Abstract
A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambda ex = 320 nm and lambda em = 420 nm) at 37 degrees C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 microM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 microM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 microM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.
Highlights
Angiotensin-I-converting enzyme (ACE, EC 3.4.15.1) is a dipeptidyl carboxypeptidase whose best-known physiological function is to cleave the C-terminal dipeptide of angiotensin I to produce the potent vasoconstrictor angiotensin II [1]
Lisinopril, trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene (E64), phenyl-methylsulfonyl fluoride (PMSF), pepstatin, N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK), N-tosyl-lysyl-chloromethyl ketone (TLCK), and ortho-phthaldialdehyde were from Sigma
We reported the development of highly efficient fluorogenic substrates for the enzyme with the general sequence Abz-peptidyl-K(Dnp)P-OH [23]
Summary
Angiotensin-I-converting enzyme (ACE, EC 3.4.15.1) is a dipeptidyl carboxypeptidase whose best-known physiological function is to cleave the C-terminal dipeptide of angiotensin I to produce the potent vasoconstrictor angiotensin II [1]. ACE inactivates the vasodilator peptide bradykinin by the sequential removal of two C-terminal dipeptides [2]. ACE is expressed as a somatic isoform (150-180 kDa) in endothelial, epithelial and neuroepithelial cells and as a smaller isoform (90-110 kDa) only in germinal cells in the testes. Somatic ACE is an ectoenzyme attached to the cell membrane composed of two highly homologous domains, N- and C-domains, each possessing a functional active site [3,4]. The germinal form of ACE is a membrane-bound enzyme which contains a single active site corresponding to the C-domain of the somatic form [5,6]. Plasma or soluble ACE is derived from proteolytic shedding from the cell membrane [7,8]
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