Kinetics of pulmonary angiotensin-converting enzyme and 5'-nucleotidase in vivo

Angiotensin-converting enzyme and 5'-nucleotidase line the luminal surface of pulmonary microvascular endothelium and participate in the synthesis and/or degradation of potent vasoactive substances. We applied Michaelis-Menten kinetics in simultaneous estimations of apparent constants Km and Amax (product of Vmax and microvascular plasma volume) of these two enzymes for the substrates 3H-labeled benzoyl-Phe-Ala-Pro and 14C-labeled 5'-AMP, respectively, in vivo. Values of angiotensin-converting enzyme for benzoyl-Phe-Ala-Pro (Km = 10-11 microM; Amax = 12-13 mumol X min-1) were somewhat higher than published estimates in vitro and changed predictably in response to the known enzyme inhibitor captopril. Kinetic values of 5'-nucleotidase for 5'-AMP (Km = 3-4 microM; Amax = 3-4 mumol/min) were substantially lower than those reported in vitro but also responded predictably to the competitive inhibitor of 5'-nucleotidase, adenosine 5'-[alpha, beta-methylene]diphosphate. These data offer in vivo estimates of enzyme kinetics that are useful in revealing enzyme behavior in their normal physiological environment and provide means of evaluating the action of pharmacological, physiological, and pathological modulators of enzyme activity, in vivo.     

Effects of acid-base imbalance on pulmonary angiotensin-converting enzyme in vivo

The effects of acid-base balance disturbances on pulmonary endothelial angiotensin-converting enzyme (ACE) were studied in anesthetized mechanically ventilated rabbits. Enzyme function was estimated from [3H]benzoyl-Phe-Ala-Pro ([3H]BPAP) utilization under first-order reaction conditions during a single transpulmonary passage and expressed as 1) substrate metabolism (M), 2) Amax/Km (Amax being equal to the product of enzyme mass and the constant of product formation), and 3) (Amax/Km)/100 ml blood flow. When respiratory acidosis/alkalosis was produced by altering respiratory rate at constant airway pressure, substrate (BPAP) utilization varied proportionally to arterial pH and inversely proportionally to arterial PCO2 (PaCO2) (P less than 0.05). Percent BPAP metabolism (%M) ranged from 92 +/- 3 (respiratory alkalosis) to 85 +/- 3 (normal), 82 +/- 3 (respiratory acidosis), and 78 +/- 2% (severe respiratory acidosis). Amax/Km similarly decreased from 899 +/- 129 to 825 +/- 143, 601 +/- 74, and 450 +/- 34 ml/min, respectively, and (Amax/Km)/100 ml blood flow was reduced from 176 +/- 26 to 131 +/- 22, 111 +/- 12, and 97 +/- 5, respectively. However, when respiratory acidosis/alkalosis was produced by altering both respiratory rate and airway pressure, no changes were observed in either %M, Amax/Km or (Amax/Km)/100 ml blood flow. Similarly metabolic alkalosis or acidosis did not alter M, Amax/Km or (Amax/Km)/100 ml blood flow. These results indicate that pulmonary endothelial ACE function can be affected by acid-base disturbances, probably indirectly through changes in perfused microvascular surface area.     

Serum angiotensin-converting enzyme. Isolation and relationship to the pulmonary enzyme.

Angiotensin-converting enzyme from rabbit serum was purified almost 60,000-fold to apparent homogeneity by a procedure exploiting its affinity for antibodies prepared against the enzyme from lung. The pure serum and pulmonary enzymes exhibited identical behavior during gel filtration, sucrose gradient centrifugation, and disc gel electrophoresis in the reduced, denatured state. Their catalytic properties with hippurylhistidylleucine, angiotensin I, and bradykinin as substrates were similar and their reactivity with antilung enzyme antibody was indistinguishable as examined by immunodiffusion, inhibition dose-response curves, and radioimmunoassay. Their content of fucose, mannose, galactose, and N-acetylglucosamine was also comparable; however, N-acetylneuraminic acid was much more abundant in the serum glycoprotein. This difference may reflect selective removal of sialic acid-deficient enzyme molecules from the circulation by the hepatic lectin which has been postulated to initiate the catabolic phase for plasma glycoproteins (Ashwell, G., and Morell, A.G. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 91-128).     

In vivo measurement of coronary circulation angiotensin-converting enzyme activity in humans

Angiotensin-converting enzyme (ACE) is present on the luminal surface of the coronary vessels, mostly on capillary endothelium. ACE is also expressed on coronary smooth muscle cells and on plaque lipid-laden macrophages. Excessive coronary circulation (CC)-ACE activity might be linked to plaque progression. Here we used the biologically inactive ACE substrate (3)H-labeled benzoyl-Phe-Ala-Pro ([(3)H]BPAP) to quantify CC-ACE activity in 10 patients by means of the indicator-dilution technique. The results were compared with atherosclerotic burden determined by coronary angiography. There was a wide range of CC-ACE activity as revealed by percent [(3)H]BPAP hydrolysis (30-74%). The atherosclerotic extent scores ranged from 0.0 to 66.97, and the plaque area scores ranged from 0 to 80 mm(2). CC-ACE activity per unit extracellular space (V(max)/K(m)V(i)), an index of metabolically active vascular surface area, was correlated with myocardial blood flow (r = 0.738; P = 0.03) but not with measures of the atherosclerotic burden. These results show that CC-ACE activity can be safely measured in humans and that it is a good marker of the vascular area of the perfused myocardium. It does not, however, reflect epicardial atherosclerotic burden, suggesting that local tissue ACE may be more important in plaque development.     

Kinetic properties of pulmonary angiotensin-converting enzyme. Hydrolysis of hippurylglycylglycine.

Some of the kinetic properties of angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) purified from hog lung have been determined using hippurylglycylglycine as substrate. The effects of pH and ionic environment on enzyme activity are complex and interdependent. At 0.1 M NaCl, the pH-activity curve shows an abrupt decrease in V/Km as the pH rises from 6 to 6.5, implying that ionization of a group in the enzyme with a pK in this range aids in binding of the substrate. Chloride is required for enzyme activity; there are two phases in the effect of NaCl. At both pH 6 AND 8, THE FIRST PHASE (UP TO 0.1 M NaCl) is activation. The second phase (above 0.1 M) at pH 6 is inhibition, while at pH 8 there is further activation which appears to be dependent upon ionic strength rather than a specific Cl-effect. Activation by cobalt and inhibition by EDTA are somewhat more effective at pH 6 than at pH 8. The nonapeptide inhibitor less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro is nearly equipotent at both pH 6 and 8, but Arg-Pro-Pro is more inhibitory at pH 8 than at pH 6.     

Canine pulmonary angiotensin-converting enzyme. Physicochemical, catalytic and immunological properties.

Antiontensin-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) has been solubilized from canine pulmonary particles and purified to apparent homogeneity. A value of approx. 140000 was estimated for the molecular weight of the native and the reduced, denatured forms of the enzyme. No free NH2-terminal residue was detected by the dansylation procedure. Carbohydrate accounted for 17% of the weight of the enzyme, and the major residues were galactose, mannose and N-acetylglucosamine with smaller amounts of sialic acid and fucose. Removal of sialic acid residues with neuraminidase did not alter enzymatic activity. The enzyme contained one molar equivalent of zinc. Addition of this metal reversed stimulation and inhibition of activity observed in the presence of Co2+ and Mn2+, respectively. Immunologic homology of pure dog and rabbit enzymes was demonstrable with goat antisera. Fab fragments and intact IgG antibodies displayed similar inhibition dose vs. response curves with homologous enzyme, whereas the fragments were poor inhibitors of heterologous activity compared to the holoantibodies. The canine glycoprotein was much less active than the rabbit preparation in catalyzing hydrolysis of Hip-His-Leu. In contrast, the two enzymes exhibited comparable kinetic parameters with angiotensin I as substrate.     

Pulmonary angiotensin-converting enzyme. Interspecies homology and inhibition by heterologous antibody in vivo.

Goat antibody against pure rabbit pulmonary angiotensin-converting enzyme was used to probe homology of converting enzymes from other species. Immunologically cross-reactive material was found in detergent-solubilized extracts of lung particles from rat, guinea pig, and dog by double immunodiffusion, radioimmunoassay, and inhibition of enzyme activity. No homology was demonstrable with bovine, frog, or chicken lung extracts. Antibodies from different individual goats yielded comparable estimates of homology by immunodiffusion and radioimmunoassay. In contrast, they varied greatly in extent and specificity of their inhibitory action on heterologous enzyme activity. The vasopressor effect of angiotensin I and the vasodepressor effect of bradykinin were diminished and potentiated, respectively, in rats treated with anti-rabbit enzyme antibody. A smaller but significant immune-dependent inhibition of the vasopressor response to angiotensin II was also observed.     

In vivo and in vitro kinetics of nitrogenase.

We measured some of the kinetic parameters of nitrogenase to intact systems of Clostridium pasteurianum and Klebsiella pneumoniae to compare them with the kinetics of the enzyme in vitro. We found that the enzyme showed multiple apparent Km values for acetylene reduction in vivo, as it does in vitro. Carbon monoxide was a noncompetitive inhibitor of acetylene reduction; azide was a noncompetitive inhibitor of acetylene reduction, and nitrogen was a partial inhibitor of acetylene reduction. Cyanide was a noncompetitive inhibitor of acetylene reduction in C. pasteurianum but it was a metabolic poison in K. pneumoniae, in addition to being an inhibitor of nitrogenase. The partial nature of nitrogen inhibition was apparent in assays where both nitrogen and CO were present. Nitrogen did not alter the apparent Ki for CO, nor did the presence of CO enhance the competitive effectiveness of nitrogen. By using recombined nitrogenase fractions, we found that the ability of nitrogen to inhibit hydrogen evolution or acetylene reduction varied with the ratio of protein components. The in vivo inhibition of acetylene reduction by dinitrogen was comparable to that obtained with an excess of the Fe protein in vitro. We conclude that there is an effective excess of the Fe protein available under active growth conditions in vivo.     

Effects of gas composition and pH on kinetics of lung angiotensin-converting enzyme

Given the pH dependence of enzymes in general and the potential importance of a blood and alveolar gas composition dependency on the interpretation of changes in the hydrolysis of angiotensin-converting enzyme (ACE) substrates by pulmonary endothelial ACE, we examined the influence of Pco2 and Po2 on the hydrolysis of a synthetic ACE substrate (benzoyl-phenylalanyl-alanyl-proline, BPAP) on passage through isolated rabbit lungs. Perfusate pH values of about 7.1, 7.4, and 7.9 were obtained by ventilating the lungs with gas containing different CO2 concentrations and Po2 values of approximately 110 and approximately 10 Torr were obtained by varying the concentration of O2 in the ventilating gas mixture. In the range studied neither acidosis nor alkalosis produced any significant changes in BPAP hydrolysis or in the kinetic parameters, Vmax and Km, for the hydrolysis process. On the other hand, a reduction in BPAP hydrolysis was detected when the Po2 was reduced from 110 to 10 Torr. The Vmax for BPAP hydrolysis by the lung was inversely correlated with the magnitude of the hypoxic vasoconstriction that occurred, suggesting that the reduced BPAP hydrolysis with hypoxia was due to the loss of perfused surface area due to the vasoconstriction. The results suggest that correlations between Pco2 and/or pH and whole-lung ACE activity that might occur in diseased lungs do not imply causalty. The hemodynamic consequences of changing Po2 (i.e., hypoxic vasoconstriction) may alter whole-organ ACE activity in the sense of changing the perfused surface area (i.e., the amount of ACE in contact with flowing perfusate).     

Pulmonary angiotensin-converting enzyme kinetics after acute lung injury in the rabbit

Depression of lung endothelial cell metabolic function may be an early and sensitive indicator of lung damage. When such functions are measured in vivo, substrates injected usually must be limited to "trace" doses due to the significant hemodynamic effects of high doses of substrate. Under first-order conditions (i.e., trace doses) the enzyme or transport system rate constant Vmax/Km may be calculated, but independent estimates of each variable (Vmax and Km) are not available. We therefore used multiple indicator-dilution methods and higher substrate concentrations to apply a mathematical model, based on saturable kinetics that yield independent estimates of the apparent kinetic parameters Vmax and Km for pulmonary angiotensin-converting enzyme (ACE). We used the ACE substrate, [3H]benzoyl-phenylalanyl-alanyl-proline ([3H]BPAP) and made these measurements and also estimates of serotonin [5-hydroxytryptamine (5-HT)] removal, before and after acute lung injury induced by intratracheal administration of phorbol myristate acetate (PMA). PMA significantly depressed the percent 5-HT removal (62 +/- 3 to 44 +/- 4%) and BPAP percent metabolism (74 +/- 2 to 66 +/- 2), when trace amounts of either compound were injected as a bolus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary angiotensin-converting enzyme antienzyme antibody.

A method has been developed for quantitating anticatalytic activity in antibody preparations made in goats against pure solubilized angiotensin-converting enzyme from rabbit pulmonary membranes. Anticatalytic activity was purified about 90-fold from a single batch of serum by a procedure including diethylaminoethylcellulose chromatography and elution from Sepharose columns containing covalently bound pure enzyme. Antiholoenzyme antibody was fractionated with respect to charge and binding affinity; however, these different populations each inhibited enzymatic hydrolysis of hippurylhistidylleucine, angiotensin I, and bradykinin. The inhibition dose-response curves were similar for hydrolysis of hippurylhistidylleucine and angiotensin I despite the difference in molecular weight of these substrates. Evidence is presented suggesting that a single molecule of antibody can bind two molecules of enzyme and that at least 18% of the total antiholoenzyme antibody population is directed against determinants which influence catalytic activity. A competitive immunoassay was developed with radioiodinated pulmonary enzyme as displaceable antigen. The anticatalytic and radioimmune assays were used to examine immunological properties of converting enzymes in various rabbit organs and fluids. Kidney, brain, and serum were found to contain converting enzymes which were immunologically identified with that in rabbit lung. Converting enzyme in seminal plasma was similar to the lung enzyme in the anticatalytic assay, but showed lower immunoreactivity in the radioimmune assay.     

Effects of alveolar pressure on lung angiotensin-converting enzyme function in vivo

Angiotensin-converting enzyme lines the luminal surface of pulmonary capillary endothelial cells. The metabolism of its synthetic substrate, 3H-benzoyl-L-phenylalanyl-L-alanyl-L-proline ([3H]BPAP) has been used as an indicator of pulmonary microvascular function. Because the flow-volume status of the pulmonary capillaries is dependent on intra-alveolar pressure, we have studied the effects of airway pressure on endothelial plasmalemmal angiotensin-converting enzyme function in rabbit lungs in vivo. Static inflation of the lungs to a pressure of 0 or 5 Torr did not change percent transpulmonary metabolism and Amax/Km ratio (defined as E X Kcat/Km and thus, under normal conditions, an indirect measure of perfused endothelial luminal surface area) compared with control measurements during conventional mechanical ventilation. When the inflation pressure was increased to 10 Torr, percent metabolism of [3H]BPAP remained unaltered but Amax/Km decreased to 60% of the control value. This decrease was in close relation to the decrease in pulmonary blood flow. Addition of 5 cmH2O positive end-expiratory pressure (PEEP) to the mechanical ventilation also decreased Amax/Km values and pulmonary blood flow but did not influence percent metabolism of [3H]BPAP. These results suggest that the detected alterations in apparent enzyme kinetics were more likely due to hemodynamic changes than to alterations in angiotensin-converting enzyme function. Thus high static alveolar pressures as well as PEEP probably reduced the fraction of perfused microvessels as reflected in changes in Amax/Km ratios. This information should prove useful in interpreting the response of pulmonary endothelial enzymes to injury.     

Tripeptidyl carboxypeptidase activity of kininase II (angiotensin-converting enzyme)

The degradation of des-Arg9-brady kinin and its analogues by highly purified preparations of hog lung and kidney kininase II (angiotensin-converting enzyme; peptidyldipeptide hydrolase, EC 3.4.15.1) was studied. The degradative peptides fragments were separated and isolated by high performance liquid chromatography and identified by amino acid analysis. Both enzymes released C-terminal tripeptides from des-Arg9-bradykinin, des-Arg9-(Leu8)-bradykinin, Pro-Pro-Gly-Phe-Ser-Pro-Phe, Pro-Gly-Phe-Ser-Pro-Phe, Gly-Phe-Ser-Pro-Phe, Bz-Gly-Ser-pro-Phe and Bz-Gly-Ala-Pro-Phe. Hydrolysis of Phe-Ser-Pro-Phe, Bz-Gly-His-Pro-Phe, Bz-Gly-Phe-Pro-Phe and Bz-Gly-Gly-Pro-Phe by both enzymes was negligible. These data indicate that kininase II can release C-terminal tripeptides of substrates having a proline residue in the penultimate position such as des-Arg9-bradykinin and its analogues, and that this enzyme is able not only to act as a dipeptidyl carboxypeptidase but also acts as a tripeptidyl carboxy-peptidase. The tripeptidyl carboxypeptidase enzyme was sensitive to inhibition by kininase II inhibitors.     

Expression of angiotensin-converting enzyme activity in cultured pulmonary artery endothelial cells

Angiotensin-converting enzyme (EC 3.4.51.1) is a carboxyterminal dipeptidyl peptidase. The enzyme catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II. In addition, the enzyme catabolizes bradykinin. Because of these actions, the enzyme is of pivotal importance in blood pressure homeostasis. Numerous investigators have demonstrated the presence of the enzyme in association with endothelial cells but relatively little is known concerning the factors controlling the expression enzyme activity by endothelial cells in culture. We have demonstrated that endothelial cells in culture do not express significant amounts of enzyme activity until several days after growth ceases due to high cell density. This is important because it demonstrates a change in function with stage of growth in culture and a possible difference in functional capabilities between nondividing endothelial cells and cells that are dividing in response to injury. Since density-dependent expression of differentiated traits does not appear to be unique to endothelial cells an understanding of the mechanisms underlying this phenomenon may provide a general explanation for the expression of differentiated traits by cultured cells.