The International Journal of Biochemistry & Cell Biology
Angiotensin converting enzymes from human urine of mild hypertensive untreated patients resemble the N-terminal fragment of human angiotensin I-converting enzyme
Introduction
Angiotensin I-converting enzyme (peptidyl dipeptidase A (ACE) (EC 3.4.15.1; kininase II) is a zinc metalopeptidase that catalyzes the hydrolysis of dipeptides from the carboxyl terminus of polypeptides. Among the most important functions of ACE is its role in the regulation blood pressure; ACE converts angiotensin I (AI) into the potent vasoconstrictor angiotensin II and inactivates the vasodilator nonapeptide bradykinin [1], [2]. ACE has a broad specificity in vitro and it is able to hydrolyze peptides such as substance P and luteinizing hormone-releasing hormone (LH-RH) [3].
ACE exists in at least two different forms: somatic ACE, having an apparent mass of 170 kDa, found at highest concentrations in the lungs and kidneys; and testicular ACE with a molecular mass 110 kDa, exclusively found in the testis [3], [4], [5]. Cloning of cDNA encoding the human somatic ACE indicated that the enzyme is organized in two homologous domains, the N-domain and the C-domain, that contain a potential catalytic site [6]. The testicular form of human ACE has been shown to contain the C-domain of somatic ACE except for a unique 67 residue sequence constituting its NH2-terminus [5], [7].
ACE is found as a membrane-bound enzyme anchored by its hydrophobic carboxyl-terminal segment and as a circulating molecule in body fluids [8].
Deddish et al. [9] reported the detection of a naturally occurring ACE with molecular weight 108 kDa, having only the N-domain active site in ileal fluid. A low molecular weight form of catalytically active angiotensin converting enzyme (91 kDa) has been observed in rat lung homogenate,[10] human lung,[11] hog kidney,[12] and human kidney [13]. ACE was solubilized during purification when treatment with strong alkali was used; in some case, enzymes with low molecular weight (LMW) were detected (86–90 kDa) [14], [15]. Lantz et al. [16] reported three different molecular forms of ACE (150, 80 and 40 kDa) characterized in human cerebrospinal fluid.
Significant ACE activity has been found in urine. Ryan et al. [17] described two forms of ACE from human urine, the first similar to somatic ACE and the second with a molecular weight of 90 kDa, and Kokubo et al. [18] found two different forms of ACE of high molecular weight and another of 140 kDa. Casarini et al. [19] described high and low molecular weight ACEs (190 and 65 kDa, respectively) purified from normal human urine. ACE activity in urine derives from renal tubules [20] and has been used as an index of renal tubular damage. [21] [22]
Despite the description by Deddish et al. of a naturally occurring N-domain ACE in ileal fluid, the function of the N-domain in vivo is until now not clear. Based on the fact that there are many descriptions of ACEs in human urine, but no N-domain ACE has been found, we report in this study on the purification and characterization of a naturally occurring form of human ACE from urine of mild hypertensive untreated patients, consisting of two low molecular weight fragments of ACE that resemble N-domain of ACE. One of them (90 kDa), differing from the ACEs purified from normal human urine, could have an important role in development of hypertension.
Section snippets
Methods
Initially, isolated urine samples from 20 hypertensive patients were analyzed using DEAE-cellulose chromatography. Since the chromatographic profile was similar in the whole population, the subsequent tests have been carried out with a pool of urine (three samples in each). The total number of urine samples collected during the study was provided by 80 patients (male and female). The hypothesis of possible hydrolysis of urinary ACE by proteases was tested. The human urine from mild hypertensive
Results
The urine samples of 20 mild hypertensive patients were submitted to DEAE-cellulose chromatography, isolated and the same profile obtained for each one (data not shown). When the urine of one patient was chromatographed in the presence and absence of protease inhibitors, the elution profile was not modified (data not shown).
Human urine from mild hypertensive untreated patients was chromatographed on a DEAE-cellulose column with linear gradient elution, and two peaks with ACE activity were
Discussion
The human urine from one mild untreated hypertensive patient collected in the presence and absence of protease inhibitors presented the same profile when submitted to a DEAE-cellulose chromatography. The results obtained suggest that the ACEs found in urine were not a product of hydrolysis by proteases during the collect.
Using DEAE-cellulose chromatography with linear gradient elution, we found in urine of mild untreated hypertensive patients the same two peaks described for urine from normal
Acknowledgements
D.E. Casarini received research support from ‘Fundação de Amparo a Pesquisa do Estado de São Paulo’ (grant number 95/9168-1). The authors are grateful to Dr Luiz Juliano, head of Biophysics Division, for the sequence of enzymes.
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