Amintas Fabiano de Souza Figueiredo

Intramolecularly quenched fluorogenic peptide substrates for human renin

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe8 to His13, His6 to His13, and Tyr4 to His13 of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC50 = 7.8 x 10(-9) M), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707-2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 microGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.

Rat uterine contraction by kallikrein and its dependence on uterine kininogen.

Smooth muscle responses to kallikrein (EC 3.4.21.8) are generally considered to result from kinin formation. In the present study, this premise was reexamined with respect to the isolated rat uterus. Rat submandibular gland kallikrein produced contractions of the rat uterus but the contractions disappeared after successive additions of the same dose of the enzyme to the preparation. Kallikrein-induced rat uterine contractions as well as bradykinin-induced contractions were enhanced by rat submandibular gland bradykinin potentiating factor. The incubation of kallikrein with rat uterine extract in the presence of a kininogen-depleted rat uterus produced kinin which elicited the uterine contraction. An extract from uterine horns previously depleted of kininogen was prepared. Incubation of this extract with kallikrein in a bath containing a kininogen-depleted rat uterus did not evoke uterine contraction. The incubation of four rat uterine horns with kallikrein in the presence of a uterine horn previously depleted of kininogen elicited contractions of the depleted uterus. These results suggest that the contraction produced by kallikrein involves kinin release from the uterus.

Microalbuminuria in Nondiabetic and Nonhypertensive Systolic Heart Failure Patients

The American Diabetes Association and the National Kidney Foundation define microalbuminuria as an albumin (microg)/creatinine (mg) ratio (ACR) between 30 and 300 microg/mg regardless of sex. Microalbuminuria is associated with increased cardiovascular risk. The authors evaluated the prevalence of microalbuminuria in nondiabetic and nonhypertensive systolic heart failure (SHF) patients. Twenty-seven SHF patients, 18 years and older, with New York Heart Association functional classes II through IV and left ventricular ejection fraction < or =40%, who were nondiabetic and nonhypertensive and not receiving angiotensin-converting enzyme inhibitors, were selected. Twenty-seven healthy individuals, paired according to sex, ethnicity, and age, were used as controls. Early-morning midstream urine was used. Data are expressed as medians. Excretion of albumin in SHF patients (39 microg/mL urine) was significantly higher than in controls (26 microg/mL urine). Creatinine excretion was not significantly different between patients and controls. ACR was significantly higher in patients (54 microg/mg) than in controls (24 microg/mg). The results indicate that microalbuminuria was significantly present in nondiabetic and nonhypertensive SHF patients.

Increased tissue kallikrein amidase activity in urine of patients with type 1 diabetes under insulin therapy, and in those with gestational diabetes mellitus not under insulin therapy.

Human tissue kallikrein (hK1) is reduced in hypertension, cardiovascular and renal diseases. There is little information on the participation of hK1 in type 1 diabetes mellitus (DM), type 2 DM, and gestational diabetes mellitus (GDM), respectively. The aim of this study was to evaluate the roles of insulin and hyperglycemia on urinary hK1 activity in type 1 DM and in GDM. Forty-three type 1 DM patients (5-35 years, disease duration ≤ 5years, receiving insulin, HbA(1c)>7.6%) were selected. Forty-three healthy individuals, paired according to gender and age, were used as controls. Thirty GDM patients (18-42 years, between the 24th and 37th week of pregnancy, recently diagnosed, not under insulin therapy) were also selected. Thirty healthy pregnant (18-42years, between the 24th and 37th week of pregnancy) and 30 healthy non-pregnant women (18-42years) were selected as controls. Random midstream urine was used. hK1 amidase activity was estimated with D-Val-Leu-Arg-Nan substrate. Creatinine was determined by Jaffes method. hK1 specific amidase activity was expressed as μM/(minmg creatinine) to correct for differences in urine flow rate. hK1 specific amidase activity was significantly higher in the urine of type 1 DM than in controls, and in the urine of GDM patients than in healthy pregnant women and healthy non-pregnant women, respectively. The data suggest that hyperglycemia, rather than insulin, is involved in the mechanism of increased hK1 specific amidase activity in both type 1 DM and GDM patients, respectively.

Perfil peptídico de hidrolisados enzimáticos de leite em pó desnatado

Sete hidrolisados de leite em po desnatado foram preparados, visando a producao de hidrolisados proteicos, como suplemento dietetico para fenilcetonuricos. Para isso, utilizaram-se uma protease do Aspergillus oryzae (AO), a papaina (PA) e a pepsina (PE), isoladamente ou em associacao, em diferentes relacoes enzima:substrato (E:S), sendo o tempo total de reacao de 5 h e a temperatura de 50 oC. Adotou-se como criterio para a avaliacao nutricional dos hidrolisados, o estudo da distribuicao dos peptideos nas amostras, de acordo com o tamanho da cadeia. Inicialmente, os hidrolisados foram fracionados por cromatografia liquida de alta eficiencia de exclusao molecular (SE-HPLC) e, para a quantificacao dos componentes das fracoes cromatograficas, empregou-se o metodo rapido da Area Corrigida da Fracao (ACF). Os resultados indicaram que, de modo geral, a acao isolada das tres enzimas foi mais vantajosa em relacao ao perfil peptidico em comparacao com as associacoes estudadas. Entre todas as preparacoes testadas, a acao isolada de AO e de PA, e a associacao destas enzimas (relacao E:S de 1% e 2%, respectivamente) produziram hidrolisados com perfis peptidicos semelhantes, mais adequados nutricionalmente, ou seja, com maior teor de di- e tripeptideos e menor proporcao de aminoacidos livres.

The effect of cations on the amidase activity of human tissue kallikrein: 1-linear competitive inhibition by sodium, potassium, calcium and magnesium. 2-linear mixed inhibition by aluminium.

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide by human tissue kallikrein (hK1) was studied in the absence and in the presence of increasing concentrations of the following chloride salts: sodium, potassium, calcium, magnesium and aluminium. The data indicate that the inhibition of hK1 by sodium, potassium, calcium and magnesium is linear competitive and that divalent cations are more potent inhibitors of hK1 than univalent cations. However the inhibition of hK1 by aluminium cation is linear mixed, with the cation being able to bind to both the free enzyme and the ES complex. This cation was the best hK1 inhibitor. Aluminium is not a physiological cation, but is a known neurotoxicant for animals and humans. The neurotoxic actions of aluminium may relate to neuro-degenerative diseases.

Kallikrein-induced rat uterus contraction is dependent on kinin release.

Glandular kallikrein (EC.3.4.21.8) releases lisyl-bradykinin (kallidin) from kininogen substrates by limited proteolysis (Chao et al, 1981). The responses of the smooth muscle to kallikrein are attributed to kinin formation and a subsequent peptide-receptor interaction (Barabe et al,1977; Fritz et al, 1979; Odya and Goodfriend, 1979). Beraldo et al.(1966) reported that rat urinary kallikrein induced rat uterine contractions in the absence of added kininogen. Nustad and Pierce (1974) suggested that uterine contractions elicited by rat urinary kallikrein depend on kinin liberation from some uterine kininogen. Beraldo et al.(1976) demonstrated the presence of kininogen in the rat uterus. Chao et al.(1981) reported that rat glandular kallikrein can cause the contraction of isolated rat uterus independent on kinin formation. As can be seen, the mechanism of kallikrein-induced uterine contraction was uncertain. We used pure rat submandibular gland kallikrein in order to explain this mechanism.

Human Tissue Kallikrein Activity in Angiographically Documented Chronic Stable Coronary Artery Disease

Background Human tissue kallikrein (hK1) is a key enzyme in the kallikrein–kinin system (KKS). hK1-specific amidase activity is reduced in urine samples from hypertensive and heart failure (HF) patients. The pathophysiologic role of hK1 in coronary artery disease (CAD) remains unclear. Objective To evaluate hK1-specific amidase activity in the urine of CAD patients Methods Sixty-five individuals (18–75 years) who underwent cardiac catheterism (CATH) were included. Random midstream urine samples were collected immediately before CATH. Patients were classified in two groups according to the presence of coronary lesions: CAD (43 patients) and non-CAD (22 patients). hK1 amidase activity was estimated using the chromogenic substrate D-Val-Leu-Arg-Nan. Creatinine was determined using Jaffé’s method. Urinary hK1-specific amidase activity was expressed as µM/(min · mg creatinine) to correct for differences in urine flow rates. Results Urinary hK1-specific amidase activity levels were similar between CAD [0.146 µM/(min ·mg creatinine)] and non-CAD [0.189 µM/(min . mg creatinine)] patients (p = 0.803) and remained similar to values previously reported for hypertensive patients [0.210 µM/(min . mg creatinine)] and HF patients [0.104 µM/(min . mg creatinine)]. CAD severity and hypertension were not observed to significantly affect urinary hK1-specific amidase activity. Conclusion CAD patients had low levels of urinary hK1-specific amidase activity, suggesting that renal KKS activity may be reduced in patients with this disease.

A simple, large scale process for purifying human urinary kallikrein, based on reverse osmosis and ammonium sulfate precipitation.

A simple, large scale process for the purification of human urinary kallikrein is described which is based upon concentration by reverse osmosis, ammonium sulfate precipitation, and gel filtration on a column of Sephadex-G-150. The yield in protein is higher than any reported in the literature to this date; the purified enzyme seems to be identical to that reported by Figueiredo and Mares-Guia at the Kinin-Symposium in Paris, 1978, based on the procedure of Hial, et al., (1974).