Purification and analysis of lung and plasma angiotensin I-converting enzyme by high-performance liquid chromatography.

We purified angiotensin I-converting enzyme (ACE) from pig and human lung and plasma for comparison of some physicochemical properties between the endothelial membrane-bound form and the soluble form of the enzyme. After affinity chromatography on Sepharose CL-4B/lisinopril, gel-filtration HPLC on Superose 12 achieved homogeneity for both forms as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Whatever the source of ACE, the molecular weight was 300 +/- 40 kDa after calibration of Superose 12 with standard globular proteins and 172 +/- 4 kDa by SDS-PAGE, with or without reduction, a result suggesting interactions between the glycopolypeptide chain and the chromatographic gel possibly related to the overall shape and sugar content of the enzyme. Ion-exchange HPLC analysis on TSK-DEAE showed that the membrane-bound and soluble forms of ACE are not isoenzymes, although isoelectrofocusing did show that the isoelectric point of soluble ACE was lower than those of tissue ACE, suggesting a different glycosylation. No significant difference between porcine and human ACE appeared. HPLC methods seem to be of particular interest for the purification of ACE with a high yield and for the analysis of its putative differently glycosylated isoforms.

A reliable radiometric assay for the determination of angiotensin I-converting enzyme activity in urine.

We present a radiometric assay for the determination of urinary angiotensin-converting enzyme activity, using benzoyl-[1-14C]glycyl-L-histidyl-L-leucine as the substrate. An optimal pH of 8.3, an optimal chloride concentration of 0.375 mol/l and complete inhibition by EDTA-Na2, captopril and enalaprilat confirm the specificity of the assay. Comparison of dialysis and ultrafiltration for concentration of urine showed the existence of angiotensin-converting enzyme inhibitors in human urine. Dialysis against water was the more effective method for avoiding enzyme inhibition. After dialysis of urine, the assay was linear with time and with enzyme concentration; it was highly sensitive (60 mU/l) and showed good reproducibility. Under our technical conditions, we found angiotensin-converting enzyme activity in urine samples with quantitatively abnormal protein contents, but not in normal urine. Urinary angiotensin-converting enzyme did not correlate with proteinuria nor with water-salt parameters or creatinine. We confirm the kidney tubular epithelial origin of the enzyme, and propose the use of our assay to study urinary angiotensin-converting enzyme as a marker of renal tubular damage.

Serum angiotensin-converting enzyme in healthy and sarcoidotic children: comparison with the reference interval for adults.

Angiotensin-converting enzyme (ACE) was measured in serum of 187 healthy children between the ages six months and 18 years. Results were pooled for five-year age intervals and compared with the reference values for adults that we previously determined [Clin Chem 1986;32:884-6). Results for each age group were also studied as a function of sex. Children had higher ACE activities in serum than did adults (P less than 0.001), but these activities were age-related only from age four to 18 years. Adolescents showed sex-related differences, with higher serum ACE activities in boys than in girls (P less than 0.05). Both sex- and age-related differences may be related to a steroid hormonal regulation of ACE biosynthesis. We also verified that children with sarcoidosis (n = 20) had significantly increased serum ACE activity. Such physiological variations in serum ACE activity must be taken into account for diagnosing sarcoidosis in children, for following the course of the disease, and for evaluating the accuracy of therapy.

Serum angiotensin-converting enzyme: an endothelial cell marker. Application to thromboembolic pathology.

Aa a plasma marker of an endothelial abnormality, the serum activity of angiotensin-converting enzyme (ACE) was investigated at rest and after stimulation either by local venostasis or infusion of an analogue of lysine-vasopressin (desmopressin acetate). Desmopressin acetate did not induce any significant change in ACE, in contrast to the effect of venostasis. Searching for an endothelial abnormality implicated in the genesis of deep vein thrombosis, we used the local venostasis test in patients affected by recurrent deep vein thrombosis. Patients, divided in three groups (group I, documented history of recurrent deep vein thrombosis; group II, only one deep vein thrombosis or recurrent superficial venous thrombosis; group III, history of arterial thromboembolism), and controls were screened for basal and stimulated levels of serum ACE, together with fibrinolytic activity and von Willebrand factor level. Two types of abnormalities of serum ACE activity were found: low basal level in group I, and low response to venostasis in groups I and III; group II did not differ from controls. Measures of fibrinolytic and ACE activities are not redundant because the two types of ACE abnormalities were not individually encountered in the same patients and were independent from abnormalities of the fibrinolytic system. These findings suggest that an endothelial lesion could participate in the pathogenesis of some forms of recurrent deep vein thrombosis and support interest in the measurement of serum ACE to discriminate some patients at high risk of deep vein thrombosis.

Molecular weight and shape of angiotensin-I converting enzyme. A neutron scattering study.

The molecular weight of angiotensin-I converting enzyme from pig lung has been determined to be 112000 (+/- 6000) by neutron scattering. This is somewhat lower than the value determined by SDS-PAGE and than previous estimates which, however, show a very wide range of values. A quantitative analysis of the amino acid and carbohydrate content was made in order to determine the enzyme concentration. The small angle neutron scattering technique also gives information on the molecular shape, yielding a radius of gyration of 44.5 +/- 1.5 A which, for the observed molecular weight, indicates that the angiotensin converting enzyme molecule is clearly elongated in aqueous solution. Dynamic light scattering experiments confirm this conclusion.

Automated kinetic assay of angiotensin-converting enzyme in serum.

We have developed and validated an automated kinetic method for angiotensin-converting enzyme (EC 3.4.15.1) on the Olli C + D analyzer, modified from that of Ronca-Testoni (Clin Chem 1983;29:1093-6) with N-[3-(2-furyl)-acryloyl]-L-phenylalanylglycylglycine used as substrate. We have determined appropriate reaction conditions for the assay, verified the principal analytical reliability criteria (repeatability, reproducibility, sensitivity), and established normal reference intervals (mean +/- SD) for the enzyme's activity, using serum of normal adults (100 +/- 35 U/L, n = 150), newborns (130 +/- 27 U/L, n = 10), women taking oral contraceptives (103 +/- 30 U/L, n = 10), smokers (109 +/- 38 U/L, n = 27), and patients with sarcoidosis (220 +/- 48 U/L, n = 15).

Early monitoring of human renal transplantations by N-acetyl-beta-D-glucosaminidase isoenzyme activities in urines.

Monitoring of variations in N-acetyl-beta-D-glucosaminidase (NAG) urinary activity, following renal transplantation, has been proposed for the early diagnosis of rejection episodes. In this study, the measurement of urinary NAG-B activity was conducted as a complement to total NAG (A + B) measurement, which is normally used alone. Selective measurement of NAG-B activity is carried out after fixation of NAG-A on ion exchanger in test tubes. Results of NAG (A + B) activity confirm that the assay of urinary NAG is a useful indicator of rejection, but a positive correlation between NAG-B and NAG (A + B) activities was observed during the various complications which can occur after transplantation. The specific measurement of this isoenzyme does not, therefore, seem to provide additional information in the early monitoring of human renal transplantations. Apart from rejection episodes, other factors are likely to produce marked NAG-B excretion, e.g. gentamicin therapy.

Distribution of N-acetyl-beta-D-glucosaminidase isoenzymes along the rabbit nephron.

Total N-acetyl-beta-D-glucosaminidase (NAG) activity has been measured in microdissected glomeruli (G) and tubular segments [proximal convoluted tubule (PCT), pars recta (PR), medullary thick ascending limb (MAL), and cortical collecting tubule (CCT)] of the rabbit kidney, by a fluorimetric method using synthetic substrate. Selective activity of the isoenzyme NAG B was also determined. Isoenzyme profiles of NAG were obtained by electrofocusing on each segment. Characterization of the isoenzymes was performed by chromatofocusing and thermosensitivity experiments on PCT. Total NAG activity, mainly composed of NAG A, was low in glomeruli and two and one-half to four times higher in PCT than in other segments, in which comparable activities were found. NAG B was detectable all along the nephron. It represented a very small fraction of total NAG, except in PCT where it was more abundant (20 to 30%). Electrofocusing revealed the presence of a minor form (NAG I) all along the nephron. Chromatofocusing and thermosensitivity studies indicated that NAG I could represent imperfectly solubilized NAG A rather than a well defined entity. From these results, it could be suggested that the reported increase in urinary excretion of NAG B after renal injury may reflect the intensity of proximal tubular lesions.

Elevation of the bronchoalveolar concentration of angiotensin I converting enzyme in sarcoidosis.

The concentration of angiotensin converting enzyme (ACE) and that of albumin (AIb) were assayed in the serum (SACE, SAlb) and in bronchoalveolar lavage fluid (LACE, LAlb). Three groups of patients were studied: 14 healthy volunteers (Group I), 45 patients with active sarcoidosis (Group II), and 7 patients with sarcoidosis in remission (Group III). The SACE in Group II (4,466 +/- 2,202 U/100 ml, mean +/- SD) was higher (p less than 0.001) than in Group I (2,470 +/- 547 U/100 ml) or in Group III (2,640 +/- 610 U/100 ml); LACE was higher in Group II (65.2 +/- 48.4 U/100 ml, p less than 0.001) than in Group I (21.1 +/- 14.7 U/100 ml), or in Group III (25.7 +/- 14.6 U/100 ml). The SAlb was found to be, respectively, 3,908 +/- 385 mg/100 ml, 3,982 +/- 965 mg/100 ml, and 3.613 +/- 222 mg/100 ml in Groups I, II, and III. The LAlb in Group II (8.2 +/- 6.2 mg/100 ml) was higher (p less than 0.01) than in Group I (2.5 +/- 1.4 mg/100 ml) or in Group III )4.1 +/- 1.0 mg/100 ml). The LACE in Group II increased with the number of alveolar lymphocytes, in nonsmokers (4 = + 0.56, df = 34, p less than 0.001) and in smokers (4 = + 0.88, df = 7, p less than 0.01). In the smokers in this group, LACE was higher with respect to the number of lymphocytes than in the nonsmokers. We conclude from this study (1) that the permeability of the alveolocapillary membrane to albumin and to ACE is increased in active pulmonary sarcoidosis, (2) that LACE increases during sarcoidosis and returns to normal when the disease is cured, and (3) that the concentration of ACE in alveolar fluid increases with tobacco use.

Trehalase activity in genetically diabetic mice (serum, kidney, and liver).

Trehalase activity was determined in serum, liver, and kidney in alloxan treated Swiss mice and in homozygous (Ob/Ob, Db/Db) and heterozygous (Ob/+, Db/m+) diabetic mice. Both alloxan and genetic diabetic mice exhibited a large increase in serum and liver trehalase activity with no change in kidney trehalase activity. The heterozygotes (Ob/+, Db/m+) showed only a slight increase of enzyme activity. Further quantitative differences were noticed between the genetic and alloxan diabetic animals. The liver enzyme activity increased from 10- to more than 20-fold in the liver of the homozygous Ob/Ob and Db/Db strains and only 3-fold (not significant compared to controls) in the alloxan treated animals. The above results suggest a regulatory relationship between the genes coding for trehalase and the enzymes of glucose metabolism activity involved in the development of the metabolic anomalies of diabetes. The structural gene for trehalase may well have survived elimination of selective pressure during phylogenesis and remained part of a co-regulated group of glucose metabolising enzymes. This could explain its sensitivity to mutations affecting glucose metabolism and its sensitivity to insulin directed regulatory mechanisms.

[Increased concentration of angiotensin I converting enzyme in the alveolar fluid of patients with sarcoidosis]. / Augmentation de la concentration de l'enzyme de conversion de l'angiotensine I dans le fluide alvéolaire de sujets sarcoidiens.

Angiotensin converting enzyme (ACE) was assayed both in serum (SACE) and in bronchoalveolar fluid lavage (LACE) in 14 healthy controls and in 45 patients with sarcoidosis with mediastinal and pulmonary involvement. Concentration of SACE was 4466 +/- 2202 U x 100 ml-1 (mean +/- SD) in sarcoidosis and 2470 +/- 547 U x 100 ml-1 (chi +/- SD) in sarcoidosis and 2470 +/- 547 U . 100 ml-1 in controls. Concentrations of LACE were 65.2 +/- 48.4 U . 100 ml-1 and 21.1 +/- 14.7 U . 100 ml-1 respectively in sarcoidosis and in controls. These results are in favor of an intraalveolar secretion of ACE in sarcoidosis. LACE could be a better criterium than SACE for the evaluation of the pulmonary activity of sarcoidosis.

Comparative specificities of trehalases from various species.

1. Using derivatives or non-symmetrical analogs of alpha,alpha-trehalose, we studied the catalytic specificities of trehalases from various species: Pseudomonas fluorescens, Melolontha vulgaris, porcine and human kidneys. 2. alpha,Beta-trehalose, beta,beta-trehalose, 6,6'dideoxy alpha,alpha-trehalose, alpha-D-xylopyranosyl alpha-D-xylopyranoside were shown to be neither substrates nor inhibitors. 3. 6'deoxy alpha,alpha-trehalose, alpha-D-glucopyranosyl alpha-D-xylopyranoside, alpha-D-allopyranosyl alpha-D-glucopyranoside and alpha-D-galactosyl alpha-D-glucopyranoside, which all possess an intact alpha-D-glucopyranosyl residue, were split by all these trehalases. 4. alpha-D-glucopyranosyl alpha-D-mannopyranoside, alpha,alpha-trehalosamine are competitive inhibitors. 5. These results show the importance of the primary alcohol group at C-6, of the equatorial configuration of the OH groups at C-2, C-3 and C-4 and of the modification of the structure at C-2 of the substrate for the catalytic activity.