Inactivation of angiotensin converting enzyme by sodium nitroprusside

Angiotensin I converting enzyme is rapidly inactivated by sodium nitroprusside and that inactivation is suppressed in the presence of chloride ion and by the presence of L-alanyl-L-proline or glycyl-L-tryptophan, which are both competitive inhibitors of its catalytic activity. The inactivation by sodium nitroprusside appears to result from the modification of an unusually reactive lysine residue in or near the active site.     

Purification of bovine angiotensin converting enzyme

A change has been made in the commonly used lisinopril affinity gel procedure for purifying angiotensin converting enzyme. The new method greatly decreases the time required and greatly increases the yield of pure enzyme. All of the enzyme in various bovine tissues was extracted with 0.5% triton X-100 and applied to the affinity column; 70% was trapped and all of the trapped enzyme was released as the apoenzyme by EDTA. The holoenzyme was recovered by dialysis against zinc containing buffer. The turnover numbers were precisely the same for enzyme from lung, atrium, kidney, striatum and blood. The tissue concentrations of ACE were very different but the final specific activities were the same.     

Inhibition of angiotensin converting enzyme by phosphoramidates and polyphosphates

N alpha-Phosphoryl-L-alanyl-L-proline is a reversible competitive inhibitor of angiotensin converting enzyme with a Ki of 1.4 nM. Alkylation of one phosphate oxygen with methyl, ethyl, or benzyl does not change the Ki. The high activity of the O-alkylated inhibitors demonstrates that the two phosphate oxygen anions do not constitute a bidentate ligand of the active site zinc ion. Substitution of valyltryptophan, glycylglycine, or delta-aminovaleric acid for alanylproline in the phosphoramidate raises the Ki to 12 nM, 25 microM, and 178 microM, respectively. Methylation of the alanine nitrogen in phosphorylalanylproline raises the Ki to 29 microM. Polyphosphates inhibit converting enzyme with the following Ki's: phosphate, approximately 300 mM; pyrophosphate, 2 mM; tripolyphosphate, 18 microM; tetrapolyphosphate, 150 microM. The inhibition by tripolyphosphate appears to be competitive and is unaffected by the addition of excess zinc ion. Since the Ki of tripolyphosphate is nearly 10-fold lower than that of N-phosphoryl-delta-aminovaleric acid and is near that of N alpha-phosphorylglycylglycine, its terminal phosphates may bind the zinc site and the cationic site on the enzyme, thus spanning the S1' and S2' sites.     

Triiodothyronine increases serum angiotensin converting enzyme

To determine whether elevated thyroid hormone is responsible for increased serum angiotensin converting enzyme in hyperthyroidism, 5 to 40 micrograms of 3,5,3'-triiodo-L-thyronine was administered orally and subcutaneously to female Swiss-Webster mice. Serum angiotensin converting enzyme was significantly increased in all animals given triiodothyronine compared to controls. Lung and kidney enzymes were moderately reduced in specific activity but unchanged in total activity due to increase in size of these organs. The results indicate that in hyperthyroidism, elevated thyroid hormone per se rather than the disease of the thyroid is responsible for elevated serum angiotensin converting enzyme.     

The angiotensin I converting enzyme.

The angiotensin I converting enzyme (kininase II; peptidyl dipeptidase; EC3.4.15.1) has a dual function: it converts angiotensin I to angiotensin II and it inactivates bradykinin. Lung, kidney, guinea pig plasma and testicles are among the richest sources of the enzyme. Vascular endothelial cells and bursh borders of renal proximal tubular cells contain high concentrations of the enzyme. The availability of synthetic peptide inhibitors was a great help in establishing the function of converting enzyme in normal and pathological conditions.     

New potent inhibitors of angiotensin converting enzyme

Using an earlier model of the favoured orientation of binding functions of angiotensin converting enzyme (ACE) inhibitors, it has been possible to postulate a new, 7,6-bicyclic system, based on hexahydropyridazine, which might be expected to have high potency. Some members of this system which have been synthesised have been shown to be very active ACE inhibitors, in vitro and in vivo.     

The angiotensin converting enzyme (ACE)

Angiotensin converting enzyme 1, found widely throughout the animal kingdom, is an integral membrane bound protein whose active sites are directed to the extracellular spaces. Two isoforms are expressed in mammals, a single domain germinal isoform required for male fertility, and a double domain somatic isoform which has a key role in the renin-angiotensin system. Both somatic domains are active with different substrate affinities. Mouse knockout experiments, and comparative work with invertebrate homologues, suggest that the two domains have clearly distinct roles. The importance of therapies involving inhibition of angiotensin converting enzyme are undisputed, but our understanding of how and why these therapies work is now being informed by the tools of genomic and comparative biology.     

Novel substrates for angiotensin I converting enzyme

Homogenous human angiotensin converting enzyme (EC 3.4.15.1) cleaves dipeptides from the C-terminus of substrates containing a free carboxyl group. In this study we demonstrate that peptides containing a C-terminal nitrobenzylamine are also cleaved by the enzyme. The hydrolysis of these substrates is inhibited by the specific converting enzyme inhibitors captopril and MK421 as well as by anti-converting enzyme antibody. Sodium chloride accelerates the rate of hydrolysis forty-fold. The product of the reaction, an amino acid nitrobenzylamide, was identified by thin layer chromatography and high performance liquid chromatography. These results suggest that the carboxyl group is not an absolute requirement for substrate hydrolysis.     

Assays for angiotensin converting enzyme inhibitory activity

A colorimetric method and a capillary electrophoresis procedure were developed for quantifying histidyl-leucine and hippurate, respectively. The colorimetric method is sensitive (extinction coefficient = 7.5 mM(-1) cm(-1)) and reproducible (CV = 1.7%, n = 5), which is based on a selective chromogenic reaction for histidyl-leucine (lambda(max) = 390 nm) using o-phthalaldehyde. For samples containing unusually high levels of histidine and/or histidyl peptides, the separation-based approach is preferable. The capillary electrophoresis method makes use of an in-capillary microextraction technique; complicated samples can be measured in less than 4 min without pretreatment. Protocols using both methods to measure angiotensin converting enzyme inhibitory activity were proposed.     

Inhibition of angiotensin converting enzyme by aldehyde and ketone substrate analogues

Three classes of carbonyl-containing substrate analogues and partial substrate analogues have been tested for their ability to inhibit angiotensin converting enzyme. (4-Oxobutanoyl)-L-proline is proposed to occupy the S1' and S2' subsites on the enzyme, thus locating its aldehyde carbonyl group at the position of the active site zinc atom. This aldehyde is 70% hydrated in aqueous solution and could mimic a tetrahedral intermediate occurring during enzyme-catalyzed substrate hydrolysis, but its Ki is only 760 microM. Carbobenzoxy-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalaninal is proposed to occupy the S1 through S4 subsites on the other side of the zinc atom. Its weak Ki of 60 microM is nearly equipotent to its parent peptide terminating in phenylalanine. However, ketoace, (5RS)-(5-benzamido-4-oxo-6-phenylhexanoyl)-L-proline [Almquist, R.G., Chao, W.R., Ellis, M.E., & Johnson, H.L. (1980) J. Med. Chem. 23, 1392-1398], one of the third class of inhibitors proposed to occupy subsites S1 through S2' on both sides of the zinc atom, has a Ki of 0.0006 microM under our assay conditions, orders of magnitude more potent than its parent peptide. The carbonyl carbon of ketoace is less than 3% hydrated in aqueous solution as determined by carbon-13 nuclear magnetic resonance spectroscopy. If the hydrate is the species bound to converting enzyme, its Ki must be less than 18 pM. Ketoace is a slow-binding inhibitor of converting enzyme, but its overall Ki is dependent on its concentration and therefore prevents calculation of kinetic constants for slow binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Esterase activity of rabbit pulmonary angiotensin converting enzyme

A series of depsipeptides have been synthesized and used to demonstrate the esterase activity of rabbit pulmonary angiotensin converting enzyme. Among the esters studied, Bz-Phe-OPhe-Ala was found to have the highest $\text{k}{}_{\mathrm{cat}}\text{K}{}_{\mathrm{m}}$ which is about $\text{1}\text{5}$ that of its exact peptide analog, Bz-Phe-Phe-Ala. Esters such as Bz-Gly-OGly-Phe, Bz-Gly-OPhe-Phe and Bz-Gly-OLeu-Ala were also hydrolyzed but at much lower rates. Normal Michaelis-Menten behavior is observed and the kinetic parameters obtained indicate that the esters and their peptide analogs bind to the enzyme equally well, but that peptides are hydrolyzed at much higher rates. Studies on the pH-rate profiles, chloride ion effect, inhibition and chemical modifications detect no mechanistic differences between ester and peptide hydrolysis.     

A metabolite of aspartame inhibits angiotensin converting enzyme

Aspartame (L-aspartyl-L-phenylalanine methyl ester, is a widely used artificIal sweetener. In humans and other animals aspartame is initially hydrolyzed to L-aspartyl-L-phenylalanine by intestinal esterases. L-Aspartyl-L-phenylalanine inhibits angiotensin converting enzyme purified from rabbit lungs with a Ki of 11 +/- 2 microM, equipotent to the IC50 of 12 microM for 2-D-methyl-succinyl-L-proline which has been reported to be an orally active antihypertensive agent in rats. Thus the possibility exists that L-aspartyl-L-phenylalanine inhibits angiotensin converting enzyme in humans consuming large quantities of aspartame. Both aspartame itself and the diketopiperazine formed from it, 3-carboxymethyl-6-benzyl-2,5-diketopiperazine, are weak inhibitors with Ki's greater than 1 mM.     

Downregulation of angiotensin converting enzyme by TNF-alpha in differentiating human macrophages

OBJECTIVE: To investigate regulation of angiotensin converting enzyme (ACE) by tumour necrosis factor alpha (TNF-alpha) in differentiating human peripheral blood monocytes (PBM). METHODS: Human PBM were allowed to differentiate to macrophages for 0-7 days and ACE amount was measured during differentiation. Experiments with TNF-alpha were performed after 2 days of differentiation. Cell cultures were incubated with TNF-alpha (0.5-10ng/ml) without or with SB 202190 (5microM), or PD 98059 (40microM). ACE amounts were measured by an inhibitor binding assay (IBA) and ACE mRNA levels by RNase protection assay (RPA). Activated p44/42 and p38 MAP kinases were measured by Western Blot analysis using phospho-p44/42 and -p38 MAPK antibodies. RESULTS: ACE amount increased by 40-fold along with macrophage differentiation. TNF-alpha caused dose dependent suppression of the amount of ACE and decreased levels of ACE mRNA. TNF-alpha activated p44/42 and p38 MAP kinases, which was inhibited by the specific inhibitors of these kinases, PD98059 or SB202190, respectively. Pretreatment of the cells with SB 202190, or PD 98059 both partly reversed TNF-alpha induced ACE suppression. CONCLUSIONS: TNF-alpha downregulated ACE, which effect was probably mediated by both p44/42 and p38 MAPK pathways. Local downregulation of ACE by TNF-alpha may be a counterbalancing mechanism in inflammatory processes.