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.     

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.     

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.     

A continuous spectrophotometric assay for angiotensin converting enzyme.

Furanacryloyl tripeptides conforming to the known substrate specificity of the angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1) have been employed to provide a continuous spectrophotometric assay for this peptidase in the visible region. The assay is based on a blue shift of the absorption spectrum that occurs upon hydrolysis of the substrate to produce a furanacryloyl-blocked amino acid and a dipeptide. Of the various furanacryloyl tripeptides tested, furanacryloyl-l-phenylalanylglyeylglycine exhibits the most suitable characteristics for routine assays of angiotensin converting enzyme.     

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.     

Ketomethylureas. A new class of angiotensin converting enzyme inhibitors

The design rationale for a new series of tripeptide derived angiotensin converting enzyme (ACE) inhibitors, which we term "ketomethylureas", is described. Analogs of tripeptide substrates (i.e. N-benzoyl-Phe-Ala-Pro) in which the nitrogen atom of the scissile amide bond and the adjacent asymmetric carbon atom of the penultimate amino acid residue are formally transposed give rise to this novel class of inhibitors. The most potent ketomethylureas inhibit ACE with I50 values in the nM range.     

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.     

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.     

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.     

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.     

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.     

The history of inhibitors of angiotensin converting enzyme.

This review paper by Sir John Vane, The Nobel Prize Laureate for the first time reveals the insides of discovery of inhibitors of angiotensin converting enzyme (ACE-1), presently known as important drugs for the treatment of hypertension, congestive heart failure and coronary artery disease.