Neurotensin receptors on the rat liver plasma membranes

Neurotensin (NT) is now classified as a brain-gut peptide in the central nervous system and gastrointestinal tract. In the present study, we characterized the NT receptors on the rat liver plasma membranes. The specific binding of [3H]NT was time dependent, reversible, and saturable. Scatchard analysis of the specific binding data yielded two classes of binding sites, a high affinity site and a low affinity site. The average maximum number of binding sites (Bmax) amounted to 13.3 +/- 1.1 fmol/mg protein at high affinity site and 122.3 +/- 21.5 fmol/mg protein at low affinity site, respectively. The dissociation constant (Kd) had values of 0.39 +/- 0.01 nM at high affinity site and 8.1 +/- 1.1 nM at low affinity site, respectively. The amount of specifically bound [3H]NT was significantly reduced in the presence of mono and divalent cations, EDTA, EGTA and a peptidase inhibitor bacitracin, NT1-13 competed with [3H]NT for its binding site with an IC50 of 0.19 nM at high affinity site (0.2 nM concentration of [3H]NT) and 0.7 nM at low affinity site (4.0 nM concentration of [3H]NT). Xenopsin, a NT analogue separated from the skin of Xenopus laevis, was equipotent (IC50 0.75 nM) with NT1-13 at 4.0 nM concentration of [3H]NT. C-terminal sequence of NT contains the structure necessary for interaction with NT binding sites whereas N-terminal sequence had no binding activity. Since NT has a hyperglysemic and a hypercholesterolemic effects in rats, these NT receptors on the rat liver plasma membranes may be involved in the hyperglycemia and/or hypercholesteroremia induced by NT.     

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4

Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near‐native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4‐M23) was expressed in the X. laevis oocytes following their injection with AQP4‐M23 cRNA. AQP4‐M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4‐M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over‐expressed AQP4‐M23, the membranes from AQP4‐M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher‐order arrays of AQP4‐M23. In addition, but only infrequently, AQP4‐M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.     

Solubilization of ligand-stabilized vasopressin receptors from plasma membranes of bovine kidney and rat liver

The solubilization of vasopressin receptors from plasma membranes of bovine kidney and rat liver by different detergents was investigated. A prerequisite for the extraction of vasopressin receptors retaining binding affinity for their ligand was the stabilization of the receptors by the prior formation of the membrane-bound hormone-receptor complexes. The vasopressin-receptor complexes from both kidney and liver membranes were solubilized in a high yield with dodecyl-beta-D-maltoside and 3-laurylamido-N,N'-dimethylpropylaminoxide. Several other nonionic detergents including octyl-beta-D-glucopyranoside effectively extracted the hepatic vasopressin receptor. For the hormone-receptor complex solubilized from bovine kidney with dodecyl-beta-D-maltoside, a Stokes' radius of 5.8 nm was determined.     

Preparation of enriched plasma membranes from bovine gallbladder muscularis for characterization of cholecystokinin receptors

A method for the preparation of enriched plasma membranes from bovine gallbladder muscularis was developed, validated, and applied to the characterization of receptors for the gastrointestinal hormone cholecystokinin (CCK) on this target. Binding of radioiodinated CCK ligands to this preparation was rapid, reversible, temperature-dependent, saturable, and specific. Only structurally related peptides inhibited CCK binding, and good correlation existed between relative potencies for binding inhibition and for stimulating gallbladder contraction. Computer analysis of CCK-binding data using a nonlinear model-fitting program best fit a model with a single class of sites, with Kd 756 pm and binding capacity 4.5 +/- 1.3 pmol/mg of protein. This degree of enrichment for plasma membranes was adequate for the initial biochemical characterization of this CCK receptor. Affinity labeling using 125I-Bolton Hunter-CCK-33 and m-maleimidobenzoyl-N-hydroxysuccinimide ester identified proteins with Mr = 70,000-85,000, Mr = 120,000-125,000, and Mr = 200,000. Labeling was inhibited in a concentration-dependent manner, with an IC50 of 1 nM CCK-8, and the electrophoretic mobility of these bands was not different under reducing and nonreducing conditions. The major labeled band of Mr = 70,000-85,000 has a lower apparent Mr than that of the analogous band in pancreas labeled with similar methods, supporting the molecular heterogeneity of CCK receptors on these two target tissues.     

EGF receptors on plasma membranes purified from bovine mammary gland of lactating and pregnant animals

A procedure for purification of plasma membranes from bovine mammary gland has been developed. The binding capacity of EGF to the plasma membranes from mammary tissue of pregnant cows was equal to 335 fmol per mg of protein thus being twofold higher than in membranes from lactating gland. The KD values were not changed. Autoradiographs of the membrane receptor linked to [125J]-EGF revealed three labeled bands corresponding to 160 kDa, 145 kDa and 115 kDa polypeptides. The main band of 145 kDa was labeled stronger in membranes from pregnant animals. The results suggest that the EGF receptor level is enhanced in fast proliferating normal mammary tissue.     

Interfacial properties of model membranes and plasma lipoproteins containing ether lipids

The interfacial properties of synthetic ester and ether phosphatidylcholines (PCs) were investigated by using the polarity-sensitive fluorescent probes 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and pyrene. The physical state of the phospholipid matrix was determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Single-bilayer phospholipid vesicles formed by sonication and model high-density lipoproteins were studied. On the basis of a number of spectroscopic and thermodynamic criteria, the interfacial regions of PCs and their ether analogues are similar. The fluorescence properties of Prodan in model lipoproteins or single-bilayer vesicles were independent of the phospholipid fatty acyl chain length and polar head group, as well as the substitution of ether linkage for ester bonds in the phospholipid. The spectral shifts correlated mainly with the physical state of the phospholipid. The emission spectrum of Prodan appeared at shorter wavelengths upon transfer from water to liquid-crystalline phospholipid and blue shifted further when the lipid was cooled to its gel phase. The effect of cholesterol in model high-density lipoproteins on the emission spectrum of Prodan was dose dependent and, at 18 mol % cholesterol, the spectrum was similar to that observed in a pure gel-phase lipid and was independent of temperature. The quantum yield of Prodan fluorescence in an ether-PC matrix was similar to that observed in water, whereas in an ester-PC matrix it was enhanced by a factor of about 5. Phospholipid-water partition coefficients of Prodan were independent of the physical state of 1,2-dimyristoyl-sn-glycero-3-phosphocholine or 1,2-tetradecyl-sn-glycero-3-phosphocholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Existence of glucocorticoid-specific receptors in the plasma membranes of rat liver

It was found that isolated plasma membranes whose purity was assayed by determinations of marker enzyme activities, specifically bind dexamethasone. The association constant and the number of binding sites were found to be equal to (7,03 +/- 4,05) . 10(9) M-1 and (1,6 +/- 0,18) . 10(-14) mol/mg protein, respectively. It was assumed that lipoprotein components of plasma membranes are involved in this binding.     

Covalent labeling of neurotensin receptors in rat gastric fundus plasma membranes

Neurotensin receptors from plasma membranes of rat gastric fundus smooth muscle were specifically and covalently labeled either by using the photoreactive analogue ¹²⁵I-labeled azidobenzoyl (Trp¹¹)-neurotensin or by cross-linking (monoiodo-Tyr³)neurotensin to the membrane preparation by means of disuccinimidyl suberate. Analysis of plasma membranes by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the same protein band with an apparent molecular weight of 110,000 was specifically labeled by both methods. This band consisted of a single chain protein since its apparent size was found to be the same with or without reduction of membrane samples before electrophoresis. Only neurotensin and its biologically active analogues were able to protect plasma membranes against specific labeling of the protein band of molecular weight 110,000. Comparison of these results with those obtained from rat brain synaptic membranes shows that although rat central and peripheral neurotensin receptors exhibit similar specificities towards a series of neurotensin analogues, their subunit structures are different.     

Binding to specific receptors on oocyte plasma membranes by serum phosvitin-lipovitellin

Specific binding sites for the serum complex of phosvitin and lipovitellin have been shown to exist on the outer surface of rapidly growing chicken oocytes. The existence and specificity of these sites were demonstrated by competition for binding to unfixed oocyte membrane fragments and by displacement of already bound and labeled phosvitin-lipovitellin from formaldehyde-fixed membranes. Only unlabeled phosvitin-lipovitellin competed with the ¹²⁵I-labeled complex for binding to the fragments or displacement of bound label; IgG isolated from egg yolks and bovine serum albumin were ineffective.     

Glycoproteins from ulva lactuca

Ulva lactuca yielded glycoprotein materials which differed significantly in their carbohydrate and protein moieties. The electrophoretic patterns of the isolated materials showed the presence in each of glycoprotein and polysaccharide components. All the glycoprotein components moved towards the cathode.The glycoprotein material isolated by extraction with NaOH was fractionated on a DEAE-cellulose column into four glycoprotein components and one protein component. All the glycoprotein components contained glucuronic acid, xylose, and rhamnose in different proportions, and in addition two of them contained glucose.     

Isolation of plasma membranes from Xenopus embryos

Summary Plasma membranes were isolated in high yield from Xenopus gastrulae by repeated sedimentation in discontinuous sucrose gradients. Most of the yolk was separated by lowspeed sedimentation before centrifugation on the discontinuous sucrose gradients. The isolation of plasma membranes was followed by covalent labelling of the surface of dissociated gastrula cells with diazoniobenzene sulphonate, by electron microscopy and the distribution of enzymatic markers. The isolated plasma membranes have a low neural inducing activity as compared to other cell constituents.     

H+-adenosine triphosphatases from plasma membranes

New data are presented on the organization of H+-pumps in plasma membranes of cells of bacteria fungi, plants and animals. It is shown that H+-ATPase of bacteria differs in principle from H+-ATPases of plasma membranes of other organisms. The transport H+, K+-ATPase functioning in cells of mucous membrane of the animal stomach as an electroneutral H+-pump is similar by its properties to Na+, K+-ATPase of plasma membranes of animal cells. H+-ATPase of plasma membranes in cells of fungi and higher plants which functions as an electrogenic H+-pump differs essentially from H+-ATPases of F0 X F1-type. Distribution of H+-ATPases in cells of different organisms and their evolution are under discussion.     

Study of insulin receptors on liver and hepatoma cell plasma membranes]

Plasma membranes (PM) of helathy rats as well as those hepatomas and PM of tumor-bearing rats were found (radioreceptor assay) to have apparently two groups of receptors for insulin with a high and low affinity for the hormone respectively and different insulin-binding capacities. Kass values of the receptors with a high affinity for insulin are drastically decreased in the PM of ascites Zajdela hepatoma (AZH) and of solid hepatoma 27 (SH-27) as well as in the liver of SH-27-bearing animals, but not in the liver of the AZH-bearing rats. Kass values of the receptors with a low affinity for insulin in the PM of AZH and in those of the liver of AZH-bearing rats appear nearly normal. In contrary, above Kass the receptors in the PM of SH-27 and SH-27-bearing animals are significantly decreased. The insulin-binding capacity of the receptors with a high affinity for the hormone in the PM of SH-27, AZH and the liver of both tumor-bearing rats is shown to be significantly higher than that in the PM of normal animals. The same property of the receptors with a low is affinity in the PM of the hepatomas and theliver of their hosts is also increased, especially in the PM of SH-27 and of the liver of SH-27-carring rats.     

Characterization of transferrin receptors on plasma membranes of lactating rat mammary tissue

Little is known about the transport of iron into the mammary secretory cell and the process of milk iron secretion. The concentration of iron in milk is remarkably unaffected by maternal iron status, suggesting that the uptake of iron into the mammary gland is regulated. It is known that iron enters other cells via transferrin receptor-mediated endocytosis. This study was designed to isolate and characterize the mammary gland transferrin receptor in lactating rat mammary tissue using immunochemical techniques. The existence of functional mammary gland transferrin receptors in lactating rodents was demonstrated using radiolabel-binding techniques. Isolation of mammary transferrin receptors by affinity chromatography was confirmed using immunoelectrophoresis and slot blot analysis. The intact transferrin receptor was found to have a molecular weight of 176 kd as determined by Western blotting followed by scanning densitometry. Reduction of the receptor with beta-mercaptoethanol gave a molecular weight of 98 kd. An additional immunoreactive band of 135 kd was observed. The presence of transferrin receptors in normal lactating rat mammary tissue is likely to explain iron transport into mammary tissue for both cellular metabolism and milk iron secretion.     

Diacylglycerol kinase in plasma membranes from wheat

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg²⁺ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl₂. The Mg²⁺ requirement could be substituted only partially by Mn²⁺ and not at all by Ca²⁺. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.