scholarly journals Column chromatography of human small-intestinal maltase, isomaltase and invertase activities

1969 ◽  
Vol 111 (2) ◽  
pp. 139-146 ◽  
Author(s):  
A. Dahlqvist ◽  
U. Telenius
Keyword(s):  
Ion Exchange ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Heat Inactivation ◽  
Enzyme Preparations ◽  
Small Intestinal ◽  
Two Peaks ◽  
Second Peak ◽  
Intestinal Disaccharidases

1. The maltase, isomaltase and invertase (sucrase) activities of solubilized mucosal preparations from human jejunum and ileum were studied with column chromatography on anion-exchange (diethylaminoethyl- and triethylaminoethyl-)cellulose and Sephadex G-200 gel. 2. On ion-exchange cellulose columns both kinds of enzyme preparations yielded two major disaccharidase peaks. The first peak contained maltase Ia (=isomaltase) and maltase Ib (=invertase). The second peak contained maltase II and maltase III. 3. On Sephadex G-200 gel columns jejunal preparations yielded the corresponding peaks as on ion-exchange columns, but the peaks appeared in the reverse order in the effluent. The ileal preparation studied yielded a single peak on gel columns, containing all the activities studied and eluted with the ‘void volume’. 4. Precipitation with ethanol did not affect the behaviour of the enzymes during ion-exchange chromatography. When gel filtration was performed after ethanol precipitation of the enzymes, however, two peaks were obtained also with the ileal preparation, and subfractionation of the invertase was obtained with both kinds of preparations. 5. The second peak from ion-exchange chromatograms, containing maltase II and maltase III, on concentration was found to have very weak isomaltase activity, probably exerted by these enzymes as such. This activity accounts for only about 1% of the total isomaltase activity of the mucosa. 6. The results support the concept of the specificity of the human small-intestinal disaccharidases previously described after heat-inactivation experiments. The subfractionation of the invertase that under certain conditions is seen on Sephadex G-200 columns appears most likely to be an artifact. Consequently the nomenclature for the human maltose-, isomaltose- and sucrose-splitting enzymes proposed by another research group after gel-filtration chromatography studies should be abandoned. It seems more logical to keep the nomenclature based on heat inactivation [maltase Ia (=isomaltase), maltase Ib (=invertase or sucrase), maltase II and maltase III] until increased knowledge about the specificity and structure of these enzymes makes possible a more rational nomenclature.

scholarly journals Heat inactivation and Sephadex chromatography of the small-intestine disaccharidases of the chick

1970 ◽  
Vol 116 (1) ◽  
pp. 71-78 ◽  
Author(s):  
R. C. Siddons
Keyword(s):  
Small Intestine ◽  
Gel Filtration ◽  
The Other ◽  
Void Volume ◽  
Heat Inactivation ◽  
Maltase Activity ◽  
Two Peaks ◽  
Hydrolysis Of ◽  
Second Peak ◽  
Intestinal Disaccharidases

1. The maltase, sucrase, isomaltase and palatinase activities of the chick small intestine are localized in particles that sediment when centrifuged at 100000g for 90min. 2. Solubilization of the particle-bound disaccharidases without loss of activity was achieved by digestion with papain. Trypsin was less effective and caused a preferential solubilization of the sucrase, isomaltase and palatinase activities. 3. On Sephadex G-200 columns, the solubilized preparations yielded two disaccharidase peaks. The first peak was eluted close to the void volume of the column and contained all the sucrase, isomaltase and palatinase activities and some of the maltase activity. The remainder of the maltase activity was eluted beyond the total volume of the column. 4. Precipitation with ethanol did not affect the behaviour of the disaccharidases of gel filtration. 5. The maltase activity of the second peak on rechromatography in a buffer containing 0.01m-maltose was eluted close to the void volume. 6. Similar pH optima but different Km values were obtained for the maltase activities of the two peaks. 7. Heat-inactivation studies showed that the first peak contained two disaccharidase enzymes; one hydrolysed sucrose and maltose and the other hydrolysed isomaltose, palatinose and maltose. The second peak contained three disaccharidase enzymes all specific for the hydrolysis of maltose. 8. It is proposed that the intestinal disaccharidases of the chick exist in the form of two complexes: a sucrase–isomaltase complex and a maltase complex.

Evaluation of dialysis treatment in uremic patients by gel filtration of serum

1990 ◽  
Vol 36 (11) ◽  
pp. 1906-1910 ◽  
Author(s):  
J Osada ◽  
T Gea ◽  
C Sanz ◽  
I Millan ◽  
J Botella
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Other ◽  
Control Group ◽  
Dialysis Treatment ◽  
Additional Information ◽  
Molecular Masses ◽  
Two Peaks

Abstract A group of substances of molecular masses between 300 and 1500 Da have been found to be toxic metabolites in patients with uremia. We determined the concentration in serum of these molecules in the following groups of patients: two hemodialyzed groups (one with cuprophane and the other with polyacrylonitrile dialyzers), one group treated with continuous ambulatory peritoneal dialysis, one group of nondialyzed azotemic patients, and one control group of healthy persons. Ultrafiltrates of the subjects' sera were fractionated on Sephadex G-15 followed by ion-exchange chromatography. Eluates were monitored by absorbance at 254 and 206 nm. Partially characterized peaks P1 and P2, obtained by gel filtration, correlated with the concentration of creatinine in serum; their concentrations were significantly (P less than 0.01) larger in hemodialyzed groups than in peritoneal dialyzed or in nondialyzed azotemic patients. After ion-exchange chromatography, two peaks (P'5 and P'6) correlated with serum creatinine and also were larger in hemodialyzed patients than in the other groups. Apparently, adequate discrimination is obtained by gel-filtration analysis and further analysis by ion-exchange chromatography does not provide additional information in most of the affected patients.

scholarly journals Rat small-intestinal galactosidases. Separation by ion-exchange chromatography and gel filtration

1968 ◽  
Vol 106 (4) ◽  
pp. 841-845 ◽  
Author(s):  
Nils Georg Asp ◽  
Arne Dahlqvist
Keyword(s):  
Ion Exchange ◽  
Ph Value ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Galactosidase Activity ◽  
Neutral Ph ◽  
Small Intestinal

1. The chromatography of rat small-intestinal β-galactosidase activities on gel-filtration and ion-exchange columns has been studied. Five different substrates were used to measure β-galactosidase activity (lactose, phenyl β-galactoside, o-nitrophenyl β-galactoside, p-nitrophenyl β-galactoside and 6-bromo-2-naphthyl β-galactoside) and the activity was measured at one acid and one more neutral pH value. 2. By gel filtration one acid β-galactosidase, hydrolysing lactose and the hetero-β-galactosides at about the same rate, and one more neutral β-galactosidase, hydrolysing lactose much more rapidly than the hetero-β-galactosides, were separated. 3. By ion-exchange chromatography the acid enzyme was fractionated into two components. These may be individual enzymes or different forms of the same enzyme.

scholarly journals Multiple chromatographic forms of ATP citrate lyase from rat liver

1983 ◽  
Vol 214 (2) ◽  
pp. 299-307 ◽  
Author(s):  
A P Corrigan ◽  
C C Rider
Keyword(s):  
Ion Exchange ◽  
Rat Liver ◽  
Gel Filtration ◽  
Total Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Atp Citrate Lyase ◽  
Citrate Lyase ◽  
Acidic Form ◽  
Two Peaks

ATP citrate lyase is shown to exist as multiple forms in extracts of rat liver. DEAE-Sephadex ion-exchange chromatography of liver supernatants reveals two peaks of activity. A minor, basic, component, comprising 14% of the recovered activity, is eluted without retention, whereas the major, acidic, form is eluted by a KCl gradient. Gel filtration of similar extracts shows the presence of a high-Mr form of ATP citrate lyase (Mr around 10(7) in addition to the tetrameric enzyme (Mr 4.1 X 10(5). This associated state, which represents 10% of the total activity, is unstable, breaking down to the tetramer, and appears to be disrupted by Mg2+. The basic form changes in the partially purified state to give the acidic form. Most of the high-Mr enzyme is acidic in nature. No evidence could be found for an association of the enzyme with mitochondrial or microsomal membranes. ATP citrate lyase from rat brain also shows two peaks of activity on DEAE-Sephadex ion-exchange chromatography, but the activity is distributed between the peaks in almost equal proportions. However, only the tetrameric enzyme was observed on gel filtration.

scholarly journals Rat small-intestinal β-galactosidases. Studies on the fractionation of ‘acid’ β-galactosidase with isoelectric focusing, gel filtration and ion-exchange chromatography

1970 ◽  
Vol 117 (2) ◽  
pp. 369-375 ◽  
Author(s):  
Nils-Georg Asp
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
High Molecular Weight ◽  
Isoelectric Focusing ◽  
Gel Filtration ◽  
Ion Exchange Resin ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Isoelectric Points ◽  
Small Intestinal

1. Different forms of the rat small-intestinal ‘acid’ β-galactosidase were separated by using the isoelectric-focusing technique. The isoelectric points of the different forms were at pH4.2, 4.6, 5.4, 6.1 and approx. 8. 2. The two forms of ‘acid’ β-galactosidase isoelectric at pH4.2 and 4.6 were completely excluded from the Sephadex G-200 gel, whereas the form isoelectric at pH8 had Kav. 0.4. The concentration and pH of the elution buffer influenced the distribution of enzyme activity between different forms. Thus, under certain conditions of ionic strength and pH, the enzyme seems to form high-molecular-weight aggregates with low isoelectric points. These may be homopolymeric aggregates or the result of binding of enzyme to, for example, membrane fragments. The forms isoelectric at pH5.4 and 6.1 are probably aggregates of intermediate size. 3. During ion-exchange chromatography at pH6.0 one fraction of ‘acid’ β-galactosidase was not retained on the column and was isoelectric at pH8 and another fraction was eluted when the buffer concentration in the eluate had increased to about 50mm. The main part of enzyme eluted in this second fraction was also isoelectric at pH8, indicating that the elution of this fraction is not a simple ion-exchange procedure but probably also involves a splitting of high-molecular-weight aggregates, originally retained because of their low isoelectric points. The enzyme subunits have a higher isoelectric point, and are therefore no longer bound to the ion-exchange resin.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

THE UPTAKE OF TESTOSTERONE AND ZINC IN VITRO BY THE HUMAN BENIGN HYPERTROPHIC PROSTATE

1973 ◽  
Vol 58 (3) ◽  
pp. 405-419 ◽  
Author(s):  
M. JOAN REED ◽  
S. R. STITCH
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Zinc Binding ◽  
Supernatant Fraction ◽  
Low Molecular Weight ◽  
Molecular Weight Peak ◽  
Steroid Binding

SUMMARY The uptake of 65Zn and [1,2-3H]testosterone by minced tissue of human benign hypertrophic prostates and the subcellular distribution of radioactivity were examined. The nature of steroid and 65Zn binding by the cytosol (105000 g supernatant) fraction was investigated by gel filtration, ion-exchange chromatography and electrophoresis. It was found that steroid binding after incubation at 4°C was specific. One or two regions of steroid binding were observed after gel filtration of the cytosol using Sephadex G-200, depending upon incubation conditions. Binding of 65Zn was found in the low molecular weight peak after G-200 gel filtration. Equimolar CdCl2 and 65ZnCl2 were incubated with [1,2-3H]testosterone and minced tissue and the cytosol was subjected to gel filtration. Compared with control values, without CdCl2, reduction of 65Zn binding by about 50% occurred, while binding of 3H-labelled steroid was unaffected. Electrophoresis and ion-exchange chromatography showed that 65Zn and 3H-labelled steroid were bound to different proteins. A sample of the zinc-binding protein was prepared by ion-exchange chromatography and the homogeneity was checked by electrophoresis.

PURIFICATION AND CHARACTERISATION OF FIBRINOLYTIC ENZYMES FROM ENDOPHYTIC FUNGI AND LIGNOSUS RHINOCERUS

2016 ◽  
Vol 78 (6-5) ◽  
Author(s):  
Zainon Mohd Noor ◽  
Mohd Sidek Ahmad ◽  
Zaidah Zainal Ariffin
Keyword(s):  
Ion Exchange ◽  
Endophytic Fungi ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fibrinolytic Enzymes ◽  
Molecular Weights ◽  
Ammonium Sulphate Precipitation ◽  
Fusarium Sp ◽  
Different Sources

Three enzymes FH3, S13 and LR1 from three different sources showed fibrinolytic activities. Two were from endophytic fungal cultures and one from the sclerotium of Lignosus rhinocerus mushroom (LR1). FH3, S13 cultures and LR1, the crude extract of the sclerotium were concentrated and purified by ammonium sulphate precipitation, ion-exchange chromatography and gel-filtration. The molecular weights of the FH3, S13 and LR1 purified enzymes were estimated to be approximately 34kDa, 34kDa and 10kDa, respectively. Maximum fibrinolytic activities were observed for FH3 at pH 7 and 30°C, S13 at pH 8 and 40°C and LR1 at pH 6 and 40°C.  In our earlier paper we identified FH3 as Fusarium sp. and S13 as Penicilium citrinum. 

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