Richard L. Soffer

Angiotensin-converting enzyme inhibitors: biochemical properties and biological actions

The review will cover the chemistry and biochemistry of angiotensin-converting enzyme inhibitors with emphasis on data published since the publication of previous reviews. The relative merits of each contribution will be evaluated, as well as their potential for leading to new discoveries. The biology of angiotensin-converting enzyme inhibitors will be brought up-to-date to give the reader an appreciation of the medical implications of this new type of antihypertensive agent.

The arginine transfer reaction.

Abstract The incorporation of arginine into hot trichloroacetic acid-insoluble material by a 150 000 · g supernatant fraction from sheep thyroid cytoplasm has been studied. 1. 1. Incorporated arginine contains a free α-amino group as judged by the fluorodinitrobenzene reaction. It can be recovered as free arginine after acid hydrolysis or digestion by pronase. 2. 2. Arginyl-tRNA is an intermediate and an enzyme has been partially purified which transfers arginine from tRNA to protein. This enzymatic transfer displays a partial requirement for a non-enzymatic protein. Boiled supernatant fraction, albumin and thyroglobulin fulfill this function, whereas hemoglobin, myoglobin, cytochrome c and polyaspartic acid do not. 3. 3. The transfer has a pH optimum between 9.0 and 9.8 and is highly dependent upon the presence of mercaptoethanol and upon the ionic strength of the reaction mixture. It does not require ribosomes, Mg 2+ nor GTP and it is neither inhibited by puromycin nor stimulated by poly(A, G). 4. 4. A similar system for the incorporation of arginine exists in the soluble fraction of rabbit liver cytoplasm. The data are most consistent with an addition reaction in which arginine is transferred from tRNA onto certain preexisting proteins.

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.

Dipeptidyl carboxypeptidase from seminal fluid resembles the pulmonary rather than the testicular isoenzyme

The specific activity, molecular weight and immunological behavior of pure dipeptidyl carboxypeptidase from rabbit seminal fluid were found to resemble the corresponding properties of the pulmonary rather than the testicular isozyme.

Physiologic, biochemical, and immunologic aspects of angiotensin-converting enzyme.

Angiotensin-converting enzyme (EC.3.4.15.1) is an exopeptidase that catalyzes cleavage of dipeptidyl residues from the COOH termini of peptide substrates.1 It was first detected by Skeggs et al.2 who found that the product of the action of porcine renin on crude equine angiotensinogen could be resolved into two compounds, provided the incubation was carried out in the presence of chloride ions. Subsequently,3 they established that this was due to a contaminating activity in their angiotensinogen preparation that released His-Leu from the COOH terminus of angiotensin I to yield angiotensin II, an octapeptide. Although both angiotensins were found to be vasopressor after intravenous infusion, angiotensin II was identified as the biologically active component of the renin-angiotensin system, since only it induced contraction of rabbit aortic strips in vitro4 and increased the perfusion pressure of isolated rat kidneys.5 The vasopressor response to angiotensin I was therefore assumed to be due to its conversion to angiotensin II, mediated by the plasma-converting enzyme, until 1967, when Ng and Vane6 recognized that this enzyme activity was insufficient to account for the rapidity of conversion in vivo. They found that intravenously administered angiotensin I was much more potent than the same dose given intraarterially and that substantial conversion to angiotensin II occurred during a single passage through the pulmonary circulation.6,7

The NH2-and COOH-terminal sequences of the angiotensin-converting enzyme isozymes from rabbit lung and testis

Abstract The NH 2 -and COOH-terminal sequences of the angiotensin-converting enzymes from rabbit lung and testis have been determined using less than 0.6mg of each protein. They are: (NH 2 )Thr-Leu-Asp-Pro-Gly-Leu-Leu-Pro-Gly-Asp- and -(Phe, Tyr)-Ser-Leu-Ala(COOH) for the pulmonary enzyme; and (NH 2 )Arg-Arg-Val-Ser-Asn-Asn-Gln-Ser-Ser- and -(Phe, Ala)-Glu-Leu-Ser(COOH) for the enzyme from testis.

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.

On the oligosaccharide moiety of angiotensin-converting enzyme

Abstract Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (∼1 mole/mol enzyme) contained mannose, galactose, glucose N -acetylglucosamine, N -acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (∼ 12 moles/mol enzyme, ∼ 90% of total carbohydrate), contained fucose, mannose, galactose, N -acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.

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.

Protein synthesis by sheep thyroid extracts

Abstract Protein synthesis has been demonstrated in the postmitochondrial fraction and in extracts obtained by disruption of particles which sediment between 700 and 20,000g. The postmitochondrial fraction was maximally active in a reconstituted system of ribosomes and supernatant fraction. Polyuridylic acid specifically stimulated the incorporation of l -phenylalanine into protein, and l -phenylalanyl sRNA was shown to be an intermediate in this reaction. Polyadenylic acid stimulated the incorporation of l -lysine. The system was inhibited by amino acid deprivation, puromycin, and RNase but not by DNase nor actinomycin D. A significant quantity of ribosomes could be isolated from particulate fractions by treatment with deoxycholate. These ribosomes interacted with the postmitochondrial supernatant fraction in protein synthesis. Extracts were prepared by disruption of a particulate fraction sedimenting between 700 and 20,000g. These extracts responded to synthetic template RNA and could be fractionated into ribosomal and supernatant components. The individual components were inactive alone but could function together or with the complementary postmitochondrial subfraction.