Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.5 (5'-nucleotidase)
 3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HeLa cells grown on Petri dishes were either pulse labelled with various cardiac glycosides or grown in low concentrations of them for up to 2 days; either in the presence of chloroquine or not. The cells were then homogenised and the cell free homogenate layered on a continuous sucrose gradient; and the glycoside content and that of various markers measured. In another series of experiments HeLa cells were grown on plastic beads under the above conditions and then the content of glycosides and of some marker enzymes measured. The rate of internalisation of ouabain, digoxin and digitoxin from the plasma membrane preparation produced by the bead method is at 9% hr-1, similar to the rate of loss of digoxin and digitoxin from whole cells but much faster than that of ouabain. In the sucrose gradient experiments it was found that [3H]ouabain, digoxin and digitoxin all initially co-distribute with the plasma membrane marker, 5'-nucleotidase, and then leave this fraction of the homogenate at a fast rate when kept at 37 degrees, to co-distribute with the lysosomal marker, beta-hexosaminidase. At 2 degrees the ouabain remains co-distributed with the plasma membrane marker. The rate of transfer is estimated to be some 90% hr-1, much faster than previously thought. Chloroquine causes an increased retention of digoxin and digitoxin in the lysosomal fraction of the homogenate. These results are best explained by supposing that the sodium pump-glycoside complex rapidly enters a region of the peripheral cytoplasm, and that this region then controls the subsequent exit of digoxin and digitoxin from the cell. The main barrier for ouabain occurs at a stage later than this. The consequences of this model on other aspects of pump activity is discussed.
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PMID:The rate of uptake of cardiac glycosides into human cultured cells and the effects of chloroquine on it. 294 66

The distribution of plasma membrane markers, the sodium pump [evaluated as ouabain-sensitive, potassium-stimulated p-nitrophenyl phosphatase (K+-pNPPase)], [3H]saxitoxin binding, and 5'-AMPase, was studied in the subcellular fractions prepared from the homogenates of the longitudinal smooth muscle/myenteric plexus of dog ileum. The K+-pNPPase activity and [3H]-saxitoxin binding were found to be predominantly associated with the synaptosomal fraction as indicated by the high level of these activities in the crude synaptosomal fraction and by the copurification of K+-pNPPase and [3H]saxitoxin binding, but not 5'-AMPase, with several synaptosomal markers during the fractionation of the crude synaptosomal fraction on density gradients. In contrast to the K+-pNPPase activity and [3H]saxitoxin binding, the 5'-AMPase activity was found to be concentrated in the microsomal pellet. Further fractionation of microsomes on density gradient resulted in copurification of 5'-AMPase but not K+-pNPPase or [3H]saxitoxin binding, with other smooth muscle plasma membrane-bound enzymes, such as high-affinity Ca2+-ATPase, Mg2+-ATPase, and Ca2+-ATPase. It was concluded that in the longitudinal smooth muscle/myenteric plexus, the sodium pump activity is present in higher density in the neuronal plasma membranes whereas 5'-AMPase activity is concentrated in the smooth muscle plasma membranes.
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PMID:Subcellular fractionation of the longitudinal smooth muscle/myenteric plexus of dog ileum: dissociation of the distribution of two plasma membrane marker enzymes. 304 Sep 6

Quantitative measurements of (Na+,K+)-ATPase activity and numbers of (Na+,K+)-ATPase sites in membranes from quiescent and regenerating rat liver have been made using an anticatalytic monoclonal antibody (9-A5) that binds to alpha subunits of the sodium pump (Schenk, D. B., and Leffert, H. L. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5281-5285). To validate the measurements, kinetic properties of 9-A5 binding to plasma membrane sodium pumps, specificity and requirements of the reactions, and mechanisms by which 9-A5 inhibits (Na+,K+)-ATPase were analyzed. 125I-9-A5 binding is saturable and reversible (k1 = 1.8 X 10(6) X M-1 X S-1; k2 = 2.7 X 10(-4) X S-1). At equilibrium, 9-A5 binds to a single class of sites revealed by Scatchard plots (KD[app] = 0.64 nM, Bmax = 29.3 pmol/mg of proteins; = 238,000 sites X cell-1). This binding requires monovalent cations (sodium, potassium, or lithium); is blocked by purified (Na+,K+)-ATPase; is inhibited noncompetitively by ATP (KI[app] = 0.5 mM); and is unaffected by ouabain. 9-A5 inhibits ATP-stimulated (Na+,K+)-ATPase noncompetitively by blocking sodium-dependent phosphorylation of alpha subunits of liver or kidney membrane (Na+,K+)-ATPase. Twelve h after 67% hepatectomy, maximal 125I-9-A5 binding to plasma membranes from regenerating liver falls 30 +/- 7% compared to sham-operated controls (p less than 0.01). In contrast, (Na+,K+)-ATPase activity in regenerating liver membranes rises 58 +/- 12% compared to controls (p less than 0.03). Similar experiments with particulate fractions from regenerating liver show insignificant decreases in maximal 125I-9-A5 binding (22 +/- 12%) but large increases in (Na+,K+)-ATPase activity (325 +/- 14%) compared to controls (p less than 0.001). No differences among groups are seen in KD values for 9-A5 binding or in the activities of plasma membrane 5'-nucleotidase (EC 3.1.3.5). Thus, stimulation of the sodium pump during the late prereplicative phase of liver regeneration is not accompanied by increases in the numbers of (Na+,K+)-ATPase sites. Instead, it appears that preexisting (Na+,K+)-ATPases are activated specifically before DNA replication starts.
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PMID:Use of a monoclonal antibody to quantify (Na+,K+)-ATPase activity and sites in normal and regenerating rat liver. 609 83