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T A Williams

Publications and source records attributed to T A Williams.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cleavage of arginyl-arginine and lysyl-arginine from the C-terminus of pro-hormone peptides by human germinal angiotensin I-converting enzyme (ACE) and the C-domain of human somatic ACE.

Mammalian germinal angiotensin I-converting enzyme (gACE) is a single-domain dipeptidyl carboxypeptidase found exclusively in male germ cells, which has almost identical sequence and enzymic properties with the C-domain of the two-domain somatic ACE. Mutant mice that do not express gACE are infertile, suggesting a role for the enzyme in the processing of undefined peptides involved in fertilization. A number of spermatid peptides [e.g. cholecystokinin (CCK) and gastrin] are processed from pro-hormones by endo- and exo-proteolytic cleavages which might generate substrates for gACE. We have shown that peptide hormone intermediates with Lys/Arg-Arg at the C-terminus are high-affinity substrates for human gACE. gACE from human sperm cleaved Arg-Arg from the C-terminus of the CCK5-GRR (GWMDFGRR), a peptide corresponding to the C-terminus of a CCK-gastrin prohormone intermediate. Hydrolysis of CCK5-GRR by recombinant human C-domain ACE was Cl- dependent, with maximal activity achieved in 5-10 mM NaCl at pH 6.4. C-Domain ACE cleaved Lys/Arg-Arg from the C-terminus of dynorphin-(1-7), a pro-TRH peptide KRQHPGKR, and two insect peptides FSPRLGKR and FSPRLGRR. C-Domain ACE displayed high affinity towards all these substrates with Vmax/Km values between 14 and 113 times greater than the Vmax/Km for the conversion of the best known ACE substrate, angiotensin I, into angiotensin II. In conclusion, we have identified a new class of substrates for human gACE, and we suggest that gACE might be an alternative to carboxypeptidase E for the trimming of basic dipeptides from the C-terminus of intermediates generated from pro-hormones by subtilisin-like convertases in human male germ cells.

Chlorides↗
PubMed1997

Cleavage-secretion of angiotensin I-converting enzyme in yeast.

Angiotensin I-converting enzyme (ACE) is a type I transmembrane protein composed of two domains (N and C domains) which undergoes a post-translational proteolytic cleavage in mammalian cells to release the soluble ectodomain. The protease involved in ACE cleavage-secretion (ACE-secretase) is not well characterised and eludes isolation: the presence of a yeast homologue, thus more amenable to genetic manipulation, would facilitate its identification. We have expressed a secreted form of the ACE C domain, lacking the C-terminal membrane anchor (C domain(deltaCOOH)), and the membrane-anchored C domain (C domain) in the yeast Pichia pastoris by fusion to prepro-alpha-factor. Immunofluorescent labelling localises the ACE C domain to the periphery of yeast cells but not C domain(deltaCOOH), however, expression of both C domain and C domain(deltaCOOH) produced soluble enzymes in the culture medium. Immunocharacterisation of the two soluble forms of the C domain indicates a proteolytic cleavage of the membrane-bound C domain to produce the soluble counterpart. Thus ACE undergoes a proteolytic cleavage in yeast.

Amino Acid Sequence↗
PubMed1997

Substrate dependence of angiotensin I-converting enzyme inhibition: captopril displays a partial selectivity for inhibition of N-acetyl-seryl-aspartyl-lysyl-proline hydrolysis compared with that of angiotensin I.

Angiotensin I-converting enzyme (ACE) is composed of two highly similar domains (referred to here as the N and C domains) that play a central role in blood pressure regulation; ACE inhibitors are widely used in the treatment of hypertension. However, the negative regulator of hematopoiesis, N-acetyl-seryl-aspartyl-lysyl-prolyl (AcSDKP), is a specific substrate of the N domain-active site; thus, in addition to the cardiovascular function of ACE, the enzyme may be involved in hematopoietic stem cell regulation, raising the interest of designing N domain-specific ACE inhibitors. We analyzed the inhibition of angiotensin I and AcSDKP hydrolysis as well as that of three synthetic ACE substrates by wild-type ACE and the N and C domains by using a range of specific ACE inhibitors. We demonstrate that captopril, lisinopril, and fosinoprilat are potent inhibitors of AcSDKP hydrolysis by wild-type ACE, with K(i) values in the subnanomolar range. However, of the inhibitors tested, captopril is the only compound able to differentiate to some degree between AcSDKP and angiotensin I inhibition of hydrolysis by wild-type ACE: the K(i) value with AcSDKP as substrate was 16-fold lower than that with angiotensin I as substrate. This raises the possibility of using captopril to enhance plasma AcSDKP levels with the aim of normal hematopoeitic stem cell protection during chemotherapy and a limited effect on the cardiovascular function of ACE.

Angiotensin I↗
PubMed1996

Drosophila melanogaster angiotensin I-converting enzyme expressed in Pichia pastoris resembles the C domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity.

Drosophila melanogaster angiotensin I-converting enzyme (AnCE) is a secreted single-domain homologue of mammalian angiotensin I-converting enzyme (ACE) which comprises two domains (N and C domains). In order to characterize in detail the enzymic properties of AnCE and to study the influence of glycosylation on the secretion and enzymic activity of this enzyme, we overexpressed AnCE (expression level, 160 mg/l) and an unglycosylated mutant (expression level, 43 mg/l) in the yeast Pichia pastoris. The recombinant enzyme was apparently homogeneous on SDS/PAGE without purification and partial deglycosylation demonstrated that all three potential sites for N-linked glycosylation were occupied by oligosaccharide chains. Each N-glycosylation sequence (Asn-Xaa-Ser/Thr) was disrupted by substituting a glutamine for the asparagine residue at amino acid positions 53, 196 and 311 by site-directed mutagenesis to produce a single mutant. Expression of the unglycosylated mutant in Pichia produced a secreted catalytically active enzyme (AnCE delta CHO). This mutant displayed unaltered kinetics for the hydrolyses of hippuryl-His-Leu, angiotensin 1 and N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) and was equally sensitive to ACE inhibitors compared with wild-type AnCE. However, AnCE delta CHO was less stable, displaying a half-life of 4.94 h at 37 degrees C, compared with AnCE which retained full activity under the same conditions. Two catalytic criteria demonstrate the functional resemblance of AnCE with the human ACE C domain: first, the kcat/Km of AcSDKP hydrolysis and secondly, the kcat/Km and optimal chloride concentration for hippuryl-His-Leu hydrolysis. A range of ACE inhibitors were far less potent towards AnCE compared with the human ACE domains, except for captopril which suggests an alternative structure in AnCE corresponding to the region of the S1 subsite in the human ACE active sites.

Amino Acid Sequence↗
PubMed1996

A study of chimeras constructed with the two domains of angiotensin I-converting enzyme.

Angiotensin I-converting enzyme (ACE) is composed of two highly similar domains called the N and C domains, which display some contrasting enzymatic properties. We constructed two ACE chimeras: chimera 1, comprised of the N domain containing the central 60 amino acid residues of the C domain, and chimera 2, comprised of the C domain containing the central 60 amino acid residues of the N domain. Chimeras 1 and 2 displayed Km values for Hip-His-Leu and Z-Phe-His-Leu and kcat ratios for these two substrates similar to that of the N and C domains, respectively. Thus, the short sequence exchanged between the two domains does not confer the specific properties of that domain for these two substrates but, rather, such specific properties must arise from the sequences surrounding the central region in each domain.

Amino Acid Sequence↗
PubMed1995

Cloning and expression of an evolutionary conserved single-domain angiotensin converting enzyme from Drosophila melanogaster.

Mammalian somatic angiotensin converting enzyme (EC 3.4.15.1, ACE) consists of two highly homologous (N- and C-) domains encoded by a duplicated gene. We have identified an apparent single-domain (67 kDa) insect angiotensin converting enzyme (AnCE) in embryos of Drosophila melanogaster which converts angiotensin I to angiotensin II (Km, 365 microM), removes Phe-Arg from the C terminus of bradykinin (Km, 22 microM), and is inhibited by ACE inhibitors, captopril (IC50 = 1.1 x 10(-9) M) and trandolaprilat (IC50 = 1.6 x 10(-8) M). We also report the cloning and expression of a Drosophila AnCE cDNA which codes for a single-domain 615-amino acid protein with a predicted 17-amino acid signal peptide and regions with high levels of homology to both the N- and C-domains of mammalian somatic ACE, especially around the active site consensus sequence. Northern analysis identified a single 2.1-kilobase mRNA in Drosophila embryos, and Southern analysis of Drosophila genomic DNA indicates that the insect gene is not duplicated. When expressed in COS-7 cells, the AnCE protein is a secreted enzyme, which converts angiotensin I to angiotensin II and is inhibited by captopril (IC50 = 5.6 x 10(-9) M) and trandolaprilat (IC50 = 2 x 10(-8) M). The evolutionary significance of these results is discussed.

Amino Acid Sequence↗
PubMed1995

A genetic study of angiotensin I-converting enzyme levels in human semen.

The plasma level of angiotensin I-converting enzyme (ACE) has been shown to be under genetic control. An insertion/deletion polymorphism in the ACE gene is associated with differences in the level of ACE in the plasma and inside T-lymphocytes. An ACE isoform is present in large amounts in spermatozoa and is expressed under an alternative, germ cell-specific promoter, whereas ACE present in the seminal fluid is the somatic form of ACE. We have investigated the effect associated with the I/D polymorphism on the level of ACE in seminal fluid and in spermatozoa. No differences in the level of ACE measured in the seminal fluid or in the spermatozoa were associated with the ACE I/D genotypes. We conclude that the modulation of expression associated with the I/D polymorphism is restricted to the somatic ACE promoter. These results also suggest that if one allele modulating the expression of ACE was under positive selection pressure, it was not through an effect on the semen concentration of ACE.

Alleles↗
PubMed1995

Application of dispersion modeling to indoor gas release scenarios.

Many complex models are available to study the dispersion of contaminants or ventilation effectiveness in indoor spaces. Because of the computationally complex numerical schemes employed, most of these models require mainframe computers or workstations. However, simple design tools or guidelines are needed, in addition to complicated models. A dispersion model based on the basic governing equations was developed and uses an analytical solution. Because the concentration is expressed by an analytical solution, the grid size and time steps are user definable. A computer program was used to obtain numerical results and to obtain release history from a thermodynamic source model. The model can be used to estimate three-dimensional spatial and temporal variations in concentrations resulting from transient gas releases in an enclosure. The model was used to study a gas release scenario from a pressurized cylinder into a large ventilated building, in this case, a transit parking and fueling facility.

Air Movements↗
PubMed1995

Recent advances in knowledge of the structure and function of the angiotensin I converting enzyme.

AIM: To review the structure and function of the angiotensin I converting enzyme (ACE), focusing on recent results from studies using a wide range of molecular biological techniques. ACE STRUCTURE AND FUNCTION: ACE is an ectoenzyme expressed as two isoenzymes in mammals, a larger somatic form found in endothelial, epithelial and neuronal tissues and a smaller form in germinal tissues. Both forms have similar enzymatic activities but differ in size and immunological properties. The somatic form of ACE is composed of two highly homologous domains (amino and carboxyl domains) while the germinal form contains only one domain. Somatic ACE has two functional catalytic sites, both dependent on a zinc cofactor. Each ACE domain has also been shown to interact differently with competitive inhibitors. MECHANISM OF ACE ANCHORAGE AND SOLUBILIZATION: The mechanism for anchoring ACE to the cell membrane has also been reported, and the solubilization step outlined. The relationship between the membrane-bound and soluble forms has been investigated, and the physiological relevance of this mechanism discussed. GENETIC STRUCTURE: The structure of the ACE gene has been determined and the distribution in cells and different tissues has been reported in various studies. CONCLUSION: All results have indicated that there are important functional and structural differences between the two domains, but at present ACE cannot be considered a true bifunctional enzyme, even though an exclusive substrate has been identified for the amino domain.

Animals↗
PubMed1995

Low vision rehabilitation for a patient with a traumatic brain injury.

The client described in this article had undergone intensive rehabilitation for physical and cognitive deficits that resulted from an anoxic encephalopathy. Her recovery was good in all areas except visual functioning. Her reading and writing deficits were initially thought to result from visual-perceptual problems. A low vision evaluation identified the deficits as resulting from a macular visual field loss. Warren (1993) has proposed that an intact visual field is one of the basic components of vision that must be present before higher visual processing can occur. Until the macular perimetry was performed on this client, the extent of her central field loss was unknown, and the treatment that focused on the higher level visual processing was unsuccessful. The low vision program targeted the central field loss as the probable cause for her difficulties and an effective treatment protocol was established. The client was instructed regarding the nature of her visual field deficit and was trained in methods to compensate for this deficit. Although she has not returned to work as of this writing because of financial disincentives, the training resulted in a measurable and functional improvement in the client's ability to read continuous print text, a task she had not performed in 4 years, and in her ability to perform all writing tasks needed for daily living.

Adult↗
PubMed1994

Identification of two active site residues in human angiotensin I-converting enzyme.

Angiotensin I-converting enzyme (ACE) contains two zinc-dependent catalytic domains (N and C domains) each bearing the motif HEXXH where the two histidines form two of the three amino acid zinc ligands. Sequence alignment of each ACE domain with other zinc metalloproteases, indicates a glutamate residues which putatively constitutes the third zinc ligand and an aspartate residue which may form an indirect zinc interaction. We investigated the functional roles of the glutamate and aspartate residues in the ACE C domain (Glu987 and Asp991) using a cDNA encoding an inactive N domain. We mutated Glu987 to aspartate (E987D) or valine (E987V) and Asp991 to glutamate (D991E) or alanine (D991A). Catalytically active mutants (E987D, D991E and D991A) exhibited similar Km values for hippuryl-His-Leu compared to non-mutated C domain. E987D displayed a 300-fold decrease in kcat and a 25-fold reduction in sensitivity to the ACE inhibitor trandolaprilat, whose binding is zinc-dependent. E987V was catalytically inactive and did not bind [3H]trandolaprilat. D991E and D991A exhibited a 3.8- and 22-fold decrease in kcat, respectively, and the Ki' values for trandolaprilat were increased 8- and 29-fold. These results provide strong evidence that Glu987 constitutes the third zinc ligand in the ACE C domain and suggest a role for Asp991 in positioning the C domain active site zinc ion.

Amino Acid Sequence↗
PubMed1994

Angiotensin I converting enzyme gene: regulation, polymorphism and implications in cardiovascular diseases.

Angiotensin I converting enzyme (ACE), also called dipeptidyl-carboxypeptidase I (DCP I), is a zinc metallopeptidase widely distributed on the surface of endothelial and epithelial cells. Its role in the vasoactive peptide, the metabolism of the two active peptides, angiotensin and bradykinin, and the beneficial effects of its inhibition in cardiovascular diseases, have raised considerable interest in this enzyme. The potential implications of ACE gene polymorphism, which affects the expression of the gene in cardiovascular diseases, has been widely investigated. This review summarizes the results of these studies.

Gene Expression Regulation, Enzymologic↗
PubMed1992

A comparison of the zinc contents and substrate specificities of the endothelial and testicular forms of porcine angiotensin converting enzyme and the preparation of isoenzyme-specific antisera.

Angiotensin converting enzyme (ACE; EC 3.4.15.1) was purified from porcine kidney and lung (endothelial isoenzyme) and testis (testicular isoenzyme) by affinity chromatography on lisinopril-2.8 nm-Sepharose. Atomic-absorption spectroscopy revealed that ACE purified from kidney and lung contained 2.58 and 2.35 atoms of zinc per molecule of enzyme (M(r) 147,000) respectively. In contrast, ACE purified from testis contained only 1.58 atoms of zinc per molecule of enzyme (M(r) 80,000). Thus it would appear that both putative zinc-binding sites in endothelial ACE contain zinc and may therefore be catalytically active. No differences were observed in the pattern of products generated on hydrolysis of benzoyl (Bz)-Gly-His-Leu, substance P, luteinizing-hormone-releasing hormone (LH-RH) and its analogue, des-Gly10-LH-RH-ethylamide, by kidney and testicular ACE. There was also no difference in the initial rates of hydrolysis of Bz-Gly-His-Leu or substance P by the two isoenzymes, although LH-RH and its analogue were hydrolysed twice as rapidly by kidney ACE. It is therefore unlikely that the N-terminal catalytic site in porcine endothelial ACE is predominantly responsible for the atypical cleavage of LH-RH generating the N-terminal tripeptide. Two polyclonal antisera were raised to the affinity-purified forms of pig kidney and testicular ACE. Isoenzyme-specific antisera were then isolated from these by absorbing out those antibodies recognizing determinants on the other isoenzyme. Immunoelectrophoretic blot analyses and immunofluorescent staining of sections of pig kidney were used to demonstrate the specificity of the antisera. Immunofluorescent staining of sections of pig testis with the antiserum specific to testicular ACE localized testicular ACE solely to the lumen of the seminiferous tubules, whereas the antiserum specific to endothelial ACE revealed the presence of this isoenzyme only in blood vessels. The antiserum to endothelial ACE, which recognizes determinants in the unique N-terminal domain, was investigated as a possible specific inhibitor of the N-terminal catalytic site. Although this antiserum failed to inhibit testicular ACE, the effect on the activity of endothelial ACE appeared to be due to inhibition of both the N- and C-terminal catalytic sites.

Amino Acid Sequence↗