Marc van Sande

PURIFICATION AND CHARACTERIZATION OF A NEW ARGININE CARBOXYPEPTIDASE IN HUMAN SERUM

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37 degrees C, we named it unstable carboxypeptidase (carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl-L-arginine and hippuryl-L-lysine, but at a different relative rate than carboxypeptidase N, and has no esterase activity on hippuryl-L-argininic acid. Its activity was inhibited by o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate carboxypeptidase U from carboxypeptidase N and other known carboxypeptidases.

Distribution of angiotensin converting enzyme in human tissues

Angiotensin converting enzyme (EC 3.4.15.1, dipeptidyl carboxypeptidase, ACE, Kininase II), the peptidase which transforms inactive decapeptide, angiotensin I, to the pressor octapeptide, angiotensin II, and which catalyses also the degradation of vasodilative nonapeptide bradykinin, was measured in 27 human tissue homogenates and physiological fluids. Two assays were used: one which measures the hydrolysis of the substrate hippuryl-glycyl-glycine, by means of high performance liquid chromatography and another, using a colorimetric assay measuring the cleaved glycyl-glycine after arylation with picrylsulfonic acid. All the tissues studied contained measurable converting enzyme activities which were inhibited by captopril (SQ 14.225) in low concentrations. High specific activities of converting enzyme were found in several tissues of the intestinal and urogenital tract, but the highest activity was found in benign prostatic hyperplasia. Normal prostate and prostatic adenocarcinoma have a much lower activity. Results obtained for human tissues are compared with those found in animals.

Exopeptidases in human platelets: an indication for proteolytic modulation of biologically active peptides

The determination in human platelets of four exopeptidases--aminopeptidase P, dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme--by means of fluorometric or liquid chromatography techniques was carried out. The results obtained show that the specific activities of dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme in intact and disrupted platelets are small compared to their specific activities in serum. However, for aminopeptidase P the specific activity of this enzyme is much higher in platelets than in serum. This suggests that circulating platelets may have a significant role as scavengers for circulating peptides containing bonds susceptible for aminopeptidase P.

Identification of the carboxypeptidase responsible for the post-synthetic modification of creatine kinase in human serum

The enzyme responsible for the post-translational modification of creatine kinase-MM isoenzyme was purified from human plasma. The enzymatic activity of this enzyme (modifying protein) on the synthetic substrates hippuryl-L-arginine, hippuryl-L-lysine, 3-(2-furylacryloyl)-L-arginine and 3-(2-furylacryloyl)-L-alanyl-L-lysine and the ratio of activities on these substrates are in good agreement with the enzymatic activity of the human serum carboxypeptidase N. The effect of metal ions, chelating agents, proteolytic inhibitors and carboxypeptidase N inhibitor could not differentiate the modifying protein from human serum carboxypeptidase N. Affinity chromatography on Concanavalin-A-Sepharose demonstrated the glycoprotein nature of the modifying protein. The difference in molecular weight observed between modifying protein and carboxypeptidase N can be explained by known instability characteristics and the influence of proteolytic enzymes during purification. Double immunodiffusion analysis with purified antiserum to human carboxypeptidase N confirmed the identity of the modifying protein and carboxypeptidase N.

Lactoferrin in human prostate tissue

SummaryLactoferrin levels have been determined by radialimmunodiffusion in homogenates of both human benign prostatic hypertrophy obtained at open surgery from patients, some of whom had been treated with oestrogens or antiandrogens, and also in prostatic adenocarcinoma tissue removed by transurethral resection. Results show that in untreated benign prostatic hyperplasia there is a statistically lower lactoferrin level in the median compared with the lateral lobes. In patients with benign prostatic hypertrophy treated before prostatectomy with oestrogens or antiandrogens the lactoferrin concentration is decreased. In neoplastic tissue removed by open surgery, the lactoferrin level is very low. Homogenates of tissue resected from protatic cancer patients show similarly low levels. The concentration of lactoferrin in human prostate is hormone dependent. The role of the protein is considered to be bacteriostatic.

Carboxypeptidase N: Colorimetric assay using a new substrate

A method has been developed for determining carboxypeptidase N (EC 3.4.17.3) activity by a hippuricase (EC 3.5.1.14)-assisted colorimetric assay. The method is based on the absorbance at 506 nm of a quinoneimine dye, produced by the action of carboxypeptidase N on the new substrates p-hydroxybenzoylglycine-L-Arg and p-hydroxybenzoylglycine-L-Lys. The enzyme acts on the substrates producing p-hydroxybenzoylglycine and L-Arg or L-Lys. The former is then hydrolyzed by hippuricase into p-hydroxybenzoic acid and Gly. Subsequently, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by sodium periodate forms a quinoneimine dye. The mean value of carboxypeptidase N activities in sera of 50 normal individuals was 30.8 (SD 5.9) nmol of p-hydroxybenzoylglycine released per milliliter of serum for the p-hydroxybenzoylglycine-L-Arg substrate and 137.8 (SD 28.1) for the p-hydroxybenzoylglycine-L-Lys substrate. The sensitivity of the assay is such that as little as 20 microliters of serum provides reliable and precise results (RSD% ranging from 1.8 to 4.9).

Tripeptidyl Carboxypeptidase Activity of Angiotensin-Converting Enzyme in Human Tissues of the Urogenital Tract

The tripeptidyl carboxypeptidase activity of angiotensin-converting enzyme (EC 3.4.15.1) has been determined in several human tissues of the urogenital tract. Benzoyl-glycyl-L-seryl-L-prolyl-L-phenylalanine was used as substrate. The cleaved benzoyl-glycine was measured by means of high-performance liquid chromatography. Results obtained showed a high enzymatic activity for seminal plasma, vesicula seminalis and benign prostatic hyperplasia. Normal prostate and in particular prostatic adenocarcinoma have low enzymatic activity. The activity of the enzyme is sensitive to inhibition by the angiotensin-converting enzyme inhibitor captopril.

Fluorometric assay for angiotensin converting enzyme in human serum by centrifugal analysis

Due to its action on angiotensin I and bradykinin, angiotensin converting enzyme (ACE, dipeptidyl carboxypeptidase, EC 3.4.15.1) plays a crucial role in blood pressure regulation [l]. Attention was focused on this enzyme when Lieberman observed elevated serum ACE levels in active sarcoidosis [2]. Because ACE is a sensitive index both in assessing the clinical status of this disease and in monitoring corticosteroid therapy [3-S], it has become an indispensable parameter in the difficult diagnosis of sarcoidosis [2]. Here we describe a one-step fluorometric assay for ACE, based on ACE-catalysed hydrolysis of the intramolecularly quenched fluorogenic substrate o-aminobenzoylglycyl-p-nitro-L-phenylalanyl+proline (Abz-Gly-Phe(NO*)-Pro) [6], which involves only one stable reagent. Due to the minimal amount of reagent required (100 pl), its high precision, and its simplicity, this assay is readily applicable to automated analysis by means of a fluorescence centrifugal analyser.

POST SYNTHETIC MODIFICATION OF CK-MM BY KININASE I

Cystolic creatine kinase (CK, EC 2.7.3.2.) is a dimeric enzyme exhibiting 3 isoenzymes : CK-MM, CK-MB and CK-BB. The CK-MM isoenzymes released in the serum during muscular damage and myocardial infarction. We have demonstrated that CK-MM isolated from human serum expresses multiple forms (Wevers et al., 1977) as a result of a post-modification of the M subunit. The cause of formation of these iso-forms was shown to be a removal of C-terminal lysine from both subunits (Yasmineh et al., 1981; Falter et al., 1981). Already, some investigators purified the enzyme responsible for this post-transl ational modification and determined its characteristics (Edwards and Watts, 1984; Perryman et al., 1984; Van Landeghem et al., 1985). Several names were given to this as yet unknown protein : CK conversion factor (Falter et al., 1981), modifying protein (Van Landegnem et al., 1985) and carboxypeptidase K (Edwards and Watts, 1984).

Kininase I Activity in Human Fluids and Tissues of the Urogenital Tract

Kininase I (EC 3.4.17.3) activity has been determined in human fluids and tissues of the urogenital tract. Benzoyl-glycyl-L-arginine and benzoyl-glycyl-L-lysine were used as substrates. The cleaved benzoyl-glycine was measured by means of high performance liquid chromatography. Results obtained showed a high enzymatic activity for kidney cortex. Normal prostate, benign prostatic hyperplasia and prostatic adenocarcinoma showed similar enzymic values. The activity of kininase I was higher in normal serum than in the tissues studied.