Angiotensin-converting enzyme from human tissues. Physicochemical, catalytic, and immunological properties

Angiotensin-converting enzyme was purified from human lung, kidney, testis, blood plasma, and seminal plasma using a facile two-step protocol which included affinity chromatography on Sepharose-bound lisinopril followed by either gel filtration or hydroxylapatite chromatography. Molecular mass for converting enzyme from all sources except testis was 140 kDa. That from testis consisted of both a 90- and a 140-kDa form in a 4:1 ratio. Detergent-extracted membrane-bound converting enzyme aggregated on gel filtration chromatography, while trypsin-extracted and soluble converting enzyme did not. Comparison of detergent-extracted and trypsin-extracted membrane-bound converting enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing indicated that the membrane binding sequence contributed minimally to the size and charge of the enzyme. Catalytic and kinetic properties assessed by interaction with substrates, inhibitors, and anti-converting enzyme immunoglobulin were similar for all forms and sources of converting enzyme. Enzyme-linked immunosorbent assay revealed only partial homology between the 90- and 140-kDa forms of the enzyme.     

Affinity chromatography of angiotensin I-converting enzyme from rabbit lung using hippurylhistidylleucyl-OH.

Approximately 50-fold purification of angiotensin I-converting enzyme (Peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was achieved by affinity chromatography using the synthetic substrate Hippuryl-His-Leu-OH. The specific activity of the enzyme was increased from 0.044 units/mg protein to 1.911 units/mg protein for Hippuryl-His-Leu-OH and from 0.33 nmol/min per mg protein to 13.8 nmol/min per mg protein for angiotensin I.     

Purified angiotensin converting enzyme from Rana esculenta ovary influences ovarian steroidogenesis in vitro

The aim of the present study was to purify and characterize angiotensin-converting enzyme (ACE) present in frog ovary (Rana esculenta). Detergent and trypsin-extracted enzymes were purified using a one-step process, consisting of affinity chromatography on lisinopril coupled to Sepharose 6B. The molecular mass was 150 kDa for both detergent-extracted and trypsin-extracted enzyme. The specific activity of detergent-extracted and trypsin-extracted ACE was 294 U mg(-1) and 326 U mg(-1) respectively. The optimum pH range was from 7-8.5 at 37 degrees C and the optimum temperature was 50 degrees C. Optimum chloride concentration was about 200 mM for synthetic substrate FAPGG (N-[3-(2-furyl)acryloyl] L-phenylalanyl glycyl glycine) and angiotensin I, and 10 mM for bradykinin. The Km and Kcat values for FAPGG were 0.608 +/- 0.07 mM and 249 sec(-1) respectively and I50 values for captopril and lisinopril, two specific ACE inhibitors, were 68 +/- 12.55 nM and 6.763 +/- 0.66 nM respectively. Frog ovary tissue from prereproductive period was incubated in vitro in the presence of frog ovary ACE (2.5 mU/ml), captopril (0.1 mM), and lisinopril (0.1 mM). Production of 17beta-estradiol, progesterone, and prostaglandins E2 and F2alpha was determined. The data showed a modulation of 17beta-estradiol, progesterone and prostaglandin E2 production by ovary ACE.     

Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

Angiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells.     

Large-scale purification of angiotensin I-converting enzyme from human plasma utilizing an immunoadsorbent affinity gel

Angiotensin I-converting enzyme was purified 1500-fold from human plasma utilizing an immunoadsorbent affinity gel prepared by coupling antibody to baboon lung angiotensin I-converting enzyme to CNBr-activated Sepharose 4B. The enzyme was eluted from the gel using 2 m magnesium chloride, pH 5.8. Subsequent hydroxylapatite and Sephadex G-200 chromatography yielded 2.6 mg of homogenous enzyme with a specific activity of 40 units/mg with hippuryl-l-histidyl-l-leucine as substrate from 48 liters of plasma. Use of the immunoadsorbent allowed the 48 liters of plasma to be processed in one-half the time it previously took to process 2 liters of plasma by other methods. This protocol enables us to obtain sufficient amounts of enzyme for structural studies that were previously impossible because of insufficient amounts of enzyme.     

Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

Summary Angiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells. Presented in part at the 31st Annual Meeting of the Histochemical Society, April 11–15, 1980, New Orleans, Louisiana. Wendy Baur and Ms. Jane Aghajanian for expert assistance in the preparation of the cell cultures. This work was supported by Research Grant HL 14456 and Training Grant HL 07053 from the National Institutes of Health, Bethesda, MD.     

Tissue angiotensin I-converting enzyme activity in spontaneously hypertensive hamsters.

The purpose of this study was to measure angiotensin I-converting activity in heart, kidney, lung and cheek pouch tissue homogenates of spontaneously hypertensive and normotensive hamsters. We also determined inhibitor sensitivity and the effects of chloride anion concentration on kidney angiotensin I-converting activity in these animals. We found no significant differences in angiotensin I-converting activity between hypertensive and normotensive hamsters in all tissues tested. Inhibitor sensitivity of kidney angiotensin I-converting activity with captopril and lisonopril was similar in both groups. Finally, kidney angiotensin I-converting activity increased significantly in both groups as chloride anion concentration in the assay buffer increased. Substituting chloride anion for citrate abrogated the increase in angiotensin I-converting enzyme activity.     

Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M

Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [D-Ala2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1--Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin (I50 = 0.08 microM), bestatin (9.0 microM) and puromycin (80 microM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met5-enkephalin, Leu5-enkephalin and Leu5-enkephalin-Arg6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin (1-13)). No significant hydrolysis of beta- and gamma-endorphin was detected. Km values decreased significantly with increasing peptide length (Km = 72.9 +/- 2.7, 43.6 +/- 4.7 and 21.4 +/- 0.9 microM for Met5-enkephalin, Leu5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Phe7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1-13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5-11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.     

Dipeptide-hydroxamates are good inhibitors of the angiotensin I-converting enzyme

The inhibition constants (Ki) and modes of inhibition have been determined for a series of dipeptide-hydroxamate compounds with bovine lung parenchyma angiotensin I-converting enzyme (peptidyldipeptide carboxy-hydrolase, E.C. 3.4. 15.1). The hydroxamido function was borne by aspartic, glutamic, or aminoadipic acid and extended by 2, 3 or 4 bond lengths from the proline amide bond. L-glu(NHOH)-L-pro (Ki = 3.4 microM) and D,L-aminoadipicyl (NHOH)-L-pro (Ki = 1.2 microM) were the best competitive inhibitors of the hydrolysis of benzoyl-gly-his-gly but were not effective as affinity ligands for purification of the enzyme.     

Hybridization and partial cDNA sequence analyses of bovine lung angiotensin I-converting enzyme

The mRNA encoding angiotensin I-converting enzyme, a zinc-metallo dipeptidyl carboxyhydrolase, has been identified in extracts prepared from bovine lung tissue. Bovine lung poly(A) + mRNAs were subjected to electrophoresis and northern blot hybridization analysis using a radiolabeled synthetic 24-deoxyoligonucleotide probe complementary to eight codons for amino acids at the active-site of the enzyme (Harris, R.B. & Wilson, I.B., J. Biol. Chem. 260, 2208-2211, 1985). This amino acid sequence contains the catalytic glutamic acid residue. A single RNA species (approximately equal to 4 kb) was detected which is 1 kb larger than predicted from the molecular weight of the enzyme. The excess nucleic acid composition may be due to leader and/or trailer sequences or the RNA may encode a high molecular weight precursor form of the enzyme. We have cloned an EcoR1-HindIII digest fragment (1400 bp) of the duplex cDNA derived from the bovine lung converting enzyme poly(A) + mRNA and also Bal31 deletion fragments generated from the 1400 bp clone. Several of the Bal31 clones contain the active-site sequence codons of the enzyme and the complete cDNA sequence of one of these (72 bp) has been determined. We found the amino acid sequence at the active site to be -Phe-Thr-Glu-Leu-Ala-Asn-Ser-, containing the catalytic Glu residue. This sequence is identical with the sequence that we previously determined by manual Edman degradation analysis of the appropriate active-site peptide except that we now find Asn instead of Asp. We have sequenced 670 bp of the 1400 bp clone but have not yet overlapped the active-site sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of antihypertensive peptides from an izumi shrimp hydrolysate

The izumi shrimp (Plesionika izumiae Omori, 1971) is an unused resource which can be caught off the southern coast of Tokushima Prefecture. We have previously found that an izumi shrimp hydrolysate significantly inhibited the age-associated spontaneous increase in blood pressure in stroke-prone spontaneously hypertensive rats. In this present study, two angiotensin I-converting enzyme inhibitory peptides were isolated from an izumi shrimp hydrolysate by using high-performance liquid chromatography, and their amino acid sequences were determined to be Val-Trp-Tyr-His-Thr and Val-Trp. A single oral administration of synthetic Val-Trp-Tyr-His-Thr or Val-Trp significantly decreased the blood pressure in stroke-prone spontaneously hypertensive rats. The antigenicity and allergenicity of the izumi shrimp hydrolysate against BALB/c mice were very low. These results demonstrate that the angiotensin I-converting enzyme inhibitory peptides isolated from the izumi shrimp hydrolysate had an anti-hypertensive effect on rats.     

Possible involvement of angiotensin I-converting enzyme in the inactivation of bradykinin by intact macrophages

As intact macrophages inactivated bradykinin, the subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig macrophages. The bradykinin-inactivating activity was found to be present in membrane and cytosol fractions but not in granular and nuclear fractions. The bradykinin-inactivating activity of the membrane fraction was inhibited by captopril, a specific inhibitor of angiotensin I-converting enzyme, whereas that of the cytosol fraction was hardly inhibited by various proteinase inhibitors used. Angiotensin I-converting enzyme activity was located predominantly in the membrane fraction and its activity was inhibited by captopril. Angiotensin I-converting enzyme activity measured with a synthetic substrate was competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for macrophage angiotensin I-converting enzyme. When macrophages were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent, both the bradykinin-inactivating activity and the angiotensin I-converting enzyme activity of macrophages decreased significantly without any inhibition of the cytosol bradykinin-inactivating activity. These findings seem to suggest that the angiotensin I-converting enzyme would be responsible for the inactivation of bradykinin in intact macrophages.     

Solubilization of angiotensin I-coverting enzyme from rabbit lung using trypsin treatment.

The solubilization of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was carried out using trypsin treatment. A good recovery of 76% was obtained. The enzyme from solubilized fraction was purified using colums of Sephadex G-200, hydroxyapatite and DEAE-cellulose. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 24.3 units/mg protein for hippurylhistidylleucyl hydroxide and 0.182 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment for 5 h could be divided into two components: (i) an enzyme of molecular weight 300 000 (peak II) and (ii) an enzyme of molecular weight 145 000 (peak III), by Sephadex G-200 gel filtration. The molecular weight of the denatured enzyme was found to be 155 000 by disc gel electrophoresis in the presence of sodium dodecyl sulfate. Km values of peak II and peak III fraction for Hippuryl-His Leu-OH were 2.6 mM.     

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

血管紧张素转换酶纯化与性质研究

为了深入了解猪肺血管紧张素转换酶 (angiotensin converting enzyme,ACE)的性质和功能 ,对猪肺 ACE的分离纯化以及部分酶学性质进行了研究 .猪肺组织匀浆经 1 .6～ 2 .6mol/L硫酸铵分级沉淀等步骤后 ,利用亲和胶进行亲和层析分离 .2 0 0 g猪肺组织中提纯出 0 .79mg ACE,比活力 38.8U/mg,SDS- PAGE电泳鉴定为一条带 ,分子量约 1 80 k D,等电点 (p I)为 p H4.5,糖含量约 2 3.6% ,氨基酸组成分析发现猪肺 ACE分子中含有 1 346个氨基酸 ,其中酸性氨基酸含量较高 ,碘乙酸的修饰结果表明猪肺 ACE中巯基基团未参与酶的催化反应 .酶反应动力学结果显示 ,ACE催化 Fa PGG底物反应时的最适 p H大约为 p H 7.6,反应活化能 Ea=4.37× 1 0 4 J/mol,酶活性部位附近的组氨酸和具有类似 α-氨基性质的氨基酸可能参与了 ACE催化反应 .有关猪肺 ACE的基本生化性质、氨基酸组成以及酶学性质的结果 ,为今后深入研究奠定了基础 .     

Purification of angiotensin I-converting enzyme from human lung.

Angiotensin I-converting enzyme (peptidyl dipeptide hydrolase, EC 3.4.15.1) was solubilized from the membrane fraction of human lung using trypsin treatment and purfied using columns of DE 52-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 9.5 units/mg protein for Hippuryl-His-Leu-OH and 0.665 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment (1 mg/200 mg protein) for 2 h could be divided into three components: (i) an enzyme of molecular weight 290 000 (peak I), (ii) an enzyme of molecular weight 180 000 (peak II) and (iii) an enzyme of molecular weight 98 000 (peak III), by columns of DE 52-cellulose and Sephadex G-200. Km values of peak I, II and III fraction for Hippuryl-His-Leu-OH were identical at 1.1 mM. pH optimum of the enzyme was 8.3 for Hippuryl-His-Leu-OH.     

Sequencing of an active-site peptide of angiotensin I-converting enzyme containing an essential glutamic acid residue

A glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme, a zinc-metallo peptidyl dipeptidase, was esterified with p-[N,N-bis(chloroethyl)amino]phenylbutyryl-L-[U-14C]proline (chlorambucyl-L-[U-14C]-L-proline), an affinity label for this enzyme (Harris, R.B., and Wilson, I.B. (1983) J. Biol. Chem. 258, 1357-1362). The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase high performance liquid chromatography (HPLC) contained the bound radiolabel. This active-site peptide (Mr = 16,000) was digested with trypsin and the labeled peptide formed (T-2) was further degraded with thermolysin. The thermolytic peptides were resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained (Th-1, Mr = 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu... The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of phenylthiohydantoin-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]proline in confirmation of our earlier findings. The sequence determined is homologous in 5 residues with the corresponding sequences of bovine carboxypeptidase A and B, two other mammalian zinc proteases. There is little sequence homology with thermolysin, a bacterial zinc protease that also contains an essential active-site glutamic acid residue.     

Purification and analysis of lung and plasma angiotensin I-converting enzyme by high-performance liquid chromatography

We purified angiotensin I-converting enzyme (ACE) from pig and human lung and plasma for comparison of some physicochemical properties between the endothelial membrane-bound form and the soluble form of the enzyme. After affinity chromatography on Sepharose CL-4B/lisinopril, gel-filtration HPLC on Superose 12 achieved homogeneity for both forms as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Whatever the source of ACE, the molecular weight was 300 +/- 40 kDa after calibration of Superose 12 with standard globular proteins and 172 +/- 4 kDa by SDS-PAGE, with or without reduction, a result suggesting interactions between the glycopolypeptide chain and the chromatographic gel possibly related to the overall shape and sugar content of the enzyme. Ion-exchange HPLC analysis on TSK-DEAE showed that the membrane-bound and soluble forms of ACE are not isoenzymes, although isoelectrofocusing did show that the isoelectric point of soluble ACE was lower than those of tissue ACE, suggesting a different glycosylation. No significant difference between porcine and human ACE appeared. HPLC methods seem to be of particular interest for the purification of ACE with a high yield and for the analysis of its putative differently glycosylated isoforms.     

Presence of renin, angiotensinogen, angiotensin II in the lamb anterior pituitary gland: immunocytochemical study after cryoultramicrotomy.

The presence of renin, angiotensin I-converting enzyme and angiotensin II detected by immunocytochemistry in the adult male rat anterior pituitary has suggested the existence of a pituitary renin-angiotensin system. To establish another mammalian experimental model we have investigated the presence of renin, angiotensinogen, angiotensin I-converting enzyme, and angiotensin II II in five normal lamb anterior pituitaries by immunocytochemistry after cryoultramicrotomy. Renin, angiotensinogen and angiotensin II immunoreactivities were observed only in cytoplasmic granules of lactotrophs, and the three proteins were found co-localized with prolactin in the same granules by double immunolabelling. No immunoreactive angiotensin I-converting enzyme was observed. These results suggest an activation of renin in the cytoplasmic granules of lactotrophs leading to a local synthesis of angiotensin II. Thus, the lamb anterior pituitary may provide a good experimental model for investigating the possible autocrine action of a local renin-angiotensin system on prolactin release in the human pituitary.     

Angiotensin I-converting enzyme-like activity in tissues from the Atlantic hagfish (Myxine glutinosa) and detection of immunoreactive plasma angiotensins

Using a highly sensitive fluorimetric assay, significant levels of angiotensin I -converting enzyme-like activity (ACELA) were detected in a range of tissues (branchial heart, gill, kidney with associated vasculature and archinephric duct, liver, whole brain and gut) from the Atlantic hagfish (Myxine glutinosa). The highest ACELA occurred in heart and gill (1.8 and 1.5 nmol His–Leu min⁻¹ mg protein⁻¹, respectively). The mammalian angiotensin I-converting enzyme (ACE) inhibitor, captopril, at 10⁻⁵ M was a potent inhibitor of the ACELA found in all hagfish tissues. Radioimmunoassay showed that immunoreactive angiotensins (251.8±11.8 pM) were detectable in hagfish plasma. The validity of the assay for measurement of hagfish angiotensins was indicated by the parallelism of the angiotensin II standard curve against serially diluted hagfish plasma. Measurement of immunoreactive plasma angiotensins and detection of significant levels of ACELA in a wide range of tissues gives indirect evidence for the presence of a renin–angiotensin system in hagfishes, the earliest evolved group of craniates.