IRREVERSIBLE ZINC ION INHIBITION OF (Na + -K + )-ADENOSINETRIPHOSPHATASE, Na + -PHOSPHORYLATION, AND K + -p-NITROPHENYLPHOSPHATASE OF ELECTROPHORUS ELECTRICUS ELECTROPLAX 1

Zinc ion in micromolar concentrations is an irreversible inhibitor of Electrophorus electricus electroplax microsomal (Na + -K + )-ATPase. The rate of inhibition is dependent on [ZnCl 2 ] and the extent of inhibition varies with the ratio of ZnCl 2 to microsomal protein. The same kinetics are observed for inhibition of K + - p -nitrophenylphosphatase and steady-state levels of Na + -dependent enzyme phosphorylation. The observations suggest that a Zn 2+ -sensitive conformational restraint is important to both kinase and phosphatase activities. The fact that inhibition is irreversible has implications for models seeking to relate zinc effects in tissue to inhibition of (Na + -K + )-ATPase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66028/1/j.1471-4159.1978.tb06247.x.pd

DEVELOPMENT OF (Na + -K + )-ATPase IN RAT CEREBRUM: CORRELATION WITH Na + -DEPENDENT PHOSPHORYLATION AND K + - para NITROPHENYLPHOSPHATASE 1

The activities of (Na + K + )-ATPase and its proposed partial reactions, K + - p NPPase and Na + -dependent phosphorylation, all increase tenfold relative to microsomal protein between 5 days prior to birth and 60 days postnatally in NaI-treated rat cerebral microsomes, and all reach half of their adult values between the fifth and tenth postnatal day. These increases are concurrent with the most rapid changes in cerebral wet weight. Increases in the amount of the related phosphorylatable polypeptide during development. as estimated by densitometry of Coomassie-stained polyacrylamide gels after electrophoresis of constant amounts of microsomal protein dissolved in sodium dodecylsulfate, parallel the increments in levels of Na + -dependent phosphorylation. The fraction of total phosphorylation that is Na + -dependent increases steadily during development. suggesting a precursor role for some of the Na + -independent fraction. The results are consistent with a single biosynthetic control for the enzymatic sites critical to the partial reactions of (Na + -K + )-ATPase. No changes in turnover number or affinity for substrate or ligands were found during development. Little similarity was noted among the age-related changes of Mg 2+ -ATPase activity. Mg 2+ -paranitrophenylphosphatase activity, and Na + -independent phosphorylation levels. The most rapid changes in (Na + -K + )-ATPase take place during the period corresponding to glial proliferation and neuronal arborization.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66087/1/j.1471-4159.1978.tb06577.x.pd

DEVELOPMENT OF (Na + -K + )-ATPase IN RAT HINDBRAIN: INCREMENTS IN PARALLEL WITH Na + -DEPENDENT PHOSPHORYLATION AND K + - p NITROPHENYLPHOSPHATASE 1

Rat hindbrain NaI-enriched microsomal (Na + -K + )-ATPase activity, K + - p NPPase activity, and Na + -dependent steady-state phosphorylation levels all increase approx 10-fold relative to microsomal protein between 5 days prenatally and 60 days postnatally. These activities, as well as the mean wet weight of the hindbrain, are at half of their 60 day values shortly after the 10th postnatal day. For all ages, these hindbrain activities average over twice those found in the forebrain in a companion paper (Bertoni & Siegel, 1978). Increases during development in the amount of the related phosphorylatable polypeptide, estimated by densitometry of stained polyacrylamide gels containing fixed amounts of microsomal protein dissolved in SDS, are in agreement with increases in steady state levels of Na + -dependent phosphorylation. The fraction of total phosphorylation that is Na + -dependent rises steadily during development consistent with, but not obligatorily due to, a conversion of some of the previously Na + -independent portion. Mg2 + -ATPase and Mg 2+ - p NPPase activities and steady-state Na + ,-independent phosphorylation levels do not increase in parallel during development. These observations add further support to the proposed partial reaction scheme for (Na + - K + )-ATPase. The major increments in (Na + -K + )-ATPase occur simultaneously with the deposition of specialized plasma membranes, particularly in the molecular layer of the cerebellum, as described in previous studies of rat hind brain.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65622/1/j.1471-4159.1979.tb00386.x.pd

REGULATORY EFFECTS OF POTASSIUM ON (Na + + K + )-ACTIVATED ADENOSINETRIPHOSPHATASE *

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73830/1/j.1749-6632.1974.tb19092.x.pd

Immunocytochemical Demonstration of Na + ,K + -ATPase in Internodal Axolemma of Myelinated Fibers of Rat Sciatic and Optic Nerves

We used postembedding electron microscopic immunocytochemistry with colloidal gold to determine the ultrastructural distribution of Na + ,K + -ATPase in the sciatic and optic nerves of the rat. Using a polyclonal antiserum raised against the denatured catalytic subunit of brain Na + ,K + -ATPase, we found immunoreactivity along the internodal axolemma of myelinated fibers in both nerves. This antiserum did not produce labeling of nodal axolemma. These results suggest that an important site of energy-dependent sodium-potassium exchange is along the internodal axolemma of myelinated fibers in the mammalian CNS and PNS and that there may be differences between the internodal and nodal forms of the enzyme.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66444/1/j.1471-4159.1991.tb02114.x.pd

Immunocytochemical Localization of (Na + ,K + )-ATPase in the Goldfish Optic Nerve

Antiserum to the catalytic subunit of goldfish brain (Na + ,K + )-ATPase has been employed at the electron microscopic level by means of the peroxidase-antiperoxidase immunohistochemical method. In optic nerve, an-tigenic sites are restricted to the nodes of Ranvier. No reaction product is detected in underlying internodal neurolemma. Outgrowing neurites for cultured retinal explants devoid of glial ensheathment exhibit a continuous distribution of the enzyme subunit. Antibodies against eel electroplax (Na + , K + )-ATPase cross-react with the goldfish brain enzyme and show a similar immunocytochemical distribution pattern.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65862/1/j.1471-4159.1981.tb02384.x.pd

Comparison of the distribution of Na+, K+-ATPase and myelin-associated glycoprotein (MAG) in the optic nerve, spinal cord and trigeminal ganglion of shiverer (shi/shi) and control (+/+) mice

Na+,K+ATPase and myelin-associated glycoprotein (MAG) were studied by immunocytochemistry on paraffin sections of the spinal cord, optic nerve and trigeminal ganglion of adult control (+/+) and CNS myelin-deficient shiverer (shi/shi) mice. Immunostaining for Na+,K+-ATPase outlined the periphery of nerve fibers in the spinal cord white matter, optic nerve and trigeminal ganglion of +/+ and shi/shi mice. Immunostaining for Na+,K+-ATPase appeared somewhat denser in the optic nerve and spinal cord lateral funiculi of shi/shi than in +/+ mice. In addition, immunostaining for Na+,K+-ATPase was demonstrated at the plasmalemmanof presumed satellite cells situated at the periphery of ganglion cell bodies in the trigeminal ganglion of both species of mice. Immunostaining for MAG was localized along the periphery of nerve fibers in the spinal cord funiculi (with little immunostaining within gray horns), optic nerve and trigeminal ganglion of both +/+ and shi/shi mice. The major differences between shi/shi and +/+ mice were that the number of MAG-immunostained nerve fibers was greatly reduced in the spinal cord funiculi and the density of immunostaining was slightly increased in the optic nerve of shi/shi mice. The numbers of MAG-immunostained nerve fibers in trigeminal ganglion were similar in both species. Also, the cytoplasm of some oligodendrocyte-like cell was found densely immunostained for MAG in the spinal cord and optic nerve of shi/shi mice, but not of +/+ mice. This light microscopic study provides evidence that the defective shiverer gene leads to a decrease in MAG deposition and to aggregations of MAG-like material within perikarya of oligodendrocyte-like cells in regions of the CNS.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26641/1/0000183.pd

Inhibition by lead of human erythrocyte (Na+ + K+)-adenosine triphosphatase associated with binding of 210Pb to membrane fragments

Fragmented human erythrocyte membranes were exposed to PbCl2 for 10-40 min at 23[deg]C prior to (Na+ + K+)-ATPase assay. Inhibition increased with exposure time. Enzyme activity in 5 [mu]g membrane protein was inhibited 50% after a 10-min exposure to 1.0 nmol PbCl2 (25 [mu]M final concentration) and was inhibited 100% after 40 min. When membranes at various concentrations were exposed to PbCl2 for 40 min, inhibition was linear with the ratio of PbCl2 to protein. Inhibition of 100% was obtained at 0.2 nmol PbCl2/[mu]g protein. A graph of activity vs. [protein] in the presence of PbCl2 intercepted the abscissa to the right of the origin, indicating that lead acts as an irreversible or very slowly reversible inhibitor. Addition of 1 mM 2,3-dimercaptopropanol, 1,3-dithiothreitol, -penicillamine or EDTA after 40 min exposure to 100 [mu]M PbCl2 restored 45, 64, 81 and 92% of the (Na+ + K+)-ATPase, respectively. These chelators, excluding EDTA, prevented inhibition when added before PbCl2. Two washings of the membrane fragments with water or 10 mM imidazole-HCl (pH 7.4) did not restore activity. 210Pb bound tightly to membrane fragments and beginning of saturation was observed at 0.19 nmol Pb2+ bound/[mu]g protein. This corresponded to 200 [mu]M final concentration of PbCl2 in the ATPase assay. At 0.2 nmol PbCl2/[mu]g protein (100% inhibition of ATPase), from 0.10-0.17 nmol of lead was bound per [mu]g protein. Under the same conditions, 1 mM -penicillamine removed 80% of the bound lead which correlated with its restoration of ATPase activity. Pb2+ does not appear to denature the enzyme. The irreversible kinetics may be related to sequestration of Pb2+ within vesicles that interfere with the accessibility of chelators to Pb2+ binding sites.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23197/1/0000124.pd

Immunocytochemical localization of (Na+ + K+)-ATPase in the rat hippocampus

The adult rat hippocampus was investigated by light microscopic immunocytochemistry for (Na+ + K+)-ATPase. In the CA1, CA2 and CA3 hippocampal regions, dense immunostaining for (Na+ + K+)-ATPase, exhibiting a punctate appearance, was demonstrated along the soma plasmalemma of hippocampal pyramidal cells in the stratum pyramidale, thus outlining these cells distinctly, and along dendrites extending into the stratum radiatum. (Na+ + K+)-ATPase immunostaining was dense in the neuropil of the strata oriens and radiatum of the rat hippocampus, but much lighter in the corpus callosum. Immunostaining at the periphery of pyramidal cell soma may be associated with the plexus formed by axon terminals of hippocampal basket cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28054/1/0000493.pd

(Na + +K + )-adenosinetriphosphatase in the brain of Shiverer (Shi/Shi) mice

The myelin-deficient Shiverer (Shi/Shi) mutant mouse may be a useful model in assessing the dependence of brain (Na + +K + )-ATPase concentration and composition on myelin membrane formation. Brain microsomal membranes from age-matched control (+/+) and Shiverer (Shi/Shi) mice were fractionated by differential centrifugation and sucrose gradient sedimentation. No reduction in (Na + +K + )-ATPase specific activity was measured in whole homogenates, high-and low-speed fractions or gradient fractions from brains of Shi/Shi mice as compared to those of +/+ mice. In addition, sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with antisera specific for mouse brain (Na + +K + )-ATPase revealed no significant difference in catalytic subunit composition between fractions of +/+ and Shi/Shi brains. The similar results obtained for both +/+ and myelin-deficient Shi/Shi mice suggest that myelin contributes little to total brain (Na + +K + )-ATPase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45404/1/11064_2004_Article_BF00971708.pd