scholarly journals Soluble neutral and acid maltases in the suckling-rat intestine. The effect of cortisol and development

1974 ◽  
Vol 144 (2) ◽  
pp. 281-292 ◽  
Author(s):  
G Galand ◽  
G G Forstner
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Heat Sensitivity ◽  
Adult Rats ◽  
Ph Optimum ◽  
Maltase Activity ◽  
Acid Maltase ◽  
Membrane Bound ◽  
Sepharose 4B ◽  
The Relationship

The 100000g supernatants from 13-day-old suckling-rat intestinal homogenates contained 43.5% of the total intestinal maltase activity, compared with 7.1% in weaned adult rats aged 40 days. The soluble maltase activity was separated on Sepharose 4B into two quantitatively equal fractions at pH6.0, one containing a maltase with a neutral pH optimum and the other a maltase with an acid pH optimum. The neutral maltase was shown to be a maltase–glucoamylase identical with membrane-bound maltase–glucoamylase in molecular weight, heat-sensitivity, substrate specificity, Km for maltose and Ki for Tris. The soluble enzyme was induced by cortisol, but the ratio of the soluble to bound enzyme fell during induction. Solubility of the neutral maltase was not accounted for by the action of endogenous proteinases under the preparative conditions used. It is postulated that the soluble neutral maltase is a membrane-dissociated form of the bound enzyme and that the relationship between these two forms is modulated by cortisol. The acid maltase generally resembled acid maltase of liver, muscle and kidney. It was shown to be a maltase–glucoamylase with optimal activity at pH3.0, and molecular weight of 136000 by density-gradient centrifugation. At pH3.0 its Km for maltose was 1.5mm. It was inhibited by turanose (Ki=7.5mm) and Tris (Ki=5.5mm) but not by p-chloromercuribenzoate or EDTA. Some 55% of its activity was destroyed by heating at 50°C for 10min. The acid maltase closely resembled β-glucuronidase and acid β-galactosidase in its distribution in the intestine, response to tissue homogenization in various media, and decrease in activity with cortisol treatment and weaning, indicating that it was a typical lysosomal enzyme concentrated in the ileum.

Purine nucleoside phosphorylase: Isolation and characterization

1990 ◽  
Vol 55 (12) ◽  
pp. 2987-2999 ◽  
Author(s):  
Katarina Šedivá ◽  
Ivan Votruba ◽  
Antonín Holý ◽  
Ivan Rosenberg
Keyword(s):  
Molecular Weight ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Leukemia Cells ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Isolation And Characterization ◽  
Reaction Proceeds ◽  
The Matrix ◽  
Sepharose 4B

Purine nucleoside phosphorylase (PNP) from mouse leukemia cells L1210 was purified to homogeneity by a combination of ion exchange and affinity chromatography using AE-Sepharose 4B and 9-(p-succinylaminobenzyl)hypoxanthine as the matrix and the ligand, respectively. The native enzyme has a molecular weight of 104 000 and consists of three subunits of equal molecular weight of 34 000. The results of isoelectric focusing showed that the enzyme is considerably microheterogeneous over the pI-range 4.0-5.8 and most likely consists of eight isozymes. The temperature and pH-optimum of phosphorolysis, purine nucleoside synthesis and also of transribosylation is identical, namely 55 °C and pH 7.4. The transribosylation reaction proceeds in the presence of phosphate only. The following Km-values (μmol l-1) were determined for phosphorolysis: inosine 40, 2'-deoxyinosine 47, guanosine 27, 2'-deoxyguanosine 32. The Km-values (μmol l-1) of purine riboside and deoxyriboside synthesis are lower than the values for phosphorolysis (hypoxanthine 18 and 34, resp., guanine 8 and 11, resp.). An affinity lower by one order shows PNP for (-D-ribose-1-phosphate, (-D-2-deoxyribose-1-phosphate (Km = 200 μmol l-1 in both cases) and phosphate (Km = 805 μmol l-1). The substrate specificity of the enzyme was also studied: positions N(1), C(2) and C(8) are decisive for the binding of the substrate (purine nucleoside).

Soluble neutral maltase–glucoamylase from the small intestine: separation and characterization of components with differing affinity for concanavalin A

1982 ◽  
Vol 60 (11) ◽  
pp. 1007-1013 ◽  
Author(s):  
G. Forstner ◽  
A. Salvatore ◽  
L. Lee ◽  
J. Forstner
Keyword(s):  
Concanavalin A ◽  
Gradient Elution ◽  
Soluble Form ◽  
Rat Intestine ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Neutral Ph ◽  
Membrane Bound ◽  
Sepharose 4B

Intestinal maltase with a neutral pH optimum exists in both a brush border membrane-bound form and a soluble form in suckling rat intestine. Previous experiments in our laboratory have shown that the soluble enzyme contains a component which binds much more tightly to concanavalin A (ConA) than solubilized forms of the membrane enzyme. We studied the origin of this component by subjecting neutral, soluble maltase activity to chromatography on Sepharose 4B at age 13, 18 (preweaning), and 25 (postweaning) days. At 13 days, two maltase peaks were obtained with approximate molecular weights of 400 000 (peak I) and 150 000 (peak II). Peak II was less prominent at 18 days and was absent at 25 days. At 13 days, the majority of peak I consisted of material which was bound between 0.025 and 0.05 M α-methyl mannoside on gradient elution chromatography of ConA-Sepharose. Peak II contained material which eluted between 0.075 and 0.3 M α-methyl mannoside. At 25 days, all of the soluble maltase eluted between 0.025 and 0.04 M α-methyl mannoside. Peak I and peak II maltases had similar pH optima and Km's for maltase. Peak II maltase had a fourfold greater activity toward glycogen than peak I maltase with approximately the same activity for palatinose, turanose, and trehalose. Both maltases were precipitated by an antibody raised against adult membrane-bound maltase. Soluble maltase with neutral pH activity in the suckling rat intestine, therefore, consists of two immunologically related isozymes which differ in their molecular weight, their binding by ConA, and their specificity for glycogen. The small isozyme disappears at or about the time of weaning.

PURIFICATION AND CHARACTERIZATION OF THE PROCOAGULANT OF THE VENOM OF TROPIDECHIS CARINATUS

1987 ◽  
Author(s):  
P P Masci ◽  
A N Whitaker ◽  
J J Morrison ◽  
E A Bennett
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Factor Xa ◽  
Factor V ◽  
Crude Venom ◽  
Adult Rats ◽  
Sds Page ◽  
Sepharose 4B ◽  
Blue Sepharose

Tropidechis carinatus is a venomous elapid snake distributed throughout Eastern Queensland. It has been considered as a tropical relative of Notechis scutatus and, similarly, the crude venom contains an indirect prothrombin activator, which will clot plasma provided that Factor V is present. Myotoxins and neurotoxins are also present. Envenomated patients regularly develop disseminated intravascular coagulation. The crude whole venom of T.carinatus was shown to have five major components by gel filtration, SDS PAGE and HPLC, and even more components by isoelectric focusing. The procoagulant eluted with a molecular weight of 55,000, being found in peak II on gel filtration on Sephadex-G150. The procoagulant was purified using a combination of Sephadex-G150 chromatography and ion-exchange on DEAE Sephadex-A50 and shown to migrate as a single band of molecular weight 55.000 by SDS PAGE. On reduction by β -mercaptoethanol this component was resolvec into u heavy chain of molecular weight 30.000 and a light chain of 25,000. The procoagulant was shown to bind to con A-Sepharose 4B and Blue Sepharose 4B. Coagulation studies using this purified procoagulant confirmed a factor Xa-like activity activating prothrombin in the presence of factor V. The purified fraction is unstable in buffer solutions at 4°C, probably because of trypsin - like autodigestion. Ouchterlony studies of the procoagulants of T.carinatus and N.scutatus show both lines of homogeneity and spurring, indicating similarities but also significant differences between the two proteins. The purified procoagulant was lethal to adult rats, an IV injection of 10 μg killing in 1 - 2 minutes. Death was prevented by prior heparinization, suggesting that the procoagulant is toxic in the absence of neurotoxin and other components.

scholarly journals Purification and characterization of kynurenine-2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats

1975 ◽  
Vol 151 (2) ◽  
pp. 399-406 ◽  
Author(s):  
T Noguchi ◽  
Y Minatogawa ◽  
E Okuno ◽  
M Nakatani ◽  
M Morimoto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Small Intestine ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Wide Range

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.

Purification and Partial Characterization of the Soluble NADH Dehydrogenase from the Phototrophic Bacterium Rhodopseudomonas capsulata

1984 ◽  
Vol 39 (1-2) ◽  
pp. 68-72 ◽  
Author(s):  
Toshihisa Ohshima ◽  
Matsumi Ohshima ◽  
Gerhart Drews
Keyword(s):  
Cytochrome C ◽  
Nadh Dehydrogenase ◽  
Rhodopseudomonas Capsulata ◽  
Subunit Structure ◽  
Ph Optimum ◽  
Single Polypeptide Chain ◽  
Membrane Bound ◽  
Single Polypeptide ◽  
Sepharose 4B

Abstract Soluble NADH dehydrogenase was purified to homogeneity from chemotrophically grown cells of Rhodopseudomonas capsulata by ammonium sulfate fractionation, AH -Sepharose 4B chromatography and FMN-Sepharose 6B affinity chromatography. The enzyme contains a single polypeptide chain of an apparent M, of 37000, suggesting that the subunit structure is different from that of the membrane-bound enzyme. The purified soluble NADH dehydrogenase requires flavin compounds, e.g., FMN, FAD and riboflavin, for activity. Addition of FMN and FAD. but not riboflavin, to the enzyme solution stabilized the enzyme. The pH optimum for activity was at 7.5. The enzyme was specific for NADH as an electron donor while NADPH was inert. Menadione, ferricyanide, cytochrome c and DCIP served as an electron acceptor. The M ichaelis constants for NADH. DCIP, FM N. and cytochrome c were 45, 2.9. 7.9 and 15 μM, respectively. Many properties of soluble NADH dehydrogenase were substantially different from those of the membrane-bound enzyme, suggesting different functions.

scholarly journals Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

1974 ◽  
Vol 141 (1) ◽  
pp. 211-217 ◽  
Author(s):  
Hilary A. Rolton ◽  
Hamish M. Keir
Keyword(s):  
Molecular Weight ◽  
Ethylene Glycol ◽  
Substrate Concentration ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Kidney Cells ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Hyperbolic Curve ◽  
Km Value

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a Km value for dCMP of 0.91mm. dCTP activation required the presence of Mg2+ or Mn2+ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg2+ or Mn2+ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Exterior and Interior Events in Early Stage of Thrombin-induced Platelet Aggregation

1977 ◽  
Vol 38 (03) ◽  
pp. 0630-0639 ◽  
Author(s):  
Shuichi Hashimoto ◽  
Sachiko Shibata ◽  
Bonro Kobayashi
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Early Stage ◽  
Adenyl Cyclase ◽  
Cellular Component ◽  
Primary Event ◽  
Rabbit Platelets ◽  
Membrane Bound

SummaryTreatment of washed rabbit platelets with 1 u/ml of thrombin at 37° C resulted in a disappearance from platelets of a protein with 250,000 dalton molecular weight which was shown to be originated from plasma membrane. Parallel loss of adenyl cyclase was noted, and both reactions were complete within 30 sec. From the patterns of disc electrophoretograms, the importance of quick suppression of thrombin action in demonstrating the primary event was stressed.Thrombin induced an apparent activation of membrane bound phosphodiesterase. This reaction was also complete within 30 sec. The cellular component which contained the enzyme activity was distinct from plasma membrane. Soluble phosphodiesterase was not influenced by thrombin at all.These reactions required intact platelet cells to react with thrombin, and no reaction was detected when subcellular preparations were treated with thrombin.Possibility of collaboration of changes in externally located synthetic enzyme with those in internally located degrading enzyme in the early phase of thrombin action on platelets was suggested.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

Biosynthesis of the Diguanosine Nucleotides. I. Purification and Properties of an Enzyme from Yolk Platelets of Brine Shrimp Embryos

1974 ◽  
Vol 52 (3) ◽  
pp. 231-240 ◽  
Author(s):  
A. H. Warner ◽  
P. C. Beers ◽  
F. L. Huang
Keyword(s):  
Molecular Weight ◽  
Brine Shrimp ◽  
Artemia Salina ◽  
Temperature Optimum ◽  
Small Molecular Weight ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Purification And Properties

An enzyme that catalyzes the synthesis of P1P4-diguanosine 5′-tetraphosphate (Gp4G) has been isolated and purified from yolk platelets of encysted embryos of the brine shrimp, Artemia salina. The enzyme GTP:GTP guanylyltransferase (Gp4G synthetase) utilizes GTP as substrate, has a pH optimum of 5.9–6.0, a temperature optimum of 40–42 °C, and requires Mg2+ and dithiothreitol for optimal activity. The synthesis of Gp4G is inhibited markedly by pyrophosphate, whereas orthophosphate has no effect on the reaction. In the presence of GDP the enzyme also catalyzes the synthesis of P1,P3-diguanosine 5′-triphosphate (Gp3G), but the rate of synthesis is low compared with Gp4G synthesis and dependent upon other small molecular weight components of yolk platelets.

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