Antonio C.M. Camargo

A convenient manual trinitrobenzenesulfonic acid method for monitoring amino acids and peptides in chromatographic column effluents

Abstract The trinitrobenzenesulfonic acid (TNBS) method of R. Fields (1971, Biochem. J. , 124 , 581–590) has been modified for the manual detection of amino acids and peptides in chromatographic column effluent by changing the reaction conditions to 1 m m TNBS in 0.4 m potassium borate buffer, pH 9.2, at room temperature for 30 to 50 min. The reaction with amines and the spontaneous hydrolysis of TNBS are stopped by neutralization to pH 6.25 with sodium monobasic phosphate (0.33 m ). Sodium sulfite (3 m m ) is added to increase the absorptivity of the product. The TNBS reagent blank is less than 0.100 A 420 after 50 min of reaction. Since the Δ A 420 of the reagent blank is ∼0.002/min before quenching the reaction, and zero afterward, the time required for reaction and for absorbance measurements need not be controlled precisely. Alkaline hydrolysis of peptides is carried out prior to detection to increase the sensitivity of the method. This procedure is convenient for the manual determination of 5 to 100 nmol of amino acids in the 50–100 samples required to define a chromatographic elution profile.

Bradykinin-potentiating peptides: Beyond captopril

The identification of novel endogenous and exogenous molecules acting in the complex mechanism of regulating the vascular tonus has always been of great interest. The discovery of bradykinin (1949) and the bradykinin-potentiating peptides (1965) had a pivotal influence in the field, respectively, in understanding cardiovascular pathophysiology and in the development of captopril, the first active-site directed inhibitor of angiotensin-converting enzyme, and used worldwide to treat human hypertension. Both discoveries originated from studies of envenoming by the snake Bothrops jararaca. The aim of the present article is to reveal that the snake proline-rich oligopeptides, known as bradykinin-potentiating peptides, are still a source of surprising scientific discoveries, some of them useful not only to reveal potential new targets but also to introduce prospective lead molecules for drug development. In particular, we emphasize argininosuccinate synthetase as a new functional target for one of bradykinin-potentiating peptides found in B. jararaca, Bj-BPP-10c. This decapeptide leads to argininosuccinate synthetase activation, consequently sustaining increased nitric oxide production, a critical endogenous molecule to reduce the arterial blood pressure.

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

Abstract: The subcellular and regional distribution of endooligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin‐containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane‐associated form of endo‐oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Sub‐cellular fractionation showed that ∼17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3‐([3‐cholamidopropyl]dimethylammonio)‐1‐propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three‐ to 10‐fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo‐oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo‐oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo‐oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide‐containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo‐oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.

Screening for Rabbit Brain Neuropeptide-metabolizing Peptidases. Inhibition of Endopeptidase B by Bradykinin Potentiating Peptide 9a (SQ 20881)

Abstract: Neutral thiol‐activated peptidases present in the pH 5‐soluble fraction of rabbit brain (separated by step‐elution chromatography on diethylaminoethyl cellulose) were screened for the hydrolysis of bradykinin, Lysbradykinin, Met‐Lys‐bradykinin, angiotensin I, angiotensin II, substance P, luteinizing hormone‐releasing hormone (LH‐RH) and neurotensin by bioassay. The column effluent was monitored for bradykinin inactivation and arylamidase activity and combined in six pools on the basis of bradykinin inactivation. The pools were characterized by determining the peptide fragments and amino acids released from bradykinin with an amino acid analyzer. Pools 1 through 3 contained 80% of the kininase activity and essentially all of the endopeptidase A and B activity, whereas pools 4 through 6 accounted for 98% of the recovered arylamidase activity. Bradykinin, angiotensin I, angiotensin II and substance P were inactivated by all the pools, whereas LH‐RH and neurotensin were inactivated by pools 3 and 4 and pools 3, 4 and 5, respectively. These data show that rabbit brain contains peptidases having some selectivity for the inactivation of neuropeptides. Endopeptidase B purified from pool 3 is inhibited by bradykinin‐potentiating peptide 9a (BPP9a SQ 20881) (Glu‐Trp‐Pro‐Arg‐Pro‐Gln‐Ile‐Pro‐Pro), a competitive inhibitor of the hydrolysis of bradykinin (Km= 3.5 ± 10−5m, Ki= 3 ± 10−6m) which also completely inhibits the inactivation of LH‐RH.

The Central Nervous System as Target for Antihypertensive Actions of a Proline-Rich Peptide from Bothrops jararaca Venom

Pyroglutamyl proline‐rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj‐PROs), are the first described naturally occurring inhibitors of the angiotensin I‐converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj‐PRO‐10c (

Endooligopeptidase A activity in rabbit heart: generation of enkephalin from enkephalin containing peptides

Two endopeptidases displaying similar specificities towards peptide hormone substrates but differing in molecular size have been identified in rabbit heart and isolated by a combination of ion-exchange chromatography, gel filtration and preparative gel electrophoresis. These two enzymes share several properties with the previously described rabbit brain endooligopeptidase A. They were shown to produce, by a single peptide bond cleavage, [Met5] enkephalin and [Leu5]enkephalin from small enkephalin containing peptides. They also hydrolyze the Phe5-Ser5 bond of bradykinin and the Arg8-Arg9 bond of neurotensin. Characteristically, the activity of both low and high Mr enzymes is restricted to oligopeptides. Both forms of heart endooligopeptidase A are inhibited by antibodies raised against the brain enzyme. When electrophoresed in SDS-polyacrylamide gel under denaturing conditions, the low Mr heart enzyme shows a major band of Mr = 73,000, comparable in size to the brain enzyme. The SDS-PAGE of the high and low Mr enzymes analyzed by immunoblotting with an antibody raised against low Mr brain endooligopeptidase A, showed a major antigen band corresponding to Mr = 72,000. In addition, immunoblotting has also demonstrated that a monoclonal antibody antitubulin reacts with a polypeptide corresponding to Mr = 50,000 present in the purified high Mr endooligopeptidase A. Both enzymes are activated by dithiothreitol and inhibited by thiol reagents, but are not affected by leupeptin, DFP or EDTA, thus indicating that they should be classified as nonlysosomal cysteinyl-endooligopeptidase A.

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B2 and M1 muscarinic acetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (<EKWAP), a member of this structurally related peptide family, was essential for the development of captopril, the first site-directed ACE inhibitor used for the treatment of human hypertension. Nowadays, more Bj-PROs have been identified with higher ACE inhibition potency compared to Bj-PRO-5a. However, despite its modest inhibitory effect of ACE inhibition, Bj-PRO-5a reveals strong bradykinin-potentiating activity, suggesting the participation of other mechanisms for this peptide. In the present study, we have shown that Bj-PRO-5a induced nitric oxide (NO) production depended on muscarinic acetylcholine receptor M1 subtype (mAchR-M1) and bradykinin B₂ receptor activation, as measured by a chemiluminescence assay using a NO analyzer. Intravital microscopy based on transillumination of mice cremaster muscle also showed that both bradykinin B₂ receptor and mAchR-M1 contributed to the vasodilatation induced by Bj-PRO-5a. Moreover, Bj-PRO-5a-mediated vasodilatation was completely blocked in the presence of a NO synthase inhibitor. The importance of this work lies in the definition of novel targets for Bj-PRO-5a in addition to ACE, the structural model for captopril development.

Activity of Pz-peptidase and endo-oligopeptidase are due to the same enzyme

During purification of endo-oligopeptidase from rabbit heart, activities cleaving bradykinin, a substrate of endo-oligopeptidase, and Mcc-Pro-Leu-Gly-Pro-D-Lys(Dnp), a substrate of Pz-peptidase, were found in the same fractions. The hydrolysis of both substrates was inhibited by antisera against endo-oligo-peptidase and Pz-peptidase, and reversibly inhibited by 1, 10-phenanthroline. The purified enzyme hydrolysed Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys, another substrate of Pz-peptidase. Purified Pz-peptidase from rabbit muscle degraded bradykinin and was inhibited by an antiserum against endo-oligopeptidase.

Pharmacological effects and metabolism of neurotensin and bradykinin in the isolated rat uterus

Neurotensin (NT) and bradykinin (BK) were found to cause contractions of isolated rat uterus preparations. In 97% of the experiments, acute tachyphylaxis followed soon after the initial administration of NT. Interrelation between the oxytocic effects of NT and BK was not observed. Among the NT fragments studied, only NT-(9-13) had an oxytocic effect (less than 1.0%). All NT fragments tested induced tachyphylaxis to NT regardless of their efficacy. Using HPLC analysis, NT but not BK was found to be degraded by the intact rat uterus. A major involvement of a carboxydipeptidase cleaving at Tyr11-Ile12 is suggested. Carboxyl-blocked neurotensinamide (NT-NH2) was found to be resistent to proteolysis and not develop tachyphylaxis. No cross-tachyphylaxis was observed with NT or NT-(1-11). The results suggest the existence of different receptors for NT and BK in the uterus, as well as the existence of different receptors or receptor states that interact with NT or NT-NH2 in the rat uterus.

The Snake Venom Peptide Bj-PRO-7a Is a M1 Muscarinic Acetylcholine Receptor Agonist

Proline‐rich peptides from Bothrops jararaca venom (Bj‐PRO) were characterized based on the capability to inhibit the somatic angiotensin‐converting enzyme. The pharmacological action of these peptides resulted in the development of Captopril, one of the best examples of a target‐driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj‐PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensin‐converting enzyme‐independent mechanisms. Here, we show that Bj‐PRO‐7a (