Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.5 (5'-nucleotidase)
3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasma membranes from chick embryo neuronal primary cultures were isolated after subjecting 5-day-old cells, previously surface labeled with either lactoperoxidase-catalyzed radioiodination or galactose oxidase/NaB3H4, to a freeze-thaw cycle. The cellular material adhering to the culture substratum was washed, and the "wash" fractions were pooled and centrifuged at 37,000g. The resulting pellet was resuspended in 3 ml of buffer, layered on 33 ml of 33% sucrose, and centrifuged at 105,000g. Radioactivity was recovered at the top of the gradient. Sedimentation of these fractions and biochemical studies revealed that the pellet was 20- and 12-fold enriched in (Na+,K+)-adenosinetriphosphatase and 5'-nucleotidase, respectively. The preparation was devoid of inner mitochondrial (succinate dehydrogenase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), and Golgi (UDP galactose:N-acetylglucosamine galactosyltransferase) enzymatic markers. Ultrastructural studies showed that the membrane preparation was homogeneous and lacked mitochondria endoplasmic reticulum and lysosomes. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of 11 protein components with molecular masses ranging from 120 to 300 kDa. This method for the isolation of plasma membranes probably depends on the capacity of the cellular material to adhere to the culture substratum and to entrap intracellular organelles during the freeze-thaw cycle. The membrane preparation seems suitable for studying the function of high-molecular-weight protein components of neuronal plasma membranes.
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PMID:Isolation of plasma membranes from neurons grown in primary culture. 282 51

The subunit molecular mass of chicken liver cytosol 5'-nucleotidase was earlier reported to be 51 kDa upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Naito, Y. and Tsushima, K. (1976) Biochim. Biophys. Acta 438, 159-168). By immunoblot analyses after SDS-gel electrophoresis, however, a fraction from the liver homogenized in the presence of leupeptin showed multiple bands with molecular masses around 57 kDa, and SDS-extracted proteins directly from the liver exhibited a single 70 kDa component. These results indicate that the 51 kDa peptide observed in the cytosol 5'-nucleotidase preparation might, in fact, be a proteolytic artifact.
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PMID:A reappraisal of the subunit molecular mass of chicken liver cytosol 5'-nucleotidase. 282 68

Cytosolic 5'-nucleotidase from bovine liver has been purified to homogeneity. Two affinity chromatographies on concanavalin A and 5'AMP-Sepharose columns result in a 12,000-fold purification. The sequential elution of glycoproteins from the concanavalin-A-Sepharose column with methyl alpha-D-glucoside and methyl alpha-D-mannoside greatly increases the degree of purification of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows two subunits having apparent molecular masses of 65 kDa and 57 kDa respectively, while only one band at 70 kDa is observed in the case of the membrane-bound 5'-nucleotidase. Both the Stokes radii, measured by gel exclusion HPLC, and the sedimentation coefficient, determined by density gradient ultracentrifugation, indicate that the cytosolic enzyme is a heterodimer of about 130 kDa. This contrasts with the membrane-bound 5'-nucleotidase which is a homodimer of 140 kDa. Moreover, the antibodies raised against the membrane 5'-nucleotidase inhibited the cytosolic form indicating that a common antigenic determinant(s) exists between the two isoenzymes. However, structural differences are revealed by immunoblotting. In the same way, the effect of lectins suggests that differences in the structure of the carbohydrate chains exist between the two isoenzymes. The purified cytosolic enzyme has lower affinity for the nucleotides than does the membrane enzyme. In addition, while ADP, [alpha,beta-CH2]ADP and ATP were strong competitive inhibitors of the membrane enzyme, ADP and ATP activate the cytosolic form and [alpha,beta-CH2]ADP has no effect. Moreover, two pH optima at 7.5 and 9.5 are observed in the cytosolic enzyme while only one at 7.5 occurred in the membrane form. Finally the exogenous cations, MgCl2 and MnCl2, are necessary for the maximal activity of the cytosolic but not of the membrane 5'-nucleotidase. All these observations indicate that the two isoenzymes are different.
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PMID:Purification of bovine liver cytosolic 5'-nucleotidase. Kinetic and structural studies as compared to the membrane isoenzyme. 283 Oct 62

Plasma membrane (PM), primarily from the anterior sperm head, and outer acrosomal membrane (OAM), were isolated from ejaculated bovine spermatozoa, and the major lipid classes were characterized. Whole sperm (WS) lipids were analyzed for comparison. PM was removed by nitrogen cavitation and purified by sucrose density-gradient centrifugation. The OAM was removed by centrifugation through hyperosmotic sucrose and recovered by sucrose density-gradient centrifugation. The PM contained primarily spherical vesicles from the region overlying the OAM and was enriched 9- and 13-fold in 5'-nucleotidase and alkaline phosphatase activity, respectively, compared to the original cavitate. The OAM was recovered as caplike structures with associated ground substance. Protein, phospholipid, and cholesterol (PR, PL, and CH as micrograms/5 x 10(9) sperm) were 300, 467, and 93 for PM and 276, 111, and 25 for OAM, respectively. Corresponding values for WS (mg/5 x 10(9) sperm) were 31.4, 6.63, and 0.72. The PR/PL (w/w) and CH/PL (mol/mol) ratios were 0.66 and 0.38 for PM; 2.48 and 0.26 for OAM; and 4.39 and 0.22 for WS. Cholesterol was the only free sterol detected by gas/liquid chromatography in WS, PM, and OAM, with traces of CH sulfate present in all three preparations. Glycolipid tentatively identified as sulfogalactolipid was detected by thin-layer chromatography (TLC) in PM but not OAM. Phospholipid composition of WS and membranes was determined by TLC. Cardiolipin (3% of total PL) was present in WS only. Choline, ethanolamine, and inositol phosphoglycerides (CP, EP, PI, PIP, PIPP); sphingomyelin (SP); phosphatidylserine (PS); and lysophosphatidylcholine (LPC) were present in WS, PM, and OAM. Approximately 50% of total PL was CP in all preparations; SP was 13% of PL in PM and 17% in OAM (p less than 0.05); EP was 7% of PL in PM and 10% in OAM (p less than 0.05). The differences in composition between PM and OAM is discussed with respect to capacitation and ability of sperm to undergo the acrosome reaction.
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PMID:Lipids of plasma membrane and outer acrosomal membrane from bovine spermatozoa. 283 8

A prerequisite for the purification of any protein to homogeneity is that the protein is not non-specifically associated with other proteins especially during the final stage(s) of the fractionation procedure. This requirement is not so often fulfilled when nonionic detergents (for instance Triton X-100) are used for solubilization of membrane proteins. The reason is that these detergents are not efficient enough to prevent the protein of interest from forming aggregates with other proteins upon contact with chromatographic or electrophoretic supporting media, which, due to their polymeric nature, have a tendency to induce aggregation of other polymers, for instance, hydrophobic proteins. The aggregation can be avoided if sodium dodecyl sulfate (SDS) is employed as detergent. We therefore suggest that membrane proteins should be purified by conventional methods in the presence of SDS and that the purified proteins, which are in a denatured state, are allowed to renature. There is good change to renature internal membrane proteins since they should not be so susceptible to denaturation by detergents as are water-soluble proteins because the natural milieu of the former proteins is lipids which in fact are detergents. In this paper we present a renaturation method based on the removal of SDS by addition of a large excess of G 3707, a nonionic detergent. By this technique we have renatured a 5'-nucleotidase from Acholeplasma laidlawii and a neuraminidase from influenza virus. The enzyme activities were higher (up to 6-fold) after the removal of SDS than prior to the addition of SDS.
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PMID:Purification of membrane proteins in SDS and subsequent renaturation. 283 10

When the particulate fraction from a rat liver homogenate was incubated with [3H]putrescine and calcium, the radioactive amine was incorporated into the membranes via a transglutaminase-mediated reaction. Fractionation of the membranes by isopycnic density gradient centrifugation revealed that the radioactive label was coincident with the 5'-nucleotidase and transglutaminase activities which serve as markers for the plasma membrane (Slife, C. W., Dorsett, M. D., Bouquett, G. T., Register, A., Taylor, E., and Conroy, S. Arch. Biochem. Biophys. 241, 329-336). If the labeled membranes were treated with digitonin and fractionated, the radioactivity and the plasma membrane enzyme activities coincidentally shifted to a greater density. Examination of the [3H]putrescine-labeled membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that the largest amount of radioactivity was associated with a large molecular weight material that did not enter the acrylamide gel. Pulse-chase experiments indicated that the large aggregate already was present in the native membrane, or that it was formed very rapidly during the putrescine incubation. The complex did not result from putrescine cross-linking between proteins since dansylcadaverine and [3H]histamine were also selectively incorporated into it. These data show that there are protein substrates in the plasma membrane which are accessible to the membrane-associated transglutaminase and that the substrates form a large molecular weight aggregate which is not dissociated by sodium dodecyl sulfate and disulfide reducing agents.
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PMID:Subcellular location and identification of a large molecular weight substrate for the liver plasma membrane transglutaminase. 286 32

Earlier reports suggested that the adenosine monophosphate (AMP)- and the p-nitrophenyl phosphate (pNPP)-hydrolyzing activities of Dictyostelium discoideum membrane preparations are due to different proteins. These results have been apparently contradicted by the recent purification to homogeneity of the two activities from culmination phase cells as a single protein [D. R. Armant and C. L. Rutherford (1981) J. Biol. Chem. 256, 12710-12718]. Results presented here from studies on the activities of vegetative cells support the concept of a single protein. Nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Triton X-100 extracts of cell membrane preparations of D. discoideum showed identical migration of pNPPase and AMPase activities. Furthermore, the previously reported different pH optima of the two activities was due to the fact that pH optima are dependent upon the substrate concentration, and the selective solubilization of AMPase from membrane preparations by phospholipase C can probably be accounted for by the finding that phospholipase C preparations from the same commercial source contain 5'-nucleotidase activity. Moreover, there are alterations in the Km and the stability of both AMPase and pNPPase in a strain with a mutationally altered alkaline phosphatase, further supporting the concept that the two activities are due to a single protein. Both substrates serve as transphosphorylation donors demonstrating that the enzyme activity is mechanistically an alkaline phosphatase.
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PMID:The membrane-bound alkaline phosphatase and 5'-nucleotidase activities of vegetative cells of Dictyostelium discoideum. 298 13

5'-Nucleotidase (EC 3.1.3.5) has been solubilized and purified 1200-fold from guinea-pig skeletal muscle, to a specific activity of 40 U/mg protein. The purified enzyme yields a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Guinea-pig skeletal muscle 5'-nucleotidase is extremely sensitive to inhibition by nucleoside di- and triphosphates. The inhibition is of the competitive type, and can be reversed only by strong excess of Mg2+. Nucleoside diphosphates are more powerful inhibitors than nucleoside triphosphates. The Ki values for ADP and ATP are 0.036 and 0.28 microM, respectively. The purified enzyme does not require exogenous cations for maximal activity and is inhibited by EDTA. This inhibition is reversed by divalent cations. This indicates that the enzyme contains a tightly bound metal cation.
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PMID:Isolation and kinetic properties of 5'-nucleotidase from guinea-pig skeletal muscle. 298 11

5'-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5'-nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E.M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J.P. (1984) Biochem. J. 221, 369-377). Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5'-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5'-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5'-nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver.
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PMID:An improved procedure for purifying 5'-nucleotidase from various sources. Evidence for tissue and species differences in their molecular mass and affinity for F-actin. 299 65

After solubilization with zwitterionic detergents 5'-nucleotidase was purified to homogeneity from chicken gizzard. Purified 5'-nucleotidase appeared to be composed of a single polypeptide chain of 79 kDa as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); gel filtration studies in the presence of detergents, however, indicated that the native enzyme is a homodimer. Antisera against purified chicken gizzard 5'-nucleotidase were raised in rabbits, they were shown to inhibit the enzymic activity of different 5'-nucleotidase preparations in a species and tissue specific manner. On frozen sections the antisera stained the cell periphery of chicken gizzard smooth and skeletal muscle and faintly stained cardiac muscle cells when using the indirect immunofluorescent technique. In chicken cardiac tissue, a prominent staining of the vascular system also became apparent. In avian and rat non-muscle tissues (hepatic and pancreatic tissue) the vascular system was always found to be brightly stained, i.e. the vascular smooth muscle and endothelial cells. On frozen sections of chicken liver the sinusoidal region of the hepatocytes was brightly stained, the bile canalicular region, however, only faintly. Using the immunocytochemical technique, a more prominent tissue specificity rather than species specificity of the available antisera became apparent. This may therefore reflect the existence of tissue-specific isoforms of the enzyme.
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PMID:Isolation of 5'-nucleotidase from chicken gizzard, its properties, and subcellular localization in chicken tissues using monospecific antibodies. 299 75


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