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R L Soffer

Publications and source records attributed to R L Soffer.

At least 19 recordsLinked to original sources
PubMed1989

Structure of testicular angiotensin-converting enzyme. A segmental mosaic isozyme.

The complete amino acid sequence of rabbit testicular angiotensin-converting enzyme has been deduced from the sequence of the corresponding cDNA clone. A protein of the expected molecular weight of 84,000 was translated in vitro from the mRNA encoded by this cDNA. All of the previously determined sequences of seven tryptic peptides from the enzyme are present in the deduced sequence, thus confirming the identity of the protein. From the deduced sequence it appears that the protein contains a signal peptide at the amino terminus and a hydrophobic anchoring domain near the carboxyl terminus. Northern analysis with oligonucleotide probes, whose sequences represented different regions of the cDNA, revealed not only the regions of extensive homology between the mRNAs encoding the testicular and the pulmonary isozymes but also a stretch of sequence near the 5' end unique to the testicular mRNA.

Amino Acid Sequence↗
PubMed1989

Purification and properties of a soluble angiotensin II-binding protein from rabbit liver.

An angiotensin II-binding activity has been purified almost 3,000-fold to a nearly homogenous state from the 100,000 x g supernatant fraction of rabbit liver. The responsible protein is apparently monomeric since its molecular weight was estimated to be 75,000 in the native state by glycerol gradient centrifugation and in the reduced, denatured state by gel electrophoresis. The Kd and Bmax values of the purified preparation were 7.2 nM and 15.2 nmol of angiotensin II bound per mg of protein, the latter figure agreeing well with the theoretical value of 13.3. Competition experiments with 125I-angiotensin II and unlabeled peptides revealed that the angiotensin antagonist [Sar1,Ala8]angiotensin II (saralasin) and the agonist [des-Asp1]angiotensin II (angiotensin III) were more tightly bound than angiotensin II, whereas angiotensin I and the carboxyl-terminal hexapeptide were less avidly bound. The cardiac peptide, atrial natriuretic factor, also competed for binding to the purified preparation but was about 15-fold less effective than angiotensin II. Although the binding activity was purified in the absence of detergent, a requirement for detergent in the binding reaction emerged during the isolation procedure. Binding by the purified protein exhibited an almost complete dependence upon the presence of detergent, p-chloromercuriphenylsulfonic acid and EDTA.

Animals↗
PubMed1988

Biochemical and immunological similarity of soluble angiotensin II-binding proteins in different organs.

Binding of angiotensin II has been detected in soluble extracts of rabbit liver, adrenal gland, aorta, brain, kidney and uterus. In each case, binding required p-chloromercuriphenylsulfonic acid and bound angiotensin II was released by treatment with dithiothreitol. These properties resemble those of the 75 kDa binding protein purified from liver. Immobilized guinea pig antiserum developed against the isolated hepatic protein removed binding activities from the different extracts in an immune-specific, quantitatively comparable manner. In addition, the activities were removed by a mouse monoclonal antibody which specifically recognized a protein of 75 kDa in the various preparations. An immunologically homologous angiotensin II-binding protein with similar characteristics was also identified in the soluble fraction of rat liver.

Adrenal Glands↗
PubMed1988

Isolation of cDNA clones of rabbit angiotensin converting enzyme: identification of two distinct mRNAs for the pulmonary and the testicular isozymes.

We have isolated cDNA clones of rabbit angiotensin converting enzyme. These clones were isolated by antibody-screening of a lambda gt11 expression library made from rabbit testicular mRNA. The 2.6 kb insert of one such clone was subcloned in pBR322 and used as a hybridization probe. Out of the twenty independently isolated clones only seven hybridized with this probe suggesting that these clones belong to at least two families. Northern analysis revealed the presence of a 2.6 kb mRNA in rabbit testes and a 5.0 kb mRNA in rabbit lungs which hybridized strongly with this probe. These results indicate that the two tissue-specific isozymic forms of angiotensin converting enzyme are encoded by two distinct mRNAs which share sequence homologies.

Animals↗
PubMed1988

A soluble angiotensin II-binding protein from rabbit liver.

An angiotensin II-binding activity has been detected in the 100,000 x g supernatant fraction of rabbit liver. The total amount of binding activity in this fraction was substantially greater than that which could be solubilized from hepatic particles by treatment with digitonin. The crude soluble binding activity resembled the binding protein which had been purified from the particles in several respects. First, binding required the presence of p-chloromercuriphenylsulfonic acid and bound angiotensin II was released by dithiothreitol. Second, the molecular weight of the responsible protein cross-linked to radioiodinated angiotensin II was about 75,000 in the reduced, denatured state. Finally, guinea pig antiserum raised against the binding protein that had been purified from particles reacted identically with the soluble and solubilized activities.

Angiotensin II↗
PubMed1988

Purification and properties of an angiotensin-binding protein from rabbit liver particles.

An angiotensin II-binding protein was purified more than 8000-fold after solubilization from rabbit liver particles with digitonin. The procedure comprised fractionation with ammonium sulfate, chromatography on DEAE-cellulose and Affi-Gel 501, gel filtration through Sephacryl S-200, and chromatography with hydroxylapatite. The purified preparation exhibited Kd and Bmax values of 6.7 nM and 8.4 nmol of angiotensin II bound/mg protein. The latter figure represents more than 60% of the theoretical value calculated for a protein of Mr 75,000 as estimated for the major protein component by gel electrophoresis. The purified preparation displayed comparable or slightly higher affinities for various angiotensin antagonists and angiotensin III than that for angiotensin II, whereas angiotensin I as well as the hexapeptide and smaller carboxy-terminal fragments were less tightly bound. Binding of angiotensin II by the isolated protein was highly dependent upon the presence of p-chloromercuriphenylsulfonic acid and also required ethylene diaminetetraacetic acid which could be almost completely replaced by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid but not by o-phenanthroline.

Angiotensin II↗
PubMed1988

Antiserum to angiotensin-binding protein inhibits vascular responses to angiotensin II.

This study characterizes inhibitory properties of an antiserum to an angiotensin-binding protein on vascular responses to angiotensin II. The antiserum was collected from guinea pigs that had been immunized with an angiotensin-binding protein that was isolated from a particulate fraction of rabbit liver. The bioassay system consisted of helically cut strips of rabbit renal artery suspended in organ chambers for measurement of isometric force development. After treatment with the antiserum (1:1,000-1:50 dilution), contractile responses to angiotensin II (10(-8) M) were reduced compared with those measured after treatment with nonimmune serum. At a dilution of 1:50, the magnitude of contractile responses to angiotensin II were approximately 40% of control values. This inhibitory action of the antiserum was similar to that induced by the angiotensin II antagonist, saralasin. Contractile responses to norepinephrine (5.9 x 10(-8) M) were not altered after incubation with the antiserum. These results indicate that the binding protein in hepatic cells may be similar in some respects to the membrane receptor mediating contractions to angiotensin II in rabbit renal arteries.

Angiotensin II↗
PubMed1987

Unexpected binding of an octapeptide to the angiotensin II receptor.

An octapeptide, TBI-22 (Lys-Gly-Val-Tyr-Ile-His-Ala-Leu), inhibited binding of angiotensin II by a solubilized angiotensin receptor partially purified from rabbit liver. This inhibition appears to result from competition for binding to the same receptor. Radioiodinated TBI-22, like angiotensin II, bound to the solubilized receptor with an affinity such that the binding was inhibited 50% by unlabeled TBI-22 or angiotensin II at nanomolar concentrations. The binding reaction, like that for angiotensin II, required p-chloromercuriphenylsulfonic acid and was reversed in the presence of dithiothreitol. TBI-22 and angiotensin II share the sequence Val-Tyr-Ile-His; this tetrapeptide alone, however, did not inhibit binding of angiotensin II. Replacement of the tyrosine residue by aspartic acid in TBI-22 greatly reduced the ability of the peptide to compete with angiotensin II for binding, suggesting an important contribution of this residue to the configuration required for recognition by the receptor.

Amino Acid Sequence↗
PubMed1987

Pulmonary and testicular angiotensin-converting isoenzymes.

A variant of angiotensin-converting enzyme occurs in (male) germinal cells. This testicular isozyme is catalytically similar to the widespread pulmonary-type isozyme, but contains a shorter polypeptide chain and does not appear until puberty. The two proteins differ at their NH2- and COOH-termini, but share many tryptic peptides. All antigenic determinants of the testicular form are represented in the pulmonary molecule whereas the latter contains determinants unrelated to catalysis which are lacking in the testicular species. The data indicate that the testicular isozyme corresponds closely to an internal part of the pulmonary polypeptide which includes its active site. The structural and developmental differences between the two polypeptides are pretranslationally determined since they are demonstrable in a cell-free system programmed by the appropriate mRNAs. Characterization of the molecular mechanisms responsible for the relationship of these isozymes may yield useful information regarding cell-specific protein expression.

Animals↗
PubMed1986

Immunohistochemical localization of two angiotensin I-converting isoenzymes in the reproductive tract of the male rabbit.

The male reproductive tract contains two different isoenzymes of angiotensin I-converting enzyme (ACE), i.e., pulmonary and testicular ACE. The present study shows selectively the cellular distribution of the ACE isoenzymes in the reproductive tract of male rabbit, using indirect immunofluorescence or immunoperoxidase methods. Testicular ACE was found in the seminiferous tubules of the testes in spermatocytes containing mature spermatids, and in spermatids within the epididymal tubular lumen in sexually mature, but not in immature, rabbits. Epididymal tubular cells contained pulmonary ACE. In the young rabbit, epididymal tissue contained more ACE than that in adult rabbit, since ACE was observed in principal cells in addition to basal cells. In mature rabbit, ACE was observed in basal cells only. Strong staining for pulmonary ACE was observed in cells of the vas deferens in both young and adult rabbit. Therefore, synthesis of epididymal ACE, unlike the testicular isoenzyme, was not stimulated by sexual maturation. Enzymatically active ACE in seminal fluid corresponds to the pulmonary isoenzyme. The present study indicates that this seminal fluid ACE may originate from cells of the epididymal tubules, particularly those of the vas deferens. Endothelial cells of blood vessels lying in the interstitium of both testicular and epididymal tissue contained the pulmonary isoenzyme.

Animals↗
PubMed1986

Thoughts and studies on purification of the angiotensin II receptor.

The angiotensin receptor is the only macromolecular component of the renin-angiotensin system which has not yet been purified and characterized in the isolated state. A purified preparation could be useful for identifying the amino acid residues it preferentially recognizes in various positions of defined peptide ligands, and for elucidating the proximate molecular mechanism by which the binding event is transduced into a cellular response. Such knowledge should expedite the development of receptor antagonists which might be more physiologically specific than other inhibitors of the system. This paper elaborates on these thoughts, and describes some recent progress in purification of the rabbit hepatic receptor.

Angiotensin Receptor Antagonists↗
PubMed1985

Angiotensin receptor is a desirable locus for physiologically specific inhibition of the renin-angiotensin system.

Angiotension II is the effector molecule of the renin-angiotensin system. Therefore, agents directed at the receptor that mediates its actions are likely to represent the most physiologically specific inhibitors of the system. We suggest here an approach to such drugs based on an operational analogy between peptidases and peptide hormone receptors and on the development of inhibitors of angiotensin-converting enzyme. The rationale that led to captopril, enalapril, and related inhibitors of this peptidase required identification of its cognitive and functional properties, i.e., what amino acid sequences it preferentially recognizes and its Zn2+ -dependent dipeptidyl carboxypeptidase activity. Purification of the enzyme was necessary to obtain this information. We speculate that this type of information may be equally useful for developing a receptor antagonist. As progress toward this objective, we describe briefly purification of rabbit hepatic angiotensin II receptor using chemical and immunoaffinity ligands. We hope to determine the cognitive and functional properties of this purified protein, i.e., what residues it preferentially recognizes in defined peptides and the molecular mechanism by which binding of ligand is transduced into a cellular response.

Angiotensin II↗
PubMed1984

Isolation of an angiotensin II-binding protein from liver.

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 II-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 that in its unreduced form suggest that the binding configuration of this protein may be sensitive to reduction.

Angiotensin II↗
PubMed1983

Solubilization and characterization of an angiotensin II binding protein from liver.

Binding sites with high affinity for angiotensin II were solubilized from hepatic membranes by treatment with digitonin. Binding of radioiodinated angiotensin II was assayed by gel filtration and independently by a technique exploiting the failure of activated charcoal to adsorb the bound ligand. The binding protein was partially purified using ammonium sulfate fractionation followed by gel filtration, and in the presence of protease inhibitors, the isolated binding protein preparation did not catalyze degradation of the angiotensin II. Binding to the membranes as well as to the solubilized preparation was specific and saturable. The membranes exhibited a single set of high-affinity binding sites with a Kd of 0.5 nM. The solubilized preparation, also showed the presence of a single class of high-affinity binding sites (Kd = 10.5 nM). Displacement studies using angiotensin I as well as various fragments, agonists and antagonists of angiotensin II disclosed a structure-activity profile similar to that found with intact membranes. Dissociation of angiotensin II from the soluble macromolecular complex was slow but was enhanced at non-physiological pH values or in the presence of 4.5 M urea, or 1% sodium dodecyl sulfate. Covalent binding of the radioiodinated angiotensin II to a single, specific macromolecular component was achieved by treatment with disuccinimidyl suberate. The apparent molecular weight of this reduced, denatured radioactive protein was estimated at about 68 000 by polyacrylamide gel electrophoresis.

Angiotensin II↗
PubMed1983

Rabbit pulmonary angiotensin-converting enzyme: the NH2-terminal fragment with enzymatic activity and its formation from the native enzyme by NH4OH treatment.

The NH2-terminal sequence of 22 residues of rabbit lung angiotensin-converting enzyme has been determined as (NH2)Thr-Leu-Asp-Pro-Gly-Leu-Leu-Pro-Gly-Asp-Phe-Ala -Ala-Asp-Asn-Ala-Gly-Ala-Arg-Leu-Phe-Ala-. In the course of purification of the enzyme for structural analysis a protein of Mr = 82,000 with angiotensin-converting activity was separated from the major fraction containing the native enzyme (Mr = 140,000). This low-molecular-weight enzyme catalyzed the hydrolysis of the synthetic substrate Hip-His-Leu at a rate 23% of that with the native enzyme, and exhibited a similar Km value as well as behaviors towards various effectors of angiotensin-converting enzyme. Edman degradation of both the native and the 82K enzymes revealed that they contain identical amino acid sequences from the NH2-termini. This result and those of peptide mapping and carbohydrate and amino acid analyses indicate that the 82K enzyme is a fragment derived from the NH2-terminal portion of the native enzyme, and hence contains its catalytic site. Evidence has been obtained indicating that the active fragment was formed from the native enzyme during its elution from the antibody-affinity column with NH4OH: on treatment of the native enzyme (140K Mr) with 1 N NH4OH at room temperature, a cleavage occurred and two proteins with Mr = 82K and Mr = 62K were obtained. The 82K Mr fragment was found to be enzymatically active and to contain the same NH2-terminal sequence as the native enzyme. The other fragment (62K Mr) was devoid of the activity and was shown to derive from the COOH-terminal portion of the native enzyme by the peptide mapping and terminal analyses. Cleavage of a peptide bond with NH4OH is unusual and appears to be specific for the native angiotensin-converting enzyme from rabbit lung.

Amino Acids↗
PubMed1983

Angiotensin-converting enzyme: immunologic, structural, and developmental aspects.

Immunization of dog and rat high pure rabbit pulmonary angiotensin-converting enzyme elicited, in some individuals, antibodies that inhibited their own converting enzyme. Active immunization with an immunologically related enzyme is thus a plausible approach for developing biologically based inhibitors of enzymes that are either in or accessible to the circulation. Rabbit testicular peptidyldipeptide hydrolase was purified to homogeneity and found to be a considerably smaller (Mr approximately 100,000) glycoprotein than pulmonary converting enzyme (Mr approximately 140,000). The two enzymes differed at their amino- and carboxy-termini. However, they exhibited identical catalytic properties, and antibodies prepared against either inhibited both similarly. In competition radioimmunoassays, antibodies against the pulmonary enzyme preferred it to the testicular species, whereas those against the latter did not distinguish between the two molecules. The testicular isozyme thus resembles an internal part of the pulmonary polypeptide, which includes its active site. In a reticulocyte lysate, mRNA from the lungs of immature and mature rabbits comparably primed the synthesis of a polypeptide (Mr approximately 129,000) that reacted with anticonverting enzyme antibodies. In contrast, an immunoreactive species was programed only by mRNA from the testis of mature animals, and this protein was much smaller (Mr approximately 85,000). Maturation dependence and a shorter polypeptide chain, the regulatory and structural properties that distinguish the testicular isozyme, are thus each pretranslationally determined.

Animals↗