Ropivacaine HCl br Introduction Mammalian proteases comprise
Introduction Mammalian proteases comprise both the peptidases or exopeptidases, which act at the N- or C-terminal positions of polypeptides, and proteinases or endopeptidases, which are capable of cleaving peptide bonds in the central regions of polypeptides. The endopeptidases are classified in four distinct groups: serine, thiol, carboxyl and metallo proteinases, on the basis of the chemical nature of the groups responsible for the catalytic activity (Barrett, 1980). The first two kininases designated kininase I and kininase II have been described as exopeptidases, and these proteins are identical to carboxipeptidase N and angiotensin I-converting enzyme (ACE), respectively. Carboxipeptidase N removes the C-terminal arginine from BK to form des-RBK, and the angiotensin I-converting enzyme sequentially removes the dipeptides F-R and S-P to form AII and then the pentapeptide R1-F5 (Erdos & Skidgel, 1990; Skidgel, Johnson, & Erdos, 1984; Skidgel, Schulz, Tam, & Erdos, 1987). Similarly, endopeptidases with kininase activity also hydrolyzed peptides other than kinins, as described for prolyl endopeptidase (E.C. 188.8.131.52) from lamb kidney (Koida & Walter, 1976), from human and rabbit kidney (Almenoff & Orlowski, 1983; Gafford, Skidgel, Erdos, & Hersh, 1983), from cytosolic fractions of rabbit Ropivacaine HCl (Hayashi et al., 2000) and from rat and human liver (Molina, Carmona, Kouyoumdjian, & Borges, 2000). The most extensively studied kininases from human urine were kininase I and kininase II (Marinkovic, Ward, Erdos, & Mills, 1980; Ryan, Chung, Martin, & Ryan, 1978). More recently, besides these two enzymes, Skidgel et al. (1987) purified a neutral endopeptidase; and Casarini et al., Di Marco et al. and Quinto et al. (Casarini, Alves, Araujo, & Stella, 1992; Casarini, Stella, Araújo, & Sampaio, 1993; Di Marco et al., 1998, Quinto et al., 1999 showed that four other endopeptidases occur in human urine: a post-proline cleaving enzyme, a metallo endopeptidase and two serine proteinases. Because of the importance of peptides like bradykinin, angiotensin II, substance P (SP), vassopresin (ADH), natriuretic atrial function (FNA) and others in renal function, in a previous work, we studied the localization of the enzymes that metabolized them along the rat nephron, specifically BK (Quinto, 2000); and also the excretion of kininases in human urine (Casarini et al., 1992, Casarini et al., 1993; Casarini, Boim, Stella, & Schor, 1999b; Quinto et al., 1999). In human urine we found kininases characterized as serine endopeptidases, which were characterized as chymotrypsin-like proteases and were located in the distal tubule (Quinto, 2000).
Materials and methods
Results Enzyme STH2 was purified 202.0 times as described previously (Casarini et al., 1992). Enzyme SH1 was purified 10 times until homogeneity by DEAE-cellulose chromatography and affinity chromatography on a Sepharose Mercurial column (Fig. 1), presenting a specific activity of 2945.0 and 101.2uMmin−1mg−1, respectively (Table 1). A molecular weight of 45kDa was obtained for H1 by polyacrylamide gel electrophoresis under dissociation conditions (Fig. 2), differing from STH2, which presents a molecular weight of 60kDa. When the hydrolysis products of SH1 and STH2 acting on BK were determined by HPLC, only R1-F5 and S6-R9 hydrolysis products indicating that the two enzymes hydrolyzed the peptide bond F5-S6. The same F5-S6 hydrolyses in the BK molecule is also hydrolyzed in LBK, and MLBK. Both enzymes are serine-proteinases that differ from other peptidase bonds of BK, that belong to different enzyme classes, such as metallo-proteinases (E.C. 184.108.40.206) of rat brain. Fig. 3 shows the chromatographic profile indicating the hydrolysis product of fluorogenic substrate AbzBKQEDDnp. Endopeptidase H1 cleaves peptide bond R9-Q10 and enzyme STH2 the F8-R9 peptide bond. These results were confirmed by mass spectroscopy and the chromatograms can be observed the Fig. 4.