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).     

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.     

A simple radioassay for angiotensin-converting enzyme.

Angiotensin-converting enzyme can be measured by the rate of release of 3H-labelled hippurate from p-[3H]benzoylglycylglycylglycine. The product is separable from the substrate by extraction of acidified reaction mixtures with ethyl acetate. Assay results for human serum angiotensin-converting enzyme can be obtained within 1.5 h of receipt of serum samples. Within the limits tested, the assay appears to be specific. However, interference by hitherto unrecognized enzymes of abnormal sera must be ruled out.     

Elite athletes and the gene for angiotensin-converting enzyme.

The deletion (D) allele of the gene for angiotensin-converting enzyme (ACE) is associated with higher plasma and tissue levels of the enzyme and has also been related to a variety of cardiovascular complications, particularly myocardial infarction. On the basis of indirect evidence, we hypothesized that inheritance of the D allele would contribute to elite athletic ability. Over a period of 4 yr, 120 Caucasian athletes who were national (Australian) representatives in sports demanding a high level of aerobic fitness were recruited. Their ACE genotypes were compared with those of a community control group recruited randomly from the electoral roll. There was no difference in ACE genotype frequencies between the two groups. The DD genotype frequency was 30% in athletes and 29% in the control group, and the II genotype frequency was 22.5 and 22%, respectively. The results do not exclude the possibility that ACE genotype could be related to some attribute relating to a specific type of elite athletic ability or that there may be a difference between genders. Larger studies are desirable.     

Formation of angiotensin III by angiotensin-converting enzyme.

Angiotensin III is formed from des-Asp1 -angiotensin I by angiotensin-converting enzyme. The Km (11 muM) of the reaction is one-third of that for the conversion of angiotensin I into angiotensin II. As suggested by the Km values, bradykinin, peptide BPP9a and angiotensins II and III are better inhibitors of the formation of angiotensin II than of the formation of angiotensin III.     

A structural study of the asparagine-linked oligosaccharide moiety of duck ovomucoid

Asparagine-linked oligosaccharides of duck ovomucoid were released quantitatively from the protein by digestion with glycoamidase A (from almond), the reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated using two different types of high performance liquid chromatography (HPLC) on a reversed phase column and an amide adsorption column. More than sixteen different oligosaccharides were separated and the structures were characterized by a combination of the 2-dimensional sugar mapping technique using HPLC, exoglycosidase digestion, and proton nuclear magnetic resonance measurements (1- and 2-dimensional). Furthermore, the HPLC profile of duck ovomucoid oligosaccharides was compared with previously reported profiles obtained from quail and chicken ovomucoids.Abbreviations COSY chemical shift-correlated spectroscopy - DQF-COSY double quantum filtered COSY - DSS sodium, 4,4-dimethyl-4-silapentane 1-sulfonate - Gal D-galactose - GlcNAc or GN N-acetyl-D-glucosamine - HOHAHA homonuclear Hartmann-Hahn spectroscopy - Man or M D-mannose - NOE nuclear Overhauser enhancement - ODS octadecylsilyl - PA pyridylamino - ROESY rotating frame nuclear Overhauser effect spectroscopy - SDS/PAGE sodium dodecyl sulfate/polyacrylamide gel electrophoresis     

Pulmonary angiotensin-converting enzyme. Structural and catalytic properties.

Angiotensin-converting enzyme has been solubilized from a particulate fraction of rabbit lung and purified to apparent homogeneity in 11% yield by a procedure including fractionation with DEAE-cellulose and calcium phosphate gel, elution from Sephadex G-200, and lectin affinity chromatography. The molecular weight estimated by equilibrium sedimentation was approximately 129,000, either in the absence or presence of 6 M guanidine hydrochloride. A slightly higher value of 140,000 determined for the reduced, denatured protein by gel electrophoresis in the presence of sodium dodecyl sulfate and a much higher figure derived from gel filtration are probably due to the glycoprotein nature of the enzyme. Its oligosaccharide content accounted for 26% of the weight calculated from its amino acid and carbohydrate composition. The estimated content of sugar residues per mole was: galactose, 57; N-acetylglucosamine, 53; mannose, 43; N-acetylneuraminic acid, 19; and fucose, 4. Threonine and alanine were identified, respectively, as NH2-terminal and COOH-terminal residues by the dansylation procedure and by digestion with carboxypeptidase A. The enzyme was found to contain approximately 1 g atom of zinc per mol. Km values for hydrolysis of hippurylhistidylleucine and angiotensin I were 2.3 and 0.07 mM, and the corresponding turnover numbers were 15,430 and 792 mol/min/mol at 37 degrees. Bradykinin was also a substrate, and release of its COOH-terminal dipeptide, Phe-Arg, was catalyzed at a comparable rate to that of His-Leu from the COOH terminus of angiotensin I. Enzyme activity required the presence of chloride ions and was inhibited by EDTA and by low concentrations of Bothrops bradykinin-potentiating peptides. In addition, hydrolysis of hippurylhistidylleucine was inhibited competitively by other defined peptides, including di- and tripeptides, which were not substrates.     

Structural changes in the oligosaccharide moiety of human IgG with aging

In order to elucidate the relationship between glycosylation of IgG and aging, oligosaccharide structures of human IgG purified from sera of men and women aged 18 to 73 years were investigated. Oligosaccharides were liberated quantitatively from IgG by hydrazinolysis followed by N-acetylation and were tagged with p-aminobenzoic acid ethyl ester. The oligosaccharide structures were then analyzed by HPLC in conjunction with sequential exoglycosidase digestion. All IgG samples were shown to contain a series of biantennary complex type oligosaccharides which consisted of ±Gal1-4GlcNAc1-2Man1-6(±GlcNAc1-4)(±Gal1-4GlcNAc1-2Man1-3)Man1-4GlcNAc1-4(±Fuc1-6)GlcNAc and their mono- and di-sialo glycoforms in different ratios. In female IgG samples only, the incidence of non-galactosylated oligosaccharides with non-reducing terminal GlcNAc residues increased with aging (r>0.8), whereas that of digalactosylated oligosaccharides decreased (r<–0.8). A weaker correlation was observed between aging and the incidence of neutral and monosialo oligosaccharides in female IgG (r:0.461 and r=–0.538, respectively) and between aging and the incidence of oligosaccharides with a bisecting GlcNAc in both male and female IgG samples (r=0.566 and r=0.440, respectively). In addition, a significant change with aging in the galactosylation of IgG oligosaccharides was observed in females in their thirties, fifties, and sixties (p<0.02, p<0.01, and p<0.04, respectively). These findings may contribute to our understanding of autoimmune diseases such as rheumatoid arthritis in which glycosylation is involved.     

Structural diversity of angiotensin-converting enzyme

The crystal structure of a Drosophila angiotensin-converting enzyme (ANCE) has recently been solved, revealing features important for the binding of ACE inhibitors and allowing molecular comparisons with the structure of human testicular angiotensin-converting enzyme (tACE). ACER is a second Drosophila ACE that displays both common and distinctive properties. Here we report further functional differences between ANCE and ACER and have constructed a homology model of ACER to help explain these. The model predicts a lack of the Cl(-)-binding sites, and therefore the strong activation of ACER activity towards enkephalinamide peptides by NaCl suggests alternative sites for Cl(-) binding. There is a marked difference in the electrostatic charge of the substrate channel between ANCE and ACER, which may explain why the electropositive peptide, MKRSRGPSPRR, is cleaved efficiently by ANCE with a low K(m), but does not bind to ACER. Bradykinin (BK) peptides are excellent ANCE substrates. Models of BK docked in the substrate channel suggest that the peptide adopts an N-terminal beta-turn, permitting a tight fit of the peptide in the substrate channel. This, together with ionic interactions between the guanidino group of Arg9 of BK and the side chains of Asp360 and Glu150 in the S(2)' pocket, are possible reasons for the high-affinity binding of BK. The replacement of Asp360 with a histidine in ACER would explain the higher K(m) recorded for the hydrolysis of BK peptides by this enzyme. Other differences in the S(2)' site of ANCE and ACER also explain the selectivity of RXPA380, a selective inhibitor of human C-domain ACE, which also preferentially inhibits ACER. These structural and enzymatic studies provide insight into the molecular basis for the distinctive enzymatic features of ANCE and ACER.     

Pulmonary angiotensin-converting enzyme substrate hydrolysis during exercise.

We examined exercise-induced changes in indicator-dilution estimates of the angiotensin-converting enzyme first-order kinetic parameter, the ratio of a normalized maximal enzymatic conversion rate to the Michaelis constant (Amax/Km), which, under stable enzymatic conditions, will vary with the pulmonary vascular surface area accessible to vascular substrate, the extravascular lung water (an index of the proportion of lung tissue perfused), and the central blood volume (from pulmonary trunk to aorta). Experiments were performed in 10 mongrel dogs at rest and through two increasing levels of treadmill exercise, with the use of two vascular space tracers (labeled erythrocytes and albumin), a water space tracer ([1,8-14C]-octanediol), and a vascular endothelium surface area marker, benzoyl-Phe-Gly-Pro ([3H]BPGP), which is a pharmacologically inactive angiotensin-converting enzyme substrate. The exercise-induced increase in cardiac output was accompanied by a linear increase in central blood volume, and dilutional extravascular lung water rapidly increased to an asymptotic proportion close to 100% of postmortem vascular lung water. There was an average 55% [3H]BPGP hydrolysis, which did not vary with flow, and the computed Amax/Km increased linearly with exercise. We conclude that exercise results in complete lung tissue recruitment and increases the pulmonary vascular surface area available for BPGP hydrolysis linearly with flow, so that pulmonary vascular recruitment continues after full tissue recruitment.     

Evolution of angiotensin-converting enzyme inhibitors

The renin-angiotensin system is perhaps the most important hormonal system in the regulation of blood pressure. Its influence on blood pressure is mediated by the potent vasoconstrictor angiotensin II. Since angiotensin-converting enzyme performs the last step in the biosynthesis of angiotensin II, inhibition of this enzyme has attracted the attention of many researchers as a novel approach in the control of high blood pressure. The evolution of inhibitors of this enzyme will be traced from the early snake venom peptide inhibitors to the drugs currently available for the treatment of high blood pressure and congestive heart failure.     

Structural studies on the oligosaccharide moiety of the lipopeptidophosphoglycan from Trypanosoma cruzi

The structure of the carbohydrate moiety of the lipopeptidophosphoglycan from Trypanosoma cruzi was studied by 13C NMR spectroscopy and by methylation analysis of the original and of an acid-degraded sample. An oligosaccharide, consisting of 2-O-substituted and 6-O-substituted mannoses, which is linked to the ceramide, was separated by partial acid hydrolysis from an external chain that contained 3-O-substituted mannopyranosyl residues. beta-D-Galactofuranosyl terminal units are attached to position 3 of (1----2)-linked mannopyranose. Besides the previously reported monosaccharide components (mannose, galactose, glucose and glucosamine), ribose was identified in a partial acid hydrolysate of the lipopeptidophosphoglycan. The last three sugars are minor components and their organization into the overall structure of the lipopeptidophosphoglycan has not been determined.     

Immunolocalization of the oligosaccharide trimming enzyme glucosidase II

We used immunoelectron microscopy to localize glucosidase II in pig hepatocytes. The enzyme trims the two inner alpha 1,3-linked glucoses from N-linked oligosaccharide precursor chains of glycoproteins. Immunoreactive enzyme was concentrated in rough (RER) and smooth (SER) endoplasmic reticulum but not detectable in Golgi apparatus cisternae. Transitional elements of RER and smooth membraned structures close to Golgi apparatus cisternae contained labeling for glucosidase II. Specific labeling was also found in autophagosomes. These results indicate strongly that glucosidase II acts on glycoproteins before their transport to, and processing in Golgi apparatus cisternae, and suggest that an important transitional region for glucosidase II exists between RER and Golgi apparatus cisternae. Degradation in autophagolysosomes could form a normal catabolic pathway for glucosidase II.     

Microsomal glucosidases acting on the saccharide moiety of the glucose-containing dolichyl diphosphate oligosaccharide

The microsomal glucosidases which act on the oligosaccharide that contains 2 N-acetylglucosamine, 9 mannose and 1–3 glucose residues have been studied. Two fractions were separated by differential solubility in detergent and phosphate solutions or by gel filtration. One of the fractions removed glucose from the oligosaccharide containing three glucose residues, and another fraction acted on the compounds containing one and two glucose residues. Both fractions were free of mannosidase. Some properties of the enzymes are described.     

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.     

Function of angiotensin-converting enzyme on matrices

The binding of the angiotensin-converting enzyme from bovine lung on BrCN-activated Sepharose, CH- and AH-Sepharoses as well as on AH-Sepharose via the carbohydrate fragment of the glycoprotein molecule modulates the possible microenvironment of the enzyme in vivo. It has been shown that the close interaction of the enzyme with the carbohydrate matrix may increase the absolute values of catalytic constants for the hydrolysis of certain substrates. The binding of the angiotensin-converting enzyme to the matrices markedly changes the enzyme activation by chloride ions by causing a shift in the activity optima towards lower activator concentrations.     

Study of peptides inhibiting the angiotensin-converting enzyme of human seminal plasma.

Several peptides were investigated for their inhibitory capacity against dipeptidyl carboxypeptidase (angiotensin-converting enzyme) from human seminal fluid. The strongest inhibitor was the nonapeptide SQ 20881. A marked inhibition was also shown by the compounds Phe-Ala-Pro and Boc-Phe-Ala-Pro, which behaved as competitive inhibitors. Among the peptides related to angiotensin, angiotensin III was the strongest inhibitor, followed by angiotensin II and the C-terminal hexapeptide of angiotensin II. The results indicate the dipeptidyl carboxypeptidase of human semen is very similar to pulmonary dipeptidyl carboxypeptidase in its susceptibility to peptide inhibitors. In view of these and other previously reported similarities, it is possible that both enzymes are identical.