Robert B. Harris

Purification of human serum angiotensin I-converting enzyme by affinity chromatography

Abstract A relatively simple procedure is described for purifying human serum angiotensin-converting enzyme. The enzyme was purified 130,000-fold to electrophoretic homogeneity using affinity chromatography as the principal purification step. The ligand was an immobilized competitive inhibitor, d -cysteinyl- l -proline. A six-carbon spacer arm was satisfactory for trapping the enzyme; 80% of the bound enzyme was eluted with 3 m urea-1.0 m NaCl-0.1 m Tris, pH 8.3. The specific activity was 39 units/mg protein and the molecular weight (155,000), isoelectric point (4.7), kinetic properties, and the effect of various inhibitors are in agreement with published reports.

Inhibition and affinity chromatography of human serum angiotensin converting enzyme with cysteinyl-proline derivatives.

Abstract Several structural derivatives of the dipeptide, l (and d )-cysteinyl- l -proline were synthesized and shown to be very potent competitive and noncompetitive inhibitors of human serum angiotensin I-converting enzyme. Only if the sulfhydryl group of the cysteine was blocked with benzyl, trityl, or benzyloxycarbonyl protecting groups, was the dipeptide a noncompetitive inhibitor. Compounds with free sulfhydryl groups were competitive inhibitors with K i values in the 10 −8 m range. d -Cys- l -Pro, our most potent inhibitor ( k 1 = 0.0055 μM), was an order of magnitude more potent than l -Cys- l -Pro consistent with findings of Cushman et al. (1977, Biochemistry 16 , 5484) that -CH 3 group substitution improves binding if the configuration is d but diminishes binding if the configuration is l . Zinc and calcium ions released inhibition by some of the noncompetitive, but only one, of the competitive inhibitors. The noncompetitive inhibitor, l -cysteinyl(benzyl)- l -proline, and the competitive inhibitor, l -cysteinyl- l -proline, were used as affinity ligands to obtain near homogenous (25 units/mg) enzyme from human plasma. The observation that compounds with a free sulfhydryl group are competitive inhibitors and those in which the sulfhydryl groups are blocked are noncompetitive inhibitors can be rationalized if the active site of the converting enzyme is an extended linear trench.

Atrial tissue contains a metallo dipeptidyl carboxyhydrolase not present in ventricular tissue: Partial purification and characterization

A new membrane-bound dipeptidyl carboxyhydrolase has been identified in bovine atrial tissue, and has been partially purified after extraction with Triton X-100. This enzyme, found in quantities of 0.01-0.03 units/g tissue assayed with Bz-Gly-His-Leu, is potentially capable of hydrolyzing atriopeptin II to atriopeptin I. The enzyme is located in the microsomal fraction and in sucrose density fractions enriched for atrial granules. The enzyme is completely inhibited by reagents for heavy metals such as EDTA, o-phenanthroline, dithiothreitol, and mercaptoethanol. The latter two compounds are also disulfide reagents. The atrial enzyme is also inhibited by D-2-methyl-3-mercaptopropanoyl-L-Pro(Captopril), 3-mercaptopropanoyl-L-Pro, 2-D-methylsuccinyl-L-Pro, and bradykinin potentiating factor, all inhibitors of the angiotensin I-converting enzyme. However, the atrial enzyme differs from the converting enzyme in a number of kinetic and molecular properties. Its activity increases with ionic strength, but the atrial enzyme does not have a chloride dependence for Bz-Gly-His-Leu hydrolysis; the pH optimum, 7.3, is slightly lower, and it is 5500 times less sensitive to the very potent converting enzyme inhibitor, D-Cys-L-Pro. The strokes radius of the atrial enzyme is 5.00 nm as compared to 4.10 nm, and its molecular weight is 240,000 compared to 145,000. Ventricular tissue, which does not contain the atrial peptides, does not contain the dipeptidyl carboxyhydrolase enzyme.

Synthesis of tert-butyl aminocarbonate, a new type of compound that can be used to acylate amines

Abstract tert-Butyl aminocarbonate (tert-butyloxycarbonyloxyamine) was prepared by reaction of hydroxylamine with excess di-tert-butyl dicarbonate. Amino carbonates have not previously been described. This compound rapidly acylates amines in both organic and aqueous solutions.

Conversion of atriopeptin II to atriopeptin I by atrial dipeptidyl carboxy hydrolase

We are examining the substrate specificity of atrial dipeptidyl carboxyhydrolase, a membrane-bound metallo enzyme that we isolated from bovine atrial tissue homogenates. This enzyme readily removes the dipeptide, Phe-Arg, from Bz-Gly-Ser-Phe-Arg, a stand-in substrate for atriopeptin II, one of several atrial natriuretic factors. We now report that the atrial enzyme cleaves the C-terminal dipeptide, Phe-Arg, from atriopeptin II to form atriopeptin I. The km (pH 7.5) is 25 microM and the ratio of relative Vmax/km as a measure of substrate specificity indicates that atriopeptin II is a 240-fold better substrate than Bz-Gly-His-Leu. Only Phe-Arg was detected as a hydrolysis product, indicating that sequential cleavage of Asn-Ser from atriopeptin II does not occur, and that atriopeptin I is not a substrate. Bz-Gly-Asn-Ser was as good a substrate for the atrial enzyme as Bz-Gly-His-Leu, but Bz-Cys(bzl)-Asn-Ser was not hydrolyzed. This result suggests that the presence of an intact disulfide bond or an S-alkylated residue in the P1 position of a substrate (as in atriopeptin I) prevents hydrolysis by the atrial enzyme. Comparative studies were made with the angiotensin I converting enzyme. Atriopeptin II was not a substrate. The stand-in substrates for atriopeptin I, Bz-Cys(bzl)-Asn-Ser and Bz-Gly-Asn-Ser were barely hydrolyzed, which by itself suggests that atriopeptin I is not a substrate of the angiotensin converting enzyme. Our results strongly suggest that atriopeptin II is converted to atriopeptin I and that hydrolysis is mediated by the atrial enzyme. The angiotensin I converting enzyme plays no role in processing these peptides. We suggest that the atrial enzyme be named atrial peptide convertase.

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.

Identification of an endogenous protease that processes atrial natriuretic peptide at its amino terminus

We have partially purified a thiol-dependent protease from bovine atrial tissue that cleaves the Arg98-Ser99 bond of rat natriuretic peptide (Gly96-Tyr126) to produce the natriuretic Ser99-Tyr126 peptide (cardionatrin I). This was the only hydrolytic product we detected. The existence of the atrial natriuretic peptide system implicates the mammalian heart as an endocrine organ which participates in the hormonal regulation of extracellular fluid volume, electrolyte balance and vascular tone. This enzyme appears to be part of that system. The atrial protease also hydrolyzes the Arg-2-Napthylamide bond of natriuretic peptide stand-in substrates; on the basis of relative Vmax/Km as a measure of substrate specificity, Bz-Leu-Arg-Arg-2-Napthylamide (NA) greater than Bz-Leu-Arg-2-NA greater than Arg-2-NA. There is little or no cleavage between the Arg-Arg pair of the first substrate. Since in the Gly96-Tyr126 peptide the Arg-Arg pair is not the principle cleavage site for this enzyme, it is very unlikely that it is a principle cleavage site for this enzyme in pro-atrial natriuretic factor. It is possible that it is a cleavage site for a different enzyme or the pair may serve as a signal for cleavage at Arg98.

Characterization of an enzyme that is capable of processing pro-gonadotropin-releasing hormone protein

A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-naphthylamide (NA) bond of benzoyl(Bz)-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA which contains two likely processing sites, Arg-Pro and Lys-Arg. On the basis of the ratio of Vmax to Km as a measure of substrate specificity, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA is about 50-fold better than Bz-Gly-Gly-Lys-Arg-2-NA. Bz-Leu-Arg-2-NA and Bz-Gly-Leu-Arg-Pro-Gly-Gly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (Bz-Gly-Gly-Lys-Arg-2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the Mr is approximately equal to 160,000 but is approximately equal to 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, Mr approximately equal to 70,000.

Isolation and Sequencing of an Active-Site Peptide from Angiotensin I-Converting Enzyme

A glutamic acid residue at the active-site of bovine lung angiotensin I-converting enzyme was esterified with p-[N,N-bis-(chloroethyl)amino]phenylbutyryl-L-[U-14]-Proline (chlorambucyl-L-[U-14C]-L-Proline), an affinity label for this enzyme. The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase HPLC contained the bound radiolabel. This active-site peptide (Mr approximately 16,000) was digested with trypsin, and the labeled peptide (T-2) was further degraded with thermolysin. The enzyme digest peptides were also resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained after thermolysin digestion (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 PTH-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]-Proline which confirms our earlier findings. The sequence that we determined is homologous in five residues with the corresponding sequences of 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.