Purification and characterization of angiotensin‐binding protein from porcine liver cytosolic fraction

Abstract

A protein that binds angiotensins with high affinity was found in porcine liver cytosol, purified to apparent homogeneity and characterized. The protein was named soluble angiotensin-binding protein (sABP) to distinguish it from angiotensin I1 receptors present on plasma membranes. Purification of the protein was achieved by a combination of ammonium sulfate fractionation, hydrophobic chromatography, ion-exchange chromatography, hydroxylapatite column chromatography and Mono Q ion-exchange chromatography. Specific angiotensinbinding activity, as measured using '251-angiotensin 11, was enriched more than 3400-fold. SDS/polyacrylamide gel electrophoresis of the purified sABP yielded a single 75-kDa protein band, in good agreement with the molecular mass estimated by affinity labeling. sABP was very similar to the angiotensin I1 receptor in its sensitivity to reducing agents and in its affinities for angiotensin analogues ([Sar', Ala*]angiotensin I1 > angiotensin I11 > angiotensin I1 > angiotensin I), suggesting a possible similarity between the ligand-binding sites of sABP and the angiotensin I1 receptor. To obtain a clue to its physiological role(s), we examined the tissue distribution of sABP and found that this protein is widely distributed not only in the peripheral organs but also in the brain.Angiotensin 11, an octapeptide, is the biologically active component of the renin-angiotensin system, with a broad range of physiological activities involved in the maintenance of blood pressure and fluid homeostasis [l, 21. For example, it acts on vascular smooth muscle and the adrenal glands to induce vasoconstriction and stimulation of aldosterone secretion, respectively. In addition to these peripheral actions, angiotensin I1 has several central actions including stimulation of drinking, induction of salt appetite and elevation of blood pressure [3]. These responses are triggered by the interaction of angiotensin I1 with its specific receptors on target cells. Because of its key roles in mediating angiotensin I1 actions, much effort has been devoted to biochemical characterization and purification of the angiotensin I1 receptor in various target tissues, leading to excellent characterization of membrane-bound forms of the angiotensin I1 receptor in terms of molecular mass and their affinities for angiotensin I1 analogues [4-131. However, attempts to purify the angiotensin I1 receptor, which is necessary for further characterization, have been hampered by its low abundance, instability in a detergent-solubilized state and the lack of efficient affinity gels. Purification trials in our laboratories and those of others, employing conventional column chromatography, resulted in only partial purification [4 -71.As a starting material, many investigators have chosen adrenocortical membranes, since the zona glomerulosa cells of the cortex are the classical target of angiotensin 11. Recently, liver has also been recognized as an important target organ

Cloning, amino acid sequence and tissue distribution of porcine thimet oligopeptidase

“…When we previously characterized sABP using '"I-angiotensin I1 as a ligand, we detected no peptidase activity in the sABP preparations and we therefore used the term 'binding protein' [12]. This failure to recognize the peptidase activity was due to the inclusion of EDTA and ClHgPhS0,H in our previous assay buffer as proteinase inhibitors.…”

“…The demonstration in the present study that sABP has a peptidase activity sensitive to the metal-chelating agents EDTA and 1 ,lo-phenanthroline, and the sulfhydryl reagent ClHgPh-S03H, further supports the prediction based on sequence similarity that sABP is a member of the thimet oligopeptidase family. The fact that sABP exhibits an angiotensin-binding activity only in the presence of ClHgPhS0,H [12] indicates that modification of the sulfhydryl group(s) in sABP, which is essential for sABP catalytic activity, is required for the binding activity. Mere inhibition of the peptidase activity, for example by EDTA, is not sufficient to permit binding activity.…”

“…Peptidase assay sABP was purified as described previously [12] and aliquots (1 pg) were incubated with 'Z51-a~igioten~in I1 (7 fmol) in 20 pl 0.25 mM Tris/HCI, pH 7.5, containing 85 mM NaCl and 0.05% bovine serum albumin at 30°C for 30 min in the presence or absence of the metal-chelating agents EDTA (1 mM), 1,lO-phenanthroline (1 mM) and the thiol reagent ClHgPhS0,H (1 mM). The reaction mixtures (10 pl) were spotted on an RP-18 WFL,4~ TLC plate and developed using solvent system I described by Poll et al [ 201.…”

“…the thimet oligopeptidase family [lo] (for a sequence comparison of these proteins see , and a microsomal endopeptidase with substrate specificity for processing proproteins [14]. While attempting the purification of angiotensin I1 receptors, studies by our group [12] and Kiron et al [13] revealed the presence of a binding protein showing a high affinity for angiotensin I1 in liver cytosols prepared in the presence of proteinase inhibitors such as EDTA and p-chloromercuriphenylsulfonic acid (CIHgPh- …”

“…These results suggest that there are at least two types of closely related thimet oligopeptidases in mammalian tissues. One type is the well-known thimet oligopeptidase and the other is its close relative formerly known as sABP [12,13,151 or microsomal endopeptidase [14, 161. We have also quantified and compared the mRNA levels of thimet oligopeptidase and sABP in various porcine tissues by RNase-protection analysis.…”