Isolated Liver Plasma Membranes Research Articles

The effect of zinc on insulin metabolism.

Experiments were designed to study the effect of Zn on in vivo and in vitro insulin metabolism. The in vivo experiments involved pretreating mice with either Zn or Na, followed by ip [125I]iodoinsulin injection. Pretreatment of mice with Zn resulted in an accelerated and increased magnitude of binding of [125I]iodoinsulin to the liver compared to mice pretreated with Na. Results are submitted which support the probability that the changes in the amounts of intact and degraded insulin in circulation with time are related to the binding and degradation of insulin in the liver rather than in the kidney. In vivo ip injected insulin was demonstrated to preferentially bind to the plasma membrane of the liver. Liver plasma membranes isolated from mice pretreated with Zn bound more [125I]iodoinsulin than plasma membranes of Na-pretreated mice. In vitro experiments employing isolated liver plasma membranes demonstrated that added Zn increased the binding and inhibited the degradation of insulin. Evidence is presented that supports the concept that two receptors exist, one at which degradation of [125I]iodoinsulin occurs and another at which degradation does not occur.

Isolation of a sulfobromophthalein-binding protein from hepatocyte plasma membrane

This paper deals with the isolation and partial characterization of a protein capable of high affinity sulfobromophthalein-binding from liver plasma membrane. The purification involves acetone powder of a crude preparation of rat liver plasma membrane, salt extraction and purification through two chromatographic steps. Based on sulfobromophthalein binding, the process gives a yield of approximately 40%, with a purification of about 300 times with respect to the starting homogenate. The best preparation can bind more than 100 nmol sulfobromophthalein/mg protein. The protein behaves as a single species in dodecyl sulphate polyacrylamide gel electrophoresis, with an apparent molecular weight of 1.7 · 10 5. The molecule does not contain sugars. The dissociation constant of the protein · sulfobromophthalein complex has been found to be 4 · 10 −6 M, a value in agreement with that of high affinity binding sites described on isolated liver plasma membrane.

Effect of chlorpropamide and phenformin on rat liver: The effect on plasma membrane-bound enzymes and cyclic AMP content of hepatocytes in vitro

Chlorpropamide and phenformin inhibited (Na +K +)-ATPase and stimulated a high affinity cyclic AMP-phosphodiesterase of isolated liver plasma membrane when tested in vitro. In addition, the two drugs decreased the intracellular cyclic AMP content of isolated hepatocytes without being effective on plasma membrane-bound adenylate cyclase. The results suggest that the plasma membrane plays an important role in the mechanism of action of the two hypoglycemic drugs, but do not exclude the presence of intracellular targets.

Binding of [ 3H]cytochalasin B and [ 3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([ 3H]cytochalasin B and [ 3H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [ 3H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB 3H 4. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10 −6 M [ 3H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10 −5 M [ 3H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [ 3H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes. [ 3H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [ 3H]cytochalasin B binding and cytochalasin B stimulated 3H colchicine binding. [ 3H]Colchicine also bound to intracellular membranes, especially smooth microsomes.

Effects of growth hormone on ATPase and fluorescence of isolated liver membranes utilizing the fluorescent substrate, 1, N6-etheno-adenosine triphosphate

The influence of bovine growth hormone on Mg 2+-ATPase (EC 3.6.1.4) in isolated liver plasma membranes of hypophysectomized rats has been investigated in vitro by means of spectrofluorescence measurements in parallel with Mg 2+-ATPase assays, using 1, N 6-etheno-ATP as substrate and fluorescence probe. Bovine growth hormone, at concentrations of 10 −14 m and above, enhanced significantly Mg 2+-ATPase activity in the presence of GTP at concentrations from 10 −6 m to 10 −10 m. Moreover, bovine growth hormone decreased fluorescence intensity of membrane protein at the peak at 330 nm and of 1, N 6-etheno-ATP at its peak at 395 nm as well. The greatest decrease in fluorescence intensity of 1, N 6-etheno-ATP was observed in the presence of 5 m m MgCl 2 and 10 −8 m GTP, consistent with the stimulating effect of bovine growth hormone on Mg 2+-ATPase activity. In addition, bovine growth hormone caused a small decrease in fluorescence intensity of 1, N 6-etheno-ADP, but not the corresponding fluorescent analogs of AMP, cyclic AMP, and adenosine. The decrease in fluorescence intensity of 1, N 6-etheno-ATP by bovine growth hormone was completely eliminated by addition of ATP, ADP, and cyclic AMP at concentrations five times that of 1, N 6-etheno-ATP. Neither AMP nor adenosine exerted any effects. Bovine growth hormone also increased the fluorescence polarization of 1, N 6-etheno-ATP from 0.177 ± 0.006 to 0.212 ± 0.010 at 300 nm under the same conditions employed for the Mg 2+-ATPase assay. These observations suggest that bovine growth hormone produced changes in tertiary structure of membrane proteins in general and probably Mg 2+-ATPase in particular with consequent enhanced enzyme activity.

Modulation by cyclic AMP in vitro of liver plasma membrane (Na+--K+)-ATPase and protein kinases.

Abstract The effect of cyclic AMP on (Na+—K+)-ATPase and protein kinase activities of isolated liver plasma membrane has been investigated. Theophylline and caffeine, several cyclic nucleotides (including cyclic AMP, dibutyryl cyclic AMP, cyclic GMP and cyclic UMP), and sodium butyrate reduced the activity of (Na+—K+)-ATPase. The most powerful inhibitor was dibutyryl cyclic AMP; however, its action is probably due to the combined effects of cyclic AMP and butyrate. The blocking agent propranolol prevented the action of both epinephrine and cyclic AMP. Liver plasma membranes contain protein kinase(s), which catalyze the transfer of terminal phosphate of ATP into protein of plasma membrane itself (endogenous phosphorylation) as well as into added proteins (exogenous phosphorylation). Endogenous phosphorylation was inhibited by cyclic AMP, dibutyryl cyclic AMP and cyclic GMP. On the other hand, exogenous phosphorylation was stimulated by cyclic AMP. The specific activity of the membrane enzyme catalyzing the latter reaction was found to be higher than that of the enzyme present in the 100 000 × g supernatant of rat liver. The parallel effect of cyclic AMP on (Na+—K+)-ATPase and on the phosphorylation of membrane protein, and the finding that the latter reaction involves the formation of acylphosphate bonds, may be indicative of a possible role of endogenous phosphorylation in the regulation of (Na+—K+)-ATPase activity.

24] Method for solubilization of myocardial adenylate cyclase and assessment of the role of phospholipids in hormone stimulation

Publisher Summary Sutherland has postulated that adenylate cyclase may be a lipoprotein whose functional activity in vivo may be dependent on the presence of membrane lipid. In recent years several observations have been reported demonstrating the critical role of phospholipids in hormone- responsive adenylate cyclase systems. Solubilization of membrane-bound adenylate cyclase abolishes its ability to be activated by hormones. Treatment of thyroid slices 8 and isolated liver plasma membranes with phospholipases results in a loss of hormone responsiveness to thyroid-stimulating hormone and glucagon, respectively. This Chapter describes the preparation of soluble cat myocardial adenylate cyclase and the use of specific phospholipids in restoring hormone-activation of the soluble enzyme.

A study of the hormonal control in vitro of Na +-K + activated ATPase of isolated liver plasma membrane

The activity of rat liver plasma membrane-bound Na +-K + ATPase was modulated in vitro by epinephrine, glucagon and insulin through an action probably mediated by cyclic AMP. Epinephrine, glucagon and cyclic AMP inhibited the activity of this enzyme; insulin did not influence the baseline activity but prevented partially the effect of the former hormones. Rat liver plasma membranes contain protein kinase activity. In the absence of exogenous substrate, phosphorylation of membrane constituents occurred very rapidly and was inhibited by cyclic AMP, both in the presence and absence of Ca ++ ions. When an exogenous substrate (protamine) was added to the system, phosphorylation was linear with time, at least up to 15 min; the reaction was stimulated by cyclic AMP, and the effect of this compound was abolished when Ca ++ was added to the medium. Hydroxylamine removed about 35% of labeled phosphate incorporated—both in the presence and absence of cyclic AMP—in membrane preparations incubated in the absence of protamine, while it had no effect in its presence. These results suggest that, as a consequence of endogenous phosphorylation, part of labeled phosphate was incorporated in membranes through an acyl linkage. As Na +-K + ATPase catalyzes two main reaction steps, the first being represented by a phosphate group transfer from ATP to a free carboxylic radical belonging to enzyme protein, these results very probably indicate that the marked effect exerted by cyclic AMP on ATPase activity may be ascribed to the inhibition of the formation of such a linkage, due to the cyclic nucleotide itself. The second step of ATPase reaction was investigated by model reactions. The hydrolysis of p-nitrophenyl phosphate was not modified by cyclic AMP while that of acetyl phosphate was inhibited. As in the latter case, the reaction concerns the hydrolysis of an acyl phosphate group, it seems probable that also the second step of ATPase reaction is inhibited by cyclic AMP.

Action of autonomic nerve intermediates on liver plasma membrane

Summary In previous work it was shown that the infusion of acetylcholine or carbamylcholine produced, in the isolated, perfused rat liver, an enhanced deposition of glycogen, which was paralleled by an increased activity of glycogen synthetase a. The present results indicate that acetylcholine inhibited the activity of adenyl cyclase of isolated liver plasma membrane, when the enzyme was assayed at pH 9.2, and counteracted the activation of epinephrine. Atropine prevented this effect. However, when adenyl cyclase was tested at pH 7.5, acetylcholine treatment resulted in an increase of the activity. This was very probably due to release of bound epinephrine. In fact, acetylcholine reduced the activity of adenyl cyclase of membranes from reserpinized animals at every pH value of the incubation medium. The action of acetylcholine on the release of bound epinephrine has been studied in membranes pre-labeled with 3H-epinephrine. When such membranes were incubated at pH 7.5, a liberation of the hormone occurred, which was enhanced by acetylcholine. This effect was prevented by bretylium. In a last set of experiments, isolated liver plasma membranes were incubated in vitro in the presence of 14C-acetylcholine. When labeled membranes were solubilized by sonication in the presence of deoxycholate, and submitted to chromatography in a column of Sephadex G-200, the fraction containing bound acetylcholine was separated from two fractions containing adenyl cyclase activity and from another fraction containing bound epinephrine.