Dependence of renal (Na+ + k+)-adenosine triphosphatase activity on thyroid status.

In thyroidectomized rats, a single injection of L-2,,5,2'-triiodothyronine (T3) (50mug/100 g body weight) elicited at 45% increase in (Na+ + k+)-dependent adenosine triphosphatase (NaK-ATPase) activity of the membrane-rich fraction of renal cortex at the optimal time of response, 48 h after injection. Three successive doses of T3 (50 mug/100 g body weight), given on alternate days, increased NaK-ATPase by 67% in the renal cortex but had no significant effect on the outer medulla or the papilla. Moreover, T3 had no effect on Mg2+-dependent adenosine trisphatase (MgATPase) in cortex, cedulla, or papilla. Three doses of T3 (50 mug/100 g body weight) given on alternate days to thyroidectomized rats elecited a 134, 79, and 46% increase in Vmax for ATP, Na4, and K+, respectively. There were no changes in the Km for ATP or the K1/2 values for Na+ and K+. Two methods were used to estimate the effect of T3 on the number of NaK-ATPase units (assumed to represent the number of Na+ pump sites); rat renal plasma membrane fractions were incubated with [gamma-32P]ATP, Mg2+, and Na+; the 32P-labeled membrane protein yeild was quantitatively dependent on Na+ and was hydrolyzed on addition of K+. There was a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of phosphorylated intermediate formed, in renal cortical membrane fractions from thyroidectomized rats given T3 or the diluent. There was also a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of [3H]ouabain specifically bound (Na+-, Mg2+-, APT-dependent) to the NaK-ATPase preparation. Injection of T3 resulted in a 70% increase in NaK-ATPase activity, a 79% increase in formation of the phosphorylated intermediate, and a 65% increase in the [H]ouabain specifically bound to the NaK-ATPase system. The T3-dependent increases in Vmax for ATP, Na+, and K+ and the proportionate increases in the phosphorylated intermediate and in the amount of [3H]ouabain bound indicate that T3 increases the number of NaK-ATPase units and that this increase accounts for the increase in NaK-ATPase activity.     

Relationship between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in the action of thyroid hormone on rat jejunal mucosa.

Administration of three successive doses of triiodothyronine (T3) (50 micrograms/100 g body wt), given on alternate days to thyroidectomized and euthyroid rats, stimulated oxygen consumption (QO2) and Na+ transport-dependent respiration (QO2 [5]) in the stripped jejunal mucosa, a preparation that consisted mostly of epithelial cells. The increase in QO2(t) accounted for 57% of the increment in QO2 in the transition from the hypothyroid to the euthyroid state and for 29% of the increment in the transition from the euthyroid to the hyperthyroid state. Administration of T3 to hypothyroid rats also increased the yield of epithelial cells. Injection of T3 into thyroidectomized and euthyroid rats increased the specific activity (at Vmax) of the (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) in jejunal crude membrane preparations. No significant change was recorded in the activity of Mg-ATPase in the same preparation. The ratio of QO2/NaK-ATPase and QO2(t)/NaK-ATPase in the various thyroid states remained constant, indicating proportionate increased in the respiratory and enzymatic indices. The effect of administration of T3 to thyroidectomized rats on the number of NaK-ATPase units (recovered in the crude membrane preparation) was estimated by: (a) Na+ + Mg++ + ATP-dependent binding of [3H]-ouabain to crude membrane fractions, and (b) the amount of the phosphorylated intermediate formed in the NaK-ATPase reaction from AT32P(gamma). Estimates were obtained of the maximal number of [3H]ouabain binding sites (Nm) and dissociation constants (Kd). Nm for [3H]ouabain and Nak-ATPase specific activity increased to about the same extent after T3 administration to thyroidectomized rats, with no change in the apparent Kd values. The amount of phosphorylated intermediate formed in jejunal crude membrane preparations also increased significantly. Thus, thyroid hormone administration may increase the number of active Na+pump sites in the plasma membrane. The apparent increase in the number of Na+ pump sites also correlated with the hormone dependent increases in QO2 and QO2(t).     

Thiophosphorylation of hog gastric (H+ + K+)-ATPase membranes by endogenous protein kinases

(H+ + K+)-ATPase-enriched membranes from hog stomachs were tested for their capacity to autophosphorylate using [gamma-32P]ATP or [gamma-35S]ATP[S] as phosphate donors. The radioactive polypeptides were characterized by SDS-PAGE. In the presence of Mg2+ and 5 microM [gamma-32P]ATP, rapid and transient incorporation of 32P occurred at 0 degrees C. Radioactivity was essentially found in the major polypeptide of the material, the 95 kDa subunit of (H+ + K+)-ATPase. Under the same experimental conditions, thiophosphorylation was slower and reached a plateau within 1 h. Incorporation levels were higher with manganese than with magnesium. After one hour at 0 degrees C, and in the presence of 10 mM manganese and 5 microM ATP[S], 0.58 +/- 0.06 nmoles of thiophosphate were incorporated per mg of protein. Twenty seven percent of the thiophosphorylated amino acids were acylphosphates i.e. likely to be the ATPase thiophosphointermediate. The remaining thiophosphorylated amino acids (73%) were thought to be produced by protein kinases. This was supported by the autoradiographies of membrane SDS-PAGE which indicated that, in addition to the 95 kDa ATPase subunit, other polypeptides were thiophosphorylated especially at 108, 58, 47, 45 and 36-40 kDa. A previous study had provided strong evidence that chloride transport in gastric microsomes, is modulated by a protein kinase-dependent phosphorylation (Soumarmon, A., Abastado, M., Bonfils, S. and Lewin M.J.M. (1980) J. Biol. Chem. 255, 11682-11687). In the present work, we demonstrate that the peptidic inhibitor of cAMP-dependent protein kinases decreased thiophosphorylation of a 45 kDa polypeptide. We suggest that this polypeptide could be regarded as a candidate for the role of chloride transporter or chloride transport regulator.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

Quaternary structure of (Na+ + K+)-dependent adenosine triphosphatase.

(Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) consists of two polypeptide chains, a large polypeptide with a molecular weight of about 100,000, and a sialoglycoprotein with a molecular weight of about 40,000. Cross-linking of purified NaK-ATPase with the (o-phenanthroline)2-cupric ion complex (CP) results in the reversible formation of dimers, trimers, tetramers, and pentamers of the large polypeptide and loss of NaK-ATPase activity. ATPase activity is partially recovered if NaK-ATPase is incubated with beta-mercaptoethanol after treatment with CP. In contrast to these results, if NaK-ATPase is cross-linked in crude canine kidney microsomes, only a dimer of the large polypeptide is formed. No cross-linking of the sialoglycoprotein to the large polypeptide is detected when NaK-ATPase is cross-linked in purified form. However, when NaK-ATPase is reacted with CP in either purified or microsomal form, the sialoglycoprotein cross-links to itself yielding a high molecular weight aggregate. The results show that the functional subunit structure of NaK-ATPase consists of at least two large polypeptides.     

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

The MgATP complex analogue cobalt-tetrammine-ATP [Co(NH3)4ATP] inactivates (Na+ + K+)-ATPase at 37 degrees C slowly in the absence of univalent cations. This inactivation occurs concomitantly with incorporation of radioactivity from [alpha-32P]Co(NH3)4ATP and from [gamma-32P]Co(NH3)4ATP into the alpha subunit. The kinetics of inactivation are consistent with the formation of a dissociable complex of Co(NH3)4ATP with the enzyme (E) followed by the phosphorylation of the enzyme: (Formula: see text). The dissociation constant of the enzyme-MgATP analogue complex at 37 degrees C is Kd = 500 microM, the inactivation rate constant k2 = 0.05 min-1. ATP protects the enzyme against the inactivation by Co(NH3)4ATP due to binding at a site from which it dissociates with a Kd of 360 microM. It is concluded, therefore, that Co(NH3)4ATP binds to the low-affinity ATP binding site of the E2 conformational state. K+, Na+ and Mg2+ protect the enzyme against the inactivation by Co(NH3)4ATP. Whilst Na+ or Mg2+ decrease the inactivation rate constant k2, K+ exerts its protective effect by increasing the dissociation constant of the enzyme.Co(NH3)4ATP complex. The Co(NH3)4ATP-inactivated (Na+ + K+)-ATPase, in contrast to the non-inactivated enzyme, incorporates [3H]ouabain. This indicates that the Co(NH3)4ATP-inactivated enzyme is stabilized in the E2 conformational state. Despite the inactivation of (Na+ + K+)-ATPase by Co(NH3)4ATP from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 microM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na+ + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the E1 and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na+ + K+)-transport.     

Anion dependence of Ca2+ transport and (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in rat pancreatic endoplasmic reticulum

Anion dependence of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase and its phosphorylated intermediate have been characterized in both "intact" and "broken" vesicles from endoplasmic reticulum of rat pancreatic acinar cells using adenosine 5'-[gamma-32P] triphosphate ([gamma-32P]ATP). In intact vesicles (Ca2+ + K+)-Mg2+-ATPase activity was higher in the presence of Cl- or Br- as compared to NO3-, SCN-, cyclamate-, SO4(2-) or SO3(2-). Incorporation of 32P from [gamma-32P]ATP into the 100-kDa intermediate of this Ca2+ATPase was also higher in the presence of Cl-, Br-, NO3- or SCN- as compared to cyclamate-, SO4(2-) or SO3(2-). When the membrane permeability barrier to anions was abolished by breaking vesicle membrane with the detergent Triton X-100 (0.015%) (Ca2+ + K+)-Mg2+ATPase activity in the presence of weakly permeant anions, such as SO4(2-) and cyclamate-, increased to the level obtained with Cl-. However, 32P incorporation into 100-kDa protein was still higher in the presence of Cl- as compared to cyclamate-, indicating a direct effect of Cl- on the Ca2+ATPase molecule. The anion transport blocker 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS) inhibited (Ca2+ + K+)-Mg2+ATPase activity to about 10% of the Cl- stimulation level, irrespective of the sort of anions present in both intact and broken vesicles. This indicates a direct effect of DIDS on (Ca2+ + K+)-Mg2+ATPase. K+ ionophore valinomycin influenced (Ca2+ + K+)-Mg2+ATPase activity according to the actual K+ gradient: Ko+ greater than Ki+ caused inhibition, Ko+ less than Ki+ caused stimulation. From these results we conclude that Ca2+ transport into endoplasmic reticulum is coupled to ion movements which must occur to maintain electroneutrality.     

The action of trypsin on the gastric (H+ + K+)-ATPase.

The effect of trypsin on gastric (H+ + K+)-ATPase and K+-phosphatase was studied. Loss of both enzymic activities was biphasic, consisting of a fast and slow phase. Several peptides were produced from the original 105,000-dalton region of the sodium dodecyl sulfate electrophoretic separation, but only two, 87,000 and 47,000 daltons, were labeled following incubation with [gamma-33P]ATP. After a 30-min hydrolysis, 35% of the original peptide remained unaltered and appeared to be a glycoprotein. ATP and ADP abolished the second phase of tryptic inactivation of both activities and only two peptides, of 78,000 and 30,000 daltons, were found on the acrylamide gel in addition to the original 105,000-dalton region, neither of which was labeled by [gamma-33P]ATP. The protection was specific for these nucleotides, AMP, beta, gamma-methylene ATP, TTP, and pNPP being ineffective. Na+ and K+ at high concentrations reduced the rate of loss of activity but no change in the peptides produced was found. The level of phosphoenzyme was increased 2-fold by trypsin treatment, whereas the quantity of K+-sensitive phosphoenzyme remained relatively constant. Thus, the 105,000-dalton region is heterogeneous, consisting of a catalytic subunit (the active site is on a 47,000-dalton fragment), a glycoprotein, and another 105,000-dalton peptide. The action of trypsin is initially to prevent interconversion of a K+-insensitive to a K+-sensitive form of the phosphoenzyme, thus inhibiting hydrolysis.     

Role of sulfatide on phosphoenzyme formation and ouabain binding of the (Na+ + K+)ATPase

A microsomal fraction rich in (Na+ + K+)ATPase activity has been isolated from the outer medulla of pig kidney. The ability of this preparation to form phosphoenzyme on incubation with [gamma-32P]ATP and to bind [3H]ouabain was studied when its sulfatide was hydrolyzed by arylsulfatase treatment. The K+-dependent hydrolysis of the Na+-dependent phosphorylated intermediate as well as the ouabain binding were inactivated in direct relation to the breakdown of sulfatide. Both characteristics of the (Na+ + K+)ATPase preparation, lost by arylsulfatase treatment, were partially restored by the sole addition of sulfatide. These experiments indicate that sulfatide may play a role in sodium ion transport either in the conformational transition of the K+-insensitive phosphointermediate, E1P, to the K+-sensitive intermediate, E2P, or in the configuration of the high-affinity binding site for K+ of the E2P form. In addition, this glycolipid may have a specific role in the proteolipidic subunit that binds ouabain.     

Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP.

The interaction of sarcoplasmic reticulum Ca(2+)-ATPase with the Mg.ATP analogues Rh(H2O)4ATP and Co(NH3)4ATP have been examined. Co(NH3)4ATP slowly inactivates Ca(2+)-ATPase in a first order process, with a rate constant of 1.13 x 10(-3) s-1 and an apparent inactivation constant, KI, of 32 mM. Rh(H2O)4ATP likewise inactivates sarcoplasmic reticulum Ca(2+)-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H2O)4ATP] is biphasic. The chi-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca(2+)-ATPase by Rh(H2O)4ATP of KI1 = 30 microM and KI2 = 221 microM. The corresponding values of k2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 x 10(-4)s-1. Tridentate Rh(H2O)3ATP also inhibits Ca(2+)-ATPase, but only after much longer incubation times. Ca(2+)-ATPase inactivation is accompanied by incorporation of radioactivity from gamma-32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca(2+)-dependent ATPase activity in this experiment was 2.19 x 10(-4)s-1, and the first-order rate constant for Ca(2+)-dependent E-P formation was 2.07 x 10(-4)s-1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca(2+)-ATPase by Rh(H2O)4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H2O)4ATP is binding to the catalytic nucleotide site on Ca(2+)-ATPase and producing a highly stable, phosphorylated intermediate.     

The (Na+ + K+)-ATPase of chick sensory neurons. Studies on biosynthesis and intracellular transport

The (Na+ + K+)-ATPase of cultured chick sensory neurons was studied with the aid of antibodies specific for this enzyme. Immunofluorescent labeling indicated the (Na+ + K+)-ATPase is evenly distributed on the neuronal cell surface; cell bodies, neurites, and growth cones were labeled with comparable intensity. Pulse-chase experiments with [35S]methionine, followed by immunoprecipitation, indicated concurrent synthesis and rapid association of the alpha (Mr = 105,000) and beta (Mr = 47,000) subunits. The alpha subunit is oligosaccharide-free while the beta subunit contains three Asn-linked oligosaccharide chains attached to a core peptide of 32,000 molecular weight. The time required for oligosaccharide processing of the newly synthesized beta subunit to endoglycosidase H-resistance suggests the (Na+ + K+)-ATPase takes 45-60 min to move from the site of polypeptide synthesis to the Golgi apparatus. Significantly less time was required for transport through the Golgi apparatus and insertion in the plasma membrane. From 30% to 55% of the newly synthesized (Na+ + K+)-ATPase did not appear on the cell surface but accumulated intracellularly. When tunicamycin was used to inhibit glycosylation of the beta subunit, there was no effect upon subunit assembly, intracellular transport, or degradation rate (t1/2 = 40 h).     

Phosphorylated intermediate of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in endoplasmic reticulum from rat pancreatic acinar cells

Formation and decomposition of the phosphorylated intermediate of endoplasmic reticulum (Ca2+ + Mg2+)-ATPase from pancreatic acinar cells have been studied using lithium dodecyl sulfate- and tetradecyltrimethylammonium bromide-polyacrylamide gel electrophoresis. Incorporation of 32P from [gamma-32P]ATP is Ca2+-dependent (approximate Km for free [Ca2+] = 2-3 X 10(-8) mol/liter). Formation of the 100-kDa phosphoprotein is rapid, reaching maximal 32Pi incorporation within 1 s at room temperature. At 4 degrees C, phosphorylation is slower and dephosphorylation is drastically decreased. For dephosphorylation, Mg2+ and monovalent cations such as K+ or Na+ are necessary. Vanadate inhibits both 32P incorporation and 32P liberation dose dependently (Km = 3 X 10(-6) mol/liter), whereas mitochondrial inhibitors and ouabain have no effect. The phosphoprotein is stable at pH 2 and destabilizes with increasing pH being completely decomposed at pH 9. Reduction of 32P incorporation in the presence of high concentrations of cold ATP and hydroxylamine suggests formation of acylphosphate present in the ATPase intermediate. The characteristics of Ca2+, cation, and pH dependencies of the ATPase activity are similar to those previously described for MgATP-dependent Ca2+ transport into rough endoplasmic reticulum from pancreatic acinar cells (Bayerd?rffer, E., Streb, H., Eckhardt, L., Haase, W., and Schulz, I. (1984) J. Membr. Biol. 81, 69-82). The data suggest that the 100-kDa phosphoprotein as described in this study is the intermediate of this Ca2+ transport ATPase.     

Ca2+-dependent protein phosphorylation associated with microsomal fraction of rat pancreas

Microsomes isolated from cat pancreas were incubated with [gamma-32P]ATP in the presence or absence of Ca2+. Following fractionation of phosphoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single microsomal protein with an apparent molecular mass of 77,000 dalton (77K) was found to be phosphorylated in a Ca2+-dependent mechanism. Maximal phosphate incorporation into the 77K protein was observed at 10(-6) mol/l [Ca2+] and was 4-fold higher than in the absence of Ca2+. The 77K phosphoprotein showed characteristic of a stable phosphoester rather than an acyl phosphate. Measurable phosphate incorporation into the 77K protein was noted 5 s following addition of [gamma-32P]ATP and reached maximum at 9-10th min. The lack of effect of exogenous cyclic AMP, cyclic AMP-dependent protein kinase, calmodulin, the calmodulin antagonist trifluoperazine, leupeptin and the suppression of phosphorylation by some phospholipid-interacting drugs suggested that the 77K protein is a substrate for cyclic AMP- and calmodulin-independent, Ca2+-activated phospholipid-sensitive kinase activity. Centrifugation of the pancreatic homogenate in a ficoll-sucrose density gradient indicated that both the 77K protein and enzyme were associated in a fraction enriched in rough endoplasmic reticulum.     

Synthesis of Na+/K+ ATPase by the preimplantation rabbit blastocyst

The rates of incorporation of [35S]methionine into Na+/K+ ATPase, actin (beta- and gamma-isoforms), and total protein of the preimplantation rabbit blastocyst were determined between Days 4 and 7 of development. Blastocyst proteins were metabolically radiolabelled with [35S]methionine and subsequently analysed by co-isolation with purified Na+/K+ ATPase using two-dimensional polyacrylamide gel electrophoresis, immunoprecipitation, immunoblotting, fluorography, and liquid scintillation spectroscopy. The rate of [35S]methionine incorporation into acid-soluble total protein increased 24-fold between Days 4 and 6 post coitum (p.c.), then diminished approximately 79% on Day 7. In-vitro incorporation of [35S]methionine was linear at each stage of blastocyst development. [35S]methionine incorporation rates were unaffected by low free intracellular methionine concentration (less than 0.06 mM) and stage-related differences in blastocoele volume. Analysis of beta- and gamma-actin synthesis revealed patterns of [35S]methionine incorporation rates which were similar to those of total protein. In contrast, synthesis of blastocyst Na+/K+ ATPase was characterized by a 90-fold increase (P less than 0.001) in the rate of [35S]methionine incorporation between Days 4 and 6 p.c. The results demonstrate that Na+/K+ ATPase is actively synthesized at a high and increasing rate during preimplantation development in the rabbit at a period which is characterized by rapid fluid accumulation by the blastocyst.     

Studies on the specific activity of [gamma-32P]ATP in adipose and other tissue preparations incubated with medium containing [32P]phosphate

The specific activity of the gamma-32P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717-720). The HPLC method also allowed the incorporation of 32P into the (alpha + beta)-positions of ATP to be determined. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [gamma-32P]ATP attained a steady-state value after 1-2 h incubation in medium containing 0.2 mM [32P]phosphate. Addition of insulin or the beta-agonist isoprenaline increased this value by 5-10% within 15 min. Under these conditions the steady-state specific activity of [gamma-32P]ATP was 30-40% of the initial specific activity of the medium [32P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the gamma-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [32P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.     

Disulfide of thionosine triphosphate, an ATP-analog inactivating (Na+ + K+)-ATPase.

(Na+ + K+)-activated ATPase in beef brain microsomes is inactivated by the disulfide of thionosine tri[gamma-32P]phosphate, an ATP analog. The inactivation of the enzyme, which is accompanied by an incorporation of radioactivity into the membrane protein, is abolished by ATP or dithiothreitol. Since dithiothreitol restores the activity of (Na+ + K+)-ATPase, which had previously been inactivated by this ATP analog, it is concluded that thionosine triphosphate disulfide reacts with a sulfhydryl group in the ATP binding site of (Na+ + K+)-activated ATPase.     

Characterization of a 67-kDa S6 protein kinase from rat liver.

Fractionation of rat liver cytosol on DEAE-cellulose resolved two S6 kinases eluting at 25 mM KCl (peak I) and 100 mM KCl (peak II). The apparent molecular weights of the peak I and peak II kinases are 26,300 and 67,000, respectively. The peak II kinase was further purified and characterized. Incubation of the kinase with [gamma-32P] ATP and Mg2+ resulted in the incorporation of 32P predominantly into a 67-kDa band. Optimal activity of the kinase was observed in the presence of 5 mM Mg2+ and in the pH range of 8.0-8.5. The Km for ATP and 40S subunit were 7.3 microM and 1.5 microM, respectively. The Mg(2+)-stimulated kinase activity was inhibited by various divalent metals, NaF, and polyamines. The properties of the peak II S6 kinase are very similar or identical to the previously described mitogen-activated S6 protein kinase and may represent the nonactivated form of this enzyme.     

Photoaffinity labeling of the lumenal K+ site of the gastric (H+ + K+)-ATPase

A photoaffinity label for the lumenal K+ site of the gastric (H+ + K+)-ATPase has been identified. Seven azido derivatives based upon the reversible K+ site inhibitor SCH 28080 were studied, one of which, m-ATIP (8-(3-azidophenylmethoxy)-1,2,3-trimethylimidazo[1,2-a] pyridinium iodide), was subsequently synthesized in radiolabeled form. In the absence of UV irradiation, m-ATIP inhibited K+ -stimulated ATPase activity in lyophilized gastric vesicles competitively with respect to K+, with a Ki value of 2.4 microM at pH 7.0. Irradiation of lyophilized gastric vesicles at pH 7.0 with [14C]m-ATIP in the presence of 0.2 mM ATP resulted in a time-dependent inactivation of ATPase activity that was associated with an incorporation of radioactivity into a 100-kDa polypeptide representing the catalytic subunit of the (H+ + K+)-ATPase. Both inactivation and incorporation were blocked in the presence of 10 mM KCl but not with 10 mM NaCl, consistent with interaction at the K+ site. The level of incorporation required to produce complete inhibition of ATPase activity was 1.9 +/- 0.2 times the number of catalytic phosphorylation sites in the same preparation. Tryptic digestion of gastric vesicle membranes, labeled with [14C]m-ATIP, failed to release the radioactivity from the membranes suggesting that the site of interaction was close to or within the membrane-spanning sections of this ion pump.     

Improved purification and partial characterization of (Na+, K+)-ATPase from cardiac muscle.

A method is described for purification of (Na+, K+)-ATPase which yielded approximately 60 mg of enzyme from 800 g of cardiac muscle with specific activities ranging from 340 to 400 mumol inorganic phosphate/mg protein per h (units/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a major 94 000 dalton polypeptide and four or five lesser components, one of which was a glycoprotein with an apparent molecular weight of 58 000. The enzyme preparation bound 600-700 pmol of [3H]ouabain/mg protein when incubated in the presence of either Mg2+ plus Pi, or Mg2+ plus ATP plus Na+, and incorporated more than 600 pmol 32P/mg protein when incubated with gamma-32P-labelled ATP in the presence of Mg2+ and Na+. The preparation is approximately 35% pure.