Browsing by Author "Oppong, S.Y."
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Item Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer(Biochemical Journal, 1997) Parvathy, S.; Oppong, S.Y.; Karran, E.K.; Buckle, D.R.; Turner, A.J.; Hooper, N.M.Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 microM, whereas TAPI-2 was slightly less potent (IC50 18 microM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is realted to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton-X-100 and CHAPS, but not with n-octyl beta-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane proteinItem Antioxidant Status of Sickle Cell Disease Children with and without Malaria at the Korle-Bu Teaching Hospital(University of Ghana, 2013-07) Dzigba, P.; Oppong, S.Y.; Adjei, G.O.; University of Ghana, College of Health Sciences, School of Biomedical and Allied Health Sciences, Department of Chemical PathologyBackground Sickle cell disease (SCD) is a hereditary disorder prevalent in malaria endemic areas, in which there is state of chronic vasculopathy characterized by endothelial dysfunction, and increased oxidative stress. SCD patients with malaria may experience worse outcomes because malaria infection in SCD leads to depletion of antioxidants which may complicate the disease. Few studies, however, have evaluated the antioxidant status of SCD patients with discrete clinical syndromes. There is also little information on the extent to which malaria depletes antioxidants in SCD patients. Setting The study was carried out at the Department of Child Health, Korle-Bu Teaching Hospital and the Centre for Tropical Clinical Pharmacology and Therapeutics. Aim The objective of the study was to investigate the levels of selected antioxidants in acutely ill sickle cell disease children with and without malaria. Methods Blood samples of 121 children between ages of 6 months and 13 years presenting with sickle cell and acute febrile illness were recruited. Known SCD children with confirmed malaria (n=26) or bacteremia (n=21) were enrolled. SCD children in steady state (n=21) visiting clinic for routine check- up as well as children with haemoglobin genotype, HbAA with malaria (n=26) and without malaria (n=25) attending the Korle-Bu Polyclinic were recruited. Relevant demographic data was recorded and clinical examination was undertaken. Haematological parameters as well as levels of the following antioxidants: reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) and ascorbic acid (ASC) were determined for each group. Results The mean levels of the antioxidants SOD, GSH and ASC was higher in non-SCD children, whowere malaria negative (AAM-) compared to SCD steady state children. The results for the anitoxidants were; SOD ( AAM- = 27.51, SCDSS= 25.85 ) U/ml, GSH( AAM- = 10800, SCDSS= 4465) mg/dl and ASC( AAM- = 3567, SCDSS= 2598) mg/dl. However, the reverse was found for GPx ( AAM- = 29.8, SCDSS= 259) U/, where mean activity was lower for non- sickle cell malaria negative children. Also, the mean levels of SOD, GPx and ASC in confirmed malaria positive children (SCDM+, AAM+) had lower activity than the non-malaria children (SCDSS, AAM-). Results for the antioxidant s were; SOD ( SCDSS =25.85, SCDM+=18.51 / AAM- =27.51 , AAM+= 23.99) U/ml , GPx ( SCDSS =259, SCDM+= 92.60 / AAM- =29.8, AAM+= 7.05) U/ml and ASC( SCDSS = 2598, SCDM+= 2401/ AAM- =3567, AAM+= 2514) mg/dl. However, the mean GSH ( SCDSS = 4465, SCDM+= 7941/ AAM- =10800, AAM+= 11700) mg/dl was higher for malaria positive compared to malaria negative groups.Item Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane(. Biochemical Journal, 1993) Oppong, S.Y.; Hooper, N.M.Angiotensin-converting enzyme (ACE; EC 3.4.1.15.1) exists in both membrane-bound and soluble forms. Phase separation in Triton X-114 and a competitive e.l.i.s.a. have been employed to characterize the activity which post-translationally converts the amphipathic, membrane-bound form of ACE in pig kidney microvilli into a hydrophilic, soluble form. This secretase activity was enriched to a similar extent as other microvillar membrane proteins, was tightly membrane-associated, being resistant to extensive washing of the microvillar membranes with 0.5 M NaCl, and displayed a pH optimum of 8.4. The ACE secretase was not affected by inhibitors of serine-, thiol- or aspartic-proteases, nor by reducing agents or alpha 2-macroglobulin. The metal chelators, EDTA and 1,10-phenanthroline, inhibited the secretase activity, with, in the case of EDTA, an inhibitor concentration of 2.5 mM causing 50% inhibition. In contrast, EGTA inhibited the secretase by a maximum of 15% at a concentration of 10 mM. The inhibition of EDTA was reactivated substantially (83%) by Mg2+ ions, and partially (34% and 29%) by Zn2+ and Mn2+ ions respectively. This EDTA-sensitive secretase activity was also present in microsomal membranes prepared from pig lung and testis, and from human lung and placenta, but was absent from human kidney and human and pig intestinal brush-border membranes. The form of ACE released from the microvillar membrane by the secretase co-migrated on SDS/PAGE with ACE purified from pig plasma, thus the action and location of the secretase would be consistent with it possibly having a role in the post-translational proteolytic cleavage of membrane-bound ACE to generate the soluble form found in blood, amniotic fluid, seminal plasma and other body fluidsItem Identification of the site of cleavage in angiotensin converting enzyme by its secretase(Biochemical Society, 1995) Hooper, N.M.; Oppong, S.Y.; Turner, A.J.Item Impact of blood perilipin A levels on obesity and metabolic health(. BMC Research Notes, 2022) Ofori, E.K.; Letsu, B.S.; Amponsah, S.K.; Ahenkorah, J.; Crabbe, S.; Kwao‑Zigah, G.; Oppong, S.Y.; Diaba‑Nuhoho, P.; Amanquah, S.D.Objective: Perilipin A is a common protein that coats lipid surfaces preventing them from being exposed to oxida‑ tive damage. Researchers have found little consistency in the relationship between perilipin A levels in the blood and body fat. This study was a cross-sectional observational that looked at circulating perilipin A levels and how they relate to metabolic health. Results: The participants in this study were 86 individuals with a mean age of 45.5±1.2 years. Multiple clinical and metabolic indicators (age, weight, BMI, total body fat mass, triglyceride, and HOMA-IR) were shown to be inversely associated with perilipin A levels (rho=− 0.32, − 0.37, − 0.40, − 0.45, − 0.33 and − 0.29; p<0.05 respectively). Obese persons were almost six times more likely than non-obese individuals to have lower perilipin A levels (odds ratio=6.22, CI=2.35–11.50, p<0.001). Our fndings underscore the important role of perilipin A proteins in metabolic health. KeywordItem Impact of exercise intensity on oxidative stress and selected metabolic markers in young adults in Ghana(BMC Research Notes, 2018-09) Diaba-Nuhoho, P.; Ofori, E.K.; Asare-Anane, H.; Oppong, S.Y.; Boamah, I.; Blackhurst, D.Objective This study aimed to evaluate the effect of different levels of exercise on markers of oxidative stress and selected metabolic parameters in Ghanaian young adults. Results Significant increases in a marker of oxidative stress malondialdehyde and antioxidants such as superoxide dismutase and uric acid were observed in the exercisers compared with the inactive group (p < 0.05). Total cholesterol and high density lipoprotein levels were significantly different (p < 0.05) between the two groups. Positive associations between exercise intensity, antioxidant concentration and malondialdehyde were observed within the exercise group for vigorous exercise with regards to uric acid, superoxide dismutase and malondialdehyde (r = 0.512, p = 0.004; r = 0.810, p = 0.001; r = 0.715, p = 0.001) respectively and moderate exercise vs malondialdehyde (r = 0.841, p = 0.001) compared to the inactive group. Exercise participants performed more vigorous exercise (p < 0.001), moderate exercise (p < 0.001) and more walking (p < 0.001) compared with the inactive group while the inactive group exhibited more sitting (p < 0.001). The study provides a first report on the risk associated with increase in oxidative stress and the importance of walking as a health promotion intervention among young Ghanaian adults.Item Inhibition by converting enzyme inhibitors of pig kidney aminopeptidase P(Hypertension, 1992) Hooper, N.M.; Hryszko, J.; Oppong, S.Y.; Turner, A.J.Several inhibitors of angiotensin converting enzyme were also found to inhibit aminopeptidase P, whereas inhibitors of other mammalian aminopeptidases were ineffective. Aminopeptidase P purified from pig kidney cortex was found to contain one atom of zinc per polypeptide chain, confirming its metalloenzyme nature. The concentrations of converting enzyme inhibitors required to cause 50% inhibition (I50) of aminopeptidase P were in the low micromolar range. The most potent converting enzyme inhibitors toward aminopeptidase P were the carboxylalkyl compounds, cilazaprilat, enalaprilat, and ramiprilat (I50 values of 3-12 /iM). The sulfydryl compounds captopril (l50 110 /iM) and YS980 (I50 20 /iM) were slightly less potent at inhibiting aminopeptidase P. In contrast, the carboxylalkyl compounds benazeprilat, lisinopril, and pentoprilat; the sulfydryl compound rentiapril; and the phosphoryl compounds ceranopril and fosinoprilat had no inhibitory effect against aminopeptidase P. This compares with I50 values in the 1-6 nM range for these inhibitors with angiotensin converting enzyme. Inhibition of aminopeptidase P may account for some of the effects or side effects noted with the clinical use of converting enzyme inhibitors. These results may provide the basis for the design of more selective inhibitors of angiotensin converting enzyme or mixed inhibitors of aminopeptidase P and angiotensin converting enzyme, or both.Item Purification and characterization of the angiotensin converting enzyme secretase(Biochemical Society Transactions, 1995) Connolly, C.; Oppong, S.Y.; Turner, A.J.; Hooper, M.N.Item Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites.(Biochemical Journal, 2000) Woodman, Z.L.; Oppong, S.Y.; Cook, S.; Hooper, N.M.; Schwager, S.L.U.; Brandt, W.F.; Sturrock, E.D.The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg(1137)/Leu(1138)) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg(1137). Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg(1203). These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg(1203)/Ser(1204) bond, identical with the Arg(627)/Ser(628) site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.