scholarly journals Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system

1981 ◽  
Vol 198 (2) ◽  
pp. 265-271 ◽  
Author(s):  
F Wuytack ◽  
G De Schutter ◽  
R Casteels
Keyword(s):  
Enzyme Activity ◽  
Smooth Muscle ◽  
Transport System ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Partial Purification ◽  
Total Enzyme Activity ◽  
Dependent Atpase ◽  
Specific Activities ◽  
Total Enzyme

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.

scholarly journals Hepatic microsomal Ca2+-dependent ATPase. Calmodulin-dependence and partial purification

1983 ◽  
Vol 214 (1) ◽  
pp. 69-75 ◽  
Author(s):  
P B Moore ◽  
N Kraus-Friedmann
Keyword(s):  
Affinity Chromatography ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Fold Increase ◽  
Partial Purification ◽  
Dependent Atpase

The hepatic microsomal fraction contains tightly bound calmodulin as demonstrated by affinity chromatography. When this calmodulin was partially removed by EGTA treatment (0.5 mM-EGTA), the uptake of 45Ca2+ by the microsomal vesicles was stimulated by added calmodulin and inhibited by trifluoperazine (TFP). The Ca2+-dependent ATPase was partially purified on a calmodulin column. This partial purification resulted in a 500-fold increase in the specific activity of the enzyme when measured in the presence of added calmodulin. Antibodies prepared against calmodulin prevented this stimulatory effect. The fraction eluted from the calmodulin column contained several protein bands indicating that the specific activity of the Ca2+-dependent ATPase is probably still underestimated. There are likely to be other calmodulin-sensitive processes present in the hepatic microsomal fraction.

EXTRACTION OF ENZYMES AND ASSESSMENT OF METABOLISM IN BOVINE RUMEN EPITHELIUM

1982 ◽  
Vol 62 (2) ◽  
pp. 429-438 ◽  
Author(s):  
ROY S. BUSH
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Total Activity ◽  
Small Contribution ◽  
Bacterial Protein ◽  
Total Enzyme Activity ◽  
Rumen Epithelium ◽  
Bacterial Enzyme ◽  
Protein Contamination ◽  
Total Enzyme

Papillae collected from the rumens of freshly killed cows were used to estimate the most appropriate methods for enzyme extraction from rumen epithelium and the amount of enzymes in extracts which might be of bacterial origin. Extractions of enzymes from fresh and frozen papillae were compared for the Polytron homogenizer (PT), the Potter-Elvehjem homogenizer (PE), the Waring blender, sonication and acetone powdering plus PE. PE extraction yielded solutions with the highest specific activity for each enzyme. PT extraction released the most protein and total enzyme activity into solution. PT extraction was chosen for the remaining tests because of the high total activity released. Mixed rumen bacteria were homogenized by sonication. Electrophoretic examination of epithelial and bacterial extracts showed differential migration for malate dehydrogenase. Lactate dehydrogenase from the epithelium showed four distinct isozymes whereas the bacterial enzyme showed little distinct band development. Contamination of epithelial extracts by bacterial protein was estimated to be less than 5%. The specific activities of 10 enzymes were found to be similar in epithelial and bacterial extracts so that a small amount of protein contamination would result in only a small contribution to total enzyme activity. The presence of the enzymes assayed in this study plus a number reported in the literature showed that rumen epithelial metabolism is more diverse than previously recognized. Key words: Rumen epithelium, enzymes, extraction

5α-Reductase activity in stroma and epithelium of rat prostate and epididymis. A contribution to elucidation of the mechanism for development of hyperplastic growth of prostatic tissue

1983 ◽  
Vol 103 (2) ◽  
pp. 273-281 ◽  
Author(s):  
Ole Djøseland ◽  
Nicholas Bruchovsky ◽  
Paul S. Rennie ◽  
Navdeep Otal ◽  
Sian Høglo
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Reductase Activity ◽  
Major Change ◽  
Growth Stimulation ◽  
Total Activity ◽  
Prostatic Tissue ◽  
Ventral Prostate ◽  
Total Enzyme Activity ◽  
Abnormal Growth

Abstract. The 5α-reductase activity was assayed in homogenates of stroma and epithelium in the rat ventral prostate and epididymis. Samples consisting of 0.3 mg/ml tissue protein in TES buffer, pH 7.0 were incubated at 37°C for 30 min in the presence of 50 nm [1,2-3H]testosterone and a NADPH-generating system started with 5 × 10−4 m NADP. The yield of 5α-reduced metabolites, as established by using thin-layer chromatography, gave an estimate of enzyme activity. Whereas the specific activity of 5α-reductase was highest in prostatic stroma and epididymal epithelium, most of the total enzyme activity was associated with the epithelium in both the prostate and epididymis. The effect of dihydrotestosterone on specific activity of 5α-reductase was studied by administering the hormone to 7-day castrated rats. In prostate, the specific activity of both stromal and epithelium forms of the enzyme reached a maximum after 4 days of treatment. In epididymis only the epithelial form of 5α-reductase underwent a major change in specific activity, the latter peaking after 8–12 days of treatment. Furthermore, while the total activity of 5α-reductase in the prostatic tissue fractions could be induced by as much as 4-fold the normal control values, the epididymal enzyme could not be induced above the normal level either in the stroma or the epithelium. This may explain the relative resistance of epididymis to abnormal growth stimulation under the influence of hormones.

scholarly journals Partial purification of presynaptic plasma membrane by immunoadsorption.

1982 ◽  
Vol 94 (1) ◽  
pp. 88-96 ◽  
Author(s):  
G P Miljanich ◽  
A R Brasier ◽  
R B Kelly
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Nerve Terminal ◽  
Specific Activity ◽  
Electric Organ ◽  
Partial Purification ◽  
Vesicle Membrane ◽  
Specific Activities ◽  
Active Zones ◽  
Presynaptic Plasma Membrane

During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

Studies on the effects of magnesium ion and propranolol on iris muscle phosphatidate phosphohydrolase

1984 ◽  
Vol 62 (2-3) ◽  
pp. 170-177 ◽  
Author(s):  
Ata A. Abdel-Latif ◽  
Jack P. Smith
Keyword(s):  
Smooth Muscle ◽  
Divalent Cation ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Divalent Cations ◽  
Dependent Manner ◽  
Soluble Enzyme ◽  
Microsomal Enzyme ◽  
Time Intervals ◽  
Phosphatidate Phosphohydrolase

The properties, subcellular distribution, and the effects of Mg2+ and propranolol on phosphatidate phosphohydrolase (EC 3.1.3.4) from rabbit iris smooth muscle have been investigated. The particulate and soluble (0–30% (NH4)2SO4 fraction) enzymes were assayed using aqueous phosphatidate dispersions and membrane-bound phosphatidate as substrates, respectively. When measured with aqueous substrate, activity was detected in both the particulate and soluble fractions, with the highest relative specific activity found in the microsomal fraction. Maximum dephosphorylation by the microsomal enzyme was about 1100 nmol of inorganic phosphate released/h per milligram protein and occurred at pH 7.0–7.5. In general Mg2+ inhibited the phosphohydrolase activity of the microsomal fraction and stimulated that of the soluble fraction, and the effects of the divalent cation on both of these activities were reversed by propranolol. The microsomal enzyme was slightly stimulated by deoxycholate and inhibited by the divalent cations Mg2+, Ca2+, and Mn2+ at concentrations > 0.25 mM. In contrast, the soluble enzyme was stimulated by Mg2+. Inhibition of the microsomal enzyme by Mg2+ (0.5 mM) was reversed by both EDTA, which also stimulated at higher concentrations (1 mM), and propranolol (0.1–0.2 mM). The inhibitory effect of Ca2+ on the enzyme was not reversed by propranolol. In the absence of Mg2+, the microsomal enzyme was inhibited by propranolol in a dose-dependent manner, and both in the absence and presence of the divalent cation the soluble enzyme was inhibited by the drug in a similar manner. These data suggest that the cationic moiety of propranolol may act by competing at the Mg2+-binding sites. Addition of propranolol (0.2 mM) to iris muscle prelabelled with [14C]arachidonic acid increased accumulation of [14C]phosphatidic acid at all time intervals (2.5–90 min) and brought about a corresponding initial decrease in the formation of [14C]diacylglycerol at short time intervals (2.5 min), thus implicating the phosphohydrolase as a possible site of action of the drug on glycerolipid metabolism in this tissue. In addition to reporting on the characteristics and distribution of phosphatidate phosphohydrolase in the iris smooth muscle, the data presented add further support to our hypothesis that propranolol redirects glycerolipid metabolism in the iris by exerting multiple effects on the enzymes involved in their biosynthesis.

scholarly journals Two-Step Production of Neofructo-Oligosaccharides Using Immobilized Heterologous Aspergillus terreus 1F-Fructosyltransferase Expressed in Kluyveromyces lactis and Native Xanthophyllomyces dendrorhous G6-Fructosyltransferase

Catalysts ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 673 ◽  
Author(s):  
Burghardt ◽  
Baas ◽  
Gerlach ◽  
Czermak
Keyword(s):  
Response Surface Methodology ◽  
Enzyme Activity ◽  
Reaction Time ◽  
Aspergillus Terreus ◽  
Kluyveromyces Lactis ◽  
Xanthophyllomyces Dendrorhous ◽  
Initial Concentration ◽  
Total Enzyme Activity ◽  
Glycosidic Bonds ◽  
Total Enzyme

Fructo-oligosaccharides (FOS) are prebiotic low-calorie sweeteners that are synthesized by the transfer of fructose units from sucrose by enzymes known as fructosyltransferases. If these enzymes generate β-(2,6) glycosidic bonds, the resulting oligosaccharides belong to the neoseries (neoFOS). Here, we characterized the properties of three different fructosyltransferases using a design of experiments approach based on response surface methodology with a D-optimal design. The reaction time, pH, temperature, and substrate concentration were used as parameters to predict three responses: The total enzyme activity, the concentration of neoFOS and the neoFOS yield relative to the initial concentration of sucrose. We also conducted immobilization studies to establish a cascade reaction for neoFOS production with two different fructosyltransferases, achieving a total FOS yield of 47.02 ± 3.02%. The resulting FOS mixture included 53.07 ± 1.66 mM neonystose (neo-GF3) and 20.8 ± 1.91 mM neo-GF4.

scholarly journals The effects of starvation and experimental diabetes on phosphoenol-pyruvate carboxykinase in the guinea pig

1977 ◽  
Vol 164 (2) ◽  
pp. 357-361 ◽  
Author(s):  
K R F Elliott ◽  
C I Pogson
Keyword(s):  
Enzyme Activity ◽  
Guinea Pig ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Blood Glucose Concentration ◽  
Mitochondrial Fraction ◽  
Kidney Cortex ◽  
Total Enzyme Activity ◽  
The Mean ◽  
Mean Blood Glucose ◽  
Total Enzyme

1. Approx. 85% of liver phosphoenolpyruvate carboxykinase is associated with the mitochondrial fraction in the fed guinea pig. Enzyme activity is unchanged in diabetes, but doubles during starvation. In contrast with earlier reports, both cytoplasmic and mitochondrial activities were found to be increased. 2. In kidney cortex, total enzyme activity is increased in both starved and diabetic animals. These changes are associated with increases in the cytoplasmic activity alone. 3. In diabetic animals the mean blood-glucose concentration was 23.1 mM. Other blood metabolites were lower than those in the rat, and the animals did not show significant ketosis. 4. Changes in the rates of gluconeogenesis from lactate and propionate paralleled those in phosphoenolpyruvate carboxykinase activity.

Changes in mass and catalytic activity concentrations of aspartate aminotransferase isoenzymes in serum after a myocardial infarction.

1986 ◽  
Vol 32 (3) ◽  
pp. 496-500 ◽  
Author(s):  
A E Niblock ◽  
G Jablonsky ◽  
F Y Leung ◽  
A R Henderson
Keyword(s):  
Myocardial Infarction ◽  
Enzyme Activity ◽  
Catalytic Activity ◽  
Aspartate Aminotransferase ◽  
Left Ventricular ◽  
Peak Activity ◽  
Ventricular Failure ◽  
Total Enzyme Activity ◽  
Activity Concentrations ◽  
Total Enzyme

Abstract We used an RIA and inhibition of enzyme activity to monitor the changes in mass and catalytic concentrations of the aspartate aminotransferase (EC 2.6.1.1;AST) isoenzymes in serum after myocardial infarction. Cytosolic (c-AST) and mitochondrial (m-AST) forms of AST were present in sera from all 38 of our patients. Although the immunological and catalytic concentrations of both isoenzymes correlated well with the size of the infarct, c-AST gave a better measure than did m-AST. About 20% of the total enzyme activity at peak activity was from the mitochondrial isoenzyme. Both isoenzyme activities peak at very nearly the same time, but m-AST has the longer half-life. Immunological evidence of the mitochondrial isoenzyme can be detected in serum for at least eight days after the infarct. The presence of left ventricular failure produces greater serum isoenzyme activities than in those without failure.

Alteration in the status of glutathione transferase of the water snail, Bulinus globosus, during aestivation and recovery

2010 ◽  
Vol 60 (2) ◽  
pp. 145-155 ◽  
Author(s):  
Yetunde Adedolapo Ojopagogo ◽  
Isaac Olusanjo Adewale
Keyword(s):  
Glutathione Transferase ◽  
Specific Activity ◽  
Glutathione Transferases ◽  
Partial Purification ◽  
Major Protein ◽  
High Concentration ◽  
Cibacron Blue ◽  
The Status ◽  
Bulinus Globosus ◽  
Specific Activities

AbstractThe varying status of glutathione transferases (GSTs) in water snail, Bulinus globosus, an intermediate host of disease-causing Schistosoma haematobium (Bilharz 1852) has been investigated. The expression of GST isoenzymes in the water snail appears seasonal with about three isoenzymes appearing during raining season, when the organism is active, which may reduce to a single peak of one isoenzyme during aestivation, when the organism is inactive. GST isoenzyme is present in high concentration in all the tissues investigated namely: haemolymph, foot muscle and hepatopancreas with specific activities of 0.006 ± 0.002, 0.45 ± 0.021 and 1.33 ± 0.103 units/mg protein respectively for 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. With this substrate, the specific activity of GST from the hepatopancreas appears higher than the specific activities that have been previously reported for GSTs from molluscs. Partial purification of the isoenzymes using Tris acrylic acid-based resins enabled us to observe that GST appears to be the major protein in the hepatopancreas of this organism. We also found indications for the presence of an endogenous GST inhibitor in the cytosol, whose function is yet unknown. All the traditional GST inhibitors such as cibacron blue, hematin, bromosulfophthalein and S-hexylglutathione were able to inhibit the isoenzymes effectively, with cibacron blue being the most potent. The isoenzymes however have narrow substrate specificity. We conclude that different isoenzymes of GST are expressed in the same class of molluscs, even when they belong to the same genus or species, and that the expression may depend on whether the snails are on aestivation or not.

Sign in / Sign up

Export Citation Format

Share Document