Automated kinetic determination of angiotensin-converting enzyme in serum.

1984 ◽  
Vol 30 (6) ◽  
pp. 901-902 ◽  
Author(s):  
A Harjanne
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Previous Method ◽  
Converting Enzyme ◽  
Kinetic Determination ◽  
Assay Time ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Abstract In this automated kinetic modification of a previous method (Anal Biochem 95: 540-548, 1979) for determining angiotensin-converting enzyme (EC 3.4.15.1), 3-(2- furylacryloyl )-L- phenylalanylglycylglycine is used as the substrate. The change in absorbance at 340 nm is used to monitor hydrolysis of the substrate. The rate of hydrolysis is roughly threefold greater than with previously reported substrates, so assay time and sensitivity are improved.

The Spectrophotometric Determination of Human Serum Carboxypolypeptidase with Angiotensin Converting Enzyme-Like Activity

1976 ◽  
Vol 50 (5) ◽  
pp. 321-327
Author(s):  
J. J. Summary
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Enzyme Inhibitors ◽  
Colorimetric Method ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Converting Enzyme Inhibitors ◽  
Enzyme Substrate ◽  
Chemical Assay ◽  
Hydrolysis Of

1. A colorimetric method was developed for the direct chemical assay of human carboxypeptidase A (carboxypolypeptidase; EC 3.4.12.2) with angiotensin converting enzyme-like activity in serum or plasma, with the substrate analogue glycyl-l-histidylglycine and the angiotensin converting enzyme substrate angiotensin I (A-I). This method was based on the spectrophototometric determination of histidylglycine and histidyl-leucine, products of the hydrolysis of glycyl-l-histidylglycine and A-I respectively. o-Phthalaldehyde reacted with the imidazole moiety of N-terminal histidyl peptides to produce a yellow chromophore. 2. A large number of inhibitors were tested for their effects on carboxypolypeptidase activity. The hydrolysis of Gly-His-Gly and A-I was inhibited by histidyl-leucine and angiotensin II, both products of the hydrolysis of A-I. Bothrops jararaca venom extract, EDTA, p-chloromercuribenzoate, 8-hydroxyquinoline and 2,3-dimercaptopropanol, previously reported as converting enzyme inhibitors, also inhibited carboxypolypeptidase activity. 3. Angiotensin converting enzyme activity in the serum of sixty-six adults ranged from 10 to 37 nmol of glycyl-l-histidylglycine hydrolysed in 10 min by 10 μl of serum at 37°C and pH 7·25.

Polarographic study of hydrolysis of [8-lysine]vasopressin and its derivatives with blood serum of pregnant women

1980 ◽  
Vol 45 (4) ◽  
pp. 1099-1108 ◽  
Author(s):  
Mikuláš Chavko ◽  
Michal Bartík ◽  
Evžen Kasafírek
Keyword(s):  
Blood Serum ◽  
Pregnant Women ◽  
Degree Of Hydrolysis ◽  
Polarographic Study ◽  
Ph Optimum ◽  
Lysine Vasopressin ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Vasopressin Analogues

A polarographic study of the hydrolysis of [8-lysine]vasopressin and some hormonogens of the vasopressin series with the blood serum of women in the last week of pregnancy was studied. The dependence of hydrolysis on pH (pH optimum: 7.4-7.50, substrate concentration (Km 1.2 . 10-5M), pH stability and thermal stability were determined. The rate of hydrolysis of individual vasopressin analogues decreases in the order: [8-lysine]vasopressin > Nα-glycyl-prolyl[8-lysine]-vasopressin > Nα-leucyl-[8-lysine]vasopressin > Nα-alanyl-[8-lysine]vasopressin > Nα-phenyl alanyl-[8-lysine]vasopressin > Nα-diglycyl-[8-lysine]vasopressin > Nα-prolyl-[8-lysine]vasopressin > Nα-triglycyl-[8-lysine]vasopressin > Nα-sarcosyl-glycyl-[8-lysine]vasopressin. The degree of hydrolysis gradually increases to a multiple with the length of the pregnancy in consequence of the presence of oxytocine. However, vasopressin is also hydrolysed to a small extent with the enzymes from the blood sera of non-pregnant women. Under similar analytical conditions oxytocin was not hydrolysed with the sera of non-pregnant women and therefore oxytocin is a more suitable substrate than vasopressin for polarographic determination of serum oxytocinase.

Single-reagent automated determination of angiotensin converting enzyme in serum.

1985 ◽  
Vol 31 (10) ◽  
pp. 1761-1761 ◽  
Author(s):  
D Hendriks ◽  
S Scharpé ◽  
M van Sande ◽  
J P Vingerhoed
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Converting Enzyme ◽  
Automated Determination

scholarly journals A rapid direct assay for the determination of the separate activities of the three arylsulphatases of Aspergillus oryzae

1977 ◽  
Vol 166 (3) ◽  
pp. 411-413 ◽  
Author(s):  
G R J Burns ◽  
C H Wynn
Keyword(s):  
Phenolic Compounds ◽  
Aspergillus Oryzae ◽  
Substrate Specificities ◽  
Direct Assay ◽  
Assay Procedure ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Potential Use

1. The three arylsulphatases of Aspergillus oryzae exhibit pronounced kinetic differences and substrate specificities. Arylsulphatase I hydrolyses all substrates tested, whereas arylsulphatase III will not hydrolyse tyrosine O-sulphate or phenolphthalein disulphate. Arylsulphatase II does not hydrolyse p-nitrophenyl sulphate or phenolphthalein disulphate at appreciable rates in the absence of added phenolic compounds. Phenols such as tyramine increase the rate of hydrolysis of these substances by this enzyme 1000-fold. At pH 6.9 arylsulphatase I exhibits an apparent Km of 0.1 mM for p-nitrophenyl sulphate, whereas the Km of arylsulphatase III for this substrate is 1 mM. 2. These differences were utilized to develop an assay procedure which can be used to determine the separate activities of the three enzymes present in mixtures. This assay has potential use as a means of examining the relative activities of the three enzymes in investigations of the differences in the mechanisms regulating their synthesis.

scholarly journals Novel activity of angiotensin-converting enzyme. Hydrolysis of cholecystokinin and gastrin analogues with release of the amidated C-terminal dipeptide

1989 ◽  
Vol 262 (1) ◽  
pp. 125-130 ◽  
Author(s):  
P Dubreuil ◽  
P Fulcrand ◽  
M Rodriguez ◽  
H Fulcrand ◽  
J Laur ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Chloride Ion ◽  
Peptide Bond ◽  
Major Product ◽  
Enzyme Hydrolysis ◽  
Ion Concentration ◽  
Converting Enzyme ◽  
Rabbit Lung ◽  
Hydrolysis Of

ACE (angiotensin-converting enzyme; peptidyl dipeptidase A; EC 3.4.15.1), cleaves C-terminal dipeptides from active peptides containing a free C-terminus. We investigated the hydrolysis of cholecystokinin-8 [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and of various gastrin analogues by purified rabbit lung ACE. Although these peptides are amidated at their C-terminal end, they were metabolized by ACE to several peptide fragments. These fragments were analysed by h.p.l.c., isolated and identified by comparison with synthetic fragments, and by amino acid analysis. The initial and major site of hydrolysis was the penultimate peptide bond, which generated a major product, the C-terminal amidated dipeptide Asp-Phe-NH2. As a secondary cleavage, ACE subsequently released di- or tri-peptides from the C-terminal end of the remaining N-terminal fragments. The cleavage of CCK-8 and gastrin analogues was inhibited by ACE inhibitors (Captopril and EDTA), but not by other enzyme inhibitors (phosphoramidon, thiorphan, bestatin etc.). Hydrolysis of [Leu15]gastrin-(14-17)-peptide [Boc (t-butoxycarbonyl)-Trp-Leu-Asp-Phe-NH2] in the presence of ACE was found to be dependent on the chloride-ion concentration. Km values for the hydrolysis of CCK-8, [Leu15]gastrin-(11-17)-peptide and Boc-[Leu15]gastrin-(14-17)-peptide at an NaCl concentration of 300 mM were respectively 115, 420 and 3280 microM, and the catalytic constants were about 33, 115 and 885 min-1. The kcat/Km for the reactions at 37 degrees C was approx. 0.28 microM-1.min-1, which is approx. 35 times less than that reported for the cleavage of angiotensin I. These results suggest that ACE might be involved in the metabolism in vivo of CCK and gastrin short fragments.

scholarly journals Kinetic probes for inter-domain co-operation in human somatic angiotensin-converting enzyme

2005 ◽  
Vol 391 (3) ◽  
pp. 641-647 ◽  
Author(s):  
Olga E. Skirgello ◽  
Peter V. Binevski ◽  
Vladimir F. Pozdnev ◽  
Olga A. Kost
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Enzymatic Activity ◽  
Active Site ◽  
The Other ◽  
Converting Enzyme ◽  
Human Enzyme ◽  
Independent Functions ◽  
The Common ◽  
The Individual ◽  
Hydrolysis Of

s-ACE (the somatic form of angiotensin-converting enzyme) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. Negative co-operativity between the two domains has been demonstrated for cow and pig ACEs. However, for the human enzyme there are conflicting reports in the literature: some suggest possible negative co-operativity between the domains, whereas others indicate independent functions of the domains within s-ACE. We demonstrate here that a 1:1 stoichiometry for the binding of the common ACE inhibitors, captopril and lisinopril, to human s-ACE is enough to abolish enzymatic activity towards FA {N-[3-(2-furyl)acryloyl]}-Phe-GlyGly, Cbz (benzyloxycarbonyl)-Phe-His-Leu or Hip (N-benzoylglycyl)-His-Leu. The kinetic parameters for the hydrolysis of seven tripeptide substrates by human s-ACE appeared to represent average values for parameters obtained for the individual N- and C-domains. Kinetic analysis of the simultaneous hydrolysis of two substrates, Hip-His-Leu (S1) and Cbz-Phe-His-Leu (S2), with a common product (His-Leu) by s-ACE at different values for the ratio of the initial concentrations of these substrates (i.e. σ=[S2]0/[S1]0) demonstrated competition of these substrates for binding to the s-ACE molecule, i.e. binding of a substrate at one active site makes the other site unavailable for either the same or a different substrate. Thus the two domains within human s-ACE exhibit strong negative co-operativity upon binding of common inhibitors and in the hydrolysis reactions of tripeptide substrates.

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