Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes.

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of (Mg2+ + Na+ + K+)-ATPase than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for (Mg2+ + Na+ +k+)-ATPase and 5'-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome c reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADPH cytochrome c reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome c reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.     

Preparation of chick brain synaptosomes and synaptosomal membranes

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome $\text{c}$: oxygen oxidoreductase (EC 1.9.3.1.), monoamine: oxygen oxidoreductase (deaminating) (EC 1.4.3.4), rotenoneinsensitive NADH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.3), NADPH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, $\text{(}\text{Na}{}^{\mathrm{+}}\text{?}\text{K}{}^{\mathrm{+}}\text{)-}\text{activated}$ ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

The fraction of phosphatidylinositol that activates the (Na+ + K+)-ATPase in rabbit kidney microsomes is closely associated with the enzyme protein

1. Extensive treatment of rabbit kidney microsomes with phosphatidylinositol-specific phospholipase C under various conditions never resulted in more than 75% hydrolysis of the substrate. 2. The non-degraded fraction of the phosphatidylinositol (10–12 nmol per mg microsomal protein) could be recovered only by an acidic extraction procedure. 3. The $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$ activity found in those membranes was not affected by this treatment. 4. Complete degradation of phosphatidylinositol could be easily achieved when the phospholipase was applied to rat liver microsomes which do not contain any detectable $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$ activity. 5. It is concluded that in rabbit kidney microsomes a close association exist between the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$ and that fraction of the phosphatidylinositol that is directly involved in the maintenance of its activity.     

Author index

The ionic influence and ouabain sensitivity of lymphocyte Mg²⁺-ATPase and $\text{Mg}{}^{\mathrm{2+}}\text{-(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-activated}$ ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5′-nucleotidase and Mg²⁺-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ was located inside the membrane.Concanavalin A induced an early stimulation of Mg²⁺-ATPase and $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ both on intact cells and purified plasma membranes. In contrast, 5′-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg²⁺-ATPase activity was 3–5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20 %). $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity was undetectable in thymocytes. However, in spleen lymphocytes $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity can be detected and was 30 % increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg²⁺-ATPase, in lymphocyte stimulation.     

Plasma membrane NADH dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species

Ca²⁺-dependent K⁺ transport and plasma membrane NADH dehydrogenase activities have been studied in several ‘high-K⁺’ (human, rabbit and guinea pig) and ‘low-K⁺’ (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca²⁺-dependent K⁺ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome $\text{c}$ reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca²⁺-dependent K⁺ channel activities in the species studied. Nor were any of the above activities correlated with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity.     

A high-affinity (Ca2+ + Mg2+)-ATPase in plasma membranes of rat ascites hepatoma AH109A cells

The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$, EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ had an apparent half saturation constant of $\text{77 ± 31}\text{nM}$ for free calcium, a maximum reaction velocity of $\text{9.9 ± 3.5}\text{nmol}$ ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K⁺, Na⁺, ouabain, KCN, dicyclohexylcarbodiimide and NaN₃, had no significant effect on the activity of high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$. Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol $\text{bis}\text{(β-}\text{aminoethyl ether}\text{)-N,N,N′,N′-}\text{tetraacetic acid}$. Since the kinetic properties of the high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ showed a close resemblance to those of erythrocyte plasma membrane $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$, the high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells.     

Phosphatidylinositol as the endogenous activator of the (Na+ + K+)-ATPase in microsomes of rabbit kidney

Incubation of rabbit kidney microsomes with pig pancreatic phospholipase A₂ produced residual membrane preparations with very low $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity. The activity could be restored by recombination with lipid vesicles of negatively-charged glycerophospholipids. Vesicles of pure phosphatidylcholine and phosphatidylethanolamine were virtually inactive in this respect, but could reactivate in the presence of cholate.Incubation of the microsomes with a combination of phospholipase C (Bacillus cereus) and sphingomyelinase C (Staphylococcus aureus) resulted in 90–95% release of the phospholipids. The residual membrane contained only phosphatidylinositol and still showed 50–100% of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity.     

Trichomonas vaginalis: NADH oxidase activity.

NADH oxidase activity was detected in the 105,000g supernatant (“soluble”) fraction of Trichomonas vaginalis and the enzyme was purified 50-fold by centrifugation, ammonium sulfate precipitation, Sephadex G-200, and DEAE-Sephadex A-25 chromatography. The ratio of oxygen uptake to NADH oxidation was approximately one-half. Addition of catalase did not affect the rate of oxygen uptake elicited by NADH. Since the purified fraction was free from interfering enzymes, the postulated reaction is as follows: $\text{NADH}\text{+}\text{H}{}^{\mathrm{+}}\text{+}\text{1}\text{2}\text{= NAD}{}^{\mathrm{+}}\text{+ H}{}_2\text{O}$. Among numerous substances tested, only NADH was a functional substrate, whereas NADPH was not oxidized. The purified enzyme had a V_max of 16.5 μmole of oxygen consumed/min/mg protein, and the apparent K_m for NADH was 7.4 μM. Substrate inhibition was observed at 3.7 mM NADH. The purified NADH oxidase was competitively inhibited by NAD⁺ as well as by NADP⁺ with 50% inhibition at 1 and 5 mM, respectively. The enzyme was also markedly inhibited by p-chloromercuribenzoate, hydrogen peroxide, and transient metal-chelators such as bathophenanthroline or o-phenanthroline. A flavoprotein antagonist, atebrin was slightly less inhibitory. Various quinones, flavin nucleotides and artificial dyes, except for p-benzoquinone, ferricyanide and cytochrome c, did not function in accepting electrons from NADH oxidase. These three compounds, however, were still poor electron acceptors in the enzymatic reaction suggesting that the trichomonad NADH oxidase has little diaphorase activity. All of these findings indicate that T. vaginalis has an unique NADH oxidizing enzyme in that H₂O seems to be the prdouct of oxygen reduction. This NADH oxidase appears important in the aerobic metabolism of this parasite.     

Membrane lipid fatty acids and regulation of membrane-bound enzymes. Allosteric behaviour of erythrocyte Mg2+-ATPase, (Na+ + K2+)-ATPase and acetylcholineasterase from rats fed different fat-supplemented diets

Studies were carried out to determine the Hill coefficients for the inhibition by F^? of the erythrocyte membrane-bound Mg²⁺-ATPase, $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase from rats fed with seven different diets. Five groups were fed with different natural fats or oil supplements, one with a hydrogenated fat supplement and the other with fat-free diet. The responses of the red cell fatty acids to dietary fats were recorded. The value of $\text{n}$ for the inhibition by F^? of the three enzymes revealed a particular and different behaviour in each group. Correlations between the fatty acid compositions of erythrocyte membranes and cooperativity of each enzyme were calculated. The results indicate that neither the essential fatty acid family nor the non-essential ones are particularly involved in the allosteric phenomena. The increase of the double bond index/saturation ratio of fatty acids, which is taken as indicative of membrane fluidity, was accompanied in an inverse manner by changes in allosteric transitions of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase, whereas the Mg²⁺-ATPase was not dependent on this ratio. Diminution of membrane fluidity, carried out by in vitro increase of its cholesterol content, yields confirmatory results of this regulatory mechanism since the value of $\text{n}$ for acetylcholinesterase shifted as predicted.These facts indicate that the membrane fluidity is a physiological regulator for the allosteric behaviour of the membrane-bound enzymes and that each enzyme exhibits a particular behaviour in this phenomenon.     

The presence of two (Na+ + K+)-ATPase inhibitors in equine muscle ATP: Vanadate and a dithioerythritol-dependent inhibitor

A potent inhibitor of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V₂O₅ inhibit sarcolemma $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ with an $\text{I}{}_{50}$ of 1 μM in the presence of 1 mM $\text{ethyleneglycol-bis-(β-aminoethylether)}\text{-N,N′-}\text{tetraacetic}$ acid (EGTA), 145 mM NaCl, 6mM MgCl₂, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate in increased by free Mg²⁺. The inhibition is half maximally reversed by 250 μM epinephrine. Equine muscle ATP was also found to contain a second $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg²⁺ and is half maximally reversed by 2 μM epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg²⁺-ATPase, Ca²⁺-ATPase and $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$.     

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at $\text{3000 × g}$ followed by sedimentation of a microsomal fraction at $\text{200 000 × g}$. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity and [³H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca²⁺ concentration for enzyme activity was 5–10 μM. Both Na⁺ and K⁺ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca²⁺ from the cardiac cell.     

Fractionation of membrane vesicles. II. A method for separation of membrane vesicles bearing different enzymes by free-flow electrophoresis

Free-flow electrophoresis was used to subfractionate membrane vesicles from calf thymocyte plasma membranes. The fractionation resulted in a separation of vesicle populations bearing four different enzymes: alkaline nitrophenylphosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1), γ-glutamyltransferase (EC 2.3.2.2), $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase ($\text{acyl-CoA:1-acylglycero-3-phosphocholine}\text{-O-}\text{acyltransferase}$, EC 2.3.1.23). The specific content of cholesterol and total phospholipid coincided with the distribution of membrane-bound protein. However, vesicles migrating towards the cathode had a higher molar ratio of cholesterol to phospholipid (0.75) compared to those migrating to the anode (0.55). Sodium dodecyl sulphate-gel electrophoresis of pooled vesicle fractions also demonstrates distinct differences in their protein pattern. Electron-micrographic thin sections show that the vesicle populations have a similar morphology and size distribution.These results are discussed in terms of heterogeneity of the original thymocytes, contamination with intracellular membranes and a heterogeneous structure of the plasma membrane.     

Evidence for essential disulfide bonds in the β-subunit of (Na+ + K+)-ATPase

$\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from dog kidney lost its activity when heated at 55°C in the presence of 0.3 M 2-mercaptoethanol. Either heat treatment alone or addition of reducing agent at around 25°C caused little inactivation. One disulfide bond per protomer (mol. wt. 146000) was reduced in the inactivated sample but in active samples no reduction occurred. Neither K⁺-dependent phosphatase activity nor phosphoenzyme formation in the presence of Na⁺ was detected in the inactivated sample, suggesting that the disulfide bond was essential for the catalytic cycle of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$. This essential disulfide bond belonged to the β-subunit, the glycoprotein component of the enzyme, indicating that the β-subunit may be an integral component of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ system.     

The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient.The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase in purified basolateral plasma membranes was 13-fold. F^?-activated adenyl cyclase co-purified with ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K⁺-stimulated phosphatase and 5′-nucleotidase also followed ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase during fractionation.     

Reconstitution of b-type cytochrome oxidase from Rhodopseudomonas capsulata in liposomes and turnover studies of proton translocation

Purified b-type cytochrome oxidase from Rhodopseudomonas capsulata was incorporated into phospholipid vesicles to measure proton extrusion with pulses of ferrocytochrome c for one oxidase turnover. In accordance with the pH shift of its midpoint potential, the purified oxidase showed a proton extrusion of 0.24 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ with uptake of 1 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ from the liposomes for the reduction of oxygen to water. This proton translocation could only be observed in the presence of valinomycin +K⁺ and was not inhibited by DCCD. Oxidase preparations from the first purification step, which contain other protein compounds especially a membrane-bound cytochrome c but not the ubiquinol-cytochrome c₂-oxidoreductase showed a pumping activity of 0.9 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$, which was inhibited by DCCD for nearly 75%. Inhibition of the electron transfer was not observed, which could be explained by a ‘molecular slipping’ of proton extrusion and electron transfer. Proton extrusion from two oxidase-turnovers was only 80% of that from one turnover. The proton pumping of the b-type oxidase strongly depended on the enzyme/phospholipid ratio.     

Inhibition of lymphocyte activation by ouabain Interference with the early activation of membrane phospholipid metabolism

Activation of lymphocytes by antigens and mitogens can effectively be prevented by ouabain, a known inhibitor of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$. Recently it was shown that lowering of intracellular levels of monovalent cations is not involved in the inhibitory effect of ouabain. $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$ was found to be closely associated with acylCoA: lysophosphatidylcholine acyltransferase in the plasma membrane of lymphocytes. Both enzymes are activated as an immediate consequence of mitogen binding. Human peripheral lymphocytes were stimulated with concanavalin A. Ouabain suppressed the induction of RNA and DNA synthesis in a concentration-dependent way. Increase of RNA synthesis was suppressed only if the glycoside were added within the first hours of activation. If ouabain was added later, incorporation of uridine remained at the rate that was reached at the time of glycoside administration, pointing to an early event where ouabain may be operative. Ouabain, in a dose-dependent manner similar to that affecting RNA and DNA synthesis, inhibited the increase in the incorporation of oleate into phospholipids in stimulated lymphocytes, whereas the turnover of phospholipid fatty acids in resting lymphocytes was unaffected. Increasing extracellular K⁺ concentrations reversed the binding of ouabain to lymphocytes. Simultaneously, the inhibition of stimulated RNA synthesis was decreased and the inhibition of oleate incorporation was reversed. These results suggest that the suppression of lymphocyte activation by ouabain is due to the inhibition of membrane phospholipid metabolism mediated by the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$.     

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method.$\text{→}\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed $\text{→}\text{H}{}^{\mathrm{+}}\text{O}$ with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygenand ferricyanide pulses, with endogenous substrates or added methanol as a substrte, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of $\text{2}\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.     

Characterization of the (Na+ + K+)-ATPase in a membranous preparation from the optic ganglion of the squid (Loligo pealei)

(1) A membrane fraction enriched in $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ specific activity of approx. 2 units/mg and was $\text{>95\%}$ ouabain-sensitive. (2) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ had a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of $\text{0.42 ± 0.04}\text{mM}$ and a pH optimum of 7.0. It was inhibited by ouabain with a $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of $\text{0.32 ± 0.04 μ}\text{M}$. (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with $\text{NaCl + KCl}\text{= 300}\text{mM}$. (4) The Mg²⁺ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the$\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was $\text{0.86 ± 0.10}\text{mM}$, and at 6 mM ATP, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was $\text{1.86 ± 0.44}\text{mM}$. High levels of Mg²⁺ caused inhibition of hydrolysis. (5) The interactions of Na⁺ and K⁺ were examined over a range of conditions. K⁺ levels caused modulations in the Na⁺ dependence in the range of 1–150 mM. (6) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves.     

Purification from brain of an intrinsic membrane protein fraction enriched in (Na+ + K+)-ATPase

A microsomal fraction from canine brain gray matter has been extracted with the detergent sodium dodecyl sulfate to partially purify the membrane bound $\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ adenosine triphosphatase. Phospholipid, glycolipid, and a family of other glycoproteins are also enriched by the procedure; it is proposed that the product is an intrinsic membrane protein fraction. 6–8-fold purification of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350–400 μmol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent.