Jørgen Jensen

Binding of ATP to brain microsomal ATPase Determination of the ATP-binding capacity and the dissociation constant of the enzyme-ATP complex as a function of K+ concentration

Abstract 1. 1. The binding of the ATP molecule to brain microsomal (Na+ + K+)-activated ATPase was studied by a rapid dialysis rate technique. 2. 2. The experiments were performed at 2° in the presence of 10 mM EDTA to minimize hydrolysis of ATP. The ionic strength was 0.073 M, pH 7.4. 3. 3. The specific activity of the preparations and the Na+ + K+ activity/Mg2+ activity ratio were changed by heat denaturation. 4. 4. The dissociation constant of the enzyme-ATP complex ( E- ATP ) was 0.12 μM. 5. 5. The Mg2+ requirement for binding of ATP to the enzyme, if any, was much lower than that for hydrolysis of ATP. The binding seemed to be independent of Na+. 6. 6. Increasing the concentration of K+ up to 3 mM led to an increase in the apparent dissociation constant of E- ATP towards a maximum (0.7 μM). The effect of K+ could be described by a model involving the formation of the following complexes: E- ATP , K + -E and K + -E- ATP . 7. 7. Proportionality was found between the ATP-binding capacity (nmoles/mg protein) and (Na+ + K+)-ATPase activity (μmoles P1 released per mg protein per h), suggesting that binding to (Na+ + K+)-ATPase was measured. The catalytic center activity based on this assumption was 7000 ± 470 min −1 .

Scatchard plot: Common misinterpretation of binding experiments

Abstract The widespread misinterpretation in the literature of ligand-protein binding experiments which show upward curvature in Scatchard plots is emphasized. The most commonly encountered errors are discussed and references to the correct methods of resolution of upward-curved Scatchard plots are given.

On the specificity of the ATP-binding site of (Na+ + K+)-activated ATPase from brain microsomes

Abstract In order to obtain information on the specificity of the ATP-binding site of ATPase from brain microsomes, the influence of pyrophosphate, adenine, adenosine, AMP, ADP, CTP, GTP, and ITP on ATP binding was investigated by a rapid dialysis rate technique previously described. 1. 1. Pyrophosphate, adenine, adenosine and AMP in concentrations up to about 3 orders of magnitude higher than that of free ATP did not affect the binding of ATP. 2. 2. Less ATP was bound in the presence than in the absence of ADP. Based on a model for competitive binding the affinity of the ATP-binding site for ADP was found to be 5 times smaller than for ATP. 3. 3. CTP, GTP and ITP were only able to displace ATP from the ATP-binding site in concentrations very high relative to that of free ATP. The affinity for these triphosphates was at least 250–850 times lower than for ATP, CTP having the highest affinity of the three compounds examined. 4. 4. The fact that the nucleotide specificity of the ATP-binding site is similar to the substrate specificity of (Na + + K + )-activated ATPase strongly suggests that this binding site is identical with the substrate site of (Na + + K + )-activated ATPase. 5. 5. It is concluded that the 6-amino group of the purine and pyrimidine ring as well as the β-phosphate group are essential for binding to the substrate site of (Na + + K + )-activated ATPase, and that binding of ATP also involves a link between the γ-phosphate group and the enzyme.

A new proposal regarding the subunit composition of (Na+ + K+)ATPase.

THREE types of high affinity ligands are available for studies with (Na+ + K+)ATPase: nucleotide substrates, vanadate and ouabain. Of these, the last is unique in apparently being absolutely specific for (Na+ + K+)ATPase. One of the generally accepted facts regarding (Na+ + K+)ATPase is that there are equal numbers of measurable (high affinity) binding sites for ATP and ouabain per molecule of enzyme1–5. There is a majority opinion that direct binding experiments yield binding capacities for these ligands of 1 mol site per 250–300 kg protein in the purest preparations available3,6–8. From these data, from determinations of the peptide composition and from estimates of the apparent molecular weight of solubilised enzyme the idea of (Na+ + K+)ATPase as a dimer (with respect to the large, so-called catalytic, peptide) has emerged9–14. Such a dimer would have one high affinity binding site for nucleotide and one for ouabain. The present report on binding site determinations on mammalian kidney microsomes before and after detergent treatment casts serious doubts on the general validity of the above simple relationship between sites and peptide composition.

An outbreak of Pontiac fever among children following use of a whirlpool

We investigated an outbreak of fever, most likely due to a contaminated whirlpool, among nine adults and six children residing in a summerhouse. The outbreak was characterized by a high attack rate, short incubation periods, influenza-like symptoms, and rapid recoveries, all features typical of Pontiac fever. However, the children had less-characteristic symptoms than the adults, and they did not have any sequelae. Findings on the childrens chest radiographs were unremarkable, and none of the children had leukocytosis. Evidence of Legionella pneumophila infection was found in six cases: in one case by isolation of L. pneumophila serogroup 1 and detection of legionellae by PCR, and in five cases by seroconversion to the clinical isolate. Six additional cases had presumptive evidence of legionella infection, with seroconversion to Legionella micdadei antigen; a PCR assay was also positive for legionellae for one of these cases. In contrast, two adult nonusers of the whirlpool had no symptoms and no serological evidence of infection. Serological testing and cultures for other pathogens, as well as cultures of all environmental samples, were negative. This investigation demonstrates the differences between adults and children with respect to the clinical picture of Pontiac fever; furthermore, it shows that culture and PCR assay of tracheal aspirates for legionellae can be performed in a hospital setting for rapid diagnosis, although the sensitivities of these methods are low.

Binding of ATP to Na+, K+-ATPase.

During the last years the reaction mechanism of Na, K+-ATPase has been the subject of thorough investigation in many laboratories, and a number of models for ATP-hydrolysis and its relation to Na+ and Kt transport have been proposed. The reaction schemes for Na+, K+-ATPase are based partly on kinetic studies of the overall rate of ATP-hydrolysis as influenced by a variety of activating ligands (e.g., Na, K+, Mg2+) or substances which inhibit the process, and partly on studies of possible part-reactions of the overall reaction (e.g., phosphorylation, ATP-ADP exchange, K*-dependent phosphatase activity). Observations on the physicochemical properties of the enzyme, coming from studies on highly purified preparations, have also contributed to the elucidation of Na+, K+-ATPase structure and function. In 1971 the first equilibrium studies on what may be the reaction of the enzyme with its substrate ATP were reported independently from two laboratories. p2 It is the purpose of this paper to review and discuss the studies on this reaction and to give some newly obtained results in this area. Special emphasis will be laid on the relation between the ATP-binding capacity and the hydrolytic activity of various enzyme-preparations. In this connection problems concerning the determination of the turnover number (molar activity) of Na, K+-ATPase will be discussed.

ATP binding to solubilized (Na+ + K+)-ATPase: The abolition of subunit-subunit interaction and the maximum weight of the nucleotide-binding unit

Membrane-bound (Na+ + K+)-ATPase from pig kidney outer medulla shows apparent heterogeneity in its ATP-binding site population when assays are carried out in the presence of K+. This finding has been interpreted as being due to interaction between (at least) two subunits, each containing an ATP-binding site. Treating the membrane-bound enzyme with the detergent, C12E8, has been shown to solubilize enzymatically active alpha beta-protomers. We show that in the dissolved enzyme all ATP-binding sites in the population are identical both in the absence and in the presence of K+, which would be consistent with an abolition of identical both in the absence and in the presence of K+, which would be consistent with an abolition of subunit-subunit interaction. This supports previous suggestions that enzyme solubilized by C12E8 is monomeric and that the membrane-bound enzyme is not. Differential extraction of enzyme-containing membranes with C12E8 yielded preparations with an ATP-binding capacity of up to 5.8 nmol per mg protein, measured by the method of Lowry et al. (Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275), with bovine serum albumin as standard. Evidence is presented that makes it likely that preparations with an ATP-binding capacity of 7.5 nmol per mg protein (as determined by the above-mentioned assay) will be obtainable. This corresponds to an alpha beta-protomer molecular weight of 133 000 which approximates closely to the minimum value found in the literature for an alpha beta-protomer (i.e., 126 000).

The K+-induced apparent heterogeneity of high-affinity nucleotide-binding sites in (Na+ + K+)-ATPase can only be due to the oligomeric structure of the enzyme

K+ induces an apparent heterogeneity among an otherwise homogeneous population of nucleotide-binding sites in (Na+ + K+)-ATPase preparations from pig kidney. With the help of ouabain we show that this heterogeneity cannot be due to a mixture of different and independent sites and conclude that each enzyme molecule must contain two nucleotide site-containing units that show interaction. Na+ induces an apparent heterogeneity among an otherwise homogeneous population of ouabain-binding sites. The argument is, therefore, extended to include one ouabain site on each of the structural units that bind nucleotide. All these structural units are shown to hydrolyse substrate at identical rates. Using the presently available molecular weight data, it is concluded that the enzyme is composed of two subunits each possessing one nucleotide-binding site, one ouabain-binding site, one alpha-peptide and the capacity for hydrolysing ATP and p-nitrophenyl phosphate.

Chlamydia pneumoniae in Children with Otitis Media

In this study the polymerase chain reaction was used to test for the presence of Chlamydia pneumoniae DNA in 118 middle-ear aspirates from 20 children with acute otitis media (AOM) and 53 children with otitis media with effusion (OME). C. pneumoniae was detected in 8 samples obtained from 5 children with OME and, together with Streptococcus pneumoniae, in a sample from 1 child with AOM. The mean age of these five children (6.6 +/- 1.4 years) was significantly higher than that of the 48 children with OME in whom C. pneumoniae could not be detected (4.3 +/- 1.9 years). The presence of C. pneumoniae in 9.4% of the examined children with OME suggests that C. pneumoniae might be a significant supplementary factor in the etiology of this common childrens disease.

The kinetics of the in vitro cholesterol uptake at the endothelial cell surface of the rabbit aorta.

Abstract The kinetics of [4- 14 C]cholesterol uptake by intima-media layers of thoracic aortas from normal rabbits have been studied by use of an in vitro method. 1. 1.The uptake of labelled cholesterol was followed during a 4-h period. The uptake curve shows an initial steep rise (estimated from the 15-min uptake value) followed by a step which has a less steep, obviously rectilinear course. 2. 2.The initial uptake of labelled cholesterol was found not to depend significantly on temperature (4–38°). The initial uptake is probably due to a superficial fixation of labelled cholesterol. 3. 3.The rate of [ 14 C]cholesterol uptake after the initial 15 min is, on the contrary, temperature dependent, presumably due to two processes having different temperature dependences. One of the hypothetical processes is assumed to have a temperature optimum at 20°; the other has an “activation energy” of about 18 kcal/mole. Judging from the temperature dependence, the [ 14 C]cholesterole uptake corresponding to the second phase of the uptake is thought to represent transport of cholesterol across the endothelial cell membrane. No causal explanation of the course of the uptake-temperature curve in the interval about 20° can be given at the present. 4. 4.The transfer rate of cholesterol from serum to intima-media is of the same order of magnitude as that found in vivo . 5. 5.The relationship between the rate of cholesterol transfer from serum to intima-media and total cholesterol concentration within the normal range (27 to 107 mg per 100 ml serum) is similar to the relationship between the velocity of an enzymatic reaction, showing substrate saturation phenomenon, and substrate concentration. In the model the initial uptake corresponds to the formation of enzyme substrate complex. Other interpretations of the relationship between cholesterol transfer rate and cholesterol concentration are discussed. 6. 6.Finally, the similarity of the kinetics found in this study with those for protein uptake by Ehrlich ascites-tumor cells and Amoeba proteus , assumed to be due to pinocytosis, is emphasized.