The sodium-stimulated adenosine-triphosphatase activity and other properties of cerebral microsomal fractions and subfractions

Schwartz, Arnold; Bachelard, H. S.; Wain, H McIL

doi:10.1042/bj0840626

Cited by 234 publications

(19 citation statements)

References 33 publications

(3 reference statements)

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“…Also, the activity of the sodium-plus-potassium-stimulated adenosinetriphosphatase system was virtually absent in the P4I preparation (Table V). These observations were taken to indicate that the present nuclear preparations were essentially free of contaminants from membrane fragments of microsomal nature and of nerve ending particles; the distribution of activities of acetylcholinesterase and of the adenosinetriphosphatase system in brain microsomes and in nerve ending particles has been demonstrated (7,15,22,24).…”

Section: Nucleic Acid and Protein Contentsmentioning

confidence: 72%

Isolation of Cell Nuclei From the Mammalian Cerebral Cortex and Their Assortment on a Morphological Basis

Kato

Kurokawa

1967

The Journal of Cell Biology

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An aqueous method is described for the isolation of highly purified nuclei from the ccrebral cortex of adult guinea pigs. Erythrocytes were removed by a short-time perfusion of the brain, myelin fragments by a rapid mechanical method, and blood capillaries by a centrifugal sieving through densc sucrose solutions. Thc nuclear preparation retained the activity of ATP:NMN adenylyltransfcrase. Recoveries of DNA in the P4I, P4II, PL and Ps preparations werc 30, 43, 8, and 7~, respectively. Microscopy and phase contrast microscopy showed a satisfactory removal of erythrocytes, myelin fragmcnts, capillaries, and cytoplasmic clcmcnts. Biochemical purity of samplcs was vcrificd by thc absence of several cytoplasmic enzyme activities. In thc electron microscopc, the majority of nuclei showed wcll-prescrved nuclear membrancs, with nuclcar pores, and werc providcd with a finely textured nuclcoplasm. Occasional contaminants wcrc elcmcnts of endoplasmic rcticulum and of the endothelium. Assortment of nuclci on a morphological basis showed that 55-65% and 47-53% of nuclei in the P4I and P4II preparations, rcsDcctivcly, consisted of neuronal nuclei. In thc PL preparation, the population of neuronal nuclei ranged between 72 and 83 ~, while 94--99 % of the nuclei in the Ps preparation consistcd of smaller nuclei, most likely of oligodcndroglial origin.

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Section: Nucleic Acid and Protein Contentsmentioning

confidence: 72%

Isolation of Cell Nuclei From the Mammalian Cerebral Cortex and Their Assortment on a Morphological Basis

Kato

Kurokawa

1967

The Journal of Cell Biology

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“…Our 91% pure enzyme was prepared with a modification of Jorgensen's method (5). Microsomes were isolated as described by Schwartz et al (10). Jorgensen's rapid purification procedure which uses a discontinuous sucrose gradient in an angle rotor was followed exactly.…”

Section: Methodsmentioning

confidence: 99%

Magnetic resonance and kinetic studies of the mechanism of membrane‐bound sodium and potassium ION‐activated adenosine triphosphatase

Grisham

Mildvan

1975

J Supramol Struct

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EPR and water proton relaxation rate (1/T1) studies of partially (40%) and "fully" (90%) purified preparations of membrane-bound (Na+ + K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 muM) in agreement with the kinetically determined activator constant, identifying this Mn2+-binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar high affinities and decrease 1/T1 of water protons due to a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO2-4 and CH3PO2-3 as a function of pH indicates that Na+ induces the phosphate monoanion to interact with enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding to the active site. Parallel 32Pi-binding studies show negligible formation (less than 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.8 and to 106% at pH 6.1, produced further decreases in 1/T1 of water protons. Preliminary 31P- relaxation studies of CH3PO2-3 in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 +/- 0.5 A) suggesting a second sphere enzyme-Mn-ligand-CH3PO2-3 complex. Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme but competes with Na+ at higher levels. From the paramagnetic effect of Mn2+ at the active site on the enzyme on I/T1 of 205T1 bound at the Na+ site, a Mn2+ to T1+ distance of 4.0 +/- 0.1 A is calculated, suggesting the sharing of a common ligand atomy by Mn2+ and T1+ on the ATPase. Addition of Pi increases this distance to 5.4 A consistent with the insertion of P between Mn2+ and T1+. These results are consistent with a mechanism for the (Na+ + K+)-ATPase and for ion transport in which the ionization state of Pi at a single enzyme active site controls the binding and transport of Na+ and K+, and indicate that the transport site for monovalent cations is very near the catalytic site of the ATPase. Our mechanism also accounts for the order of magnitude weaker binding of Na+ compared to K+.

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“…The ATPase from beef heart [31], beef brain [32], cat brain [el. 32], human red blood cells [33] and rabbit kidney [25] were prepared according to published procedures.…”

Section: Preparation Of Na K-atpasesmentioning

confidence: 99%

Studies concerning the possible reconstitution of an active cation pump across an artificial membrane

et al. 1972

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Summary. The cortical tissue of rat brain was fractionated through zonal centrifugation in a continuous sucrose density gradient, yielding a variety of morphologically distinct membrane fragments derived from nerve-end particles possessing variable levels of activity of Na, K-dependent Mg-sensitive ATPase (Na, K-ATPase) and other enzymes. Upon addition of certain of the zonal fractions, particularly those rich in the ATPase and acetylcholinesterase activities, to one side of planar artificial membranes, formed from mixtures of oxidized cholesterol and alkanes and bathed in a solution containing sodium, potassium, and magnesium ions, direct current membrane resistance fell from one to three orders of magnitude. Subsequent addition of ATP to the same side of the membrane to which the ATPase was added (the cis side) led to the development of net short-circuit current flow and open-circuit potential across the membrane (the cis side being negative with respect to the trans side). Development of the short-circuit current and open-circuit potential is dependent upon the presence of all the substrates of Na, KATPase as well as that of the enzyme itself. The net current flow is inhibited and the open-circuit potential discharged by the addition of ouabain to the trans side of the membrane, of phospholipase A to the cis side, or of trypsin to either side of the membrane. These observations provide circumstantial evidence for the reconstitution of the active cation pump across the artificial bilayer. Efforts to effect a similar reconstitution across membranes of this and other compositions employing Na, K-ATPase preparations from beef heart, beef brain, cat brain, human red cells, rabbit kidney, and rat brain microsomes failed.It is well established that biological membranes provide the environment for a large number of enzymatic reactions and associated processes including active transport. In these cases, membrane composition appears to be crucial in at least two respects: (1) the membrane may provide specific cofactors required for one or more enzymatic reactions; (2) specific membrane viscoelastic properties, determined by membrane composition, may be essential to proper enzymatic function [29]. These considerations focus

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The sodium-stimulated adenosine-triphosphatase activity and other properties of cerebral microsomal fractions and subfractions

Cited by 234 publications

References 33 publications

Isolation of Cell Nuclei From the Mammalian Cerebral Cortex and Their Assortment on a Morphological Basis

Isolation of Cell Nuclei From the Mammalian Cerebral Cortex and Their Assortment on a Morphological Basis

Magnetic resonance and kinetic studies of the mechanism of membrane‐bound sodium and potassium ION‐activated adenosine triphosphatase

Studies concerning the possible reconstitution of an active cation pump across an artificial membrane

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