Purification and characterization of angiotensin-converting enzyme (ACE) from sheep lung

Abstract

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) in the renin-angiotensin system regulates blood pressure by catalyzing angiotensin I to the vasoconstrictor angiotensin II. In this study, the ACE was purified and characterized from sheep lung. The kinetic properties of the ACE were designated. The inhibition effect of captopril, a specific ACE inhibitor, was determined. ACE was purified from sheep lung using the affinity chromatography method in one step. NHS-activated Sepharose 4 Fast Flow as column filler and lisinopril as a ligand in this method used. The molecular weight and purity of ACE were designated using the SDS-PAGE method. Optimum temperature and optimum pH were found for purified ACE. K M and V max values from Lineweaver-Burk charts determined. The inhibition type, IC 50 , and K i values of captopril on purified ACE were identified. ACE was 6405-fold purified from sheep lung by affinity chromatography in one step and specific activity was 16871 EU/mg protein. The purity and molecular weight of ACE were found with SDS-PAGE and observed two bands at around 60 kDa and 70 kDa on the gel. Optimum temperature and optimum pH were designated for purified ACE. Optimum temperature and pH were found as 40 °C and pH 7.4, respectively. V max and K M values were calculated to be 35.59 (µmol/ min).mL −1 and 0.18 mM, respectively. IC 50 value of captopril was found as 0.51 nM. The inhibition type of captopril was determined as non-competitive from the Lineweaver-Burk graph and the K i value was 0.39 nM. As a result, it was observed in this study that the ACE enzyme can be successfully purified from sheep lungs in one step. Also, it was determined that captopril, which is a specific ACE inhibitor, has a significant inhibitory effect with a very low IC 50 value of 0.51 nM.

Difference in the Inhibitory Effect of Thiol Compounds and Demetallation Rates from the Zn(II) Active Site of Metallo-β-lactamases (IMP-1 and IMP-6) Associated with a Single Amino Acid Substitution

“…As illustrated in Figure C,D, ethyl 3-mercaptopropionate competitively inhibited IMP-1 ( K i = 3.1 ± 0.3 μM) but noncompetitively inhibited IMP-6 ( K i = 13.0 ± 0.7 μM). This behavior has been reported in studies of angiotensin-converting enzyme (ACE) inhibitors. , ACE inhibitors such as captopril, enalaprilat, and ramiprilat are described as competitive inhibitors and have also been found to exhibit mixed and noncompetitive inhibitory effects. Therefore, an inhibitor that binds to the active site may act as a noncompetitive inhibitor, and ethyl 3-mercaptopropionate may exhibit similar behavior.…”

Investigation of inhibition effect of folic acid (vitamin B9) on angiotensin-converting enzyme activity purified from human plasma

“…In a work by Strittmatter et al, the molecule weight with SDS-PAGE of ACE from rat lung and brain corpus striatum was determined to be 175 kDa and 165 kDa, respectively (Strittmatter et al, 1985). In our before works, the molecule weight of the ACE from sheep lungs and sheep kidneys was defined to be 70 and 60 kDa with SDS-PAGE (Aydin et al, 2021;Kiylik et al, 2022). In this study, the molecule weight and purity of the ACE from human plasma were described using SDS-PAGE, and 70 kDa and 60 kDa bands were analyzed on the gel (Figure 1).…”