Biomedical subjects
Publications and source records attributed to J F Riordan.
Crystals of recombinant human angiogenin have been grown from solutions containing sodium potassium tartrate and polyethylene glycol as precipitants. They belong to the space group C222(1) (a = 83.36 A, b = 120.64 A, c = 37.72 A) and contain a single molecule in the asymmetric unit. The crystals diffract to at least 2.3 A resolution and are suitable for three-dimensional X-ray structural analysis.
The active site of angiotensin-converting enzyme (ACE) has been shown by chemical modification to contain a critical tyrosine residue, identified as Tyr-200 in human testis ACE (hTACE). We have expressed a mutant hTACE containing a Tyr-200 to Phe mutation. The mutant exhibits a marked decrease in kcat: 15-fold and 7-fold for the hydrolysis of furanacryloyl-Phe-Gly-Gly and angiotensin I, respectively, whereas its Km increases by only 1.6- and 2.2-fold, respectively. We conclude that Tyr-200 is not required for substrate binding. Instead, the effect on kcat together with a 100-fold decrease in affinity for the ACE inhibitor lisinopril indicates that Tyr-200 may participate in catalysis by stabilizing the transition state complex. Thus, Tyr-200 in hTACE has a role analogous to that of Tyr-198 in carboxypeptidase A.
The testis-specific isozyme of angiotensin-converting enzyme (ACE) is identical, from residue 68 to the C terminus, to the second half or C-terminal domain of somatic ACE. However, the first 67 residues, comprising the signal peptide and a Ser-/Thr-rich 36-residue sequence that constitutes the N terminus of mature testis ACE, are unique. We have expressed a mutant human testis ACE lacking this 36-residue N-terminal sequence and find that compared to the wild-type protein the mutant is 15 kDa smaller due to the loss of greater than 90% of all O-linked sugars, but that it retains full enzymatic activity and is stable in culture. Heavy O-glycosylation is a property of testis ACE that is not shared by the somatic enzyme and is attributable to this unique sequence.
Ante-mortem diagnosis of primary pericardial mesothelioma is very rare. We report a case which presented clinically as tuberculous constrictive pericarditis. The patient underwent pericardial resection with an immediate haemodynamic benefit, although the malignant process progressed, and he died 14 weeks later.
Daily dose schedules of 100-200 mg of almitrine bismesylate improve arterial blood gases in patients with hypoxaemic chronic obstructive airways disease (COPD) but dose related side effects are evident. In the present study, daily doses approximately half of those previously used were employed in a randomised double blind manner in 85 patients (age 35-79 years) with hypoxaemic COPD. After a one month period to check stability of arterial blood gases, patients were allocated to almitrine (A) or placebo (P) using an unequal code (60% A, 40% P). Tablets, 50-100 mg daily were stopped for one month after 3, 6 and 9 months to counteract drug accumulation. 50 patients in group A and 35 in group P were comparable on entry; mean age 65 (SD = 8) yrs., Pao2 7.8 (0.7) kPa (58.3 (5.0) mmHg), PaCO2 5.8 (0.8) kPa (43.2 (6.0) mmHg), forced expiratory volume in one second--FEV1 0.89 (0.25) l and 6 minute walking distance 296 (97) metres. The improvement in baseline PaO2 values was the same 0.8-1.3 kPa (6-9.8 mmHg) as with previous higher dose therapy. Approximately one third of patients did not respond, defined as PaO2 elevation > 0.67 kPa (5 mmHg). The sequential dosing scheme stabilised blood levels of almitrine within the therapeutic range of 280-300 ng.ml-1. After withdrawal of therapy arterial blood gases and spirometry reverted to pre-treatment levels, suggesting no permanent reversal of pathophysiology. Dose related side effects of breathlessness, indigestion and peripheral neuropathy were not observed. Nerve conduction studies revealed no difference in peripheral nerve dysfunction in hypoxaemic COPD between active and placebo therapy.(ABSTRACT TRUNCATED AT 250 WORDS)
Explore the source record for details and available documents.
The blood pressure regulating somatic isozyme of angiotensin-converting enzyme (ACE) consists of two homologous, tandem domains each containing a putative metal-binding motif (HEXXH), while the testis isozyme consists of just a single domain that is identical with the C-terminal half of somatic ACE. Previous metal analyses of somatic ACE have indicated a zinc stoichiometry of 1 mol of Zn2+/mol of ACE and inhibitor-binding studies have found 1 mol of inhibitor bound/mol of enzyme. These and other data have indicated that only one of the two domains of somatic ACE is catalytically active. We have repeated the metal and inhibitor-binding analyses of ACE from various sources and have determined protein concentration by quantitative amino acid analysis on the basis of accurate polypeptide molecular weights that are now available. We find that the somatic isozyme in fact contains 2 mol of Zn2+ and binds 2 mol of lisinopril (an ACE inhibitor) per mol of enzyme, whereas the testis isozyme contains 1 mol of Zn2+ and binds 1 mol of lisinopril. In the case of somatic ACE, the second equivalent of inhibitor binds to a second zinc-containing site as evidenced by the ability of a moderate excess of inhibitor to protect both zinc ions against dissociation. However, active site titration with lisinopril assayed by hydrolysis of furanacryloyl-Phe-Gly-Gly revealed that 1 mol of inhibitor/mol of enzyme abolished the activity of either isozyme, indicating that the principal angiotensin-converting site likely resides in the C-terminal (testicular) domain of somatic ACE and that binding of inhibitor to this site is stronger than to the second site.(ABSTRACT TRUNCATED AT 250 WORDS)
A 42-kDa bovine protein that binds bovine angiogenin [angiogenin binding protein (AngBP)] has been identified as a dissociable cell-surface component of calf pulmonary artery endothelial cells and a transformed bovine endothelial cell line, GM7373. Binding of 125I-labeled bovine angiogenin (125I-Ang) to AngBP occurs with an apparent Kd approximately 5 x 10(-10) M and is specific, saturable, and inhibited by excess unlabeled angiogenin. 125I-Ang can be crosslinked efficiently to AngBP by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide. Bovine ribonuclease A competes with the binding of 125I-Ang to AngBP, but lysozyme does not. Direct binding to AngBP of 125I-labeled bovine ribonuclease A is, however, much weaker than that of 125I-Ang. Two enzymatically active derivatives of angiogenin cleaved at residues 60-61 and 67-68, respectively, fail to induce angiogenesis and also bind to AngBP only weakly. AngBP has been isolated by treatment of cells with heparan sulfate, affinity chromatography on angiogenin-Sepharose of the material dissociated from the cell surface, and gel filtration HPLC. The results suggest that AngBP has the characteristics of a receptor that may likely function in angiogenesis.
The testis isozyme of angiotensin-converting enzyme (ACE; EC 3.4.15.1) is a membrane-bound protein that, apart from the first 35 N-terminal residues, is identical to the C-terminal half of somatic ACE and contains the same putative C-terminal membrane anchor. Stable transfection of Chinese hamster ovary (CHO) cells with an expression vector containing the full-length human testis ACE cDNA results in the expression of two forms of recombinant human testis ACE (hTACE): membrane-bound ACE and, surprisingly, large quantities (up to 3 mg/liter) of soluble hTACE in the conditioned medium. Both forms are fully active and are physicochemically similar. However, by phase separation in Triton X-114, the soluble enzyme is hydrophilic, as is an anchor-minus mutant hTACE recovered from the medium of CHO cells transfected with a vector that contains a 3'-truncated testis ACE cDNA lacking the sequence encoding the membrane anchor. In contrast, the membrane-bound hTACE is amphipathic but is converted to a hydrophilic form on treatment with trypsin. The data establish that in ACE the hydrophobic sequence near the C terminus is necessary for membrane anchoring. Moreover, in CHO cells, membrane-bound hTACE is apparently solubilized by proteolytic cleavage of this anchor. A similar mechanism may account for the release of endothelial ACE in vivo to generate serum ACE and more generally for the constitutive processing and solubilization of analogously anchored proteins such as the amyloid precursor protein, among others. The release of membrane-bound ACE in CHO cells may, therefore, provide a useful system for the study of membrane-protein-solubilizing proteases.
Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.
Angiotensin converting enzyme (ACE) is a central participant in blood pressure regulation and is strategically located within the pulmonary vasculature in order to carry out this function. It is also present in kidney, brain and a variety of other tissues where its function is unknown. The molecular weight of ACE from these sources is approximately 185,000. A smaller form of the enzyme, Mr approximately 100,000, is found in mature testis; its function is also unknown. The lung form of human ACE contains 1277 amino acids and consists of two homologous repeated domains, each of which appears to have a potential catalytic site. The testis form of human ACE contains 701 amino acids and has only a single domain which is largely identical to the carboxy-terminal half of the lung enzyme. This raises important questions such as, why does lung ACE possess two possible active sites, do each of the two bind zinc, and are they both catalytically active? To answer these questions, we have examined the binding of potent peptide inhibitors to ACE, redetermined the zinc stoichiometry and chemically modified both lung and testicular ACE with fluorodinitrobenzene (FDNB). Peptide inhibitors bind with essentially a 1:1 stoichiometry, indicative of a single active site. The zinc content of lung ACE is 1.4-1.8 g-at/mol. For testicular ACE it is 0.8-1.1 g-at/mol. FDNB modifies a single tyrosine, and to a much lesser extent a lysine, with concomitant loss of all catalytic activity. Sequence analysis identifies the specific residues modified and indicates that they occur only in the carboxy terminal half of lung ACE.(ABSTRACT TRUNCATED AT 250 WORDS)
The peptidase and esterase activities of rabbit pulmonary angiotensin converting enzyme (ACE) are rapidly abolished on reaction with 1-fluoro-2,4-dinitrobenzene (Dnp-F). Inactivation follows first-order kinetics with respect to the reagent and is accompanied by stoichiometric incorporation of 3,5-[3H]Dnp, indicating that the effect is due to a specific modification of the enzyme. Thin-layer chromatography of an acid hydrolysate of the modified enzyme indicates that most of the radioactive label is present as O-Dnp-tyrosine (65 to greater than 95%) and the rest as N epsilon-Dnp-lysine. The pH dependence of the reaction is consistent with modification of either tyrosine or lysine. The presence of a competitive inhibitor effectively protects the enzyme against inactivation by Dnp-F. Acetylation of ACE with N-acetylimidazole also protects the enzyme against modification with Dnp-F. The results indicate the presence of catalytically essential tyrosine and lysine residues at the active site of ACE.
Inactivation of rabbit lung angiotensin converting enzyme (ACE) by 1-fluoro-2,4-dinitrobenzene (Dnp-F) has been shown to be due primarily to the modification of a tyrosine residue [Bünning, P., Kleeman, S.G., & Riordan, J.F. (1990) Biochemistry (preceding paper in this issue)]. Rabbit testicular ACE is also inactivated by Dnp-F. The specific site of modification has been identified by peptide mapping of tryptic digests of the Dnp-modified protein. Two principal 340-nm-absorbing peaks, not observed with protein modified in the presence of inhibitor, have been characterized. Amino acid and sequence analyses show that these peptides contain two distinct residues that have been selectively modified. The sequence of the major (greater than 90% of the total) modified peptide is YVEFTNK with the Dnp group on tyrosine. The sequence of the second, minor peptide is KVQDLQR with the Dnp group on lysine. Identical peptides were obtained from Dnp-modified rabbit lung ACE. These modified amino acids correspond to residues 200 and 118, respectively, in testicular ACE (human enzyme numbering). Both peptides are present only in the carboxy-terminal half-domain of lung ACE, corresponding to residues 776 and 694, respectively. These results indicate that the Dnp-F sensitive, catalytically functional active site is located in the "testicular" half of lung ACE.
Limited proteolysis of carboxypeptidase A from bovine pancreas with subtilisin Carlsberg generates a stable intermediate, carboxypeptidase S, whose esterase and peptidase activities are increased and decreased, respectively, under standard assay conditions. Carboxypeptidase S was isolated by affinity chromatography. Sequence analysis shows that it is cleaved solely at the Ala154-Gly155 bond. Its enzymatic properties were determined under stopped-flow conditions with Dns-Gly-Ala-Phe and its ester analogue Dns-Gly-Ala-OPhe. For both substrates, the Km values are increased 30-40-fold. The kcat value for peptide hydrolysis is virtually unaffected whereas that for ester hydrolysis is increased 10-fold. The magnitude of the Km effect is equivalent to a loss of 9 kJ/mol of binding energy and likely reflects a disruption of the network of hydrogen bonds that links Tyr-248 and Arg-145 to the backbone carbonyls of Ala-154 and Gly-155. The difference in kcat effects for the two substrate classes is related to differences in the chemical nature of the rate-determining step. Product release is rate determining for catalytic hydrolysis of ester substrates, and hence, the increase in kcat indicates that dissociation of products is facilitated as a result of the Ala154-Gly155 bond scission. The changes in enzymatic activity accompanying limited proteolysis are due to conformational alterations in the vicinity of the active center of the molecule. The affinity of a monoclonal antibody, mAb 100, directed toward the antigenic determinant located between residues 209 and 218 in carboxypeptidase A is diminished considerably for carboxypeptidase S.(ABSTRACT TRUNCATED AT 250 WORDS)
Explore the source record for details and available documents.
Angiogenin activates the phosphoinositide-specific phospholipase C (PLC) in cultured rat aortic smooth muscle cells to yield a transient (30 s) peak of 1,2-diacylglycerol (DG) and inositol trisphosphate. Within 1 min, the DG level falls below that of the control and remains so for at least 20 min. A transient increase in monoacylglycerol indicates that depletion of DG may be the consequence of hydrolysis by DG lipase. In addition to these changes in second messengers, a rapid increase in incorporation of radiolabeled tracer into cellular cholesterol esters is observed. Stimulated cholesterol ester labeling is inhibited by preincubation with either the DG lipase inhibitor RHC 80267 or the acyl coenzyme A:cholesterol acyltransferase inhibitor Sandoz 58035. Cells prelabeled with [3H]arachidonate show a sustained increase in labeling of cholesterol esters following exposure to angiogenin. In contrast, cells prelabeled with [3H]oleate show only a transient elevation that returns to the basal level by 5 min. This suggests initial cholesterol esterification by oleate followed by arachidonate that is released by stimulation of the PLC/DG lipase pathway.
Explore the source record for details and available documents.
Explore the source record for details and available documents.