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.

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.

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.

Luminal digestion of lactoferrin in suckling and weanling rats

1987 ◽  
Vol 253 (3) ◽  
pp. G397-G403 ◽  
Author(s):  
J. R. Britton ◽  
O. Koldovsky
Keyword(s):  
Small Intestine ◽  
Polyacrylamide Gel Electrophoresis ◽  
Young Animal ◽  
Product Analysis ◽  
Weanling Rats ◽  
Total Product ◽  
Two Peaks ◽  
Hydrolysis Of ◽  
Soluble Material

The development of luminal digestion of lactoferrin was evaluated in vitro by incubating 125I-labeled lactoferrin with fluid flushed from the stomach and small intestine of 12-day-old suckling and 31-day-old weanling rats, followed by measurement of radioactivity in trichloroacetic acid-soluble material. Gastric hydrolysis of lactoferrin at pH 3.2 in the weanling was 20-fold greater than that in the suckling. In the small intestine at neutral pH, luminal degradation of lactoferrin was minimal in the suckling but increased significantly after weaning, with maximal degradative capacity demonstrable in the midjejunum. Sephadex G-75 chromatography of intestinal acid-soluble breakdown products revealed two peaks of radioactivity, each comprising 40-45% of the total product; analysis of intestinal acid-precipitable products by polyacrylamide gel electrophoresis yielded several discrete lower molecular weight species. Food deprivation for 12 h/100 g body wt decreased lactoferrin degradation in the weanling jejunum and midjejunum. Our findings suggest that lactoferrin digestion may vary with respect to postnatal age of the organism, segment of the gastrointestinal tract, and dietary state. In the young animal, lactoferrin degradation is minimal, and consequently its potential for biological function may be high.

Impact of Enterococcus faecium on specific activity of disaccharidases in small intestine of gnotobiotic pigs

Biologia ◽  
2006 ◽  
Vol 61 (6) ◽  
Author(s):  
Viktor Buleca ◽  
Soňa Gancarčíková ◽  
Rudolf Žitňan ◽  
Radomíra Nemcová ◽  
Alojz Bomba ◽  
...  
Keyword(s):  
Small Intestine ◽  
Enterococcus Faecium ◽  
Specific Activity ◽  
Constant Level ◽  
The Other ◽  
Lactase Activity ◽  
Maltase Activity ◽  
Other Hand ◽  
Gnotobiotic Pigs ◽  
Saccharase Activity

AbstractThe aim of the present work was to study the changes in the activity of disaccharidase enzymes (lactase. maltase, saccharase) in the small intestine of gnotobiotic pigs aged 0–35 days and inoculated with Enterococcus faecium. The continual decrease of lactase activity was observed from the 14th day of age up to the end of the experiment. The most significant decrease of specific lactase activity in the duodenum (2.1 µmol/mg protein/hour) was noted from the 21st to the 28th day of age. On the other hand, the specific saccharase activity increased moderately during the post weaning period and maltase activity maintained a constant level.

Malate dehydrogenase from thermophilic Humicola lanuginosa and Mucor pusillus: purification and comparative properties of the enzymes with differing thermostabilities

1984 ◽  
Vol 62 (7) ◽  
pp. 559-565 ◽  
Author(s):  
Anupam S. Wali ◽  
Autar K. Mattoo
Keyword(s):  
Malate Dehydrogenase ◽  
Conformational Changes ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
The Other ◽  
Heat Inactivation ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Other Hand ◽  
Humicola Lanuginosa

Malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) was purified from the thermophiles Humicola lanuginosa and Mucor pusillus. The H. lanuginosa enzyme was homogeneous on sodium dodecyl sulphate – polyacrylamide gels, while the M. pusillus enzyme was more than 95% pure. The two enzymes appeared to be composed of two subunits of equal size, each of 36 000 daltons (H. lanuginosa) or 33 000 daltons (M. pusillus). The native enzymes revealed molecular weights of 68 000 as determined by gel filtration. The isoelectric points of malate dehydrogenase from H. lanuginosa and M. pusillus were 3.9 and 4.2, respectively. The reduction of oxaloacetate by the H. lanuginosa enzyme was optimum at pH 8.5–9 with apparent Km's of 0.12 mM for oxaloacetate and 0.027 mM for NADH. On the other hand, M. pusillus enzyme snowed a pH optimum of 7.8–8.5 with apparent Km's of 0.075 mM for oxaloacetate and 0.1 mM for NADH. The L-malate oxidation reaction was catalyzed optimally at pH 10 by the H. lanuginosa enzyme with apparent Km's of 5.8 mM for malate and 0.1 mM for NAD, while the M. pusillus enzyme catalyzed it optimally between pH 9.5 and 10 with apparent Km's of 4.44 mM for malate and 0.16 mM for NAD. The optimum temperature for reduction of oxaloacetate was 50 °C for both the enzymes. The H. lanuginosa enzyme was resistant to heat inactivation at 40 °C, but lost 60% of its activity after 15 min at 50 °C. Mucor pusillus enzyme, on the other hand, retained 90% activity at 60 °C after 10 min. The two enzymes were protected from heat inactivation by monovalent cations (viz Na+, K+, and NH4+), as well as citrate, which may possibly involve conformational changes.

scholarly journals Praline metabolism by germinating Lilium longiflorum pollen. I. Labelling of cytoplasmic, wall and culture medium molecules

2014 ◽  
Vol 50 (1-2) ◽  
pp. 51-66 ◽  
Author(s):  
W. V. Dashek ◽  
R. R. Mills
Keyword(s):  
Growth Medium ◽  
Culture Medium ◽  
Gel Filtration ◽  
Lilium Longiflorum ◽  
Paper Electrophoresis ◽  
Void Volume ◽  
Molecular Weights ◽  
The Common ◽  
Two Peaks ◽  
Salt Extract

Radioactivity occurs in trithloroacetic acid (TCA)-soluble and precipitable, cytoplasm and salt-washed walls following germination of <em>Lilium longiflorum</em>, cv. 'Ace' pollen in medium containing [<sup>14</sup>C]-proline (Pro). Sephadex gel filtration on G-25 through G-100 was employed to determine whether radioactivity in cytoplasm, wall and growth medium from pollen fed [<sup>14</sup>C]-Pro or [<sup>3</sup>H]=Pro plus [<sup>14</sup>C]-arafbinose (Ara) was contained within molecules possessing molecular weights of 5,000 to 100,000 daltones or greater. G-25 elution profiles of a crude cytoplasmic fraction (15,000 X g supernatant) from [<sup>14</sup>C]-Pro labelled pollen yielded a radioctive void volume peak and a retarded peak. The void volume peak contained hydroxyproline (Hyp), and exhibited a coincidence of [<sup>3</sup>H]-Pro and [<sup>14</sup>C] -Ara labelling when pollen was double labelled with the two isotopes. This peak also contained radioactivity when pollen was germinated in 2-[<sup>3</sup>H]-myo-inositol. Germination in medium supplemented with 100 µM 2,2'-dipyridyl eliminated radioactivity from 2-[<sup>3</sup>H]-myo-inositol or [<sup>14</sup>C]-,Pro in the peak. Filtratian on G-25 of aTCA-soluble fraction of a salt-extract of walls from [<sup>14</sup>C]-Pro labelled pollen resulted in void volume and two retarded peaks. Void volume and two retarded peaks were also obtained upon G-25 filtration of a cellulase-digest of walls from [M]-Pro labeled pollen. The void volume peak contained Hyp, Lys, Gly, Ala, Ser, Glu and Asp acids, Val, Tyr, Leu or lieu and Pro. Sephadex G-90, 75, and 100 elution profiles of cellulasedigests of walls from [<sup>3</sup>H]-,Pro and [<sup>14</sup>C]-Ara labelled pollen yielded radioactive retarded and Hyp-containing void volume peaks with a coincidence of [<sup>3</sup>H] and [<sup>14</sup>C] labelling. Label in the void volume was obtained when either rhozyme P11- or pepsin-digests of walls from [<sup>14</sup>C]-Pro labelled pollen were gel filtered on G-50. Paper electrophoresis coupled with paper chromatography of acid hydrolyzates of salt-washed wall fractions demonstrated 15 of the common amino acids. Gel filtration on G-25 of growth medium in which pollen was germinated resulted in two peaks, one of which eluted in the void volume. contained Hyp and excluded during subsequent gel filtration on G-100.

Regulation of two aspartokinase isozymes in Streptococcus bovis

1994 ◽  
Vol 40 (3) ◽  
pp. 224-227 ◽  
Author(s):  
E. O. Kalcheva ◽  
M. M. Faiziev ◽  
V. O. Shanskaya ◽  
S. S. Maluta
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Growth Conditions ◽  
Growth Cycle ◽  
The Other ◽  
Streptococcus Bovis ◽  
Gel Filtration Chromatography ◽  
Regulatory Properties ◽  
Second Peak

Streptococcus bovis has been found to contain two distinct aspartokinases that can be separated by gel filtration chromatography. One of these isozymes elutes on Sephadex G-200 gel filtration at a molecular weight greater than 250 000. The molecular weight of the other isozyme is approximately 125 000. The earlier peak of aspartokinase activity is slightly inhibited by meso-diaminopimelate, while the second peak is sensitive to inhibition by lysine. The latter aspartokinase is not formed when the organism is grown in a medium containing more than 1 mM lysine. The level of lysine-sensitive aspartokinase is decreased during the growth cycle, whereas diaminopimelate-sensitive activity is little affected by the growth conditions. The regulatory properties of the two aspartokinases suggest that they may play different physiological roles.Key words: aspartokinase activity, isozymes, repression, inhibition.

scholarly journals Proteins of the kidney microvillar membrane. The amphipathic forms of endopeptidase purified from pig kidneys

1983 ◽  
Vol 211 (3) ◽  
pp. 743-753 ◽  
Author(s):  
I S Fulcher ◽  
A J Kenny
Keyword(s):  
Chelating Agents ◽  
Gel Filtration ◽  
Small Region ◽  
The Other ◽  
Kidney Cortex ◽  
Dimeric Molecule ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Hydrolysis Of ◽  
Positively Charged

The purification of detergent-solubilized kidney microvillar endopeptidase (EC 3.4.24.11) by immuno-adsorbent chromatography is described. The product (the d-form) was 270-fold purified compared with the homogenate of kidney cortex and was obtained in a yield of 5%. It was free of other peptidase activities and homogeneous by electrophoretic analyses. It contained about 15% carbohydrate and one Zn atom/subunit. Two trypsin-treated forms were also characterized. One (dt-form) was obtained by treatment of the d-form. The other (tt-form) was the result of solubilizing the membrane by treatment with toluene and trypsin. All three forms had apparent subunit Mr values of approx. 89 000, but the d-form appeared to be slightly larger than the other two. Estimates of Mr by gel filtration showed that of the tt-form to be 216 000 whereas those of the other forms were 320 000. An estimate of the detergent (Triton X-100) bound to the d- and dt-forms accounted for this difference. By several criteria, including charge-shift crossed immunoelectrophoresis and hydrophobic chromatography, the d- and dt-forms were shown to be amphipathic molecules. In contrast, the tt-form was hydrophilic in its properties. Differences in ionic properties were also noted, consistent with the loss, in the case of the dt-form, of a positively charged peptide. The results indicate that the native endopeptidase is a dimeric molecule, each subunit being anchored in the membrane by a relatively small region of the polypeptide close to one or other terminus. The d- and dt-forms had similar enzyme activity when assayed by the hydrolysis of 125I-insulin B-chain. Chelating agents and phosphoramidon inhibited the endopeptidase. The kinetic constants were determined by a new two-stage fluorimetric assay using glutarylglycylglycylphenylalanine 2-naphthylamide as substrate and aminopeptidase N (EC 3.4.11.2) to hydrolyse phenylalanine 2-naphthylamide. The Km was 68 microM and Vmax. 484nmol X min-1 X (mg of protein)-1.

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.

scholarly journals The assay and partial characterization of macromolecular heparin depolymerase activity in rat small intestine

1979 ◽  
Vol 180 (3) ◽  
pp. 587-596 ◽  
Author(s):  
E Young ◽  
A A Horner
Keyword(s):  
Small Intestine ◽  
Total Radioactivity ◽  
Gel Chromatography ◽  
Rat Small Intestine ◽  
Ph Optimum ◽  
Tris Maleate ◽  
Two Peaks ◽  
And Storage ◽  
Second Peak

Homogenates of rat small intestine can depolymerize macromolecular rat skin heparin (RS heparin) to products similar in size to commercial heparin [Horner (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3469–3473]. This activity is attributed to an enzyme provisionally named ‘macromolecular heparin depolymerase’. An assay for macromolecular heparin depolymerase activity in rat small intestine has been developed, based on the action of the enzyme on 35S-labelled macromolecular RS heparin. The depolymerized products are separated into two peaks by gel chromatography through columns of Bio-Gel A-15m. The amount of label in the second peak, expressed as a percentage of the total radioactivity, is the index of enzyme activity. The pH optimum was found to be 6.0 and the temperature optimum 45 degrees C. The enzyme was shown to be most stable in 50mM-Tris/maleate buffer containing 1 mM-EDTA. Macromolecular heparin depolymerase activity measured as a function of time and substrate concentration produced curves typical of an enzymic reaction. Evidence was obtained demonstrating that the activity did not originate from bacteria in the intestine. Macromolecular heparin depolymerase activity was increased by dilution and storage at 7 degrees C for 24 h. This suggests that homogenates of rat small intestine contain an unstable inhibitor of the enzyme.

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