scholarly journals The purification and properties of carbonic anhydrases from guinea-pig erythrocytes and the mucosae of the gastrointestinal tract

1970 ◽  
Vol 120 (4) ◽  
pp. 797-808 ◽  
Author(s):  
M. J. Carter ◽  
D. S. Parsons
Keyword(s):  
Gastrointestinal Tract ◽  
Carbonic Anhydrase ◽  
Guinea Pig ◽  
High Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Carbonic Anhydrases ◽  
Low Activity

Procedures for isolating carbonic anhydrase (EC 4.2.1.1) enzymes from the erythrocytes and the mucosae of the gastrointestinal tract of guinea pigs are described. From a haemolysate, haemoglobin was removed by the addition of ammonium sulphate, and also by two other methods, namely by gel filtration or by adsorption on DEAE-Sephadex. The crude enzyme thus obtained was resolved into the different isoenzymes by chromatography with DEAE-cellulose. From particle-free supernatants of homogenates of some gastrointestinal tissues, carbonic anhydrases were purified by ammonium sulphate fractionation, gel filtration, and ion-exchange chromatography with DEAE-cellulose. The major isoenzymes from blood, stomach, proximal colonic mucosa and caecal mucosa were homogeneous during ion-exchange chromatography, acrylamide-gel electrophoresis, and centrifugal examination. From these tissues, carbonic anhydrase was isolated as two major isoenzymes. They resemble the pairs of isoenzymes discovered in the bloods of other species. The carbon dioxide hydratase activity of one isoenzyme (`high activity' carbonic anhydrase) was 40 times that of the other isoenzyme (`low activity' carbonic anhydrase), as measured at a single substrate concentration. Two other minor components of the enzyme are also found in guinea-pig erythrocytes. All of the enzymes isolated had molecular weights of nearly 30000 (sedimentation equilibrium). `High activity' carbonic anhydrases from blood and gastrointestinal tissues were indistinguishable according to some chemical, physical and kinetic measurements; similarly `low activity' carbonic anhydrases from those tissues were indistinguishable. `High activity' carbonic anhydrase was markedly different from the `low activity' carbonic anhydrase with respect to its amino acid composition, chromatographic behaviour and isoelectric pH value. Marked differences were also found in the tissue concentrations of the major isoenzymes. It is suggested that the characteristic and selective distribution of the different forms of carbonic anhydrase in the guinea-pig tissues is related to the specific and different physiological functions of the enzymes.

Separation of Heparin into Subtractions by DEAE-Cellulose Chromatography

1979 ◽  
Vol 42 (05) ◽  
pp. 1452-1459 ◽  
Author(s):  
Robert H Yue ◽  
Toby Starr ◽  
Menard M Gertler
Keyword(s):  
High Activity ◽  
Uronic Acid ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Net Charge ◽  
Uronic Acid Content ◽  
Successful Separation ◽  
Salt Gradient ◽  
Structure And Activity ◽  
Low Activity

SummaryCommercial porcine heparin can be separated into three distinct subtractions by using DEAE-cellulose chromatography and a stepped salt gradient. Gram quantities of heparin can be fractionated by this technique. All three heparin subtractions can accelerate the inhibition of thrombin by antithrombin III with different efficiency. The specific activities of the high activity heparin, intermediate activity heparin and low activity heparin are 228 units/mg, 142 units/mg and 95 units/mg, respectively. Both the uronic acid content and the quantity of N-SO4 for all three heparin subfractions have been evaluated. The high activity heparin has the lowest uronic acid and N-SO4 content. The successful separation of commercial heparin into three distinct subfractions by means of ion-exchange chromatography suggests that the net charge on these three heparin components will serve as a model system in the elucidation of the structure and activity relationship to the biological function of heparin.

scholarly journals The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

1972 ◽  
Vol 130 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Colin H. Self ◽  
P. David J. Weitzman
Keyword(s):  
Molecular Weight ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Low Concentrations ◽  
Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

scholarly journals Carbonic anhydrase isoenzymes in the erthrocytes and dorsolateral prostate of the rat

1969 ◽  
Vol 114 (3) ◽  
pp. 463-476 ◽  
Author(s):  
J. E. A. McIntosh
Keyword(s):  
Carbonic Anhydrase ◽  
High Activity ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Efficient Catalyst ◽  
Molecular Weights ◽  
Hydrolysis Of ◽  
Low Activity

1. Three forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from the erythrocytes of the rat and two forms from the dorsolateral prostate of the rat. Several additional minor components were observed but not isolated. Separation of the isoenzymes was achieved by ion-exchange chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing. 2. The general properties of the isolated isoenzymes, their molecular weights and their contents of zinc were closely similar. As catalysts of the hydration of carbon dioxide, however, they were distinctly different. The two most abundant isoenzymes of the erythrocytes, which were found in equal proportions, differed 70-fold in specific activity, whereas the isoenzymes of the dorsolateral prostate were similar to one another and resembled the high-activity component of the erythrocytes. The inhibition of the latter by acetazolamide (5-acetamido-1-thia-3,4-diazole-2-sulphonamide) was mainly competitive, whereas in identical conditions the low-activity erythrocyte component and the dorsolateral prostate isoenzymes were non-competitively inhibited. 3. The use of chloroform–ethanol to remove haemoglobin from the rat haemolysate was found (a) to bring about changes in the kinetic properties of the soluble isoenzymes and (b) to cause the appearance of an additional isoenzyme. 4. The actions were compared of the inhibitors acetazolamide, 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was an efficient catalyst of the hydrolysis of β-naphthyl acetate whereas the high-activity forms were much less active towards this ester. Neither of the isoenzymes present in the dorsolateral prostate catalysed this reaction. 6. Carbonic anhydrase in the rat dorsolateral prostate accounts for no more than 5% of the unusually high content of zinc in this organ.

THYROID-STIMULATING AND LIPOLYTIC ACTIVITIES OF PURIFIED PREPARATIONS OF HUMAN THYROID-STIMULATING HORMONE

1972 ◽  
Vol 53 (1) ◽  
pp. 95-100 ◽  
Author(s):  
ANNE STOCKELL HARTREE ◽  
MARJORIE THOMAS ◽  
BRIDGET E. FURNIVAL ◽  
T. W. BURNS ◽  
P. LANGLEY
Keyword(s):  
Lipolytic Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Protein Molecule ◽  
Thyroid Stimulating Hormone ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Thyroid ◽  
Rat Adipose Tissue ◽  
Stimulating Hormone

SUMMARY A partially purified fraction of human thyroid-stimulating hormone (DEAE-II) was further purified by ion-exchange chromatography on IRC-50, gel-filtration on Sephadex G-100 and finally chromatography on DEAE-cellulose. Two fractions were obtained which were high in thyroid-stimulating activity (8·3 and 7·3 units human Research Standard A/mg) and were comparable in potency to other preparations of the human hormone reported in the literature. They were also electrophoretically heterogeneous as were the preparations of other workers. Lipolytic activity toward cells obtained from human or rat adipose tissue was demonstrated for all fractions containing thyroid-stimulating activity, the two activities being roughly parallel. It is concluded that both thyroid-stimulating and lipolytic activities are probably present in the same protein molecule, but it is unlikely that the latter activity is of physiological significance.

Purification of glysojanin, an antifungal protein, from the black soybean Glycine soja

2003 ◽  
Vol 81 (6) ◽  
pp. 387-394 ◽  
Author(s):  
Patrick H.K Ngai ◽  
T B Ng
Keyword(s):  
Liquid Chromatography ◽  
Ion Exchange ◽  
Glycine Soja ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fast Protein Liquid Chromatography ◽  
Antifungal Protein ◽  
Black Soybean

A monomeric protein, with a molecular mass of 25 kDa and an N-terminal sequence resembling a segment of chitin synthase, was isolated from the seeds of the black soybean Glycine soja. The protein, designated glysojanin, demonstrated potent antifungal activity against the fungi Fusarium oxysporum and Mycosphaerella arachidicola. It inhibited HIV-1 reverse transcriptase with an IC50 of 47 µmol/L, [methyl-3H]thymidine incorporation by mouse spleen cells with an IC50 of 175 µmol/L, and translation in the rabbit reticulocyte lysate with an IC50 of 20 µmol/L. Glysojanin was purified using a procedure that involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography by fast protein liquid chromatography on Mono S, and gel filtration by fast protein liquid chromatography on Superdex 75.Key words: antifungal protein, seeds, soybean, purification.

Comparative studies of manganese(II)-, nickel(II)-, zinc(II)-, copper(II)-, cadmium(II)-, and iron(III)-binding components in human cord and adult sera

1984 ◽  
Vol 62 (6) ◽  
pp. 449-455 ◽  
Author(s):  
Show-Jy Lau ◽  
Bibudhendra Sarkar
Keyword(s):  
Molecular Weight ◽  
Trace Metals ◽  
Ion Exchange ◽  
Comparative Studies ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Low Molecular Weight ◽  
High Molecular Weight Proteins

The binding of six trace metals, Mn(II), Ni(II), Zn(II), Cu(II), Cd(II) and Fe(III), to human cord serum has been studied by Sephadex G-100 gel filtration at physiological pH, using radioisotopes as tracers. The results are compared with those obtained from adult serum. In both cord and adult sera, extensive amounts of the metals are bound to high molecular weight proteins. Among them, Fe(III) is mostly bound to transferrin; Ni(II), Zn(II), Cu(II), and Cd(II) are bound to albumin and other macro-molecules. The binding of Mn(II) either to transferrin or albumin is not resolved. Small fractions of Zn(II), Cu(II), and Cd(II) and large fractions of Mn(II) and Ni(II) are found to be associated with low molecular weight components of both sera. The distribution varies from metal to metal. However, the low molecular weight component of the size 1500 – 10 000 is present in all the metals studied. Further purification of this component was attempted by DEAE-cellulose ion-exchange chromatography. The possible identity as well as the biological role played by this particular component of serum in the transport of metals in blood and across membranes is discussed.

scholarly journals Carbonic anhydrase in skeletal and cardiac muscle from rabbit and rat

1992 ◽  
Vol 282 (1) ◽  
pp. 165-171 ◽  
Author(s):  
C Geers ◽  
D Krüger ◽  
W Siffert ◽  
A Schmid ◽  
W Bruns ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Cardiac Muscle ◽  
High Activity ◽  
Skeletal Muscles ◽  
Carbonic Anhydrases ◽  
Striated Muscles ◽  
Membrane Bound ◽  
Triton X ◽  
Dextran Solution ◽  
Low Activity

We have studied the distribution of carbonic anhydrases (CA) in several skeletal muscles of the hindlimb of rabbits and rats and in cardiac muscle of the rabbit. To remove erythrocyte CA, hindlimbs and hearts were thoroughly perfused with dextran solution, and the effectiveness of the perfusion was in most cases assessed by determining the contamination of the muscles with radioisotopes that had been used to label the erythrocytes before the perfusion was started. We observed three forms of CA: (1) cytosolic (sulphonamide-resistant) CA III; (2) a cytosolic sulphonamide-sensitive CA, probably isoenzyme II; (3) a membrane-bound form that was extracted from the particulate fraction using Triton X-100. These CA isoforms were distributed as follows. (1) CA III is located in the cytoplasm of slow, oxidative skeletal muscles and is absent from or low in fast skeletal and cardiac muscle; this holds for rabbits and rats and is identical with the pattern previously described for several other species. (2) The cytosolic sulphonamide-sensitive CA is present in fast rabbit muscles and absent from slow muscles of this species. In contrast, all skeletal muscles of the rat studied here lack, or possess only very low, activity of this isoenzyme. (3) The membrane-bound form of CA is present in all rabbit muscles studied; its activity appears somewhat higher in fast than in slow skeletal muscles. (4) Cardiac muscle constitutes an exception among all striated muscles of the rabbit as it possesses no form of cytosolic CA but a high activity of the membrane-bound form.

scholarly journals Resolution of myocardial phospholipase C into several forms with distinct properties

1983 ◽  
Vol 215 (2) ◽  
pp. 325-334 ◽  
Author(s):  
M G Low ◽  
W B Weglicki
Keyword(s):  
Ion Exchange ◽  
Phospholipase C ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Elution Profile ◽  
Close Correspondence ◽  
Molecular Weights ◽  
Low Activity

Phospholipase C activity capable of hydrolysing phosphatidylinositol in bovine heart was resolved into four forms (I-IV) by ion-exchange chromatography. Some of these forms could only be detected if the assay was performed at acidic pH (I and IV) or in the presence of deoxycholate (II). Gel-filtration chromatography indicated that the four forms had different molecular weights in the range 40000-120000. I, II and III all had pH optima in the range 4.5-5.5. However, the major form (III) also had substantial activity at pH 7.0 and above. The activities of I, II and III at pH 7.0 were stimulated by deoxycholate; this effect was most marked with I and II, which had very low activity at this pH. All forms of the enzyme were inhibited by EGTA and required 2-5 mM-CaCl2 for maximal activity. When the fractions eluted from the ion-exchange and gel-filtration columns were assayed with polyphosphoinositides as substrates there was a close correspondence to the elution profile obtained with phosphatidylinositol as substrate; there was no evidence for the existence in heart of phospholipase C activities specific for individual phosphoinositides.

Isolation of deoxyribonuclease II from bovine intestinal mucosa

1980 ◽  
Vol 58 (9) ◽  
pp. 749-753 ◽  
Author(s):  
D. Stephen Keys ◽  
S. H. Zbarsky
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Degraded Dna ◽  
Major Peak ◽  
Dnase Ii ◽  
Minor Peak ◽  
Supernatant Solution ◽  
Bovine Small Intestine

Mucosa from bovine small intestine was homogenized in Krebs–Ringer phosphate buffer, pH 7.8, the homogenate centrifuged at 16 300 × g, and the supernatant solution filtered through cheesecloth to remove lipid material. The filtrate was centrifuged at 105 000 × g and the supernatant solution chromatographed on DEAE-cellulose. The major peak of DNase II activity, eluted with 20 mM phosphate – 10 mM EDTA buffer, pH 7.8, was purified further by ion-exchange chromatography on CM-cellulose and gel filtration on Sephadex G-100. The enzyme was purified 78-fold in 13% yield. Evidence was adduced to indicate that the second minor peak of DNase II activity, eluted from the DEAE-cellulose by a potassium chloride gradient in the 20 mM phosphate – 10 mM EDTA buffer, was an artifact arising from the presence of significant amounts of DNA in the 105 000 × g supernatant. The enzyme degraded DNA endonucleolytically to 3′-PO4, 5′-OH oligonucleotides and is similar in its properties to DNase II from other tissues.

scholarly journals Biochemical properties and positional specificity of Lipase from hepatopancreas of Tra catfish (Pangasius)

2013 ◽  
Vol 16 (4) ◽  
pp. 85-91
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Ion Exchange ◽  
Lipase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Biochemical Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Positional Specificity ◽  
Tra Catfish ◽  
Ph Range

Lipase from the hepatopancreas of Tra catfish (Pangasius) was precipitated by ammonium sulfate fractionation, purified by ion-exchange chromatography on DEAE cellulose and gel filtration on Sephadex G- 75. On substrate triolein, purified lipase has Km= 1.381 mM and Vmax= 0.063 mM/min. The lipase was stable at a pH range of 7.0- 9.0 and in temperatures of 35-50°C. At 500C the enzyme loosed 44,7% activity after 120 min. The enzyme was specific for the α- positions (1, 3) of triglyceride. In bile salt solution of 0.015M NaTC, lipase activity of the enzyme increased in 3.08 folds in comparison of sample without NaTC.

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