John F. Dixon

Studies on the characterization of the sodium—Potassium transport adenosine triphosphatase: Purification and properties of the enzyme from the electric organ of Electrophorus electricus☆

Abstract The (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) from the electric organ of Electrophorus electricus has been purified by a procedure similar to that previously worked out in this laboratory. This consists of extraction of microsomes with the nonionic detergent, Lubrol WX, under optimal concentrations of detergent and protein, in the presence of ATP, and stabilization of the Lubrol extract further with Na and a commercial phospholipid preparation, Asolectin. This is followed by further purification by zonal centrifugation, which also removes free Lubrol. The final step is a novel ammonium sulfate fractionation (1, 4). The purified enzyme has a specific activity of 1200 μmoles phosphate/mg protein/hour. Unlike the microsomes or the Lubrol extract, the purified enzyme is highly stable, losing only 20–25% of its activity on storage at 0 °C for 30 days and losing no activity on freezing and storing at −70 °C. Polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate shows that 90–95% of the protein in the purified enzyme consists of a 93,000 molecular-weight protein and a 47,000 molecular-weight protein. The higher molecular-weight protein is the catalytic subunit of the enzyme, since it becomes phosphorylated on incubation with Na, Mg, and [γ- 32 P]ATP. The lower molecular-weight protein is a glycoprotein, since it stains with periodic acid-Schiff reagent. Milligram quantities of each protein can be obtained in pure form by chromatography on Sephadex G-150 equilibrated with 0.1% sodium dodecyl sulfate. The yield of purified enzyme from the microsomes is 50%. The kinetic parameters of the purified enzyme are reported in some detail and they resemble very closely those of the purified enzyme from the rectal gland of Squalus acanthias and the partially purified enzyme of beef brain (16), in spite of the fact that these three species are widely separated in evolution.

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. V. Partial purification of the lubrol-solubilized beef brain enzyme.

Abstract The Lubrol-solubilized Na-K adenosinetriphosphatase from NaI-treated beef brain microsomes has been partially purified. This purification involves salt and isoelectric precipitation, ultracentrifugation at 114,000 g for 20 hr, and chromatography on carboxymethylcellulose. The final enzyme preparation is water-clear and is relatively stable if stored at 0 ° in the presence of Na and ATP. Most of the Lubrol is removed by the purification procedure. The partially purified enzyme has a specific activity which is ten times that of the original microsomes. A single, somewhat diffuse, ouabain sensitive ATPase band runs on electrophoresis in 3% polyacrylamide of Lubrol extracts of NaI-treated microsomes. However, partial aggregation occurs during purification, as attested by the appearance of a Na-K ATPase peak in the void volume (molecular weight > 2 × 10 6 ) on chromatography on 6% agarose and a ouabain-sensitive ATPase band which does not enter 3% polyacrylamide on disc gel electrophoresis. The enzyme can be disaggregated with Lubrol into a single species which, on polyacrylamide gel electrophoresis, runs as the enzyme in the original Lubrol extracts; however, concentrations of Lubrol required to disaggregate the enzyme lead to partial inactivation. The various proteins in the preparation do not separate as discrete bands on disc gel electrophoresis unless the proteins are solubilized in phenol-acetic acid-urea. It is estimated that the Na-K ATPase in the present partially purified preparation accounts for approximately 10% of the protein.

A novel action of lithium: Stimulation of glutamate release and inositol 1,4,5 trisphosphate accumulation via activation of the N-methyl D-aspartate receptor in monkey and mouse cerebral cortex slices

Beginning at therapeutic concentrations (1-1.5mM), the anti-manic-depressive drug, lithium, stimulated the release of the major excitatory central neurotransmitter, glutamate, in monkey cerebral cortex slices in a time- and concentration-dependent manner, and this was associated with increased inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] accumulation. (+/-)-3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphoric acid (CPP), dizocilpine (MK-801), ketamine, and Mg(2+)-antagonists to the N-methyl D-aspartate (NMDA) receptor/channel complex selectivity inhibited lithium-stimulated Ins(1,4,5)P3 accumulation. Antagonists to cholinergic-muscarinic, alpha 1-adrenergic, 5-HT2-serotoninergic and H1-histaminergic receptors had no effect. Antagonists to non-NMDA glutamate receptors had no effect on lithium-stimulated Ins(1,4,5)P3 accumulation. Possible reasons for this are discussed. Similar results were obtained in mouse cerebral cortex slices. Carbetapentane, which inhibits glutamate release, inhibited lithium-induced Ins(1,4,5)P3 accumulation in this model. It is concluded that the primary effect of lithium in the cerebral cortex slice model is stimulation of glutamate release, which, via activation of the NMDA receptor, leads to Ca2+ entry. Ca2+ entry, in turn, activates phospholipase C. These effects may have relevance to the therapeutic action of lithium in the treatment of manic-depression, as well as its toxic effects, especially at lithium blood levels above 1.5mM. A general conclusion which can be drawn from these studies and earlier studies in our laboratory is that lithium potentiates the action of phospholipase C, whether this enzyme is activated by lithium-induced presynaptic release of neurotransmitter, such as glutamate, or by the addition of an exogenous neurotransmitter, such as acetylcholine. However, this does not appear to be due to a direct activation of phospholipase C.

A simple procedure for the preparation of highly purified (sodium + potassium) adenosinetriphosphatase from the rectal salt gland of Squalus acanthias and the electric organ of Electrophorus electrieus☆

Abstract A simple purification procedure for the Na,K-ATPase from membranes of the rectal gland of Squalus acanthias or crude microsomal fractions from the electric organ of Electrophorus electricus is presented here. The purification procedure consists of solubilization of the Na,K-ATPase with the nonionic detergent. Lubrol WX, chromatography of the diluted Lubrol extract on aminoethyl cellulose, and ammonium sulfate fractionation (1) of the concentrated eluate from the aminoethyl cellulose column. The yields of final purified enzyme are comparable to the earlier purification (1–4) involving the expensive and cumbersome zonal centrifugation stop. The purity of the final enzyme, as attested to by specific activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is as great or greater than that previously reported for the enzyme purified by the procedure involving zonal centrifugation. The simplicity of the present procedure, coupled with the ready commercial availability of electric eels which are quite hardy on shipment, makes purification of the Na,K-ATPase widely available to workers in the field.

Lithium Enhances Accumulation of [3H]Inositol Radioactivity and Mass of Second Messenger Inositol 1,4,5-Trisphosphate in Monkey Cerebral Cortex Slices

Abstract: We previously reported that lithium, in the presence of acetylcholine, increased accumulations of inositol 1,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate in brain cortex slices from the guinea pig, rabbit, rat, and mouse. In the mouse and rat, the Li+‐induced increases required supplementation of the medium with inositol. This probably relates to the following facts: (a) Brain cortices of the mouse and rat contain in vivo concentrations of inositol half of that of the guinea pig. (b) Incubated rat brain cortex slices are depleted of inositol by 80%. (c) The slices require 10 mM inositol supplementation to restore in vivo concentrations. We now show that in monkey brain cortex slices, therapeutic concentrations of Li+ increase accumulation of inositol 1,4,5‐trisphosphate. The inositol 1,3,4,5‐tetrakisphosphate level is not increased. Neither inositol nor an agonist is required. The same effects are seen whether inositol 1,4,5‐trisphosphate is quantified by the [3H]inositol prelabeling technique or by mass assay, although mass includes a pool of inositol 1,4,5‐trisphosphate that is metabolically inactive. Thus, in a therapeutically relevant model for humans, Li+ increases inositol 1,4,5‐trisphosphate levels in brain cortex slices, as was previously seen in lower mammals at non‐rate‐limiting concentrations of inositol.

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase: XI. Comparison of kinetic properties of the purified with the impure membrane-bound enzyme from Squalus acanthias

Abstract A variety of kinetic parameters have been compared in the membrane-bound and purified forms of the (sodium + potassium)-activated adenosinetriphosphatase (NaK ATPase) from the rectal gland of the spiny dogfish, Squalus acanthias . The kinetic parameters which have been studied have been temperature optima, pH optima, Mg-activation curves, optimum ATP/Mg ratios, K m for ATP, ouabain-inhibition curves, and Na and K-activation curves. All kinetic parameters were remarkably similar for both forms of the enzyme. This encourages us to believe that information obtained from the pure enzyme can be extrapolated to the enzyme in its native membrane environment and should throw light on the molecular mechanism of Na and K transport.

Inositol 1,2-cyclic 4,5-trisphosphate is formed in the rat parotid gland on muscarinic stimulation

Hawkins et al. [Hawkins, P.T., Berrie, C.P., Morris, A.J., and Downes, C.P. (1987) Biochem J. 243, 211-218] were unable to find any formation of inositol 1,2-cyclic 4,5-trisphosphate on muscarinic stimulation of rat parotid slices, contrary to what has been found in mouse pancreas and in platelets. We have repeated the studies of Hawkins et al. using [3H]inositol-prelabelled rat parotid minilobules and our improved HPLC method for clearly separating the three inositol trisphosphates. Substantial amounts of inositol 1,2-cyclic 4,5-trisphosphate formed on muscarinic stimulation of rat parotid minilobules, amounting to 5% of inositol 1,4,5-trisphosphate at 10 sec and one third of inositol 1,4,5-trisphosphate at 5 min.

Chapter 37: The phosphoinositide signalling system. I. Historical background. II. Effects of lithium on the accumulation of second messenger inositol 1,4,5-trisphosphate in brain cortex slices

Publisher Summary The interaction of neurotransmitters, growth factors, and hormones with cell surface receptors often stimulates intracellular processes by activating membrane enzymes, usually via a GTP-binding protein, to produce “second messengers” that diffuse to intracellular targets. In recent years, it has become clear that on binding of a variety of agonists to their receptors, inositol lipids in cell membranes are cleaved to inositol phosphates and diacylglycerol (DAG). There are three main phosphoinositides in the cell membrane: phosphatidylinositol (PtdIns), which comprises about 95% of membrane inositol lipid, phosphatidylinositol Cphosphate [PtdIns(4)P] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ]. Activation of an appropriate receptor stimulates cleavage of PtdIns(4,5)P 2 to form DAG and inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ]. Ins (1,4,5) P3 diffuses to Ca 2+ storage sites in the endoplasmic reticulum (ER), binds to the Ins( 1,4,5)P 3 receptor in the ER membrane and releases Ca 2+ through a channel into the cytosol. Ca 2+ then activates physiological responses via Ca 2+ /calmodulin protein kinases and possibly by other mechanisms. DAG activates protein kinase C, which phosphorylates a different set of proteins, leading to a variety of biological responses. Diacylglycerol is also formed in some systems by agoniststimulated phosphodiesteratic cleavage of phosphatidylcholine and phosphatidylethanolamine.

Agonist-Stimulated Inositol Polyphosphate Formation in Cerebellum

Abstract: The accumulation of inositol polyphosphates in the cerebellum in response to agonists has not been demonstrated. Guinea pig cerebellar slices prelabeled with [3H]inositol showed the following increases in response to 1 mM serotonin: At 15 s, there was a peak in 3H label in the second messenger inositol 1,4,5‐trisphosphate [Ins(l,4,5)P3], decreasing to a lower level in about 1 min. The level of 3H label in the putative second‐messenger inositol 1,3,4,5‐tetra‐kisphosphate [Ins(l,3,4,5)P4] increased rapidly up to 60 s and increased slowly thereafter. The accumulation of 3H label in various inositol phosphate isomers at 10 min, when steady state was obtained, showed the following increases due to serotonin: inositol 1,3,4‐trisphosphate [Ins(l,3,4)P3], eightfold; Ins(l,3,4,5)P4, 6.4‐fold; Ins(l,4,5)P3, 75%; inositol 1,4‐bisphosphate [Ins(1,4)P2, 0%; inositol 3,4‐bisphosphate, 100%; inositol 1‐phosphate/inositol 3‐phosphate, 30%; and inositol 4‐phosphate, 40%. [3H]Inositol 1,3‐bisphosphate was not detected in controls, but it accounted for 7.2% of the total inositol bisphosphates formed in the serotonin‐stimulated samples. The fact that serotonin did not increase the formation of Ins(1,4)P2 could be due to the fact that Ins(1,4)P2 is rapidly degraded or that Ins(l,4,5)P3 is metabolized primarily by Ins(1,4,5)P3‐3’ kinase to form Ins(1,3,4,5)P4. In the presence of pargyline (10 nM), [3H]Ins(l,3,4,5)P4 and [3H]Ins(l,3,4)P3 levels were increased, even at 1 μM serotonin. Ketanserin (7 μM) completely inhibited the serotonin effect, indicating stimulation of serotonin2 receptors. Quisqualic acid (100 μM) also increased the levels of [3H]Ins(l,4,5)P3, [3H]Ins(l,3,4,5)P4, and [3H]Ins(l,3,4)P3, but the profile of these increases was different. The quisqualic acid‐stimulated formation of inositol phosphate isomers was not affected by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, indicating that it was not due to the ionotropic properties of the quisqualate receptor. Similar results were obtained on stimulation of the labeled slices with glutamate, but the magnitudes were less. The data show that in the guinea pig cerebellum, stimulation of the serotonin2 and metabotropic quisqualic acid receptors leads to the initial formation of Ins(l,4,5)P3, but its subsequent metabolism varies, presumably owing to two kinds of receptors, localized on different cell types in the cerebellum with varying levels of inositol phosphate‐metabolizing enzymes.

Biochemical aspects of the phosphoinositide signalling system with special reference to the formation of inositol cyclic phosphates and arachidonic acid and metabolites on agonist stimulation

Several aspects of the phosphoinositide signalling system recently studied in our Laboratory are considered here. 1. The formation of inositol 1:2-cyclic-4,5-trisphosphate (IcP3) and inositol 1:2-cyclic-4-bisphosphate (IcP2) have been shown here to occur in pancreatic minilobules stimulated with carbamylcholine. Identification is based on mobility on ionophoresis on paper and on HPLC, acid lability, and conversion of the inositol cyclic phosphates to their respective non-cyclic inositol phosphates on treatment with acid. The levels of inositol 1:2-cyclic phosphate (IcP), IcP2, and IcP3 were 0.7%, 6.8%, and 29.8% of their respective non-cyclic inositol phosphates. The level of IcP3 is sufficient to evoke release of calcium from the endoplasmic reticulum. 2. In a previous study, we demonstrated that on agonist stimulation of pancreatic minilobules prelabelled with [14C]arachidonate, [14C]stearate, or [3H]glycerol, there was a substantial release of all three of these compounds, amounting to approximately 50% of the total PI loss, which was up to 70% of the total cellular PI (7). It was shown that this loss in PI was due to the sequential actions of phospholipase C and diacylglycerol (DG) lipase. Evidence against the phospholipase A2 pathway was no formation of lysophosphatidylinositol. Further evidence against the phospholipase A2 pathway shown here is the lack of stimulation by agonist of glycerophosphorylinositol formation. We also show here that the stimulation of PI loss in guinea pig brain cortex slices is likely also to be via the sequential actions of phospholipase C and DG-lipase, i.e., there was an increase in the steady-state level of monoacylglycerol and a rise in free arachidonate on stimulation with acetylcholine. The formation of prostaglandin E and prostaglandin F was also increased in brain cortex, corpus striatum, and hippocampus. The effects of acetylcholine were abolished by atropine. 3. Previous studies showed that the DG-lipase inhibitor, RHC 80267, inhibited agonist-stimulated formation of glycerol and fatty acids and raised the steady-state level of DG (7). We have now used RHC 80267 as a tool to elevate the level of DG and to lower the level of arachidonate to see if either of these products might modulate the carbamylcholine-stimulated cGMP levels in pancreatic minilobules. RHC 80267 inhibited formation of cGMP. Addition of arachidonate did not affect this inhibition, nor did addition of free arachidonate to control minilobules have any effect, thus suggesting that liberation of free arachidonate by carbamylcholine was not responsible for the carbamylcholine-induced rise in cGMP.(ABSTRACT TRUNCATED AT 400 WORDS)