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

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 K(m) 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.     

Cyclic nucleotide phosphodiesterase activities from pig coronary arteries. Lack of interconvertibility of major forms

DEAE-cellulose chromatography, with or without dithiothreitol and over a pH range of 6.0 to 8.5, resolved two phosphodiesterase activities (peaks I and II) from the soluble fraction of pig coronary arteries. The activity of peak I was increased by calmodulin (3-7-fold), whereas that of peak II was not. Chromatography of peak I on Biol-Gel A-0.5 m columns resolved two peaks of phosphodiesterase activity (peaks Ia and Ib). Peak Ia was eluted in the presence or absence of 0.1 M KCl and was relatively insensitive to calmodulin. Peak Ib was eluted only in the presence of KCl and was sensitive to calmodulin. The substrate specificity and kinetic behavior were the same for peaks I, Ia, and Ib. Repeated gel chromatography of either peak Ia or Ib, under appropriate conditions, yielded a mixture of peaks Ia and Ib. Peak Ia appears to be a reversible aggregate of peak Ib. Gel chromatography of peak II resolved only one phosphodiesterase activity, which was eluted without KCl, was highly specific for cyclic AMP, was not sensitive to calmodulin and migrated differently on the gel column than either peak Ia or Ib. Sucrose density gradient centrifugation of the soluble fraction from pig coronary arteries in the presence or absence of dithiothreitol resolved two peaks of phosphodiesterase activity (6.6 S and 3.6 S) which were similar to peaks I and II separated by DEAE-cellulose chromatography with regard to their substrate specificity and their sensitivity to calmodulin. Upon recentrifugation, each of the two peaks of phosphodiesterase activity gave a single peak of activity which migrated with the same S value as did its parent. These results indicate that the two major forms of phosphodiesterase of pig coronary arteries, which are representative of those found in many tissues, are not interconvertible in cell-free systems.     

Intestinal brush border membrane-bound disaccharidases of the American alligator, Alligator mississippiensis

1. The disaccharidase activities of the small intestines of American alligators (Alligator mississippiensis) were studied in epithelial scrapes and brush-border membrane preparations. 2. Maltase, isomaltase and trehalase activities were found. Activities of these enzymes were higher in the proximal small intestine and decreased distally. 3. Disaccharidase activities were enriched 12-15 times in brush-border membrane preparations, compared with mucosa/enterocyte crude homogenates and were co-enriched with the brush-border membrane marker alkaline phosphatase. 3. The pH optima were: maltase 6.5; isomaltase 5.6; and trehalase 5.8. The Q10 of maltase, the most active enzyme, was equal to 1.82. 4. In reptiles, as in mammals, disaccharidase activities may be correlated with feeding habits. The co-occurrence of sucrase and isomaltase may not be a common feature of vertebrates.     

Purification and Properties of Proteases from a Marine-psychrophilic Bacterium

Protease which was found in the culture fluid of Pseudomonas sp. No. 548 was fractionated into four components with protease activity by a two step chromatography using DEAE-cellulose. Each protease was further purified by gel filtration on Sephadex G-100 and/or G-75. The protease of Ia was obtained in crystalline form and was shown to be homogeneous by analysis with electrophoresis, while the other three enzymes were also highly purified. The enzymatic properties of the proteases were investigated. All of the four enzymes were inactivated by ethylene diamine tetraacetate. Proteases Ia, Ib, and IIb were inactivated by diisopropylfluorophosphate. The optimum activity of protease Ia was shown to be at pH 10.0, and that of the other enzymes were at pH 7.0 to 8.0. The proteases of Ia, Ib, and IIb were stabilized by calcium ion. The effect of temperature, pH, and metal ions on the activity of the enzyme were also investigated.     

Polypeptide antibiotic 26a from Bacillus subtilis. II. Isolation and purification procedures.

Both the analytical and preparative methods by which the preparations of 26a bydrochloride salt with a high antibacterial activity and 20--30% recovery have been obtained from the fermentation fluids of Bacillus subtilis are presented. On an industrial scale the antibiotic can be yielded by absorption of bioactivity on Amberlite CG-50I column and precipitation with picric acid of crude substance from active elutes as adduct which was divided on equilibrated CM--cellulose and finally purified by gel filtration on Sephadex G-25 column. The purified preparation gave a single band by SDS-polyacrylamide gel electrophoresis and one ninhydrin-positive spot by thin-layer chromatography on silica gel G plates corresponding to single zones of bioactivity on bioautograms, and moved as a single peak of almost constant antibacterial activity on Sephadex G-75, G-100 and G-200 columns. The potency of the purest preparations, lot Sephadex G-25, was 6,500--7,000 arbitrary units/mg, and were characterized as follows: purification factor, 57; purity of 98--100% by densitometer scans of SDS-polyacrylamide gels; MIC for Sarcina lutea by twofold agar dilution assay, 0.306 microgram/ml.     

The fractional composition of immunoglobulin preparations studied by gel filtration on different carriers and by high-pressure liquid chromatography]

The fractional composition of immunoglobulin preparations produced by different manufacturing enterprises of this country has been studied by gel chromatography in columns packed with different carriers (Sephadex G-200 and ultragel AcA-34) and by high-performance liquid chromatography (HPLC). This study has revealed the nonstandard character of immunoglobulin preparations produced according to the same technological procedure (modified Cohn's method). The fractionation of immunoglobulins on different carriers with the use of different methods has yielded similar results confirmed by the statistical processing of the data. The results obtained in the study of the fractional composition of immunoglobulin preparations evidence that gel filtration with the use of ultragel and HPLC have greater resolving capacity in comparison with the method of gel filtration on traditionally used Sephadex G-200.     

Purification and characterization of a pig intestinal alpha-limit dextrinase

Mucosa from the duodenal and jejunal regions of pig small intestine was repeatedly freeze-thaw treated to solubilize an enzyme preparation, enriched in maltase, glucoamylase and alpha-limit dextrinase activities; isomaltase and sucrase remained essentially insoluble during the treatment. Chromatographic procedures, including ion-exchange, gel filtration and hydroxylapatite chromatography of the solubilized preparation, brought to homogeneity an alpha-glucosidase active towards maltose, alpha-limit dextrins and starch in decreasing order, with only a very weak capacity to hydrolyse alpha-1,6-linkages. Michaelis constants and maximal velocities, as well as relative rates of hydrolysis of several substrates, including maltodextrins and alpha-limit dextrins, were determined and served to characterize what seems to be a rather specific alpha-1,4-glucosidase. The participation of this enzyme in the hydrolysis of alpha-limit dextrins and more generally in pathways for starch breakdown in the pig digestive tract is examined and discussed.     

Purification and some properties of an extracellular maltase from Bacillus subtilis.

Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor. Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose. A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal.     

Extensive charging of transfer ribonucleic acid by bean leaf extracts in vitro

1. Phenol was effectively removed from aqueous extracts of RNA by chromatography on Sephadex G-50. 2. Elution of tRNA from Sephadex G-50 columns at pH7.6 was shown to remove 91% of the endogenously bound amino acids. 3. tRNA prepared without recourse to ethanolic precipitation was capable of accepting much greater amounts of amino acids than could redissolved samples of precipitated tRNA. 4. Aminoacyl-tRNA synthetase enzymes were partially purified with calcium phosphate gel. Elution of enzymes from the gel at pH6.5 yielded a fraction having phenylalanine- and alanine-charging activity, but no aspartate-, lysine- or proline-charging activity, whereas elution at pH7.6 gave a fraction having aspartate-, lysine- and proline-charging activity but no phenylalanine- or alanine-charging activity. 5. By using partially synthetase enzymes and tRNA eluted from DEAE-Sephadex A-50 columns, 52% of the theoretical maximum of aminoacyl-tRNA synthesis was obtained in vitro.     

Acidic phospholipases A2 from the venom of common sea snake enhydrina schistosa

Two acidic phospholipases A have been purified from the venom of common sea snake (Enhydrina schistosa) by chromatography on Sephadex G-75 gel media, Bio-Rex 70 ion-exchanger followed by repeated Chromatography on DEAE-Sephadex A-25 ion-exchanger. The two preparations were shown to be homogeneous by polyacrylamide gel electrophoresis and ion-exchange chromatography. The enzymes were shown to be specific for the ‘two’ position of egg yolk lecithin. The molecular weight of both enzymes determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was approx. 14000. Both enzymes were non-lethal. Amino acid composition data indicated high contents of aspartic acid, glycine and alamine in both enzymes.     

Purification of multiple forms of adenosine deaminase from rabbit intestine.

Two forms of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), differing in molecular size, have been purified and obtained in homogeneous form from rabbit intestine. The purification procedures involved extraction with acetate buffer, pH 5.5, precipitation and fractional reextraction with (NH4)2SO4, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 and Sephadex G-200. Gel filtrations analysis gave molecular weight estimates of 265 000 and 32 000 for the large and small deaminases respectively. The two enzymes forms had similar pH optima and pH stability ranges.     

Hexokinase microheterogeneity in rabbit red blood cells and its behaviour during reticulocytes maturation

Hexokinase in rabbit reticulocytes is present in two molecular forms (hexokinase Ia and Ib) separable by ion-exchange chromatography on DE-52 columns. By the use of ion-exchange HPLC we have been able to show that the isozymic form we previously called hexokinase la can be resolved into two peaks of activity one of which is (Ia) soluble, the other (Ia*) particulate. Hexokinase Ia* can be solubilized by detergents like saponine and Triton X-100 and disappears during ‘in vivo’ reticulocytes maturation. This new hexokinase micro-heterogeneity is not caused by different oxidized forms of the enzyme nor influenced by the presence of proteolytic inhibitors during lysate preparation.     

Phylogenetic analysis reveals key residues in substrate hydrolysis in the isomaltase domain of sucrase-isomaltase and its role in starch digestion

BackgroundStarch constitutes one of the main sources of nutrition in the human diet and is broken down through a number of stages of digestion. Small intestinal breakdown of starch-derived substrates occurs through the mechanisms of small intestinal brush border enzymes, maltase-glucoamylase and sucrase-isomaltase. These enzymes each contain two functional enzymatic domains, and though they share sequence and structural similarities due to their evolutionary conservation, they demonstrate distinct substrate preferences and catalytic efficiency. The N-terminal isomaltase domain of sucrase-isomaltase has a unique ability to actively hydrolyze isomaltose substrates in contrast to the sucrase, maltase and glucoamylase enzymes.MethodsThrough phylogenetic analysis, structural comparisons and mutagenesis, we were able to identify specific residues that play a role in the distinct substrate preference. Mutational analysis and comparison with wild-type activity provide evidence that this role is mediated in part by affecting interactions between the sucrase and isomaltase domains in the intact molecule.ResultsThe sequence analysis revealed three residues proposed to play key roles in isomaltase specificity. Mutational analysis provided evidence that these residues in isomaltase can also affect activity in the partner sucrase domain, suggesting a close interaction between the domains.Major conclusionsThe sucrase and isomaltase domains are closely interacting in the mature protein. The activity of each is affected by the presence of the other.General Significance: There has been little experimental evidence previously of the effects on activity of interactions between the sucrase-isomaltase enzyme domains. By extension, similar interactions might be expected in the other intestinal α-glucosidase, maltase-glucoamylase.     

Some properties of monkey intestinal sucrase

A detergent solubilised sucrase from monkey small intestine has been purified 388-fold to gel electrophoretic homogeneity with an overall recovery of 36%. The molecular weight of the enzyme was 263 kDa by gel filtration. Electrophoresis in the presence of SDS indicates that the enzyme is a hetero-dimer. Mixed substrate inhibition studies and inhibition by PCMB and Tris suggest the presence of two catalytically active sites in the form of maltase and sucrase with isomaltase activity being common to both sites. Polyclonal antiserum against the purified enzyme showed a single continuous precipitin line with the purified antigen.     

Purification of xanthine dehydrogenase from rat liver: a rapid procedure with high enzyme yields

Xanthine dehydrogenase (EC 1.2.1.37) was purified approximately 1000-fold from liver homogenates of adult male Sprague-Dawley rats. Enzyme recovery was good (greater than 20% of the starting activity was obtained), and the homogeneously pure enzyme had a molecular mass of approximately 300,000 Da. The purified protein exhibited a specific activity of 2470 units/mg protein and spectral properties identical to those of the best preparations of this enzyme reported by other investigators. Routine preparations of this enzyme also possess higher dehydrogenase:oxidase ratios (typically between 5 and 6) than do other xanthine dehydrogenase preparations so far reported in the literature. Maximum dehydrogenase:oxidase ratios, greater than 10, could be obtained from this procedure if only peak dehydrogenase fractions from the chromatography columns were saved. The present small-scale purification method, which can be completed in 48-60 h, utilizes ammonium sulfate fractionation, Sephadex G-200 column chromatography, Blue Dextran-Sepharose column chromatography, and preparative gel electrophoresis.     

Multiple prolactin-releasing activity in the bovine hypothalamic extract.

The prolactin (PRL)-releasing activity (PRA) in the bovine hypothalamic extract (BHE) was compared to that of known substances with PRA and further characterized by gel filtration and reversed-phase high performance liquid chromatography (RP-HPLC). Crude BHE produced marked dose-dependent stimulation of PRL secretion from the cultured rat adenohypophysial cells. Among the synthetic substances examined, vasoactive intestinal peptide (VIP), thyrotropin-releasing hormone (TRH) and beta-endorphin (END) showed significant PRA. However, the flatter dose-response slope for TRH compared with BHE or the small amounts of VIP and END in BHE suggested that these peptides could not account for the major active elements of BHE. Oxytocin and interleukin-1beta were also tested, but they exhibited no PRA in our assay system. Gel filtration of BHE on the Sephadex G-100 column yielded two peaks of PRA distinct from TRH, VIP and END. One eluted in the void and the other in more retarded fractions. The latter fractions were pooled and subjected to the two-step RP-HPLC. The PRA was separated into three peaks designated peaks I, II and III in the first RP-HPLC experiment. Furthermore, the second RP-HPLCs with finer resolution revealed that peak II as well as peak III consisted of three peaks, while peak I eluted as a single peak. Most of these seven PRA peaks exhibited different RP-HPLC profiles from those of the newly characterized PRL-releasing peptides. These findings again provide confirmatory evidence that BHE contained unique factors different from the above known substances.     

Fractionation of the mycobacillin-synthesizing enzyme system.

The mycobacillin-synthesizing enzyme system was highly purified by fractionation at 30-55% (NH4)2SO4 saturation. The enzyme concentrate on Sephadex G-200 gel chromatography was resolved into three distinct fragments. Each of the fragments on further purification by DEAE-cellulose ion-exchange chromatography behaved as a single-component system, as clearly indicated by the sharpness of the peaks in the elution diagram. None of the fragments alone nor any two of them in all possible combinations possessed mycobacillin-synthesizing activity, which was restored only when the three fragments were used together in the test system.     

Density-Gradient and Chromatographic Fraction of Leptospiral Lipase

Fractionation of leptospiral lipase by CsCl density gradients and G-200 Sephadex chromatography yielded five active protein peaks. Two were obtained from the density gradients and three from G-200 Sephadex columns. Esterase activity of these fractions was demonstrated by electrophoretic examination. Several protein bands were visible when disc electrophoresis was performed on the respective fractions. Lipolytic and esterolytic activities were both present, and the overlapping of these activities was discussed.     

Presence of sucrase in the yolk sac of the chick

The presence of sucrase in the yolk sac of the chick was studied biochemically and immunologically. The sucrase was partially purified from the yolk sac of hatched chicks and was compared with the sucrase purified from the small intestine. Immunodiffusion with antiserum against intestinal sucrase and characterization of the activity revealed that the two enzymes were almost identical. However, the size of the yolk sac sucrase was found to be slightly smaller than that of the intestinal enzyme by chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis. Immunocytochemical studies showed that the sucrase was located on the free surface of yolk sac endodermal cells, but the sucrase may also be present in the cytoplasm.