Angiotensin-converting enzyme: vascular endothelial localization.

Fluorescein-labeled antibody to rabbit pulmonary angiotensin-converting enzyme localized in the vascular endothelium of rabbit lung, liver, adrenal cortex, pancreas, kidney, and spleen. Epithelial cells of the renal proximal tubules were the only parenchymal cells among the organs studied that demonstrated immunoreactivity.

Canine pulmonary angiotensin-converting enzyme. Physicochemical, catalytic and immunological properties.

Antiontensin-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) has been solubilized from canine pulmonary particles and purified to apparent homogeneity. A value of approx. 140000 was estimated for the molecular weight of the native and the reduced, denatured forms of the enzyme. No free NH2-terminal residue was detected by the dansylation procedure. Carbohydrate accounted for 17% of the weight of the enzyme, and the major residues were galactose, mannose and N-acetylglucosamine with smaller amounts of sialic acid and fucose. Removal of sialic acid residues with neuraminidase did not alter enzymatic activity. The enzyme contained one molar equivalent of zinc. Addition of this metal reversed stimulation and inhibition of activity observed in the presence of Co2+ and Mn2+, respectively. Immunologic homology of pure dog and rabbit enzymes was demonstrable with goat antisera. Fab fragments and intact IgG antibodies displayed similar inhibition dose vs. response curves with homologous enzyme, whereas the fragments were poor inhibitors of heterologous activity compared to the holoantibodies. The canine glycoprotein was much less active than the rabbit preparation in catalyzing hydrolysis of Hip-His-Leu. In contrast, the two enzymes exhibited comparable kinetic parameters with angiotensin I as substrate.

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.

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.

Enzymatic arginylation of beta-melanocyte-stimulating hormone and of angiotensin II.

Porcine beta-melanocyte-stimulating hormone and angiotensin II were examined as acceptors in the reaction catalyzed by arginyl-tRNA-protein transferase. Both inhibited enzymatic transfer of [14C]arginine from tRNA to bovine albumin. Inhibition was competitive with albumin and the K-i values were, respectively, 15 and 0.8 muM. The expected arginylated compounds were isolated and characterized. Beta-melanocyte-stimulating hormone and its arginylated product had identical activities in the frog epithelium bioassay. In contrast, the biological activity of angiotensin II was diminished by enzymatic arginylation. The pressor effect of the arginylated derivative on anesthetized rats and its activity on the isolated rat uterus were, respectively, approximately 60% and 20% of those found for the unmodified peptide.

Escherichia coli mutants defective in dipeptidyl carboxypeptidase.

Two independent mutants of Escherichia coli deficient in dipeptidyl carboxypeptidase activity (Dep-) were isolated after mutagenesis with ethyl methanesulfonate. Mating experiments and introduction of specific episomes indicated that the responsible gene was located at approximately 27--31 min on the E. coli chromosome. The Dep- mutants differed from the parental strain in their inability to grow with N-acetylalanylalanylalanine as the sole nitrogen source. Revertants selected for growth on this substrate of the enzyme were found to have reacquired the activity. Enzyme activity was highly sensitive to inhibition by 1-(D-3-mercapto-2-methylpropanoyl)-L-proline (SQ 14225), a potent inhibitor of mammalian dipeptidyl carboxypeptidase (angiotensin-converting enzyme, peptidyl dipeptidase, EC 3.4.15.1). This compound also reduced the rate of growth of the wild type with N-acetylalanylalanylalanine but not with ammonium sulfate. A fraction of the enzyme was released into the medium by osmotic shock, indicating that its presence in the periplasmic space may account for growth with N-acetylated peptides that cannot be taken up by E. coli. In addition to providing information about the specific role of this exopeptidase in E. coli, the Dep- mutants may prove useful for delineating the regulation and cellular function of dipeptidyl carboxypeptidases in higher organisms.

Pulmonary angiotensin-converting enzyme antienzyme antibody.

A method has been developed for quantitating anticatalytic activity in antibody preparations made in goats against pure solubilized angiotensin-converting enzyme from rabbit pulmonary membranes. Anticatalytic activity was purified about 90-fold from a single batch of serum by a procedure including diethylaminoethylcellulose chromatography and elution from Sepharose columns containing covalently bound pure enzyme. Antiholoenzyme antibody was fractionated with respect to charge and binding affinity; however, these different populations each inhibited enzymatic hydrolysis of hippurylhistidylleucine, angiotensin I, and bradykinin. The inhibition dose-response curves were similar for hydrolysis of hippurylhistidylleucine and angiotensin I despite the difference in molecular weight of these substrates. Evidence is presented suggesting that a single molecule of antibody can bind two molecules of enzyme and that at least 18% of the total antiholoenzyme antibody population is directed against determinants which influence catalytic activity. A competitive immunoassay was developed with radioiodinated pulmonary enzyme as displaceable antigen. The anticatalytic and radioimmune assays were used to examine immunological properties of converting enzymes in various rabbit organs and fluids. Kidney, brain, and serum were found to contain converting enzymes which were immunologically identified with that in rabbit lung. Converting enzyme in seminal plasma was similar to the lung enzyme in the anticatalytic assay, but showed lower immunoreactivity in the radioimmune assay.

Modification of a specific ribosomal protein catalyzed by leucyl, phenylalanyl-tRNA: protein transferase.

Escherichia coli ribosomes washed with 1 M NH(4)Cl were found to function as acceptor for leucine and phenylalanine in the reaction catalyzed by leucyl, phenylalanyl-tRNA:protein transferase. When isolated subunits were acylated with [(14)C]phenylalanine and reisolated by gradient centrifugation, the recovered 30S particles had a specific radioactivity nearly 30 times that of similarly treated 50S particles. Autoradiography of gels, which contained protein from acylated 30S particles, that had been subjected to electrophoresis in 8 M urea and in sodium dodecyl sulfate, suggested that acceptor activity was largely due to a single protein with a molecular weight of about 12,000. Leucine and phenylalanine residues that had been transferred to ribosomal protein were reactive with fluorodinitrobenzene and were released as leucyl- or phenylalanylarginine after treatment with trypsin. The results indicate that leucyl, phenylalanyl-tRNA: protein transferase catalyzes the addition of these amino acids to an NH(2)-terminal arginine residue of a specific ribosomal protein on the 30S subunit.

A mutant of Escherichia coli defective in leucyl, phenylalanyl-tRNA-protein transferase.

A mutant of E. coli that lacks leucyl,-phenylalanyl-tRNA-protein transferase (EC 2.3.2.6) has been isolated. Ability to produce the two activities could be introduced into the mutant from an F' strain whose episome contains genetic material located between 45 and 54 min on the E. coli chromosome. When grown into stationary phase and resuspended in minimal medium with glycerol, the mutant exhibited a marked lag before resuming growth. Revertants that did not show this lag were selected and were found to have regained both transfer activities. Extracts of wild-type, mutant, and revertant strains were compared as acceptors for the enzymatic transfer of radioactive phenylalanine. Analysis of the labeled polypeptides by disc gel electrophoresis indicated that certain potential acceptors may be preferentially acylated in vivo. These data provide genetic confirmation that the same enzyme protein catalyzes the transfer of leucine and phenylalanine and suggest that leucyl,phenylalanyl-tRNA-protein transferase is involved in a growth regulatory mechanism.

Pulmonary angiotensin-converting enzyme. Structural and catalytic properties.

Angiotensin-converting enzyme has been solubilized from a particulate fraction of rabbit lung and purified to apparent homogeneity in 11% yield by a procedure including fractionation with DEAE-cellulose and calcium phosphate gel, elution from Sephadex G-200, and lectin affinity chromatography. The molecular weight estimated by equilibrium sedimentation was approximately 129,000, either in the absence or presence of 6 M guanidine hydrochloride. A slightly higher value of 140,000 determined for the reduced, denatured protein by gel electrophoresis in the presence of sodium dodecyl sulfate and a much higher figure derived from gel filtration are probably due to the glycoprotein nature of the enzyme. Its oligosaccharide content accounted for 26% of the weight calculated from its amino acid and carbohydrate composition. The estimated content of sugar residues per mole was: galactose, 57; N-acetylglucosamine, 53; mannose, 43; N-acetylneuraminic acid, 19; and fucose, 4. Threonine and alanine were identified, respectively, as NH2-terminal and COOH-terminal residues by the dansylation procedure and by digestion with carboxypeptidase A. The enzyme was found to contain approximately 1 g atom of zinc per mol. Km values for hydrolysis of hippurylhistidylleucine and angiotensin I were 2.3 and 0.07 mM, and the corresponding turnover numbers were 15,430 and 792 mol/min/mol at 37 degrees. Bradykinin was also a substrate, and release of its COOH-terminal dipeptide, Phe-Arg, was catalyzed at a comparable rate to that of His-Leu from the COOH terminus of angiotensin I. Enzyme activity required the presence of chloride ions and was inhibited by EDTA and by low concentrations of Bothrops bradykinin-potentiating peptides. In addition, hydrolysis of hippurylhistidylleucine was inhibited competitively by other defined peptides, including di- and tripeptides, which were not substrates.

Regulation of proline catabolism by leucyl,phenylalanyl-tRNA-protein transferase.

A mutant of Escherichia coli lacking leucyl,phenylalanyl-tRNA:protein leucyltransferase, EC 2.3.2.6) exhibited several abnormal growth characteristics relative to the wild type or a revertant when grown with glycerol as a carbon source. All three strains were auxotrophic for proline. The mutant required higher levels of this amino acid than did the other strains to attain a normal growth yield and metabolized exogenous [14C]proline more rapidly. The greater rate of proline utilization was associated with a 4-fold increase in specific activity of proline oxidase. When glucose rather than glycerol was employed as a carbon source, proline oxidase activity was reduced by catabolite repression and the growth ccharacteristics of the mutant were similar to those of the parental and revertant strains. These results suggest that the mutant growth phenotype is due to an altered rate of proline catabolism and constitue evidence for regulation of a specific metabolic pathway by leucyl,phenylalanyl-tRNA-protein transferase.

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.

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-converting enzyme from rabbit pulmonary particles.

Angiotensin-converting enzyme has been solubilized from rabbit pulmonary particles and purified to homogeneity. The molecular weight of the native enzyme was estimated to be about 136,000 by glycerol gradient centrifugation, and a value of 140,000 was obtained for the reduced denatured protein by disc-gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol. The enzyme was found to be a glycoprotein with carbohydrate accounting for approximately 16% of its dry weight. The major sugar residues were identified as galactose, N-acetylglucosamine, and mannose, with smaller amounts of fucose and sialic acid. The homogeneous enzyme catalyzed the release of His-Leu from the COOH-terminus of angiotensin I and of Phe-Arg and Ser-Pro from that of bradykinin.