The activity of angiotensin I-converting enzyme (ACE) was determined in tubular fluid collected from several portions of the rat nephron and urine and in total and efferent arteriolar blood using hippuryl-L-His-Leu as substrate. ACE activity decreased 30% from the pre- to the postglomerular arterioles (P < 0.001), suggesting a role of the glomerulus in ACE clearance. The enzyme activity was found to be present throughout the rat nephron. However, the highest activities were found in the proximal tubule and urine (0.692 +/- 0.007 and 1.05 +/- 0.015 pmol x microl(-1) x min(-1), respectively). Compared with other segments, ACE activity decreased from the initial portion of the proximal tubule to the distal nephron and increased again in the urine. Along the proximal tubule, ACE was secreted and degraded and/or reabsorbed and then secreted again into the collecting duct; no ACE activity was found in the late distal tubule, but a high level was detected in the urine, indicating a potential physiological role in the inactivation of the kinins formed by kallikrein beyond the connecting tubules. Moreover, the possible role of mesangial cells (MC) in the decrease of intraglomerular ACE was also evaluated. The analysis of ACE gene showed that MC in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on MC function.

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The activities of serine endopeptidase, prolyl endopeptidase and neutral endopeptidase were determined in tubular fluid collected from several portions of the rat nephron as well as in urine. The enzyme activities were measured by HPLC using bradykinin (BK) as substrate. Free residual peptides of BK obtained by the action of these enzymes on the locally produced BK were also determined. The endopeptidase activities were found to be present throughout the nephron. Equimolar fragments of BK were detected in the early proximal tubule (Arg1-Pro7, Phe8-Arg9, Arg1-Gly4, Phe5-Arg9, and BK), late proximal tubule (Arg1-Phe5, Arg1-Pro7, Gly4-Pro7, Gly4-Arg9, and BK), late distal tubule (Arg1-Gly4, Phe5-Arg9, Arg1-Phe5, Ser6-Arg9, Gly4-Arg9, BK, and [des-Arg9]BK) and urine (Phe8-Arg9, Phe5-Arg9, Arg1-Phe5, Ser6-Arg9, Arg1-Pro7, Gly4-Pro7, Gly4-Arg9, BK, and [des-Arg9]BK). Our data suggest that the endopeptidases and exopeptidases are secreted by the nephron. Early proximal tubules secrete angiotensin converting enzyme and neutral endopeptidase, differing from late distal tubules that produce prolyl endopeptidase, serine endopeptidase, carboxypeptidase, and also neutral endopeptidase. All enzymes detected along the rat nephron were found in the urine. The existence of endopeptidases and carboxypeptidase in the distal nephron may have a potential physiological role in the inactivation of the kinins formed by kallikrein in the kidney and also in the inactivation of additional peptides other than BK.

SUMMARY Plasma levels of kininogen, kallikrein, and prekalllkrein were determined in patients with malignant hypertension (MH) and compared to nonnotensive controls (NC) and patients with mild to moderate essential hypertension (EH). Also, a recently described kinin potentiating factor (KPF) was estimated by dividing the value of kininogen determined by trypsln (Kgn-Try) by that of kininogen determined by human urinary kallikrein (Kgn-HuUK). No significant alterations were detected among plasma values of prekallikrein and kallikrein of MH as compared to NC. However, Kgn-HuUK values were significantly lower in MH (1.9 ± OJ MgLBK/ml) as compared to EH and NC (2.7 ± 0.1 MgLBK/ml and 3.0 ± 0.2 MgLBK/ml respectively,p < 0.05). Furthermore, KPF values were also low (p < 0.05) in MH (1.6 ± 03) when compared with similar values obtained in EH and NC (3.0 ± 0.2 and 2.8 ± 0.1, respectively). Adequate control of blood pressure levels for 90 days in MH group caused no significant alterations in plasma levels of kininogen and KPF. It is suggested that diminished kininogen levels as well as a decrease in a kinin potentiation KPF that is generated in plasma by trypsln may be involved in the patbogenesis of human malignant hypertension. which several humoral alterations ofvasopressor systems like renin-angiotensin system, 1 catecholamines,* and more recently vasopressin 8 have been implicated in the pathogenesis of the vascular lesion. The kallikrein-kinin system has been implicated in several hypertensive states, since kinins are vasodilators 4 and also influence water and electrolyte excretion.8 -• A defective kallikrein-kinin system has been recently suggested in malignant, but not benign, hypertension in the rat.'In the present work we evaluated the levels of some plasma precursors of kallikrein-kinin system in malignant hypertension, compared to "benign" hypertension and normal subjects, by studying plasma levels of kininogen, kallikrein, and prekallikrein. Also, a newly described kinin potentiating factor* was estimated in these subjects since it is known that several potentiating substances of both exogenous' 110 or endogenous origin 11 ' " may influence the activity of the kallikrein-kinin system.

A NEP-like enzyme was purified from human urine. Based on the mol. wt of the isolated NEP-like enzyme, it was concluded that this enzyme was produced in the kidney. In the kidney, this enzyme may cleave the kinins filtered through the glomerulus and also the kinins produced in the distal nephron. An internally quenched fluorogenic peptide, Abz-FDQ-EDDnp, was selectively hydrolysed by NEP-like and by recombinant NEP.

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The isolation procedure for horse urinary kallikrein was considerably improved by the introduction of two new purification steps: a) removal of mucoproteins and concentration of the urine by ultrafiltration and b) affinity chromatography on benzamidine-Sepharose conjugate. The homogeneity of the enzyme preparations, regarding their protein moiety, was demonstrated by: 1)3) a single symmetric peak on DEAE-Sephadex chromatography, with constant values for 28oM26o ratios, esterolytic and amidolytic specific activities; a single band, although dispersed, on gelelectrophoresis at pH 8.3, also in the presence of sodium dodecyl sulfate, and a unique sequence for the six amino-terminal residues.The isolated enzyme was shown to be a single chain glycoprotein ( -kallikrein), similar to human urinary and porcine-pancreatic kallikreins regarding the protein moiety molecular mass, amino-acid composition, and partial aminoterminal sequence; differences were found in their total sugar content and even more conspicuously in their carbohydrate composition. In contrast to porcine pancreatic ß-kallikrein, horse urinary kallikrein was not substrate-activated and unlike other -kallikreins, did not present the biphasic time-course in benzoyl-L-arginine ethyl ester hydrolysis. The specificity constants (^cat/^m ) f°r ester an< 3 4-nitroanilide substrates were lower for horse urinary than for pancreatic 0-kallikrein and as observed with the latter enzyme, were affected by NaCl. Pferdeharn-Kallikrein, I: Vollständige Reinigung und CharakterisierungZusammenfassung: Die Isolationsmethode für l) Pferdeharn-Kallikrein wurde durch zwei zusätz-liche Reinigungsschritte beträchtlich verbessert: a) Entfernung der Mucoproteine und Einengen des Urins durch Ultrafiltration; b) Affinitäts-chromatographie an einem Benzidin-SepharoseKonjugat. Der Proteinanteil der Enzympräpara-2) tion war homogen. Dafür sprechen drei Kriterien:Mit DEAE-Sephadex-Chromatographie wurde nur ein einziger symmetrischer Gipfel erhalten, dessen Fraktionen konstante Werte für das^4 2 8oA426o'Verhältnis sowie die spezifischen Aktivitäten der Esterolyse und Amidolyse ergaben; Gelelektrophorese bei pH 8.2 lieferte nur eine, wenn auch verbreiterte Bande, auch in Gegenwart von Natriumdodecylsulfat;

Angiotensin I-converting enzyme (ACE) plays a key role in the renin-angiotesin aldosterone cascade. We analysed the secondary structure and structural organization of a purified 65kDa N-domain ACE (nACE) from Wistar rat mesangial cells, a 90 kDa nACE from spontaneously hypertensive rats and a 130 kDa somatic ACE. The C-terminal alignment of the 65 kDa nACE with rat ACE revealed that the former was truncated at Ser(482), and the sequence of the 90 kDa nACE ended at Pro(629). Protein's secondary structure consisted predominantly of alpha-helices. The 90 and 65 kDa isoforms were the most stable in guanidine and at low pH, respectively. Enzymatic activity decreased with loss in secondary structure, except in the case of guanidine HCl where the 90 kDa fragment loses its secondary structure faster than its enzymatic activity. We identified and characterized the activity and stability of these isoforms and these findings would be helpful on the understanding of the role of nACE isoforms in hypertension.