Distribution and metabolism of angiotensin I and II in the blood vessel wall.

The demonstration of all components of the renin-angiotensin system in vascular tissue has raised questions as to the precise location of the local angiotensin II generation within the vascular wall. We investigated the metabolism of angiotensin I to angiotensin II in the vascular wall in the isolated rabbit thoracic aorta. Angiotensin I (3 x 10(-9) M) applied into the aortic lumen was partially converted to angiotensin II (14% after 60 minutes), but most of the luminal angiotensin I was degraded to peptide fragments or diffused as intact angiotensin I, peptide fragments, or both, into the vessel wall. Incubation studies with [3H]angiotensin I revealed that angiotensin I or angiotensin I fragments mainly diffused into the medial layer of the aorta and to a lesser degree into the adventitia and the endothelium. After removal of the endothelium, angiotensin II generation could no longer be detected. Addition of the angiotensin converting enzyme inhibitor ramiprilat (10(-7) M) to the incubation medium led to a complete blockade of angiotensin II generation by endothelial angiotensin converting enzyme. Our results underline the importance of the endothelium for conversion of angiotensin I to angiotensin II and provide evidence that conversion of angiotensin I to angiotensin II is predominantly achieved by endothelial cells. They also support the concept of an endocrine versus autocrine/paracrine renin-angiotensin system where the endothelium of the vasculature is the critical target site for angiotensin II production by both systems and, thus, the most important site for the actions of angiotensin converting enzyme inhibitors.

ACE inhibitor effect on bradykinin metabolism in the vascular wall.

In the isolated rabbit thoracic aorta the ACE inhibitor ramiprilat attenuated bradykinin degradation by enzymes localized on vascular endothelial cells as well as on vascular smooth muscle cells by 26% and 32%, respectively. We conclude that the ACE inhibitor can attenuate vascular bradykinin degradation not only by inhibition of endothelial ACE but also by inhibition of other bradykinin degrading enzymes in deeper layers of the vascular wall.

Peptide inhibitors and the active site(s) of angiotensin converting enzyme.

Angiotensin converting enzyme (ACE) is a central participant in blood pressure regulation and is strategically located within the pulmonary vasculature in order to carry out this function. It is also present in kidney, brain and a variety of other tissues where its function is unknown. The molecular weight of ACE from these sources is approximately 185,000. A smaller form of the enzyme, Mr approximately 100,000, is found in mature testis; its function is also unknown. The lung form of human ACE contains 1277 amino acids and consists of two homologous repeated domains, each of which appears to have a potential catalytic site. The testis form of human ACE contains 701 amino acids and has only a single domain which is largely identical to the carboxy-terminal half of the lung enzyme. This raises important questions such as, why does lung ACE possess two possible active sites, do each of the two bind zinc, and are they both catalytically active? To answer these questions, we have examined the binding of potent peptide inhibitors to ACE, redetermined the zinc stoichiometry and chemically modified both lung and testicular ACE with fluorodinitrobenzene (FDNB). Peptide inhibitors bind with essentially a 1:1 stoichiometry, indicative of a single active site. The zinc content of lung ACE is 1.4-1.8 g-at/mol. For testicular ACE it is 0.8-1.1 g-at/mol. FDNB modifies a single tyrosine, and to a much lesser extent a lysine, with concomitant loss of all catalytic activity. Sequence analysis identifies the specific residues modified and indicates that they occur only in the carboxy terminal half of lung ACE.(ABSTRACT TRUNCATED AT 250 WORDS)

Essential residues in angiotensin converting enzyme: modification with 1-fluoro-2,4-dinitrobenzene.

The peptidase and esterase activities of rabbit pulmonary angiotensin converting enzyme (ACE) are rapidly abolished on reaction with 1-fluoro-2,4-dinitrobenzene (Dnp-F). Inactivation follows first-order kinetics with respect to the reagent and is accompanied by stoichiometric incorporation of 3,5-[3H]Dnp, indicating that the effect is due to a specific modification of the enzyme. Thin-layer chromatography of an acid hydrolysate of the modified enzyme indicates that most of the radioactive label is present as O-Dnp-tyrosine (65 to greater than 95%) and the rest as N epsilon-Dnp-lysine. The pH dependence of the reaction is consistent with modification of either tyrosine or lysine. The presence of a competitive inhibitor effectively protects the enzyme against inactivation by Dnp-F. Acetylation of ACE with N-acetylimidazole also protects the enzyme against modification with Dnp-F. The results indicate the presence of catalytically essential tyrosine and lysine residues at the active site of ACE.

Sulfate potentiation of the chloride activation of angiotensin converting enzyme.

The angiotensin converting enzyme (ACE)-catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions, notably chloride. This activation is enhanced by sulfate; at pH 7.5, the effect is maximal at 0.8 M sulfate and is mediated through a specific interaction of the divalent anion with the enzyme, not through an increase in ionic strength. Sulfate decreases the apparent binding constant for chloride which manifests as a decrease of the apparent KM value, but it does not change kcat. Thus, at pH 7.5, sulfate solely affects substrate binding in accord with the ordered bireactant mechanism of chloride activation that pertains with this substrate [Bünning, P., & Riordan, J.F. (1983) Biochemistry 22, 100-116]. Increasing the pH from 6 to 9 in the absence of sulfate increases the apparent binding constant for chloride almost 60-fold from 3.3 to 190 mM. In the presence of 0.8 M sulfate, however, the change is only about 6-fold, from 0.7 to 4.2 mM. Over the same pH range, the apparent KM for furanacryloyl-Phe-Gly-Gly obtained with saturating chloride concentrations shifts from 0.14 to 0.48 mM, while in the presence of 0.8 M sulfate about 3-fold lower apparent KM values are obtained. Sulfate does not appear to affect the pK of a group on the enzyme that controls the mechanism of chloride activation but rather decreases the apparent KM by reducing the apparent binding constant for chloride.

Kinetic properties of the angiotensin converting enzyme inhibitor ramiprilat.

The interaction of angiotensin converting enzyme (ACE) with ramiprilat was studied at pH 7.5 in the presence of 300 mmol/l sodium chloride with furanacryloyl-Phe-Gly-Gly as substrate. Ramiprilat inhibits ACE with a Ki value of 7 pmol/l. It is both a slow- and tight-binding inhibitor; the mode of inhibition is fully competitive. Binding of ramiprilat to ACE proceeds by a two-step mechanism E + I in equilibrium EI in equilibrium EI* in which the inhibitor rapidly binds to enzyme to form an initial enzyme-inhibitor complex, which then undergoes a slow isomerization. The interaction of ramiprilat with ACE is compared to that of two other potent inhibitors, captopril and enalaprilat.

Synthesis and secretion of alpha 2-macroglobulin by human lung fibroblasts.

Synthesis and secretion of alpha 2-macroglobulin were studied with the human lung fibroblast cell line GM 1379. After incubation with [3H]leucine the cells secreted radioactively labeled alpha 2-macroglobulin consisting of subunits with a molecular weight of 180,000. When the cells were treated with tunicamycin the unglycosylated alpha 2-macroglobulin subunit exhibited a molecular weight of 160,000. Poly(A) +RNA was isolated from the cultured cells and translated in a rabbit reticulocyte lysate. From the translation products an alpha 2-macroglobulin species with a molecular weight of 160,000 was immunoprecipitated. The addition of pancreatic microsoms to the translation mixture resulted in the synthesis of an alpha 2-macroglobulin subunit which had molecular weights of 180,000. Thus, a size of approximately 160,000 for the protein moiety and 20,000 for the carbohydrate portion can be estimated for a subunit of alpha 2-macroglobulin from human lung fibroblasts.

Characteristics of angiotensin converting enzyme and its role in the metabolism of angiotensin I by endothelium.

Angiotensin converting enzyme (ACE) was isolated from rabbit lung tissue by affinity chromatography on an N-(1-carboxy-5-aminopentyl)-L-alanylglycine resin. The molecular weight of the enzyme was determined to be 140,000, and upon isoelectric focusing a set of bands between pH 4.3-4.7 was observed. When the enzyme was treated with endoglycosidase F the molecular weight decreased to 100,000, and a virtually identical molecular weight was found for the in vitro translated form of ACE. The ACE-catalyzed hydrolysis of angiotensin I was followed by high-performance liquid chromatography separation and fluorescence monitoring of substrate and products. The kinetic parameters of the angiotensin I hydrolysis were KM = 17 mumol/L and kcat = 420 min-1 at pH 7.5. By the same analytical method the metabolism of angiotensin I by cultured endothelial cells and by the isolated, perfused rabbit aorta was investigated. Only a small fraction of angiotensin I was converted to angiotensin II; the larger part was directly degraded to small peptides. In the presence of ACE inhibitors no angiotensin II was formed but the rate of the angiotensin I hydrolysis was virtually unchanged. Thus, the action of ACE is critical to the generation of angiotensin II but not to the degradation of angiotensin I, and during ACE inhibition no accumulation of angiotensin I occurs.

The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism.

Zinc is essential to the catalytic activity of angiotensin converting enzyme. The enzyme contains one g-atom of zinc per mole of protein. Chelating agents abolish activity by removing the metal ion to yield the inactive, metal-free apoenzyme. Zinc does not stabilize protein structure since the native and apoenzymes are equally susceptible to heat denaturation. Addition of either Zn2+, Co2+, or Mn2+ to the apoenzyme generates an active metalloenzyme; Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ fail to restore activity. The activities of the metalloenzymes follow the order Zn greater than Co greater than Mn. The protein binds Zn2+ more firmly than it does Co2+ or Mn2+. Hydrolysis of the chromophoric substrate, furanacryloyl-Phe-Gly-Gly, by the active metalloenzymes is subject to chloride activation; the activation constant is not metal dependent. Metal replacement mainly affects Kcat with very little change in Km, indicating that the role of zinc is to catalyze peptide hydrolysis.

Rat lung tissue is a site of alpha 1-proteinase inhibitor synthesis: evidence by cell-free translation.

Poly(A) +RNA isolated from lungs of normal rats and of rats suffering from experimental inflammation was translated in a cell-free translation mixture from rabbit reticulocytes. The translation products were immunoprecipitated with specific antisera against alpha 1-proteinase inhibitor and alpha 2-macroglobulin. Comparable levels of mRNA for alpha 1-proteinase inhibitor were found in rat lung tissue from control and experimentally inflamed animals. alpha 2-Macroglobulin mRNA could not be detected in rat lung tissue.

Inhibition of angiotensin converting enzyme by 2-[N-[(S)-1-carboxy-3-phenylpropyl]-L-alanyl]-(1S,3S,5S)-2-azabicyclo [3.3.0]octane-3-carboxylic acid (Hoe 498 diacid). Comparison with captopril and enalaprilat.

The interaction of angiotensin converting enzyme (ACE) with 2-[N-[(S)-1-carboxy-3-phenylpropyl]-L-alanyl]-(1S,3S,5S) - 2 - azabicyclo[3.3.0]octane - 3 - carboxylic acid (Hoe 498 diacid) and its ester 2-[N-[(S)-1-ethoxycarbonyl-3 - phenylpropyl] - L - alanyl]-(1S,3S,5S)- 2-azabicyclo[3.3.0]octane-3-carboxylic acid (Hoe 498) was studied at pH 7.5 in the presence of 300 mmol/l sodium chloride with furanacryloyl-Phe-Gly-Gly as substrate. Hoe 498 diacid inhibits ACE with a Ki value of 7 pmol/l. It is both a slow-and tight-binding inhibitor; the mode of inhibition is fully competitive. Binding of Hoe 498 diacid to ACE proceeds by a two-step mechanism E+I in equilibrium EI in equilibrium EI* in which the inhibitor rapidly binds to enzyme to form an initial enzyme-inhibitor complex which then undergoes a slow isomerization. The interaction of Hoe 498 diacid with ACE is compared to that of the two other potent inhibitors, captopril and enalaprilat.

Substrate specificity and kinetic characteristics of angiotensin converting enzyme.

Furanacryloyl-Phe-Gly-Gly has been shown to be a convenient substrate for angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1). A detailed kinetic analysis of the hydrolysis of this substrate indicates normal Michaelis-Menten behavior with kcat = 19000 min-1 and KM = 3.0 x 10(-4) M determined at pH 7.5, 25 degrees C. The enzyme is inhibited by phosphate and activated by chloride; maximal activity is observed with 300 mM NaCl. In the absence of added zinc, activity is lost rapidly below pH 7.5 due to spontaneous dissociation of the metal, but in the presence of zinc, the enzyme remains fully active to about pH 6. The pH-rate profile indicates two groups on the enzyme with apparent pK values of 5.6 and 8.4. The substrate specificity of the enzyme has been examined in terms of the fundamental specificity quantity kcat/KM as well as the separate constants by using a series of furanacryloyl-tripeptides. The activity toward furanacryloyl-Phe-Gly-Gly has been compared with that toward the physiological substrates angiotensin I and bradykinin.

Activation of angiotensin converting enzyme by monovalent anions.

The angiotensin converting enzyme catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions in the order C1- greater than Br- greater than F- greater than NO3- greater than CH3COO-. In the alkaline pH region, increasing anion concentrations decrease the KM but do not change the kcat. This behavior is characteristic of an ordered bireactant mechanism in which the anion binds to the enzyme prior to the substrate. At acidic pH values, however, the anion activation is a result of both a decrease in KM and an increase in kcat, implying a bireactant mechanism in which anion and substrate bind randomly. For both the ordered and the bireactant mechanisms the anion serves as an essential activator. The effect of chloride on enzyme activity was studied over the pH range 5-10 under kcat/KM conditions and demonstrates that the apparent chloride binding constant increases from 3.3 mM at pH 6.0 to 190 mM at pH 9.0. The kcat vs. pH profile exhibits two pK values of 5.6 and 9.6, while the variation of KM with pH is characterized by a pK of 8.9 and a 2-fold increase between pH 6.5 and 7.5. The chloride activation of the hydrolysis of furanacryloyl-Phe-Gly-Gly is compared with that of the physiological substrates angiotensin I and bradykinin.

The catalytic mechanism of angiotensin converting enzyme.

A detailed investigation of the catalytic mechanism of angiotensin converting enzyme was undertaken in order to establish a molecular basis for its mode of action. These studies include the characterization of the kinetic properties of the enzyme. In particular, a mechanism for the anion activation, a characteristic feature of angiotensin converting enzyme, has been established. The active site of the enzyme contains a catalytically essential zinc atom which appears to be directly involved in the hydrolytic step of catalysis. Further components of the active site are the carboxyl group of an aspartyl or glutamyl residue, tyrosyl, arginyl and lysyl residues. The latter one is involved in mediating the anion activation. These results have enabled us to compare the active site of angiotensin converting enzyme with those of two other zinc peptidases and, thereby, deduce a mechanism for its mode of action. These investigations have confirmed a hypothetical model of the active site of angiotensin converting enzyme which has served to construct potent inhibitors of the enzyme now being used as antihypertensive agents.

Characteristics and functions of proteinase A and its inhibitors in yeast.

A purification and some properties of proteinase A from yeast are described. A specific macromolecular inhibitor of proteinase A from yeast cytosol has been isolated and shown to be a protein (molecular weight 7,700) consisting of a majority of polar amino acids. Proline, arginine, cysteine and tryptophan were not detected in the inhibitor. Possible biological functions of proteinase A and the proteinase A-inhibitor (and of other yeast proteinases and their inhibitors) in the following processes are discussed: general protein turnover, catabolite inactivation of enzymes, enzyme degradation at starvation and at transition to spore formation, and activation of pre-enzymes and precursor proteins by limited proteolysis.