Characterisation of a Carboxypeptidase in Human Serum Distinct from Carboxypeptidase N (original) (raw)
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A Simple Spectrophotometric Assay of Carboxypeptidase N (Kininase I) in Human Serum
cclm, 1983
Kininase I (carboxypeptidase N; EC 3.4.17.3) consists of carboxypeptidase NI (CNi) and carboxypeptidase N2 (CNi); these two enzymes can be differentiated by their activities towards hippuryl-L-arginine and hippuryl-L-lysine, respectively. A Spectrophotometric assay for both carboxypeptidases in human serum is described and the biochemical behaviour of these enzymes investigated. The pH optima are found to be 8.4 for CNi and CN 2. The Michaelis-Menten constants are: CNi 4.59 ± 0.03 mmol/i; CN 2 37.26 ± 3.49 mmol/1. CN 2 can be inhibited by EDTA (76%), dimercaprolum (97%) and phenanthroline (98%). Diisopropylfluorophosphate has no influence on both enzymes. Elevated haemoglobin only interferes with CNi measurements, and high bilirubin concentfations slightly alter the activity of both enzymes. High CNi activities were found in sera of patients with sarcoidosis, and elevated CN 2 activities were found in lung cancer. Eine einfache spektralphotometrische Methode zur Bestimmung der Carboxypeptidase N (Kininase I) im menschlichen Serum Zusammenfassung: Die Carboxypeptidase N (CN; EC 3.4.17.3), auch Kininase I genannt, läßt sich aufgrund ihres unterschiedlichen biologischen Verhaltens in CNi mit Hippuryl-L-arginin und CN 2 mit Hippuryl-Llysin als Enzymsubstrat unterscheiden. Beide im Serum enthaltenen Enzyme spalten die terminalen Aminosäuren ab und lassen dadurch Hippursäure entstehen, deren Konzentration spektralphotometrisch gemessen wird. CNi und CN? wurden biochemisch charakterisiert. Das pH Optimum lag bei 8,4. Die Michaelis-Menten-Konstante betrug: CN! 4,59 4 0,03 mfnol/1, CN 2 37,29 ± 3,49 fnmol/1. CN 2 wurde zu 76% durch EDTA, Dimercaprol (97%) und Phenanthrolin (98%) inhibiert. Diisopropylfluorophosphat hatte keinen Einfluß auf beide Enzyme. Erhöhtes Hämoglobin interferierte bei der CNi, Bilirubin zeigte nur eine geringe Interferenz. Klinische Bedeutung scheint der CNi bei der Lungensarkoidose zuzukommen, bei der die höchsten Aktivitäten im Gegensatz zu anderen Lungenerkrankungen gefunden wurden. Beim Lungenkarzinom war CN 2 erhöht.
Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen
Journal of Biological Chemistry, 1994
A novel basic carboxypeptidase clearly different from carboxypeptidase N has been isolated from human plasma. It circulates as an enzymatically inactive precursor enzyme bound to plasminogen. During fibrinolysis, it can be converted to its active form, carboxypeptidase U, through the action of plasmin. The active enzyme has an apparent molecular weight of 63,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzes the synthetic peptides hippuryl-L-arginine and hippuryl-L-lysine but, in contrast to other human basic carboxypeptidases, has only a limited esterase activity. After its activation, carboxypeptidase U tends to be very unstable. The role of basic carboxypeptidases, i.e. enzymes that cleave COOH-terminal basic amino acids lysine and arginine, has gained renewed interest in medical science since it became evident that this type of enzyme can be involved in peptide hormone maturation. Most peptide hormones are initially synthesized as precursor proteins that are frequently cleaved at pairs of basic amino acids such as Arg-Arg or Lys-Arg, and carboxypeptidase B-like enzymes working in concert can generate the correct product from some of these precursors (1). In the plasma compartment, this family of enzymes is represented by carboxypeptidase N (arginine carboxypeptidase, kininase I, anaphylatoxin inactivator, serum carboxypeptidase B, EC 3.4.17.31, which is synthesized in the liver (2) and circulates in plasma as an M, 280,000 tetrameric complex. It is composed of two identical high molecular weight subunits (M, 83,0001, which are heavily glycosylated and lack enzymatic activity, and two identical low molecular weight subunits (M, 55,000), which lack carbohydrate but contain the active center (3,4). The high molecular weight subunit functions to stabilize the active subunit at body temperature and keep it in the circulation (4). Physiologically interesting substrates for this enzyme are the kinins bradykinin and kallidin (2), the anaphylatoxins C,, and C,, (5,6), the fibrinopeptides 6Aand 6D (7), the creatine kinase MM isoenzyme (8, 9), the hexapeptide enkephalins (lo), and the atrial natriuretic peptide atriopeptin I1 (11). Its most likely physiological function is to protect the organism from the actions of potent peptides that may escape from tissues or be released in the circulation. Recently, we have described the important differences in arginine carboxypeptidase activities between fresh serum and older serum or heparinized plasma, depending on the substrate * This work was supported in part by the Belgian National Fund for Scientific Research, Brussels, Belgium and the Belgian Program on Interuniversity Poles of Attraction. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Analytical Biochemistry, 2006
In plasma, two basic carboxypeptidases are known: carboxypeptidase N (CPN, EC 3.4.17.3) 1 and procarboxypeptidase U (proCPU, TAFI, EC 3.4.17.20). proCPU is a zymogen that must be cleaved at Arg92 by thrombin or plasmin to become a fully active enzyme. It is widely assumed that the basic carboxypeptidase activity in plasma is solely attributable to the constitutively active CPN [1]. Most of the assays described in the literature to measure basic carboxypeptidase activity make use of the synthetic substrate benzoyl-glycyl-L-arginine (Bz-Gly-Arg, hippuryl-L-arginine) . With the aim to discover more sensitive substrates for plasma CPN activity measurement, we screened 13 diVerent Bz-Aa-Arg peptides as potential CPN substrates using a recently developed kinetic assay . In our search for new substrates, we discovered that the zymogen proCPU shows intrinsic enzymatic activity depending on the substrate used. This intrinsic enzymatic activity of proCPU interferes with the quantiWcation of CPN activity in plasma. Our data indicate that newly introduced substrates for measuring CPN activity should be screened for intrinsic activity toward proCPU.
Analytical Biochemistry, 2005
Carboxypeptidase U (CPU, TAFIa) is a novel determinant of the fibrinolytic rate. It circulates in blood as an inactive zymogen, procarboxypeptidase U, which is activated during the process of coagulation and fibrinolysis. CPU has a very short half-life at 37°C. Its intrinsic instability complicates the determination of kinetic parameters of different substrates using an endpoint method. We developed a fast kinetic assay for measuring continuously the release of the C-terminal arginine by CPU independent of the nature of the substrate peptide used, allowing us to perform substrate specificity studies of CPU. This method uses arginine kinase, pyruvate kinase, and lactate dehydrogenase as auxiliary enzymes. The CPU activities measured using this kinetic assay were in the range of 97-103% of those determined with our HPLC-assisted reference assay, and the obtained K m and k cat values for hippuryl-Larginine and bradykinin were in good accordance with those described in the literature. As expected, no arginine cleaving was seen using dipeptides and peptide substrates with a proline in the penultimate position. The presented kinetic assay enables the fast screening of substrates with a C-terminal arginine and is a valuable new tool for the kinetic evaluation of both synthetic and physiological substrates of CPU.
Interaction of carboxypeptidase a with monoclonal antibodies
Mol Immunol, 1984
Several mouse monoclonal antibodies to carboxypeptidase A (CPA) were prepared and purified, and their interaction with the enzyme was investigated. CPA is a well-characterized zinccontaining exopeptidase exhibiting peptidase as well as esterase activity. The antibodies obtained could be classified as follows: antibodies inhibiting mainly the peptidase activity of the enzyme, antibodies inhibiting mainly its esterase activity, antibodies affecting both activities, and antibodies which bind to the enzyme but have no marked effect on its catalytic properties. Binding constants of _ 1 O6 M -' were obtained for most of the antibody-enzyme complexes tested. Additional information on the effect of the monoclonal antibodies on the active site of CPA was obtained by determining the change in the circular dichroism spectra of arsanilazotyrosine-248 carboxypeptidase A occurring as a result of the interaction of the enzyme with the antibodies studied. These findings suggest that CPA possesses at least three different specific antigenic sites, and that the active site of the enzyme for its peptidase activity differs from that for its esterase activity, though both sites seem to overlap to a considerable extent.
Isolation and characterization of a basic carboxypeptidase from human seminal plasma
Archives of Biochemistry and Biophysics, 1988
A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent M, 98,000 and after purification with an il!l, 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at M,. 53,000 was seen which was converted to two smaller bands (M, 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (6'7.8 pmol/min/mg) with the ester substrate benzoyl (Bz)-Glyargininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 PM, kcat = 43 min-l), Arg'-Met5-enkephalin (Km = 103 PM, kcat = 438 min-'), and Lys'-Met5-enkephalin (Km = 848 PM, kcat = 449 min-l). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction. 0 1988Academic press, I~~.
Purification and properties of five different forms of human procarboxypeptidases
European Journal …, 1989
Three different procarboxypeptidases A and two different procarboxypeptidases B have been isolated for the first time, in a pure and native state, from human pancreatic extracts. These proteins were purified in one or two quick steps by anion-exchange HPLC. All these forms have been biochemically characterized. Two of the procarboxypeptidases A, the A1 and A2 forms, are obtained in a monomeric state while the other, the A3 form, is obtained as a binary complex of a procarboxypeptidase A with a proproteinase E. This complex is stable in aqueous buffers at various ionic strengths and develops carboxypeptidase A and proteinase E activities in the presence of trypsin. The A3 and A2 forms show clear differences in electrophoretic mobility in SDS/polyacrylamide gels, isoelectric point, proteolytic activation process with trypsin and susceptibility to thermal denaturation. In contrast, these properties are similar in the A1 and A3 (binary complex) forms. On the other hand, with respect to the properties listed above, the B1 and B2 forms differ from each other mainly in isoelectric point. An overall comparison of the above properties reveals the unusual character of the A2 form, midway between the other A and B forms. N-terminal extended sequence analysis carried out on these proenzymes confirm that they constitute different isologous forms.
Substrate specificity of human carboxypeptidase A6
The Journal of biological chemistry, 2010
Carboxypeptidase A6 (CPA6) is an extracellular matrixbound metallocarboxypeptidase (CP) that has been implicated in Duane syndrome, a neurodevelopmental disorder in which the lateral rectus extraocular muscle is not properly innervated. Consistent with a role in Duane syndrome, CPA6 is expressed in a number of chondrocytic and nervous tissues during embryogenesis. To better characterize the enzymatic function and specificity of CPA6 and to compare this with other CPs, CPA6 was expressed in HEK293 cells and purified. Kinetic parameters were determined using a panel of synthetic carboxypeptidase substrates, indicating a preference of CPA6 for large hydrophobic C-terminal amino acids and only very weak activity toward small amino acids and histidine. A quantitative peptidomics approach using a mixture of peptides representative of the neuropeptidome allowed the characterization of CPA6 preferences at the P1 substrate position and suggested that small and acidic P1 residues significantly inhibit CPA6 cleavage. Finally, a comparison of available kinetic data for CPA enzymes shows a gradient of specificity across the subfamily, from the very restricted specificity of CPA2 to the very broad activity of CPA4. Structural data and modeling for all CPA/B subfamily members suggests the structural basis for the unique specificities observed for each member of the CPA/B subfamily of metallocarboxypeptidases.