Absence Of Potassium Ions Research Articles

An improved automated determination of Na +K +-activated adenosine triphosphatase

To increase our capacity to measure Na +K +-activated adenosine triphosphatase (NaK-ATPase) activity in large numbers of samples, we developed an automated ATPase assay. We utilized Technicon AA II technology and AA I components to measure inorganic phosphate colorimetrically. ATP was hydrolyzed by a membrane-rich fraction of rat intestinal mucosa in duplicate manifolds in the presence and absence of potassium ions. Samples were assayed at 60/hr with a high degree of precision and essentially no sample carryover. Absorbance was linear between 1 and 25 m m phosphate; and the amount of phosphate hydrolyzed by NaK-ATPase and Mg-ATPase was linear with protein from 40 to 220 μg/ml. Specific activities of these enzymes measured with this automated system reflected trends identical to those obtained with our manual ATPase assay. This automated assay has more than quadruple the output capacity of both our manual method and other automated systems, with an equivalent degree of specificity, accuracy, and precision.

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.

Changes in Ouabain-Sensitive Adenosine Triphosphatase Activity in Gills of Coho Salmon (Oncorhynchus kisutch) During Parr–Smolt Transformation

The effects of incubation temperature, pH, sodium, potassium, and ATP concentration, and ouabain on the activity of Na+–K+-activated ATPase of the gills of seawater-adapted juvenile coho salmon (Oncorhynchus kisutch) were determined. The temperature and pH optima were 40 C and 7.4, respectively. The apparent Km for ATP at equimolar Mg++ concentration was 0.2 mM at Na+ and K+ concentrations of 100 and 20 mM, respectively. Maximal enzyme activity for Na+ concentration of 10.50 and 100 mM occurred at K+ concentrations of 12.5, 15.0, and 20.0 mM, respectively. The Ki for ouabain was 2 × 10−6 M and 7 × 10−6 for K+ concentrations of 10 and 20 mM, respectively.A large portion (up to 60%) of the ouabain-sensitive ATPase activity in freshwater fish was activated by sodium ions in the absence of potassium ions (Na+-activation). Exposure to sea water resulted in a large increase in total ouabain-sensitive activity and a sharp decrease in the proportion of sodium activation. These changes occurred within 14 days after transfer to full strength sea water.On a seasonal basis, total ouabain-sensitive enzyme activity in juvenile freshwater coho was low (less than 5 μmol Pi/mg N per h) to the end of November, increased to a peak (over 125 μmol Pi/mg N per h) in mid-January, and subsequently declined by late February. A slow, steady rise in activity occurred during the smoking period of March and April and the relative contribution of sodium ions to the total activity declined in this period.

Potassium ion as an essential factor for binding of vitamin B 12 coenzyme to apopropanediol dehydratase

Holopropanediol dehydratase (EC 4.2.1.28) was resolved by gel filtration on a column of Sephadex G-25 in the absence of potassium ion, yielding the active apoenzyme almost quantitatively. Among irreversible cabamide inhibitors of propanediol dehydratase, cyano- and methylcobalamin were also dissociated from the corresponding inactive complexes with the apoprotein, resulting in the liberation of the active apoenzyme. Hydroxocobalamin-apoenzyme complex, however, was not resolved in this procedure. In both cases of the coenzyme and coenzyme analogs, no resolution was observed by the gel filtration in the presence of both the substrate and potassium ion. When the holoenzyme coexisted with potassium ion only, the resolution was found to proceed according to the kinetics of the first-order reaction. These experimental results indicate that potassium ion plays an essential role in the binding of the apoenzyme with coenzyme B 12 or its analog.

Effect of potassium ion, theophylline and propranolol on the biphasic lipolytic response to some catecholamines

The biphasic lipolytic dose-response curves to the levo-isomers of the catecholamines were investigated to determine what role cyclic AMP played in the mediation of these responses. As has been reported previously, the dose-response curve to l-epinephrine was biphasic. The first phase of the response curve has been shown to be equivalent to the lipolytic response reported many times in the literature and was, therefore, considered to be mediated by cyclic AMP. Removal of potassium ion from the incubation medium abolished the first phase of the response (Lipolysis I), but had no effect on the second phase (Lipolysis II). In the absence of potassium ion, lipolytic response to l-epinephrine was significantly augmented by the presence of sublipolytic concentrations of theophylline and significantly inhibited by the presence of the betaadrenergic blocking agent, propranolol. The lipolytic dose-response curve to the dextro-isomer of epinephrine was monophasic. Omission of potassium ion from the incubation medium had very little effect on the lipolytic response to this agent. The response to d-epinephrine closely resembled the Lipolysis II response to l-epinephrine. The lipolytic activity of d-epinephrine was significantly augmented by theophylline and inhibited by propranolol. These data are interpreted to suggest that the second phase of the biphasic dose-response curve to l-epinephrine (Lipolysis II) was mediated by cyclic AMP.

The sedimentation properties of the intestinal alpha-glucosidases of normal human subjects and of patients with sucrose intolerance.

The maltase‐isomaltase, maltase‐sucrase and glucoamylase of the human intestinal mucosa have been solubilized by Triton X‐100 and submitted to centrifugation in a mannitol gradient. Under these conditions, the three enzymes sediment together in a 10 S fraction; with one normal mucosa out of 10 which were investigated a supplementary 13 S peak of both glucoamylase and maltase‐isomaltase was observed.Several experimental conditions, like treatment of the extracts at 45° or solubilization of the α‐glucosidases by papain in the absence of potassium ions, induced an important inactivation of the maltase‐isomaltase and deep alterations in the sedimentation profile of the 3 glucosidases: the glucoamylase and the residual maltase‐isomaltase were distributed in 3 fractions with 13 S, 10 S and 6–7 S sedimentation coefficient, whereas the maltase‐sucrase was mostly present as a 7 S structure. In sucrose intolerance, the maltase‐sucrase is absent and the activities of the maltase‐isomaltase and glucoamylase are significantly reduced. The residual enzymes were ‐recovered as 13 S and 6 S structures instead of the normal 10 S species.These observations allow the conclusion that the three α‐glucosidases are naturally linked to each other in the normal mucosa and that the alteration of one of them induces a structural rearrangement of the 3 enzymes. The absence of maltase‐sucrase in patients with congenital intolerance to sucrose can be regarded as the result of a single mutation of the corresponding structural gene and as the cause of a reorganization of the two other α‐glucosidases; this reorganization can explain a partial loss of the enzymatic activity.

The Metabolism of Isolated Fat Cells: VIII. Amino Acid Transport in Ghosts

Abstract Ghosts of isolated fat cells take up α-aminoisobutyric acid (AIB) by a saturable and partially energy-dependent transport process. Of several amino acids tested, methionine and alanine were the most effective in inhibiting influx and stimulating efflux of AIB, indicating that the transport of these amino acids and AIB is shared by a common carrier-mediated system in the plasma membrane of ghosts. The uptake of AIB was stimulated by sodium ion, inhibited by ouabain, and diminished in the absence of potassium ion in the incubation medium. The initial rate of AIB influx was not correlated to the initial rate of potassium influx. The results suggest that the capacity of ghosts to accumulate and maintain a steady state content of AIB is a function of the potassium and sodium gradients across the plasma membrane but is not directly linked to active sodium-potassium transport. Sodium-potassium transport is probably the major energy-requiring process involved in the accumulation of AIB. Insulin, adrenocorticotropin, and epinephrine did not alter the rate of uptake or the steady state levels of AIB in preparations of ghosts that are sensitive, with respect to glucose transport or metabolism, to the effects of these hormones.

The ribosomes of the extremely halophilic bacterium, Halobacterium cutirubrum

The ribosomes of Halobacterium cutirubrum, a micro-organism that grows in saturated sodium chloride and probably has an internal concentration of potassium chloride near saturation, exist as 70 s particles when isolated in saturated potassium chloride containing 0·1 M -magnesium chloride at pH 7. These particles are stable in concentrations of magnesium ions as high as 0·4 M . If the potassium ion concentration is below about 4 M , or the magnesium ion concentration below 0·1 M , the ribosomes dissociate into 52 s and 31 s subunits. The requirement by the 70 s particles for potassium ions is specific; in high concentrations of ammonium or caesium ions, many of the ribosomes dissociate into subunits, while in high concentrations of sodium ions, they dissociate and aggregate in a complex way. About 53% of the RNA of the cell is accounted for by the ribosomes, which consist of 60% RNA and 40% protein. The estimated molecular weights of the ribosome and its subunits are in the ratio 3 : 2 : 1. At low concentrations of magnesium ions in the absence of potassium ions, the ribosomes lose low molecular weight RNA and up to 75% of their protein, to produce a labile 42 s component and more stable 33 s and 22 s particles, which contain about 80% RNA. It is suggested that the 42 s, 33 s and 22 s components, with molecular weights in the ratio 3 : 2 : 1, represent essentially the RNA moieties of the whole 70 s ribosome and of the 52 s and 31 s subunits respectively, together with some structural protein. Gel electrophoresis showed that the proteins which are lost are acidic. It is concluded that in these ribosomes, the stability of the ribonucleoprotein depends on potassium ions which shield negative groups, and on magnesium ions, which provide ionic cross-links between RNA and protein.