Purification and Properties of Angiotensin-Converting Enzyme from Hog Lung

Abstract

Angiotensin-converting enzyme was purified 1500-fold from a homogenate of hog lungs. The purification procedure included fractionation with ammonium sulfate, inactivation of contaminating enzymes at pH 4.7, batch treatment with CM-cellulose, and chromatography on columns of DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The enzyme was assayed with hippurylglycylglycine and, after pH 4.7 was established, with both hippurylglycylglycine and angiotensin I as substrates. The ratio of activity toward the two substrates remained constant throughout the later stages of purification. The final enzyme preparation, on analytical disc gel electrophoresis, showed one major protein band with the expected ratio of activities toward both substrates. The molecular weight was estimated to be approximately 300,000 by gel filtration. The pH optimums with hippurylglycylglycine and angiotensin I as substrates were 8.4 and 7.5, respectively, and chloride ion was required for optimal activity of the enzyme with both substrates. Hydrolysis of both substrates was inhibited by ethylenediaminetetraacetic acid (EDTA) or the pentapeptide pyroglutamyllysyltryptophanylalanylproline. It would, therefore, appear that a single enzyme, purified in the described manner, hydrolyzes both angiotensin I and hippurylglycylglycine.

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

“…This served as a major impediment to detailed characterization, and was finally solved when model tripeptide substrates such as hippuryl-His-Leu, hippurylGly-Gly, and benzyloxycarbonyl (Z)-Phe-His-Leu were developed (Cushman and Cheung, 1969;Piquilloud et al, 1970;Yang et al, 1970). These and similar assays were used to purify ACE first from pig lung (Dorer et al, 1972;Nakajima et al, 1973). By 1980, ACE had been purified from the lung of pig, rabbit, dog, and cow, and from the sera of rabbit and humans (Soffer, 1981).…”

Mechanical Stretch/Relaxation Stimulates a Cellular Renin-Angiotensin System in Cultured Rat Mesangial Cells

“…Control and stretch/relaxation mesangial cells were assayed for ACE-like activity by modification of the method of Dorer et al [23]. The cells were washed five times with buffer (0.05  M Hepes, pH 8.0, 0.1% sodium azide) and then incubated with 200 µl buffer containing [ 3 H]hippuryl-glycyl-glycine (20 mCi/mmol) for 60 min at 37°C in the presence or absence of captopril (10 µ M ).…”

“…Three hundred microliters of the upper layer was removed and counted in a liquid scintillation counter (Beckmann LS 3801). The ACE-like activity was calculated as: units = [400 nmoles/(total counts) ×(60 min) × (0.01 ml)] × [cpm sample – cpm blank] × 3.3 [23]. …”

Release of angiotensin converting enzyme by the lung after Pseudomonas bacteremia in sheep.

“…A majority of estimates are in the range of 129,000 to 140,000 daltons (39)(40)(41)(42). Other laboratories have suggested that ACE is an even larger molecule (43,44) although the measured weights (300,000 and 450,000 daltons) might result from aggregation of a smaller -150,000-dalton subunit. We found ACE activity in fractions representing molecules of 145,000 and 299,000 daltons.…”