Biomedical subjects

K B Alves

Publications and source records attributed to K B Alves.

12 recordsLinked to original sources

PubMed2000

Angiotensin converting-like enzymes from urine of untreated renovascular hypertensive and normal patients: purification and characterization.

Angiotensin converting-like enzymes (ACE) were isolated from urine of normal (P0N, P1N and P2N) and untreated renovascular hypertensive (P0, P1 and P2) patients. The urine were submitted to ion exchange chromatography. Enzymes P0 and P0N were eluted with the equilibrium buffer (0.02 M Tris-HCl, pH 7.0), while P1, P1N, P2 and P2N with ionic strength linear gradient of 0.02-0.5 M Tris-HCl, pH 7.0 in 0.7 mS and P2 and P2N in 1.2 mS conductance. The active fractions were submitted to gel filtration in Sephadex G-150, equilibrated and performed with 0.05 M Tris-HCl/0.15 M NaCl buffer, pH 8.0. All enzymes were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (molecular mass: P0, P2 and P2N about 60 kDa; P1, 95 kDa and P21N 170 kDa). The enzymes were recognized by Y1 polyclonal antibody raised against human renal ACE. The K(M) values were in millimolar order for hippuryl-L-His-Leu (HHL) while for benzyloxycarbonyl-Phe-L-His-Leu (ZFHL) they were in 10(-4) M order. The enzymes were able to hydrolyze angiotensin I (AI) (P0 and P0N about 25%, P1 and P1N about 70%, P2 100% and P2N 66%) and bradykinin (BK) (P0N 22%, P1N 81%, P2N 62%, P0 and P1 50% and P2 35%), and their activities were inhibited by captopril.

Adult↗

PubMed1999

Rabbit kidney aminopeptidases: purification and some properties.

Aminopeptidases (EC.3.4.11...) are widely distributed in nature and have medical and biological importance due to their function in the modification and degradation of protein. Two aminopeptidases were purified from rabbit kidney homogenate by ion exchange and gel filtration chromatography columns, using aminoacyl of beta-naphthylamides and p-nitroanilides as substrates. The enzymes' homogeneity was assured by SDS-PAGE. The first enzyme (P1) has an optimum of pH 7.0, a molecular mass of 70 kDa, best catalytical efficiency for methionyl-beta-naphthylamide, is 70% inhibited by 0.5 mM Zn2+ and Co2+ ions, 3.33 mM sodium hydrocortisone succinate and 0.08 mM p-hydroxymercuribenzoate, and is little or not inhibited by EDTA, amino acids, p-nitroaniline, beta-naphthylamine, deoxicholate, bestatin and puromycin. The second enzyme (P2) has an optimum of pH 7.0, a molecular mass of 54 kDa, best catalytical efficiency for Leu-beta-naphthylamide, is inhibited by 0.5 mM ions Zn2+ (45%), 0.02 mM EDTA (94%) 0.08 mM p-hydroxymercuribenzoate (70%), 3.33 mM beta-ME (13%), 1.33 mM p-nitroaniline (40%), 1.33 mM beta-naphthylamine (17%), 1.33 mM sodium deoxicholate (96%), 3.33 mM sodium hydrocortisone succinate (60%), and is 30% activated by 0.5 mM Co2+ ions. Puromycin and bestatin are competitive inhibitors with Ki values in 10(-6) and 10(-7) M order, respectively. P1 is a methionine aminopeptidase, while P2 is a leucine aminopeptidase.

Aminopeptidases↗

PubMed1999

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase.

The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ss-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ss-naphthylamide was eluted at 750 microS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/K M ratio for L-Leu-ss-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 microM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 microM), p-nitroaniline (0.25 mM) and ss-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 microM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4 degrees C and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.

Aminopeptidases↗

PubMed1996

Basic aminopeptidase from rabbit kidney: purification and partial characterization.

The aminopeptidase activity of a homogenate of rabbit kidney treated with Triton X-100 was measured using L-aminoacyl-2-naphthylamides (AA-NA). After gradient elution ion-exchange chromatography, four peaks of aminopeptidase activity were eluted. The enzyme eluted at 450 microS containing 33.5% of the activity towards Arg-NA was applied to a Superdex 75 column and presented only one protein band on 10% SDS-polyacrylamide gel electrophoresis. This enzyme has an apparent molecular mass of 78 kDa, is five-fold activated by 0.15 M NaCl and the highest Vmax/KM ratio was obtained with Arg-NA. Enzyme activity was inhibited 100% by 0.13 mM sodium p-hydroxymercuribenzoate, 20% by 0.75 mM EDTA and 100% by 0.66 mM o-phenanthroline. Puromycin and bestatin behaved like competitive inhibitors with a Ki of 0.60 mM and 5.0 microM and 5.0 microM, respectively.

2-Naphthylamine↗

PubMed1992

Angiotensin converting enzymes from urine of treated and untreated essential mild hypertensive patients (EHP) with diuretic: partial purification and characterization.

Urine of untreated EHP was eluted, on a ion-exchange chromatography, in two protein peaks with ACE activity, at 0.7 mS (BI) and 1.25 mS (BII), while urine of treated EHP, was eluted only in one peak with ACE activity (0.7 mS). BI (Mr, 88 kDa) and BII (Mr, 61 kDa) convert AI to AII, hydrolyze bradikinin, are inhibited by captopril, EDTA and metal ions.

Adult↗

PubMed1992

Endopeptidase and carboxypeptidase activities in human urine which hydrolyze bradykinin.

1. We have fractionated the bradykinin inactivating activity of human urine by stepwise elution chromatography on DEAE-cellulose and recovered 95% of the inactivating activity and 29% of the protein (absorbance at A280 nm). 2. Seven of nine fractions which presented activity were also tested for angiotensin I and II inactivating activity, angiotensin converting activity and for the hydrolysis of hippuryl-His-Leu and hippuryl-Arg. Sites of hydrolysis in bradykinin were determined by HPLC of the hydrolysates and fragments were compared with authentic peptides. 3. Cleavage sites demonstrated for Fractions A through G were: Phe8-Arg9 (A and B), Phe5-Ser6 (C and F), Pro7-Phe8 (D), Gly4-Phe5 and Pro7-Phe8 (E) and Pro3-Gly4 (G). 4. The relative molecular weight of the bradykininase activity present in each fraction, determined by gel filtration, was: 16 kDa (A), 70 kDa (B), 60 kDa (C), 88 kDa (D), 230 kDa (E), 45 kDa (F) and 49 kDa (G). 5. Bradykinin inactivating activity was inhibited 50-100% by 3 mMEDTA (A, B, D, E and G), 1 mMM 2-mercaptoethanol (A, B, C and G), 0.1 microM Hg2+ (A, C and G), 0.1 mM PMSF (C and F), 1 mM TPCK (C and F), 1 mM Zn2+ (C), 60 microM BPP5a and 40 microM BPP9a (D), 0.1 microM phosphoramidon (E) and 3 mM sodium p-hydroxymercuribenzoate (G). 6. The properties of some of these bradykinin inactivating activities correspond to enzymes previously described in urine and tissues: carboxypeptidases (Fractions A and B), angiotensin I converting enzyme (Fraction D), neutral endopeptidase (Fraction E). However, the chymotrypsin-like activity of Fractions C and F and the prolylendopeptidase activity of Fraction G have not been described before in urine and they are being purified in order to obtain a more accurate characterization.

Bradykinin↗

PubMed1992

Inhibition of aminopeptidase activity by aromatic and other cyclic compounds.

The effect of 2-naphthylamine, p-nitroaniline, o-phenanthroline, sodium deoxycholate and hydrocortisone succinate on the activity of human urine aminopeptidase, rat kidney methionyl and arginyl aminopeptidase, soybean and Enterolobium contortisiliquum seed aminopeptidase was studied using aminoacyl-2-naphthylamide and L-Leu-p-nitroanilide as substrates. Ki values ranged from 10 microM to 2.7 mM. On the basis of Ki and Km values, and catalytic efficiency for each enzyme, it is clear that the aminopeptidases from human urine and from soybean seed should be assayed with both substrates, whereas L-Leu-p-nitroaniline is a more appropriate substrate for the rat kidney aminopeptidases. Sodium deoxycholate is a better inhibitor than hydrocortisone succinate. Non-competitive inhibition was observed in all cases except for E. contortisiliquum seed aminopeptidase.

Aminopeptidases↗

PubMed1991

Purification and partial characterization of Enterolobium contortisiliquum seed arylamidase.

1. Arylamidase activity was isolated from Enterolobium contortisiliquum seeds (2 U/g) using L-Leu-2-naphthylamide as substrate to monitor the purification. 2. The enzyme preparation was purified 733-fold by ammonium sulfate precipitation, and by ion exchange and gel filtration chromatography, in 6.6% yield. 3. SDS-Polyacrylamide gel electrophoresis after fast protein liquid chromatography on a Mono Q column, showed only one protein band with a molecular weight of 35 kDa. 4. The optimum pH for arylamidase activity was 6.5. Taking the hydrolysis rate of Lys-2-naphthylamide as one, the relative rates at which the other substrates were hydrolyzed were: Leu-2-naphthylamide, 30, Met-2-naphthylamide, 18, Arg-2-naphthylamide, 2, Ala-2-naphthylamide, 1.5, and L-Leu-p-nitroanilide, 26. 5. The arylamidase activity was inhibited 50% by 0.1 mM HgCl2, 0.1 mM MnCl2, 0.1 mM ZnCl2, 0.13 mM NiCl2, 0.2 mM o-phenanthroline and 1 microM sodium p-hydroxymercuribenzoate, and activated 35% by 5.0 microM EDTA. Iodoacetate (0.67 mM), dithioerythritol and 2-mercaptoethanol (3.3 mM), and chloride ions (0.2 M) had no effect on the enzyme activity.

Aminopeptidases↗

PubMed1990

Partial purification and properties of rat urine arylamidases.

1. Arylamidases were isolated from rat urine using L-aminoacyl-2-naphthylamides and L-Leu-p-nitroanilide as substrates to monitor the purification. 2. Ion-exchange chromatography separated three peaks of activity (A, B and C). After gel filtration chromatography, the second and third peaks (B and C) were further purified to provide B1 and C1. Each behaved like a single active protein band on 7.5% polyacrylamide gel electrophoresis without SDS. The molecular weights of fractions B1 and C1 determined by SDS-PAGE were 440 and 270 kDa, respectively. 3. The pH optimum for arylamidase activity was 7.5 for both forms on all substrates tested. The maximum value for the Vmax/Km ratio was obtained using L-Leu-2-naphthylamide as substrate for both forms. 4. The arylamidase activity of B1 and C1 was not affected by the presence of chloride ions and was increased in the presence of CaCl2 and MnCl2 only when L-Glu-2-naphthylamide was used as substrate. EDTA (3.3-33.0 microM) and o-phenanthroline (0.1-1.0 mM) but not -SH (0.08-0.67 mM) or -S-S-(0.42-3.3 mM) group reagents inhibited the arylamidase activity. Hydrolysis of L-Leu-2-naphthylamide by fractions B1 and C1 was competitively inhibited by leucine (0.14-0.56 mM), indomethacin and puromycin (67-267 microM) and bestatin (8.3-33.3 microM). For each inhibitor, the Ki values were similar in the two fractions: 100 microM for L-leucine, 10 microM for indomethacin and puromycin and 1.0 microM for bestatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopeptidases↗

PubMed1989

Partial purification and characterization of soybean seed arylamidase activity.

The arylamidase activity of a soybean seed extract was measured using L-aminoacyl-2-naphthylamides and L-Leu-p-nitroanilide. The enzyme preparation was purified 42-fold with 45% yield, by precipitation with 25-75% ammonium sulfate saturation and by ion exchange and gel filtration chromatography. The highest kcat/Km ratio was that of L-Lys-2-naphthylamide (216 s-1 microM-1) and the lowest was that of L-Leu-p-nitroanilide (40 s-1 microM-1). Enzyme activity was inhibited by -SH and -S-S-group reagents while chelating agents had no effect. L-Lysine, L-leucine, puromycin and bestatin were competitive inhibitors and the Ki values found were: 3.5 mM, 0.9 mM, 0.23 mM and 0.8 microM, respectively.

Aminopeptidases↗

PubMed1987

Purification and characterization of aminopeptidase activity from rat urine.

The aminopeptidase activity of rat urine was tested using L-aminoacyl-2-naphthylamides and L-Leu-p-nitroanilide. The enzyme preparation was purified by ion-exchange and gel filtration chromatography and showed only a single active protein band on 7.5% polyacrylamide gel electrophoresis. The enzyme was characterized by studying the effects of ions (divalent cations and chloride), chelating agents and -SH and -S-S- group reagents and by determining kinetic parameters (Km and Vmax for different substrates and Ki for amino acids, antibiotics and anti-inflammatory drugs).

Aminopeptidases↗

PubMed1984

Kinin-converting aminopeptidase from human urine. Further purification and characterization through kinetic and inhibitory studies.

An aminopeptidase from human urine (HUA) able to hydrolyze L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and to convert both MLBK and LBK to BK has been further purified and characterized. The preparation now obtained showed a 3-fold higher specific activity than the previously described one and a single active protein band in 7% polyacrylamide gel electrophoresis accounting for 86% of total protein. Kinetic constants for this kinin-converting enzyme were determined using L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and LBK. The Km values for different naphthylamides were in the 10(-5) M range while that for L-Leu-p-nitroanilide was 3.6 X 10(-4) M. With LBK as substrate the aminopeptidase activity showed the highest catalytic efficiency in spite of a Km in the mM range. The enzyme was poorly inhibited by -SH and -S-S- group reagents. Some L-aminoacids, as well as mono- and diamines, indomethacin, puromycin and bestatin were equipotent competitive inhibitors of both arylamidase and aminopeptidase activities. Results obtained in this paper are compatible with our conclusion that human urine, unlike other enzyme sources, contains only one aminopeptidase, and that this enzyme displays both arylamidase and kinin-converting activities. The enzyme's action may be important in the metabolism of kinins, yielding peptides which could interact with both B-1 and B-2 kinin receptors in the kidney.

Amines↗