recent data suggest that in heparinized plasma, where physiological levels of calcium are maintained, about half of the VIII:C activity has a molecular weight of 50,000 as determined by gel filtration and ultracentrifugation (Rock, et al. Thromb. Res. 13:85, 1978). When heparinized plasma was subjected to electron irradiation in the frozen state there was a perceptible loss of VIII:C activity at 1 megarad and 80% loss with 60 megarads of irradiation. Analysis of the course of inactivation showed a biphasic curve with 73% of the VIII:C activity having a target size of 40,000 daltons while 28% had a molecular weight in excess of one million. Similar results were obtained when blood was collected in citrate and rapidly processed (∼5 min) to platelet-poor plasma. Following electron irradiation, 62% of VIII:C activity showed a target size of 35,000 daltons while the remaining 38% gave a target size of 275,000. These results provide further evidence that circulating VIII:C activity in normal plasma has a molecular weight of about 40,000 and suggest that reports of higher molecular weights are an artifact of the chelation of calcium as evidenced by the biphasic decay of VIII:C activity in citrated plasma.

scholarly journals Die Trennung der Hämolymphe-Proteine bei Vorpuppen und Puppen der Galleria mellonella L. mittels Gel-Filtration auf Sephadex G-200, Bestimmung ihrer isoelektrischen Punkte und ihrer Molekulargewichte / Separation of Haemolymph Proteins of Prepuae and Pupae of Galleria mellonella L. by means of Gel Filtration on Sephadex 200, Determination of their Isoelectric Points and their Molecular Weights

1969 ◽  
Vol 24 (6) ◽  
pp. 732-740 ◽  
Author(s):  
Milan Marek
Keyword(s):  
Gel Electrophoresis ◽  
Galleria Mellonella ◽  
Gel Filtration ◽  
Starch Gel Electrophoresis ◽  
Molecular Weights ◽  
Isoelectric Points ◽  
Starch Gel ◽  
The Individual ◽  
Naphthyl Acetate

Gel filtration on Sephadex G-200 was carried out on haemolymph proteins of prepupae. ligatured prepupae, male and female pupae and cooled pupae of Galleria mellonella L.The proteins were separated into two main fractions. The esterase activity of the eluated haemolymph was determined by means of beta-naphthyl acetate after filtration.After elution the samples were condensed and additionally separated on horizontal starch-gel electrophoresis.The “cooling protein” of pupae and the “ligature protein” of ligatured larvae of Galleria mellonella were shown by means of starch-gel electrophoresis to be new proteins, so far not described.The isoelectric point and molecular weight were determined for the individual protein fractions.They were then stained with amido black 10 B for the proof of proteins, and with alpha-naphthyl butyrate with Fast-Blue BB salt for the identification of esterases.

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