Emanuel Silverstein

Induction of angiotensin converting enzyme in human monocytes in culture

Abstract Angiotensin converting enzyme (E.C.3.4.15.1, peptidyl dipeptidase) in circulating human monocytes rose from undetectable or minimal levels in vivo to as high as 35.5 nmol/min·mgprotein (>300-fold increase) after 6 or 7 days in culture. Enzyme induction was enhanced by autologous serum and exposure for two days to 0.45 μM dexamethasone. Potent inhibition of enzyme induction by 370 μg/ml of actinomycin D and 1 μM cycloheximide suggested that new messenger RNA and enzyme biosynthesis are involved in the induction. Human monocyte and lung enzyme were similar with respect to EDTA inhibition, CoCl2 activation and inhibition by an antienzyme antiserum. Human lymphocytes had minimal or undetectable enzyme which was not induced after 4 days in culture.

Elevation of angiotensin-converting enzyme in granulomatous lymph nodes and serum in sarcoidosis: clinical and possible pathogenic significance.

A statistically highly significant elevation of serum ACE was found in a group of 58 patients with sarcoidosis (serum ACE was elevated in 34% of patients), as compared with normal controls and patients with tuberculosis and various other common diseases. The results suggest that serum ACE is a useful aid for the diagnosis of sarcoidosis when elevated, but that a normal value does not rule out the condition and may occur in more than one-half of monitored patients. There is a trend to diminution of serum ACE with increasing duration of disease with or without steroid therapy, perhaps correlating with the total body mass of active granulomas, as indirectly suggested in preliminary data by correlation of serum ACE with serum globulin in 16 sarcoidosis patients. It is not yet clear whether there is any significant steroid effect on serum ACE, but a significant number of patients on steroid therapy for more than 2-4 yr have elevated serum ACE values, which in some instances are extremely high. There was a 12-fold elevation in ACE to specific activities generally exceeding those of normal lung in granulomatous lymph nodes of 14 patients with sarcoidosis, suggesting that sarcoid granulomas may be actively synthesizing ACE and resulting in elevation of serum ACE. Extensively fibrotic sarcoid lymph nodes had normal or slightly elevated ACE, suggesting that obliteration of granulomas in sarcoid lymph nodes diminishes their ACE content and that this obliteration may be related to the tendency to diminution of serum ACE with time. ACE was not elevated in one tuberculous lymph node or in experimental granulomas, suggesting that elevation of ACE may have some specificity for the granuloma of sarcoidosis rather than being a characteristic of all granulomas. The catalytic and physical properties of ACE in serum and lymph nodes in sarcoidosis were generally similar to normal ACE with respect to pH activity, modulators, polyacrylamide-gel electrophoresis, and Sephadex G-200 gel filtration. However, sarcoid lymph node ACE appeared to be more heat labile than normal lung or lymph node ACE, suggesting the possibility that an abnormal ACE may be present in sarcoidosis. If an abnormal enzyme is indeed present, it might be coded for by a host gene that is not normally expressed or a nonhost gene or it might be a normal ACE that has been altered. No ACE activity was found in circulating white blood cells in sarcoidosis or in control subjects, suggesting that circulating white blood cells may not contain the epithelioid cell precursor or that ACE synthesis (or less likely, uptake) may be turned on at a later stage in the transformation. Lysozyme activity was also elevated in sarcoid lymph nodes. Serum ACE and serum lysozyme were significantly positively correlated in 16 sarcoidosis patients, suggesting a relationship between the two...

Elevated serum and spleen angiotensin converting enzyme and serum lysozyme in Gaucher's disease

In adult chronic non-neuronopathic (Type 1) Gauchers disease significant (p less than 0.001) elevations of angiotensin converting enzyme in serum (93.3 +/- 14.8 nmol/min/ml; number elevated, 8/11; normal control 32.2 +/- 1.30, n = 58) and spleen (5.62 +/- 0.35 nmol/min/mg protein, n = 2; control, 0.431 +/- 0.101, n = 4) and serum lysozyme (15.6 +/- 3.37 mug/ml; number elevated, 4/5) were observed. The KM for hippuryl-L-histidyl-L-leucine of Gaucher (1.31 mM) and normal (1.23 mM) serum angiotensin converting enzyme were similar. The increased angiotensin converting enzyme (ACE) in Gauchers disease may be related to the genetic defect resulting in increased ACE synthesis in Gaucher cells, or perhaps generally, while high lysozyme may reflect an increased body mass of reticuloendothelial cells. These enzyme elevations may be of use in suggesting the possible presence of Gauchers disease and perhaps in assessing the magnitude of pathologic involvement.

Separation and purification of oxidized and reduced nicotinamide adenine dinucleotides

Abstract Sharp column chromatographic separations of NAD and NADH at neutral and acid pH and of NADP and NADPH at neutral pH by discontinuous elution with volatilizable eluents are described. The presence of impurities in NAD and NADH was elucidated by DEAE-cellulose chromatography, by which purification may be readily achieved. The detrimental effect of freezing or lyophilizing of NADH is described. The ratio of the absorbance at 260 mμ to that at 340 mμ of the purest NADH preparations obtained is 2.24. The molar absorbance of NADH at 260 mμ is estimated at 14.0 × 10 3 × liter −1 × mole −1 and that for NAD at 17.4 × 10 3 × liter −1 × mole −1 .

Human Lung Angiotensin Converting Enzyme PURIFICATION AND ANTIBODY PREPARATION

To enable its immunohistologic localization, angiotensin converting enzyme (EC 3.4.15.1) from human lung was solubilized by trypsinization and purified approximately 2,660-fold to apparent homogeneity from a washed lung particulate fraction. The specific activity of pure enzyme was estimated to be 117 mumol/min per mg protein with the substrate hippuryl-l-histidyl-l-leucine. Consistent with previously described lung enzyme studies, catalytic activity was strongly inhibited by EDTA, O-phenanthroline, SQ 20,881, and SQ 14,225 and increased by CoCl(2). SQ 20,881 was a somewhat more potent inhibitor than SQ 14,225, unlike rabbit lung enzyme. The Michaelis constant (K(m)) with hippuryl-l-histidyl-l-leucine was 1.6 mM. The molecular weight was estimated at 150,000 from sucrose density gradient centrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single polypeptide chain estimated at 130,000 daltons. Rabbit antibody to human lung enzyme was prepared by parenteral administration of pure angiotensin-converting enzyme in Freunds adjuvant. Rabbit antibody to human lung angiotensin-converting enzyme appeared to crossreact weakly with the rabbit enzyme and strongly inhibited the catalytic activity of the enzymes from human serum, lung, and lymph node. The specificity of the rabbit antibody and purity of the final human lung enzyme preparation was suggested by the single precipitin lines obtained by radial double immunodiffusion, and by the coincidence of enzyme catalytic activity and immunoreactivity on polyacrylamide gel electrophoresis, with both relatively pure and highly impure enzymes. Generally applicable sensitive analysis of acrylamide gels for immunoreactivity (and subsequently for any other activity) by use of intact gel slices in radial double immunodiffusion was devised. Human lung enzyme was very tightly bound to and catalytically active on anti-human enzyme antibody covalently bound to Sepharose 4B, and could not be readily dissociated without inactivation. Antibody to human lung angiotensin converting enzyme has permitted tissue localization of the enzyme, which appears to be clinically useful in diseases associated with abnormal abundance of angiotensin-converting enzyme in tissues, such as sarcoidosis.

Immunofluorescent detection of angiotensin-converting enzyme (ACE) in Gaucher cells

The cellular localization of the elevated angiotensin-converting enzyme (ACE) in the spleen of a patient with Gauchers disease was examined by immunofluorescence using an antibody prepared against highly purified human lung enzyme. Intense ACE-specific cytoplasmic fluorescence was observed in Gaucher cells, but not in various controls, indicating the localization of large quantities of enzyme in these cells. These results and the demonstrated capability of mononuclear phagocytes for marked induction of this enzyme suggest the possibility that induction of active synthesis of ACE in Gaucher cells may be responsible for the elevated enzyme levels in the serum and spleen of patients with Gauchers disease.

Equilibrium kinetic study of the mechanism of mitochondrial and supernatant malate dehydrogenases of bovine heart

Abstract The kinetics at equilibrium have been examined for mitochondrial and supernatant malate dehydrogenases ( l -malate:NAD+ oxidoreductase, EC 1.1.1.37) from bovine heart at catalytic concentration by means of isotopic exchange of reactants to compare their catalytic mechanisms. In accord with previous studies of lactate, alcohol and pig heart mitochondrial malate dehydrogenases the rate of substrate interchange exceeded the NAD+ ⇌ NADH rate, in some cases by more than 100-fold, except at lowest substrate concentration. At pH 8.0 a marked depression in the NAD+ ⇌ NADH rate to near zero without inhibition in the oxaloacetate ⇌ malate rate was compatible with a compulsory binding order with substrate binding to E (enzyme)-coenzyme complexes, but not to free enzyme. At pH 9.0 only a moderate depression in the NAD+ ⇌ NADH rate was found, indicating a partially compulsory pathway in which coenzyme dissociation occurs from both E- coenzyme and E- coenzyme-substrate complexes, but is more rapid from the binary complex. Kinetics with variable concentration of non-reactive pairs of substrate and coenzyme suggested the formation of the abortive complex E- NADH malate, but not E- NAD + -oxaloacetate . The initial rate of reduction of oxaloacetate was in all cases greater than the equilibrium rates. Minimum estimates were obtained for some dissociation constants. Despite considerable structural differences, the similar kinetic behavior at equilibrium of the mitochondrial and supernatant enzymes suggests a uniform catalytic mechanism which includes a compulsory binding order at pH 8.0 with coenzyme binding prior to substrate, a partially compulsory pathway at pH 9.0, non-rate-limiting chemical transformation, and formation of the abortive complex, E- NADH-malate . This mechanism is also in accord with that previously found with pig heart mitochondrial malate dehydrogenase.

Increased Serum Angiotensin-Converting Enzyme in Chronic Renal Disease

Serum angiotensin-converting enzyme (SACE) was significantly elevated in 16 of 48 patients with various types of chronic renal disease not on hemodialysis [45.6 +/- (SD) 16.7 nmol/min/ml, p less than 0.001] and in 17 of 52 patients with chronic renal disease on maintenance hemodialysis (43.2 +/- 13.8, p less than 0.001) in comparison with 58 healthy adult controls (32.2 +/- 9.8). There was no significant relationship between SACE and renal function as indicated by serum creatinine and creatinine clearance, or the presence or absence of maintenance hemodialysis, except for a transiently increased elevation of SACE immediately post-dialysis due to hemoconcentration. There was a statistically insignificant positive correlation between SACE and 24-hour urinary protein excretion (r = 0.254, n = 21). Mean blood pressure measured in patients on maintenance hemodialysis was inversely related to SACE (p less than 0.05) following dialysis. Chronic renal disease irrespective of severity and therapy tends to be associated with elevated SACE and must be considered in the evaluation of sarcoidosis.

Elevated serum angiotensin-converting enzyme in hyperthyroidism.

Serum angiotensin-converting enzyme was elevated in patients with hyperthyroidism (72 +/- 31 nmol/minute/ml, n = 12, p less than 0.001) but not in patients with hypothyroidism (38 +/- 3, n = 3) or thyroiditis (26, n = 1), and was positively correlated in 23 patients with serum thyroxine concentration (r = 0.60, p less than 0.01) and triiodothyronine resin uptake (r = 0.56, p less than 0.01). Triiodothyronine failed to enhance the synthesis of angiotensin-converting enzyme in rabbit alveolar macrophages or in human monocytes in culture, suggesting that the increased serum enzyme is a consequence of an effect other than increased angiotensin-converting enzyme synthesis. Hyperthyroidism should be considered in the evaluation of serum angiotensin-converting enzyme for the diagnosis and management of sarcoidosis.

Malate dehydrogenase and aspartate aminotransferase of Phycomyces blakesleeanus.

Abstract Malate dehydrogenase has been isolated from Phycomyces blakesleeanus in electrophoretically distinct supernatant and mitochondrial forms. The supernatant enzyme which comprises at least 88% of total malate dehydrogenase has been purified 190-fold by ammonium sulfate fractionation and diethylaminoethyl-cellulose and carboxymethyl-cellulose chromatography, and characterized kinetically in comparison with mitochondrial enzyme with respect to oxalacetate, malate and nicotinamideadenine dinucleotide and its analogues. Malate dehydrogenase and aspartate aminotransferase activities exist on separate macromolecules estimated at 70,000 and 81,500 molecular weight, respectively, on the basis of partial separation on Sephadex G-100. Different heat inactivations, pH-activity profiles, and recoveries on purification support this conclusion.