Isolation of an angiotensin II-binding protein from liver.

Abstract

A protein that specifically binds angiotensin II has been isolated in nearly homogeneous form by two independent approaches after solubilization from rabbit liver particles by treatment with digitonin. The protein purified by either of these methods resembles in size the single radioactive macromolecular component made by using disuccinimidyl suberate to crosslink radioiodinated angiotensin II with its receptor in the solubilized extract. In the first technique, angiotensin II as an affinity ligand specifically extracted the protein from a preparation that had been freed of angiotensin-degrading activity. In the second approach, the angiotensin II-protein complex was specifically precipitated by anti-angiotensin II antibodies and staphylococcal protein A-Sepharose. The protein could be eluted from the affinity column with angiotensin II or 4 M MgCl2. The angiotensin TI-protein complex dissociated in the presence of sulfhydryl-containing reagents, and these could therefore be used to elute it from either the chemical or the immunoaffinity-based matrix. This effect of sulfhydryl-containing reagents and the paradoxical observation that the isolated protein after denaturation exhibited a slower electrophoretic mobility in its reduced form than in its unreduced form suggest that the binding configuration of this protein may be sensitive to reduction.Angiotensin II is the biologically active component of the renin-angiotensin system (1, 2). Several mechanisms may contribute to its role as an important vasopressor agent. Among these are a contractive effect on blood vessels (1), a stimulation of cells of the adrenal zona glomerulosa to elaborate aldosterone (3-6), and a direct action on the central nervous system (7). These and other effects of angiotensin II are thought to be mediated by specific receptors on the surface of its target cells (8). Such receptors have been detected and their binding properties have been characterized in particulate fractions from various cell types (reviewed in ref. 9). In addition, they have recently been solubilized in active form from bovine adrenal (10) and rabbit hepatic (11) membranes. However, despite the fact that it is thought to mediate directly and thus determine the specificity of action of angiotensin II, the responsible receptor is the only component of the renin-angiotensin system that has not been purified and characterized in the isolated state. It has therefore not yet been possible to address directly certain important questions such as whether or not a chemically identical receptor molecule is responsible for the action of angiotensin II on different cell types.As a result of the development of inhibitors of angiotensinconverting enzyme (12, 13), it has already become apparent that suppression of the renin-angiotensin system represents an important approach to antihypertensive therapy (reviewed in ref. 14). However, this enzyme is a dipeptidyl carboxypeptidase capable of acting on many peptide substrates in addition to angiotensin I (reviewed in ref. 15). Ther...

The ADP/ATP carrier is the 32-kilodalton receptor for an NH2-terminally myristylated src peptide but not for pp60src polypeptide.

“…On the basis of this evidence, we have used the MGYsrc peptide as a probe in membrane cross-linking studies to identify potential pp6Qsrc binding proteins. This experimental rationale is based on the ability to use radiolabelled small ligands as probes to identify membrane-bound receptors and has been successfully exploited by other investigators to identify the receptors for, e.g., gamma interferon, erythropoietin, angiotensin II, and parathyroid hormone (21,29,31,39).…”

Hydrodynamic properties of the angiotensin II receptor from bovine adrenal zona glomerulosa

“…An essential step towards comprehension of the mechanism of action of All is the elucidation of its receptor structure. The major drawback to the physical characterization and purification of the All receptor has been the difficulty in solubilizing the protein in a sufficiently stable and active form (Brecher et al, 1974;Devynck et al, 1974;Forget & Heisler, 1979;Capponi & Catt, 1980;Sen et al, 1984). Some groups of workers have been able to recover binding activity after solubilization, but their methods have not led to further purification or characterization of the receptor (Chang & Lotti, 1981;Capponi et al, 1983;Sen et al, 1983;Akiyama et al, 1984).…”

Review: Novel roles of intracrine angiotensin II and signalling mechanisms in kidney cells

“…Indeed, Ang II has been shown to bind to a high-affinity cytoplasmic Ang II binding protein in non-renal tissues, although the consequences of this interaction remain to be determined. 22,78,109,110 To determine whether intracellular or internalised Ang II plays any physiological role in proximal tubule cells, microinjection of Ang II directly into the cells may be one of the best approaches available, because it can differentiate the intracellular effects mediated by the microinjected Ang II from those mediated by extracellular Ang II via membrane receptors. Using this novel approach, we recently demonstrated that microinjection of Ang II into proximal tubule cells induced intracellular [Ca 2+ ] i responses and the responses were blocked by co-microinjection with the AT 1 -receptor agonist losartan or by pre-treatment of the cells with thapsigargin to deplete intracellular calcium stores or with U-73122 to inhibit phospholipase C. …”