Otto Hansen

Active Na—K transport and the rate of ouabain binding. The effect of insulin and other stimuli on skeletal muscle and adipocytes

1. The effect of stimulation or inhibition of active Na—K transport on [3H]ouabain binding has been investigated in isolated soleus muscles and adipocytes.

A simple and rapid method for the determination of the number of 3H-ouabain binding sites in biopsies of skeletal muscle

Summary Based on vanadate facilitation of 3H-ouabain binding to Na-K-ATPase, a method has been developed allowing the measurement of the number of 3H-ouabain binding sites in small rat muscle biopsies (2–14 mg). Comparison with results obtained using intact fibres showed close agreement over the wide range of values for 3H-ouabain binding sites (100–900 pmol per g wet weight) associated with variations in age, thyroid status and K-deficiency.

Ouabain binding and Na+K+ transport in rat muscle cells and adipocytes

Abstract 1. 1. The accumulation and the release of [3H]ouabain has been characterized in isolated intact soleus muscles and free fat cells of the rat. The number of ouabain-binding sites was determined and compared with the rates of active Na+K+ transport. 2. 2. In soleus muscle [3H]ouabain is bound to the surface of the plasma membrane by a reversible and saturable process, which is inhibited by K+, Li+, 2,4-dinitrophenol, the omission of Na+ or the addition of digoxin. 3. 3. The release of [3H]ouabain from preloaded muscles is accelerated by K+, 2,4-dinitrophenol and unlabelled ouabain or digoxin, presumably becuase of diminished binding of the glycoside. 4. 4. Under steady-state conditions for ouabain binding a kinetic analysis indicates that soleus muscles contain 7.2 · 10−1 moles binding sites per g wet wt or 3350 per μm2 of sarcolemma surface area. An apparent dissociation constant of 2.1 · 10−7 M was found. 5. 5. Measurement of 22Na efflux and 42K influx under basal conditions indicate that the number of Na+ and K+ transported per ouabain-binding site correspond to respectively 500 and 325 per min. 6. 6. In isolated fat cells ouabain binding showed qualitatively the same characteristics as in soleus muscle, although the rate was considerably faster and the affinity higher (apparent dissociation constant: 1.7 · 10−8 M. 7. 7. The fat cells were estimated to contain 2.0 · 10−11 moles ouabain-binding sites per ml of cells or 66 per μm2 of plasma-membrane surface. The ouabain-sensitive component of 42K influx corresponds to 3450 ions per site per min. 8. 8. It is concluded that in intact muscle cells and adipocytes, which constitutes the major portion of total body weight in mammals, the binding of ouabain is qualitatively closely similar to that described in microsomal Na + + K + - activated ATPase.

The relationship between g-strophanthin-binding capacity and ATPase activity in plasma-membrane fragments from ox brain

Abstract 1. 1. Binding of labelled g-strophanthin to plasma-membrane fragments from ox brain has been studied using a filtration technique. ATPase-containing particles were separated from medium after incubation with 5 mM Mg2+, 2 mM EDTA, 10 mM Na+, 3 mM ATP, 60 mM Tris, pH 7.4 (37°), and varying [3H]g-strophanthin concentrations. 2. 2. g-Strophanthin binding was studied as a function of time and concentration The variation of the equilibrium level of bound g-strophanthin (EG) with the concentration of unbound g-strophanthin ( G ) fits the following simple model for the reaction E + G ⇄ k−1 k1 EG where E represents the available part of the enzyme system, i.e. E = EG max − EG . 3. 3. Addition of unlabelled g-strophanthin, K+ or EDTA when a steady-state level of g-strophanthin binding was established decreased the binding towards a new equilibrium indicating reversibility of the binding process. 4. 4. Based on the model presented, the binding capacity ( EG max ) was calculated for a series of enzyme preparations with varying activity. A linear relationship between g-strophanthin-binding capacity and ATPase activity was obtained indicating a constant catalytic centre activity per g-strophanthin binding site.

Non-uniform populations of g-strophanthin binding sites of (Na+ + K+)-activated ATPase Apparent conversion to uniformity by K+

Abstract 1. (1) [ 3 H]g-strophanthin (ouabain) binding to (Na + + K + )- activated ATPase from ox brain was studied as a function of the P i concentration with the (Mg 2+ +P i )-facilitated pathway. Scatchard-type plots of bound versus bound/free g-strophanthin at equilibrium of binding resulted in curved lines. The non-linearity was most easily seen at low P i concentration or after addition of the nucleotide analogue β,γ-methyl-eneadenosine triphosphate (ADPCP) or suramin at high P i concentration. 2. (2) The curved lines obtained with (Mg 2+ +P i )-supported g-strophanthin binding are converted to straight lines at a certain, not very high K + concentration. This effect seems specific for K + and is apparently not due to a delay of the time of equilibrium. 3. (3) The experiments are explained by assuming the existence of two (or more) populations of enzymes with different affinities for substrates and ligands affecting g-strophanthin binding. The sites with apparently high affinity for g-strophanthin may be less dependent on P i for g-strophanthin binding or they may have a higher affinity for P i . On the other hand, they are more affected by K + such that all enzyme sites exhibit homogeneity with respect to g-strophanthin affinity after addition of K + .

(Na+ + K+)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K+-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K+-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K+-dependent phosphatase activities in the range 0.19-0.37 mumol X (g wet weight)-1 X min-1 were obtained, the value decreasing with age and K+-deficiency. Complete inhibition of the K+-dependent phosphatase was obtained with 10(-3) M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K+-dependent phosphatase activity and (Na+ + K+)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min-1 was estimated from simultaneous determination of K+-dependent phosphatase activity and [3H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [3H]ouabain binding sites measured in intact muscles or biopsies hereof.

The effect of magnesium, ATP, Pi, and sodium on the inhibition of the (Na+ + K+)-activated enzyme system by g-strophanthin

Abstract The influence of magnesium, sodium, ATP, and P i on the reaction of the (Na + + K + )-activated enzyme system with g-strophanthin has been investigated. Magnesium is required for the reaction, but with magnesium alone the rate of reaction with g-strophanthin is very low. With magnesium, ATP has an effect both on the rate and on the steady-state level of inhibition. With ATP in concentrations which are low relative to the concentration of magnesium, it increases the rate of inhibition. With a non-limiting concentration of magnesium, the concentration of ATP for half-maximum inhibition by 1 μM g-strophanthin is about 1 μM. When the ATP concentration is increased above the magnesium concentration, i.e. with decreasing concentrations of uncomplexed magnesium (Mg 2+ ), both the rate and the steady-state level of inhibition is decreased. With magnesium, P i increases the rate of inhibition just as ATP, but the concentration of P i to obtain a certain inhibition by 1 μM g-strophanthin is higher than the concentration of ATP. The maximum rate of inhibition obtained with magnesium and P i is higher than with magnesium and ATP. P i in concentrations higher than the concentration of magnesium (3 mM with 2.5 mM magnesium and 2 mM EDTA) did not decrease the rate and the steady-state level of inhibition as did ATP. With ATP, sodium decreases the requirement for magnesium for the inhibition. With 1 μM g-strophanthin the concentration of Mg 2+ for 50 % inhibition and the MgATP/ATP i × K ratio is decreased by a factor of 10 3 –10 4 by an increase in the sodium concentration from 0 to 10 mM (Mg 2+ decreased from 4 μM to 0.7 nM). Sodium increases the rate of inhibition and with sub-optimal concentrations of magnesium it also increases the steady-state level of inhibition. The maximal rate of inhibition with magnesium, ATP, and sodium is faster than with magnesium and P i , and also than with magnesium and ATP; but even under optimal conditions for the reaction the rate of inhibition with 1 μM g-strophanthin is a relatively slow process. Without ATP and with or without P i , sodium has the opposite effect. It decreases the requirement for Mg 2+ for a given inhibition. With 1.5 mM P i , the concentration of Mg 2+ necessary for 50 % inhibition by 1 μM g-strophanthin is increased from 5 μM to 3 mM when the sodium concentration is increased from 0 to 50 mM. The concentration of g-strophanthin to give 50 % inhibition under steady-state conditions with 3 mM ATP and 100 mM sodium decreases when the Mg 2+ concentration is increased and goes towards a minimum value of 10 nM. With non-limiting concentrations of magnesium and P i for inhibition, the value was 11 nM and with magnesium, sodium and P i 440 nM. The results suggest that with ATP, sodium increases the affinity for Mg 2+ while with P i , sodium decreases the affinity for g-strophanthin.

Facilitation of ouabain binding to (Na+ + K+)-ATPase by vanadate at in vivo concentrations

In the presence of Mg2+ vanadate was shown to facilitate ouabain binding to (Na+ + K+)-ATPase in much the same way as Pi does. Thus the hypothesis that vanadate interacts with the phosphate site of the enzyme seems to be supported by ouabain binding experiments. At given ouabain concentrations maximum binding is achieved at microM concentrations of vanadate whereas mM concentrations of Pi are needed. Na+ as well as K+ counteract ouabain binding but some cardiac glycoside binding is still possible at in vivo concentrations of these cations. A minor contamination of the enzyme preparations with vanadate could explain the in vitro binding of ouabain that can be obtained with Mg2+ and in the absence of Pi.

Effect of age, potassium depletion and denervation on specific displaceable [3H]ouabain binding in rat skeletal muscle in vivo

1. Following intraperitoneal injection of [3H]ouabain in rats, the isotope is rapidly distributed in blood plasma available for binding to the Na‐K‐ATPase in the plasma membranes of most tissues. In skeletal muscle tissue excised and washed 4 × 30 min in ice‐cold buffer, 95% of the 3H activity retained was shown to be [3H]ouabain using a specific binding assay.

Antibodies to pig kidney (Na++K+)-ATPase inhibit the Na+ pump in human red cells provided they have access to the inner surface of the cell membrane

Antisera from rabbits that had been immunized with a highly active membrane preparation of (Na+ + K+)-ATPase from the outer medulla of pig kidney strongly inhibited (Na+ + K+)-ATPase activity in various tissues. When the antiserum was incorporated into released human red cell ghosts, the ouabain-sensitive efflux of Na+ into both 15 mM K+ and K+-free high Na+ media was completely abolished. This effect was not observed when non-immune serum was used, or when the immune serum was allowed access only to the outer surface of the red cell membranes.