Angiotensin‐converting enzyme inhibition studies by natural leech inhibitors by capillary electrophoresis and competition assay

Abstract

A protocol to follow the processing of angiotensin I into angiotensin II by rabbit angiotensin-converting enzyme (ACE) and its inhibition by a novel natural antagonist, the leech osmoregulator factor (LORF) using capillary zonal electrophoresis is described. The experiment was carried out using the Beckman PACE system and steps were taken to determine (a) the migration profiles of angiotensin and its yielded peptides, (b) the minimal amount of angiotensin II detected, (c) the use of different electrolytes and (d) the concentration of inhibitor. We demonstrated that LORF (IPEPYVWD), a neuropeptide previously found in leech brain, is able to inhibit rabbit ACE with an IC 50 of 19.8 lM. Interestingly, its cleavage product, IPEP exhibits an IC 50 of 11.5 lM. A competition assay using p-benzoylglycylglycylglycine and insect ACE established that LORF and IPEP fragments are natural inhibitors for invertebrate ACE.Fifty-four percent of insect ACE activity is inhibited with 50 lM IPEP and 35% inhibition with LORF (25 mM). Extending the peptide at both N-and C-terminus (GWEIPEPYVWDES) and the cleavage of IPEP in IP abolished the inhibitory activity of both peptides. Immunocytochemical data obtained with antisera raised against LORF and leech ACE showed a colocalization between the enzyme and its inhibitor in the same neurons. These results showed that capillary zonal electrophoresis is a useful technique for following enzymatic processes with small amounts of products and constitutes the first evidence of a natural ACE inhibitor in invertebrates.Keywords: capillary electrophoresis; invertebrate; leech; natural angiotensin-converting inhibitor.In mammals, angiotensin-converting enzyme (ACE) is a well known zinc-metallopeptidase that converts angiotensin I to the potent vasoconstrictor angiotensin II and degrades bradykinin, a powerful vasodilator, both for regulation of vascular tone and cardiac functions [1,2]. Synthetic substrates were developed for the determination of ACE activity in various biological fluids, mostly human plasma, for the diagnosis of sarcoidosis and other granulomatous diseases [3]. After the successful use of captopril, the first ACE inhibitor in the treatment of hypertension, a number of molecules have been synthesized and used in the treatment of congestive heart failure and for preventing cardiac impairment after myocardial infarction [2][3][4]. The development of this class of anti-hypertensive drugs benefited from structural data on carboxypeptidase active sites [5]. In the last two decades, the ACE gene has been cloned allowing the identification of two isoenzymes: somatic ACE resulting from gene duplication and primarily expressed in endothelial cells, and the germinal or testicular ACE, resulting from the transcription in the male reproductive system from intragenic promoter of a hydrophobic C-terminal peptide for membrane-anchoring, specifically cleaved by a metalloprotease to release soluble forms of both isoenzymes [6]. Recently, a new ACE, termed ACE2, has been characterized [7][8][9]. Th...

Evidence for an Annelid Neuroendocrine System

“…Complementary to these findings, evidences of natural ACE inhibitors have been demonstrated in leeches [22]. In fact, the leech osmoregulator Factor (IPEPYVWD, see the LORF section), a neuropeptide found in both central nervous system and sex ganglia of leeches is involved in water retention control through inhibition of ACE with an IC 50 of 19.8 µM for rabbit ACE.…”

“…Competition assay using p-[ 3 H]benzoylglycylglycylglycine and insect ACE established that LORF and IPEP are natural inhibitors for invertebrate ACE. 54% of insect ACE activity is inhibited with 50 µM d'IPEP and 35% with 25 µM de LORF like [22].…”

Angiotensin‐converting enzyme inhibitory and antioxidant activities of enzymatically synthesized phenolic and vitamin glycosides

“…The importance of ACE inhibitors in the chronic treatment of various cardiovascular diseases such as congestive heart failure [5], myocardial infarction [6], diabetic nephropathy [7] and renal dysfunction [8] is now well established. Several synthetic drugs such as enalapril, captopril, perindopril, ceranopril, ramipril, quinapril and fosinopril [9,10] and certain naturally occurring ‘biologically active peptides’ show ACE inhibitory activities [11,12]. Several naturally occurring glycosides also show ACE inhibitory activities which include phenylpropanoid glycosides from chou wu tong ( Clerodendron trichotomum ) [13] and some Brazilian plants [14], monascus ( Monascus purpureus Went) from fermented soya bean extracts [15] and glycosides from white Forsythia ( Abeliophyllum distichum ) [16].…”