Molecular isoforms of Na+/K(+)-ATPase in the nervous system and kidney of the spontaneously diabetic BB/Wor rat.

Na+/K(+)-ATPase was evaluated in the retina and kidney of the spontaneously diabetic BB/Wor rat after 1 and 4 months of insulin dependency. Retinal synthesis of the Na+/K(+)-ATPase was measured during a 2-h intravitreal pulse of [35S]methionine and analyzed by SDS-PAGE and scintillation counting. Synthesis of the alpha-1 and 'alpha(+)' (includes both alpha-2 and alpha-3) isoforms of the catalytic subunit was increased 123% and 69%, respectively at 4 months. Increases were also suggested at 1 month, but were not significant. The diabetes-dependent peak of synthesis in long-term diabetic rats turned over rapidly and by 3 days after intravitreal labeling, radioactively labeled enzyme was equal in both control and diabetic retinae. The amount of axonally transported, labeled enzyme recovered from endings of the optic nerve in the superior colliculus paralleled retinal labeling. Significant renal hypertrophy (48%) was noted at 4 months, but not at 1 month. The strophanthidin-inhibition constant for diabetes-induced renal enzyme was the same as for control enzyme (approx. 10(-4) M), indicating that diabetic renal hypertrophy does not induce a Na pump isozyme that is more sensitive to cardiotonic steroids. SDS-PAGE of the renal enzyme also failed to indicate more than one isoform of the alpha subunit.

The catalytic subunits of the (Na+,K+)-ATPase alpha and alpha(+) isozymes are the products of different genes.

The sequences of the first 14 amino acids of the (Na+,K+)-ATPase catalytic subunits from rat kidney (alpha) and rat brain axolemma (alpha(+)) have been determined. They are: (alpha), NH2-Gly-Arg-Asp-Lys-Tyr-Glu-Pro-Ala-Ala-Val-Ser-Glu-His-Gly; (alpha(+)), NH2-Gly-Arg-Glu-Tyr-Ser-Pro-Ala-Ala-Glu-Val-Ala-Glu-Val-Gly. Although they are highly homologous, it is clear these sequences are also sufficiently different to conclude they are the products of different genes, or at least different exons of the same, differentially spliced, gene. Among mammals, the amino terminal sequence of the kidney alpha chain is essentially invariant. Thus this section of the (Na+,K+)-ATPase molecule is more highly conserved in one tissue between several species than between different tissues in the same species. This may reflect upon the difference in function of the alpha and alpha(+) isozymes of (Na+,K+)-ATPase.

Partial purification and characterization of the (Ca2+ + Mg2+)-ATPase from squid optic nerve plasma membrane.

A membrane fraction enriched in axolemma was obtained from optic nerves of the squid (Sepiotheutis sepioidea) by differential centrifugation and density gradient fractionation. The preparation showed an oligomycin- and NaN3-insensitive (Ca2+ + Mg2+)-ATPase activity. The dependence of the ATPase activity on calcium concentration revealed the presence of two saturable components. One had a high affinity for calcium (K1 1/2 = 0.12 microM) and the second had a comparatively low affinity (K2 1/2 = 49.5 microM). Only the high-affinity component was specifically inhibited by vanadate (K1 = 35 microM). Calmodulin (12.5 micrograms/ml) stimulated the (Ca2+ + Mg2+)-ATPase by approx. 50%, and this stimulation was abolished by trifluoperazine (10 microM). Further treatment of the membrane fraction with 1% Nonidet P-40 resulted in a partial purification of the ATPase about 15-fold compared to the initial homogenate. This (Ca2+ + Mg2+)-ATPase from squid optic nerve displays some properties similar to those of the uncoupled Ca2+-pump described in internally dialyzed squid axons, suggesting that it could be its enzymatic basis.

Regulation of (Na+, K+) adenosinetriphosphatase of nerve ending membranes: action of norepinephrine and a soluble factor.

Norepinephrine added in vitro to nerve ending membranes from rat cerebral cortex stimulates the activity of (Na+, K+) adenosinetriphosphatase (ATPase) only in the presence of the soluble brain fraction. In its absence norepinephrine inhibits the enzyme. (Mg2+)ATPase also showed stimulation by norepinephrine in the presence of the soluble fraction, but of lesser magnitude. The activation of (Na+, K+)ATPase by norepinephrine is not reproduced by cyclic AMP and is not antagonized by either alpha- or beta-adrenergic blocking agents. These results suggest that the stimulation caused by norepinephrine is a direct effect on the enzyme and is not mediated by cyclic AMP or adrenergic receptors.

Isolation of non-myelin plasma membranes unique to white matter.

A procedure is described for isolating two membrane fractions from rabbit spinal-cord white matter enriched with 5'-nucleotidase, a nonspecific plasma membrane marker, 2',3'-cyclic nucleotide phosphohydrolase, an oligodendroglial plasma membrane marker, and acetylcholinesterase, an axonal plasma membrane marker. While the two membrane fractions exhibited similar enrichments with respect to cyclic nucleotide phosphohydrolase, enrichments of 5'-nucleotidase and acetylcholinesterase were significantly greater in the heavier membrane fraction. Selected enzyme markers for cyto- and mitochondrial membranes were not detected. Moreover, gray matter did not yield homologous membrane fractions in the gradient when subjected to the identical procedure, indicating that the two membrane fractions were unique to white matter. While electronmicroscopic examination revealed that both membrane fractions were comtaminated with myelin, the heavier fraction was least contaminated and exhibited a fair degree of homogeneity with respect to single membrane vesicular profiles. It was concluded that both membrane fractions were enriched with oligodendroglial and axonal plasma membranes, with the heavier fraction containing significantly more axolemma.

Cytochemical detection of nucleoside diphosphatase activity in the central nervous system of the rat.

The cytochemical localization of nucleoside diphosphatase and thiamine pyrophosphatase occurs within the "mature face" of the Golgi apparatus and over the neurilemma in neurons of the cerebellum, the cerebral cortex and the brain stem. The hydrolytic reaction product of the brain enzyme differs from that of the liver in that it is not found in the endoplasmic reticulum or nuclear envelope. Hydrolysis of IDP, UDP or GDP is not greater than that of ADP or CDP in brain homogenates, in contrast to that found in the liver. The NDPase activity of brain homogenates is optimal at pH 7.2, stimulated by heavy metals and inhibited by uranyl nitrate. Thick section cytochemistry suggests that the reaction product is restricted to a network of polygonally shaped compartments. NDPase activity on the neurilemma may reflect the role of this enzyme in the synthesis of glycoproteins involved in neuronal surface recognition.

Localization of acetylcholinesterase in granule-containing cells of glomus-like bodies in pre- and postnatal rabbits by electron microscopy.

The distribution of acetylcholinesterase (ACHE) was studied in the granule-containing cells which constitute the glomus-like bodies found near the origin of the great vessels in pre- and postnatal rabbits. Karnovsky's method for localization of ACHE at the electron-microscope level was used and suitable controls were carried out. In the granule-containing cells, ACHE reaction product was evident in the perinuclear cisternae and cisternae of the rough endoplasmic reticulum as well as at the cell membrane. ACHE activity was also localized at the axolemma of unmyelinated axons found near the granule-containing cells and around afferent synaptic terminals to these cells. Possible functions of ACHE associated with the monoamine-storing granule-containing cells are presented.

The localization of acetylcholinesterase at the autonomic neuromuscular junction.

Acetylcholinesterase has been localized at the autonomic neuromuscular junction in the bladder of the toad (Bufo marinus) by the Karnovsky method. High levels of enzyme activity have been demonstrated in association with the membranes of cholinergic axons and the adjacent membranes of the accompanying Schwann cells. The synaptic vesicles stained in occasional cholinergic axons. After longer incubation times, the membrane of smooth muscle cells close to cholinergic axons also stained. Axons with only moderate acetylcholinesterase activity or with no activity at all were seen in the same bundles as cholinergic axons, but identification of the transmitter in these axons was not possible.