An improved fluorometric assay of rat serum and plasma converting enzyme.

Abstract

SUMMARYThe most sensitive nonradiometric routine assay for angiotensin-converting enzyme (ACE) activity uses fluorometry to detect His-Leu released from Hip-His-Leu. Our results indicate that, in contrast to human serum, rat serum and plasma contain large and variable amounts of dipeptidase activity that lead to a subestimation of the ACE activity measured in 0.1 M potassium phosphate buffer, pH 8.3, containing 0.3 M NaCI, the most commonly used assay for human serum and tissue ACE. We describe and validate an assay for 1 to 10 /xL rat and human serum or plasma using 5 mM Hip-His-Leu in 500 fiL of 0.4 M sodium borate buffer, pH 8.3, containing 0.9 M NaCI at 37°C that reduced the subestimation error to ^ 3% (rat serum) and ^ 0.1% (human serum) and increased the ACE activity twofold to threefold. The K m and V,^ are reported for rat serum ACE

N-Domain Angiotensin I-Converting Enzyme With 80 kDa as a Possible Genetic Marker of Hypertension

“…21 Both enzymes (S-1 and S-2 ACEs) were inhibited by the competitive inhibitors enalapril and captopril on the order of 2 mol/L and also by 1.6 mol/L of EDTA, as described in the literature for ACE purified from different sources. 19,44,45 S-1 and S-2 ACE have a K m of 10 Ϫ3 mol/L with HHL used as substrate, similar to the K m described in the literature for rat serum, 46 human kidney, 17 human urine, 47,19 and mesangial cells. 40 The activity of purified ACE from urine of SHR was maintained at temperatures Ͻ4°C and 37°C but was decreased at high temperatures, similar to the results described in the literature by Nishmura et al 48 and Andrade et al 40 AI and BK, the physiologically important substrates of ACE, were hydrolyzed in a similar manner by ACE S-1 and S-2 and by all purified ACE forms in this study.…”

Biochemical evaluation of the renin-angiotensin system: the good, bad, and absolute?

“…ACE plays a key role in the formation of ANG II, but the peptidase metabolizes other peptides including bradykinin, substance P, acetyl-SerAsp-Lys-Pro, and ANG-(1-7) (16,145). ACE activity is typically measured by small peptide substrates such as hippurylHis-Leu or furylacrylol-Phe-Ala-Gly-Gly in a buffer containing chloride (10 -200 mM) and zinc (1-100 M) for optimal peptidase activity (24,122). Quenched fluorescent substrates Mca-Ser-Phe-Leu-Tyr-DNP or Mca-Tyr-Val-Ala-Arg-Ala-PheLys-DNP have also been applied for ACE measurement; however, these are not specific substrates and appropriate assay conditions that include an inhibitor cocktail to prevent nonspecific hydrolysis by other peptidases must be considered, as well as separate samples sets for an ACE inhibitor (i.e., captopril, lisinopril) to confirm assay specificity (81).…”

Reduced cortical renal GLUT1 expression induced by angiotensin-converting enzyme inhibition in diabetic spontaneously hypertensive rats

“…ACE activity was determined using the fluorometric assay (24). One kidney was quickly harvested, rinsed, blotted and homogenized in 0.4 M sodium borate buffer, pH 7.2.…”