Tolerability during double-blind randomized phase I trials with the house dust mite allergy immunotherapy tablet in adults and children.

BACKGROUND AND OBJECTIVE: The orodispersible house dust mite (HDM) sublingual immunotherapy (SLIT)-tablet (ALK, Denmark) is being developed for the treatment of HDM respiratory allergic disease. The objective of the 2 phase I trials was to investigate tolerability and the acceptable dose range of HDM SLIT-tablet treatment in adults and children with HDM respiratory allergic disease. PATIENTS AND METHODS: The trials were randomized, multiple-dose, dose-escalation, double-blind, placebo-controlled phase I trials including patients with HDM-induced asthma, with or without rhinoconjunctivitis. Both trials were registered in EudraCT (Trial 1: 2005-002151-41; Trial 2: 2007-000402-67). Trial 1 included 71 adults (18-63 years) and trial 2 included 72 children (5-14 years). Both trials included 6 dose groups that were randomized 3:1 to active treatment or placebo once daily for 28 days. Adverse events (AEs) were coded in MedDRA (version 8.1 or later). Immunological variables included specific IgE and IgE-blocking factor. RESULTS: No serious AEs were reported. In trial 1 (maximum dose, 32 development units [DU]), 1 patient in the 16 DU group discontinued due to AEs. The entire 32 DU group was discontinued as 1 patient had a severe adverse reaction. In trial 2 (maximum dose, 12 DU), no patients discontinued prematurely. The most frequently reported AEs were mild application-site related events. The total number of events was dose-related within each trial. HDM SLIT-tablet treatment induced changes in immunological parameters in a dose-dependent manner. CONCLUSIONS: These trials demonstrate that doses up to 12 DU of HDM SLIT-tablet were tolerated in the selected populations, and thus are suitable for further clinical investigations in adults and children with HDM respiratory allergic disease.

Clinical evolution of patients with respiratory allergic disease due to sensitisation to Alternaria alternata being treated with subcutaneous immunotherapy.

INTRODUCTION: Sensitisation to Alternaria is a cause of respiratory disease in Spain, particularly in childhood, but it is also a significant marker of the severity of this disease. Therefore, the use of an aetiological treatment (allergen specific immunotherapy) is essential, and both subjective and objective clinical parameters should be used to follow up this treatment. OBJECTIVE: This open-label, uncontrolled, observational, prospective study was designed in order to study the evolution of these patients on allergen specific immunotherapy therapy in daily clinical practice and to assess the use of different monitoring tools. MATERIAL AND METHODS: A total of 99 patients were included. They were monosensitised to this perennial allergen and treated with subcutaneous allergen specific immunotherapy. After one year of follow-up, these patients were assessed for the presence of symptoms, use of medication, clinical incidents, quality of life and asthma control. RESULTS: After one year of treatment a significant fall was observed in the use of concomitant medication (ß2-agonists: p=0.0278, inhaled corticosteroids: p=0.0007, anti-leukotrienes: p=0.0495), nasal symptoms (p=0.0081), quality of life (PAQLQ, p<0.0001) and asthma control (ACQ, p<0.0001). Twenty-one patients had to attend emergency department due to exacerbation of their allergic disease, and only one of them had to be admitted to hospital. CONCLUSION: respiratory allergic disease due to Alternaria alternata is a disease which is hard to control, and in our daily practice, the use of specific subcutaneous immunotherapy can be of significant benefit in our paediatric patients.

Localization of intracellular compartments that exchange Na,K-ATPase molecules with the plasma membrane in a hormone-dependent manner.

BACKGROUND AND PURPOSE: Dopamine is a major regulator of sodium reabsorption in proximal tubule epithelia. By binding to D1-receptors, dopamine induces endocytosis of plasma membrane Na,K-ATPase, resulting in a reduced capacity of the cells to transport sodium, thus contributing to natriuresis. We have previously demonstrated several aspects of the molecular mechanism by which dopamine induces Na,K-ATPase endocytosis; however, the location of intracellular compartments containing Na,K-ATPase molecules has not been identified. EXPERIMENTAL APPROACH: In this study, we used different approaches to determine the localization of Na,K-ATPase-containing intracellular compartments. By expression of fluorescent-tagged Na,K-ATPase molecules in opossum kidney cells, a cell culture model of proximal tubule epithelia, we used fluorescence microscopy to determine cellular distribution of the fluorescent molecules and the effects of dopamine on this distribution. By labelling cell surface Na,K-ATPase molecules from the cell exterior with either biotin or an epitope-tagged antibody, we determined the localization of the tagged Na,K-ATPase molecules after endocytosis induced by dopamine. KEY RESULTS: In cells expressing fluorescent-tagged Na,K-ATPase molecules, there were intracellular compartments containing Na,K-ATPase molecules. These compartments were in very close proximity to the plasma membrane. Upon treatment of the cells with dopamine, the fluorescence labelling of these compartments was increased. The labelling of these compartments was also observed when the endocytosis of biotin- or antibody-tagged plasma membrane Na,K-ATPase molecules was induced by dopamine. CONCLUSIONS AND IMPLICATIONS: The intracellular compartments containing Na,K-ATPase molecules are located just underneath the plasma membrane.

Insulin-induced stimulation of Na+,K(+)-ATPase activity in kidney proximal tubule cells depends on phosphorylation of the alpha-subunit at Tyr-10.

Phosphorylation of the alpha-subunit of Na+,K(+)-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K(+)-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K(+)-ATPase alpha-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the alpha-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat alpha-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive (86)Rb uptake in opossum kidney cells expressing mutant rat alpha1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive (86)Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K(+)-ATPase alpha-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Can customized implants correct enophthalmos and delayed diplopia in post-traumatic orbital deformities? A volumetric analysis.

The purpose of this study was to determine whether orbital reconstruction with customized implants can correct post-traumatic orbital deformities such as late enophthalmos and delayed diplopia. The hypothesis proposed was that an overcorrection of the orbital volume is needed to resolve enophthalmos. A retrospective observational descriptive study was conducted. Patients with a major trauma who required customized orbital implants for the delayed treatment of unilateral orbital fractures that had initially been operated on using titanium mesh and/or osteosynthesis plates were included. The orbital volumes of the unaffected contralateral side, of the affected orbit after initial reconstruction with mesh, and of the affected orbit subsequently reconstructed with the customized implant were calculated. All of the patients included in this study had diplopia in the gaze position prior to the installation of the implant. In addition, they all had severe enophthalmos. After surgery, no patient with a customized implant showed diplopia. The enophthalmos was corrected in all but one case. On average, orbits reconstructed with customized implants had lower volumes compared to the unaffected contralateral side. In cases where the enophthalmos was resolved, the volume was reduced by an average of 8.55%. Further studies using a larger number of cases and with controlled volumetric corrections using CAD/CAM are needed.

Inhibition of (Na+,K+)-ATPase by magnesium ions and inorganic phosphate and release of these ligands in the cycles of ATP hydrolysis.

The kinetic data of magnesium and inorganic phosphate inhibition of the (Na+,K+)-dependent ATP hydrolysis are consistent with a model where both ligands act independently and their release in the ATPase cycle is an ordered process where inorganic phosphate is released first. The effects of magnesium on the stimulation of the ATPase activity by Na+, K+ and ATP, and the inhibition of that activity by inorganic phosphate, are consistent with Mg2+ acting both as a 'product' and as a dead-end inhibitor. The dead-end Mg-enzyme complex would be produced with an enzyme form located downstream in the reaction sequence from the point where Mg2+ acts as a 'product' inhibitor. In the absence of K+, Mg2+ inhibition was reduced when either Na+ or ATP concentrations were increased well beyond those concentrations needed to saturate their high-affinity sites. This ATP effect suggests that the dead-end Mg-enzyme complex formation is affected by the speed of the E2-E1 conformational change. The present model is consistent with the formation of an Mg-phosphoenzyme complex insensitive to K+ which could become K+-sensitive in the presence of high Na+ concentrations. These Mg-enzyme complexes appear as intermediaries in the Na+-ATPase activity found in the absence of external Na+ and K+. These results can be interpreted on the basis of Mg2+ binding to a single site in the enzyme molecule. In addition, these experiments provide kinetic evidence indicating that the stimulation by external Na+ of the ATPase activity in the absence of K+ is due to a K+-like action of Na+ on the external K+ sites.

Predictive factors in infraorbital sensitivity disturbances following zygomaticomaxillary fractures.

The aim of this study was to define if the alterations in sensory modalities could be a predictive factor in the prognostic recovery of the ION. Ten patients that had suffered facial trauma, associated with sensitivity alterations of the ION were evaluated prospectively. Touch detection thresholds (TD) were measured using Von Frey's filaments aesthesiometer. A warm/cold discrimination (W/C) was also done to the patients, on the same areas. The patients were examined in both sides of the face, using the non-traumatized side as control. The tests were done before surgery and several times postoperatively. For statistical analysis of the results, the two-sample t test was used. A significant difference (P < 0.0001) in the mean tactile recovery time between the areas without thermal sensitivity before surgery and those with normal thermal sensitivity before surgery was observed. Therefore, we propose that during the preoperative examination, the surgeon examines the thermal discrimination in order to establish prognosis and approximate recovery times.

PKC-beta and PKC-zeta mediate opposing effects on proximal tubule Na+,K+-ATPase activity.

Dopamine (DA) inhibits rodent proximal tubule Na+,K+-ATPase via stimulation of protein kinase C (PKC). However, direct stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) results in increased Na+,K+-ATPase. LY333531, a specific inhibitor of the PKC-beta isoform, prevents PMA-dependent activation of Na+,K+-ATPase, but has no effect on DA inhibition of this activity. A similar result was obtained with a PKC-beta inhibitor peptide. Concentrations of staurosporine, that inhibits PKC-zeta, prevent DA-dependent inhibition of Na+,K+-ATPase and a similar effect was obtained with a PKC-zeta inhibitor peptide. Thus, PMA-dependent stimulation of Na+,K+-ATPase is mediated by activation of PKC-beta, whereas inhibition by DA requires activation of PKC-zeta.

Inactivation of the Na,K-ATPase by modification of Lys-501 with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS).

The sodium pump or Na,K-ATPase, maintains the Na+ and K+ gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K-ATPase with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) inhibited the ATPase activity. Both the labeling of the protein and the loss of ATPase activity were prevented by co-incubation with ADP (acting as an ATP analog) or KCl. Only the alpha-subunit was labeled by SITS. The alpha-subunit from the inhibited enzyme was extensively digested with trypsin, and SITS-labeled peptides were purified by reverse-phase HPLC and sequenced. The amino acid sequence determined, His-Leu-Leu-Val-Met-X-Gly-Ala-Pro-Glu, indicated that SITS modifies Lys-501 (X) on the alpha-subunit of Na,K-ATPase.

Evaluation of paediatric tolerance to an extract of Alternaria alternata under two treatment regimes. A multicentre study.

In order to evaluate the efficacy and safety of an extract of Alternaria alternata in a paediatric population, a two phase study plan has been elaborated that in the first place consists of a retrospective analysis of tolerance under the standard treatment regimes used by the clinical groups involved. This was achieved by analysing the records of 94 patients that have been treated with this extract, these being consecutive patients included at 7 clinics over a period of 6 months. Two regimes were used: a conventional short regime of 7 doses and a cluster regime. Under neither of these two regimes were any serious reactions registered. The percentage of local reactions was significantly greater using the short conventional regime than with the cluster regime (1.9% and 0.4% respectively, p = .035). In contrast, no significant differences were observed with respect to the systemic reactions (0.5% and 1.2%), these percentages also being similar to those registered with other extracts in which identical regimes have been used. In conclusion, we can confirm that a very satisfactory tolerance profile was observed, with the advantage that through using shorter regimes than the conventional regime of 13 doses, a considerable saving is made both in the number of visits and the doses necessary to reach the maintenance dose.

Kinetic mechanism of inhibition of the Na+-pump and some of its partial reactions by external Na+ (Na+o).

The effects of external Na+ on the activity of the Na+-pump are complex. The first-order rate constant for Na+-efflux is reduced in the presence of very low external Na+ concentrations, and this inhibition is reversed when the Na+ level is raised. The same pattern has been observed for Na+-ATPase activity; however, it is not apparent from the current reaction mechanisms at which site (or sites) external Na+ binds to cause inhibition. In this paper, the effect of external Na+ on Na+-pump activity was studied by simulation, using a model similar to the Post-Albers scheme. Curves similar to those experimentally observed were obtained assuming that: (i) after phosphorylation, three Na+ ions are translocated and consecutively released to the external medium with decreasing dissociation constants; (ii) external Na+, with low affinity, binds to the K+o (external) sites stimulating dephosphorylation. These assumptions also permit one to explain the experimental observation that external Na+ (with both high and low affinities) competes with K+, inhibiting the K+ influx due to the Na+-pump, and the kinetically similar behavior of Na+-ATPase and ATP/ADP exchange reactions at low variable Na+ concentrations. The experimental evidence available that supports the present hypothesis is discussed.

Inhibition of Na(+)-pump expression by impairment of protein glycosylation is independent of the reduced sodium entry into the cell.

Previous studies indicate that inhibition of protein N-glycosylation reduces Na(+)-pump activity. Since this effect is preceded by an inhibition of the entry of sodium into the cell, it is unclear whether the reduced Na(+)-pump is produced by the inactivation of protein glycosylation per se or by the lower intracellular sodium concentration. We compared the effects of tunicamycin, which inhibits protein glycosylation, and amiloride, which inhibits the entry of sodium into the cell, on the expression of the Na(+)-pump activity in A6 cells. The short-circuit current across A6 epithelia, which corresponds to sodium ions transported through the Na+ channel and the Na(+)-pump, was almost totally inhibited after 24-hr treatment with 1 microgram/ml tunicamycin. The maximal Na(+)-pump activity, measured after permeabilizing the apical cell membrane with amphotericin B, was only 30% inhibited. This inhibition increased to 80% after 72-hr treatment with tunicamycin. Thus, tunicamycin inhibits the activities of both the apical Na+ channel and the basolateral Na(+)-pump. However, the reduced number of Na(+)-pump molecules, as well as the inhibition of the Na(+)-pump activity, were not observed when the Na+ channel was inhibited for 72-hr with amiloride. Thus, the reduced Na(+)-pump expression produced by inactivation of protein glycosylation is not secondary to reduced entry of sodium into the cell.

Chemical modification as an approach to elucidation of sodium pump structure-function relations.

Chemical modification of specific residues in enzymes, with the characterization of the type of inhibition and properties of the modified activity, is an established approach in structure-function studies of proteins. This strategy has become more productive in recent years with the advances made in obtaining primary sequence information from gene-cloning technologies. This article discusses the application of chemical modification procedures to the study of the Na(+)-K(+)-ATPase protein. A wide array of information has become available about the kinetics, enzyme structure, and various conformational states as a result of the combined use of inhibitors, ligands, modifiers, and proteolytic enzymes. We will review a variety of reagents and approaches that have been employed to arrive at structure-function correlates and discuss critically the limits and ambiguities in the type of information obtained from these methodologies. Chemical modification of the Na(+)-pump protein has already provided a body of data and will, we anticipate, guide the efforts of mutagenesis studies in the future when suitable expression systems become available.

Inhibition and derivatization of the renal Na,K-ATPase by dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate.

Treatment of purified renal Na,K-ATPase with dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) produces both reversible and irreversible inhibition of the enzyme activity. The reversible inhibition is unaffected by the presence of saturating concentrations of the sodium pump ligands Na+,K+, Mg2+, and ATP, while the inactivation is prevented by either ATP or K+. The kinetics of protection against inactivation indicate that K+ binds to two sites on the enzyme with very different affinities. Na+ ions with high affinity facilitate the inactivation by H2DIDS and prevent the protective effect of K+ ions. The H2DIDS-inactivated enzyme no longer exhibits a high-affinity nucleotide binding site, and the covalent binding of fluorescein isothiocyanate is also greatly reduced, but phosphorylation by Pi is unaffected. The kinetics of inactivation by H2DIDS were first order with respect to time and H2DIDS concentration. The enzyme is completely inactivated by the covalent binding of one H2DIDS molecule at pH 9 per enzyme phosphorylation site, or two H2DIDS molecules at pH 7.2. H2DIDS binds exclusively to the alpha-subunit of the Na,K-ATPase, locking the enzyme in an E2-like conformation. The profile of radioactivity, following trypsinolysis and SDS-PAGE, showed H2DIDS attachment to a 52-kDa fragment which also contains the ATP binding site. These results suggest that H2DIDS treatment modifies a specific conformationally sensitive amino acid residue on the alpha-subunit of the Na,K-ATPase, resulting in the loss of nucleotide binding and enzymatic activity.

Carbodiimide inactivation of Na,K-ATPase, via intramolecular cross-link formation, is due to inhibition of phosphorylation.

We have recently shown that inactivation of renal Na,K-ATPase by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide occurs via an intramolecular cross-link formed between an activated carboxyl group and an endogenous nucleophile (Pedemonte, C.H., and Kaplan, J.H. (1986) J. Biol. Chem. 261, 3632-3639). The modified enzyme shows the same level of Rb+ binding as untreated enzyme: 3.16 and 2.93 ATP-sensitive mumol of Rb+ binding/mumol of phosphoenzyme, respectively. Thus, the Rb+ binding site and the transition accomplished by low affinity nucleotide binding which accelerates de-occlusion are not greatly affected by the carbodiimide inactivation. 1 mM K+ reduces the ADP binding to the high affinity nucleotide binding site to the same extent in normal and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-treated enzyme and Na+ counteracts this effect. Thus, the competition between Na+ and K+ ions for binding to the free enzyme are also largely unaltered by the modification. Phosphorylation from ATP (microM) in the presence of Na+ and Mg2+ ions and from inorganic phosphate in the presence of Mg2+ ions (in the absence or presence of ouabain) is greatly inhibited (85%) following carbodiimide treatment. The extent of inhibition of phosphorylation quantitatively correlates with the residual Na,K-ATPase activity (15%). Consequently, the rate of inactivation by carbodiimide is reduced when a greater proportion of the enzyme is in the phosphorylated form. Fluoroscein isothiocyanate, which inhibits the Na,K-ATPase by covalently modifying a lysine residue close to the high affinity binding site for ATP in the alpha-subunit does not bind to the carbodiimide-inactivated enzyme. Since high affinity nucleotide binding is only partially inhibited by the modification produced by the carbodiimide this suggests that the lysine residue to which fluoroscein isothiocyanate binds is not specifically required for competent nucleotide binding.

Carbodiimide inactivation of Na,K-ATPase. A consequence of internal cross-linking and not carboxyl group modification.

Irreversible inhibition of Na,K-ATPase and K+-dependent p-nitrophenylphosphatase activities was produced by incubation of purified Na,K-ATPase enzyme with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EPC). Inhibition was time and [EPC] dependent and displayed first order kinetics with respect to time. The [EPC] to reduce the enzyme velocity by 50% for Na,K-ATPase and phosphatase activities was 1.6 and 2.2 mM, respectively. Analysis of the kinetics of inhibition by EPC indicated that reaction at one site was sufficient to produce inhibition. Inhibition was greatly reduced by the presence of Mg2+, Na+, K+, choline, or Tris (decreasing order of effectiveness); ATP was without effect. This suggests that cation-bound enzyme forms were less reactive with the carbodiimide than free enzyme; ATP-bound enzyme was as reactive. Apparently the cations Na+, Mg2+, Tris, and choline stabilize E1 forms of the enzyme which are different from the E1 form stabilized by ATP. Addition of [14C]glycine ethyl ester (Gly-OEt) resulted in incorporation of radioactivity into both alpha and beta subunits that was dependent upon the presence of EPC, and the incorporation was reduced by the cations which reduced the inhibition due to EPC. Simultaneous addition of Gly-OEt with EPC prevented inhibition, although 14C incorporation still took place. If Gly-OEt addition was delayed the initial inactivation was not affected, but little subsequent inactivation occurred. The protection against inactivation by EPC occurs on the addition of other exogenous nucleophiles, such as aminoethane or ethylenediamine. Dicyclohexylcarbodiimide, a more potent hydrophobic carbodiimide inhibitor, shows similar effects; the inhibition due to dicyclohexylcarbodiimide is also prevented by the simultaneous presence of a nucleophile. After treatment with a carbodiimide and exogenous nucleophile the Na,K-ATPase has modified carboxyl residues but is not inhibited. Thus, modification of the cation-protectable carboxyl groups does not by itself cause inhibition. It seems likely that the inhibition of activity due to carbodiimide alone is not due to the modification of a carboxyl group per se but to the formation of an intramolecular bond between the carbodiimide-activated carboxylic acid and an endogenous nucleophile. The formation of such bonds suggests the close juxtaposition of amine and carboxyl groups in the secondary structure of the enzyme.

Effects of ATP and monovalent cations on Mg2+ inhibition of (Na,K)-ATPase.

The hydrolysis of ATP catalyzed by purified (Na,K)-ATPase from pig kidney was more sensitive to Mg2+ inhibition when measured in the presence of saturating Na+ and K+ concentrations [(Na,K)-ATPase] than in the presence of Na+ alone, either at saturating [(Na,Na)-ATPase] or limiting [(Na,0)-ATPase] Na+ concentrations. This was observed at two extreme concentrations of ATP (3 mM where the low-affinity site is involved and 3 microM where only the catalytic site is relevant), although Mg2+ inhibition was higher at low ATP concentration. In the case of (Na,Na)-ATPase activity, inhibition was barely observed even at 10 mM free Mg2+ when ATP was 3 mM. When (Na,K)-ATPase activity was measured at different fixed K+ concentrations the apparent Ki for Mg2+ inhibition was lower at higher monovalent cation concentration. When K+ was replaced by its congeners (Rb+, NH+4, Li+), Mg2+ inhibition was more pronounced in those cases in which the dephosphorylating cation forms a tighter enzyme-cation complex after dephosphorylation. This effect was independent of the ATP concentration, although inhibition was more marked at lower ATP for all the dephosphorylating cations. The K0.5 for ATP activation at its low-affinity site, when measured in the presence of different dephosphorylating cations, increased following the sequence Rb+ greater than K+ greater than NH+4 greater than Li+ greater than none. The K0.5 values were lower with 0.05 mM than with 10 mM free Mg2+ but the order was not modified. The trypsin inactivation pattern of (Na,K)-ATPase indicated that Mg2+ kept the enzyme in an E1 state. Addition of K+ changed the inactivation into that observed with the E2 enzyme form. On the other hand, K+ kept the enzyme in an E2 state and addition of Mg2+ changed it to an E1 form. The K0.5 for KCl-induced E1-to-E2 transformation (observed by trypsin inactivation profile) in the presence of 3 mM MgCl2 was about 0.9 mM. These results concur with two mechanisms for free Mg2+ inhibition of (Na,K)-ATPase: "product" and dead-end. The first would result from Mg2+ interaction with the enzyme in the E2(K) occluded state whereas the second would be brought about by a Mg2+-enzyme complex with the enzyme in an E1 state.

A monosaccharide is bound to the sodium pump alpha-subunit.

We have recently reported that the Na pump alpha-subunit has cytosolic-oriented oligosaccharides which were sensitive to cleavage by an enzyme specific for hydrolysis of N-linked glycans [Pedemonte et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9789-9793]. We now describe experiments that characterize the saccharides and further substantiate our previous findings. Bovine milk galactosyltransferase has been used in conjunction with radiolabeled UDP-galactose to label N-acetylglucosamine residues on the protein. The Na pump alpha-subunit contains some O-linked carbohydrates; however, the bulk (> 80%) of the radioactivity was found in oligosaccharides sensitive to peptide:N-glycosidase F degradation but not to alkaline hydrolysis. Alkaline hydrolysis produced degradation of the protein, and the [3H]Gal radiolabeled carbohydrates remained bound to peptides and were released by subsequent peptide N-glycosidase F treatment. The exogenously galactosylated sugars cleaved by the glycosidase were analyzed by liquid chromatography and had elution volumes identical to a galactose-N-acetylglucosamine disaccharide standard. Since the galactose was exogenously added, we propose that the N-linked glycans on the alpha-subunit of the Na pump are composed of a single sugar residue, which is probably N-acetylglucosamine.

Ca2+-stimulated ATPase: inactivation by Ca2+ and mechanism.

The preincubation of the rat blood cell membranes in the presence of low Ca2+ levels causes an irreversible inhibition of the Ca2+-stimulated ATPase activity. The inactivation is dependent on the Ca2+ concentration and the apparent Ki is identical to the Ca2+ concentration needed to reach the half-maximal activity of the enzyme. This fact and the energy of activation (Ea = 13.8 K cal/mol) for the inhibition suggest that Ca2+ inactivates the Ca2+-stimulated ATPase by binding to the same site which it normally occupies to activate the enzyme. It is concluded that the Ca2+-stimulated ATPase is in a dynamic equilibrium between two states: a stable ATP-bound state and an unstable ATP-free state.

Kinetic studies and characteristics of a modifier of Ca2+-stimulated ATPase.

1. The thermostable modifier increases the Mg2+-stimulated ATPase with a parallel decrease of Ca2+-stimulated ATPase. 2. The modifier inhibited Ca2+-stimulated ATPase by diminishing the velocity without any significant effect on the apparent affinity of binding of Ca2+. 3. The inhibition of Ca2+-stimulated ATPase was independent of Ca2+ concentration and could not be reversed by increasing Ca2+ levels. 4. Monovalent cations and calmodulin increased the Mg2+-stimulated ATPase in the presence of the modifier.