ATPase In Liver Plasma Membranes Research Articles

Stimulatory effect of hormonal signaling factors on (Ca(2+)-Mg2+)-ATPase activity in rat liver plasma membranes: cross talk with regucalcin.

The effect of hormonal signaling factors on (Ca(2+)-Mg2+)-ATPase activity in rat liver plasma membranes was investigated. The presence of inositol-glycan (10(-7)-10(-5) M), dibutyryl cAMP (10(-4) and 10(-3) M) or inositol 1,4,5-trisphosphate (IP3; 10(-6) and 10(-5) M) in the enzyme reaction mixture produced a significant increase in (Ca(2+)-Mg2+)-ATPase activity. These effects were completely inhibited by the presence of vanadate (10(-4) M), an inhibitor of the enzyme phosphorylation, and N-ethylmaleimide (5 x 10(-3) M), a SH group modifying reagent. Meanwhile, regucalcin, a Ca(2+)-binding protein isolated from rat liver cytosol, increased the enzyme activity by binding to the SH groups of (Ca(2+)-Mg2+)-ATPase in liver plasma membranes. The presence of regucalcin (0.25 microM) with an effective concentration completely inhibited the effect of inositol-glycan (10(-5) M) to increase (Ca(2+)-Mg2+)-ATPase activity, while the effect of dibutyryl cAMP (10(-3) M) or IP3 (10(-5) M) was not altered. The inositol-glycan effect was not modulated by the presence of dibutyryl cAMP or IP3. Now, the preincubation of the plasma membranes with regucalcin did not modify the effect of inositol-glycan on the enzyme activity, suggesting that regucalcin competes with inositol-glycan for the binding to the plasma membranes. The present results suggest that there may be a cross talk with regucalcin and hormonal signaling factors in the regulation of (Ca(2+)-Mg2+)-ATPase activity in liver plasma membranes.

The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles.

The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.

Effects of alcohol on alpha-adrenergic receptor regulation of calcium ATPase in liver plasma membranes

The effects of α 1- and α 2-adrenergic agonists, viz., phenylephrine and clonidine, respectively, were studied on rat liver plasma membrane Ca ++-ATPase. Phenylephrine produced a 23% inhibition of enzyme activity at 5 μM. Prazosin, an alpha 1 antagonist, completely prevented the effect of phenylephrine. Clonidine produced a comparable inhibition of Ca ++-ATPase, but was not reversed by the antagonist yohimbine, suggesting a lack of functionally significant α 2 receptors as previously reported. The results support the role of high-affinity Ca ++-ATPase in liver plasma membranes in the control of cytosolic free Ca ++ levels through regulation by α 1-adrenergic receptors. In vitro and acute ethanol exposure produced inhibition of plasma membrane Ca ++-ATPase. In addition, ethanol treatment significantly reversed the inhibitory effect of phenylephrine on Ca ++-ATPase. Chronic ethanol exposure for four weeks increased Ca ++-ATPase activity over control and increased enzyme activity in the presence of phenylephrine. These results demonstrate that ethanol alters the α-adrenergic receptor interaction with Ca ++-ATPase resulting in reduced receptor regulation of cytosolic Ca ++ levels. These changes may prevent the liver from maintaining Ca ++ levels for second messenger functions, such as glycolysis and gluconeogenesis.

Thallium replaces potassium in activation of the (Na+,K+)-ATPase of rat liver plasma membranes.

The capacity of thallium to substitute for K+ in activation of (Na+,K+)-ATPase of liver plasma membranes was studied. Our results indicate that T1+ can replace K+ in the activation of the (Na+,K+)-ATPase of liver plasma membranes. In the presence of Na+, similar activation is obtained with T1+ concentrations only 1/10 of those of K+. In all other aspects, the (Na+,K+)- and (Na+,T1+)-ATPases were found to be identical.

Characterization of the ouabain-insensitive ATPase activity of rat liver plasma membranes.

Very little is currently known about the ouabain-insensitive ATPase activity of the liver plasma membrane; we have therefore characterized it in plasma membranes from rat liver prepared using two different isolation techniques. Greater than 85% of ATPase activity in both preparations was ouabain-insensitive. Based on the effects of multiple inhibitors, including dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide (NEM), vanadate, and oligomycin, the ouabain-insensitive ATPase activity of rat liver plasma membranes consists of a family of ATP-hydrolysing enzymes. Ouabain-insensitive ATPase activity was stimulated by HCO3-in both plasma membranes (13%) and mitochondria (69%) over the range of 7.5 to 9.0, and HCO3--stimulation was similarly inhibited by oligomycin in both preparations. A fraction of the ouabain-insensitive ATPase of liver plasma membranes is inhibited by DCCD and is resistent to inhibition by oligomycin; these characteristics are similar to those of the non-mitochondrial H+-ATPases recently described in lysosomes, endosomes, clathrin-coated vesicles, and Golgi from liver and other cell types.

Properties of Na +, K +-ATPase of liver plasma membranes in the hamster

This study was designed to establish the properties of liver plasma membranes (LPM) Na+,K+-ATPase in the hamster and to determine whether a similar assay may be used to measure enzyme activity in the hamster and in the rat. Maximal Na+,K+-ATPase activity was obtained when the assay medium contained 5 mM Mg APT2- with or without 1 mM free Mg2+, 120 mM Na+, 12,5 mM K+. The incubation must be performed at 37 degrees C, pH 7.4. In the absence of free Mg2+, the saturation curve with respect to the substrate Mg ATP2- resulted in biphasic complex kinetics with a maximal activity at a substrate concentration of 5 mM. In the presence of 1 mM free Mg2+ activation of Na+,K+-ATPase and modification of the kinetics were observed: the biphasic curve tended to disappear and to become of the Michaelis-Menten type. The apparent Km for Mg APT2- was 0.36 mM and the Vmax 34.5 mumol.h-1.mg protein-1. In the presence of 10 mM free Mg2+ a decrease in the Vmax was observed without any effect on the apparent Km for Mg APT2-. It is concluded that the same incubation medium may be used to assay LPM N+,K+-ATPase from hamster and rat and that the addition of 1 mM free Mg2+ to the incubation medium is recommended to obtain Michaelis-Menten kinetics in order to eliminate complex kinetics due to the absence of free Mg2+.

Hormone and prostaglandin E 1 control of potassium and cyclic AMP levels in isolated rat liver cells

The effect of hormones and prostaglandins on the uptake of potassium and on the level of cyclic AMP has been investigated in rat hepatocytes isolated by Seglen's modification of the procedure of Berry and Friend. Hepatocytes contained about 100 μEq K +/g; the concentration fell by about 34% when the cells were kept at 4°C, and rose to the initial value after incubation at 37°C. This uptake of potassium was significantly reduced by epinephrine and glucagon; simultaneously, the level of cyclic AMP was enhanced. Insulin prevented both hormonal effects. Monobutyryl-cyclic AMP mimicked the action of epinephrine and glucagon on potassium uptake. Prostaglandin E 1, which has been recently found to enhance the activity of liver adenylate cyclase, produced a rise of the level of cyclic AMP; in addition, it reduced the uptake of potassium and stimulated glycogenolysis. Prostaglandins E 2, F 1α and F 2α were much less effective. These data suggest that increased levels of cyclic AMP result in a reduced uptake of potassium and confirm previous findings about a cyclic AMP mediated inhibition of (Na +-K +)ATPase of liver plasma membranes.