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C Southan

Publications and source records attributed to C Southan.

At least 37 records · Page 2Linked to original sources
PubMed1989

Glutathione transferases in the tapeworm Moniezia expansa.

Four forms of GSH transferase were resolved from Moniezia expansa cytosol by GSH-Sepharose affinity chromatography and chromatofocusing in the range pH 6-4, and the presence of isoenzymes was further suggested by analytical isoelectric focusing. The four GSH transferase forms in the cestode showed no clear biochemical relationship to any one mammalian GSH transferase family. The N-terminal of the major GSH transferase form showed sequence homology with the Mu and Alpha family GSH transferases. The major GSH transferase appeared to bind a number of commercially available anthelmintics but did not appear to conjugate the compounds with GSH. The major GSH transferase efficiently conjugated members of the trans-alk-2-enal and trans,trans-alka-2,4-dienal series, established secondary products of lipid peroxidation.

Amino Acid Sequence↗
PubMed1989

Sequence similarity between dopamine beta-hydroxylase and peptide alpha-amidating enzyme: evidence for a conserved catalytic domain.

A comparison of human dopamine beta-hydroxylase (EC 1.14.17.1) with bovine peptide C-terminal alpha-amidating enzyme (EC 1.14.17.3), revealed a 28% identity extending throughout a common catalytic domain of approximately 270 residues. The shared biochemical properties of these two enzymes from neurosecretory granules suggests that the sequence similarity reflects a genuine homology and provides a structural basis for a new family of copper type II, ascorbate-dependent monooxygenases.

Amino Acid Sequence↗
PubMed1989

Inactivation of dopamine beta-hydroxylase by beta-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide.

beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Inactivation of dopamine beta-hydroxylase by p-cresol: isolation and characterization of covalently modified active site peptides.

Recently, p-cresol has been shown to be a mechanism-based inhibitor of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) [Goodhart, P. J., DeWolf, W. E., Jr., & Kruse, L. I. (1987) Biochemistry 26, 2576-2583]. This inactivation was suggested to result from alkylation of an active site residue by an aberrant 4-hydroxybenzyl radical intermediate. In support of this hypothesis, we report here the isolation and characterization of two modified tryptic peptides from DBH inactivated by p-cresol. Using a combination of automated Edman sequencing, mass spectroscopy (MS), and tandem MS, we have determined the sequence of the putative active site peptides, identified the site of attachment of p-cresol, and defined the chemical nature of the adduct formed. Both modified peptides are the same primary sequence: Ala-Pro-Asp-Val-Leu-Ile-Pro-Gly-Gln-Gln-Thr-Thr-Tyc-Trp-Cys-Tyr-Va l-Thr-Glu- Leu-Pro-Asp-Gly-Phe-Pro-Arg, where Tyc is an amino acid residue with the in-chain mass of a cresol-Tyr adduct (106 + 163 Da). Gas-phase deuterium exchange studies (employing N2H3-DCI MS) of the isolated phenylthiohydantoin (Pth) derivatives of modified residue 13 demonstrate that p-cresol forms two chemically distinct covalent adducts and support the hypothesis that a (4-hydroxyphenyl)methyl radical is generated during catalysis. Rearrangement to a (4-methylphenyl)oxy radical may also occur prior to inactivation.

Alkylation↗
PubMed1988

The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2).

A nitroreductase enzyme has been isolated from Walker 256 rat carcinoma cells which can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986; Knox RJ et al., Biochem Pharmacol 37: 4661-4669, 1988). The enzyme has now been identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) by comparison of partial protein sequences, coenzymes, substrate and inhibitor specificities, and spectroscopic data. 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and 5(4)-aminoimidazole-4(5)-carboxamide were shown to be inhibitors of the isolated Walker cell enzyme. This observation could explain the reported antagonistic action of the aminoimidazole carboxamides to the antitumour effects of CB 1954.

Amino Acid Sequence↗
PubMed1988

The glutathione transferase activity and tissue distribution of a cloned Mr28K protective antigen of Schistosoma mansoni.

A protective Mr28K antigen of Schistosoma mansoni, expressed from its cDNA, has been purified in a single step and shown to possess glutathione (GSH) transferase activity as predicted from sequence homologies with two mammalian GSH transferase multigene families. It is notable for its high 1-chloro-2,4-dinitrobenzene GSH transferase and linoleic acid hydroperoxide GSH peroxidase activities. The major GSH transferase of S. mansoni has been purified and its subunit is identical to this Mr28K antigen by criteria of Mr, immunochemistry, substrate specificity and peptide sequence analysis. In the parasite, the antigen is present in the tegument, protonephridial cells and subtegumental parenchymal cells. No significant immunological cross-reactivity between the S.mansoni and mammalian (human and rat) GSH transferases was observed.

Amino Acid Sequence↗
PubMed1987

The separation of glutathione transferase subunits by using reverse-phase high-pressure liquid chromatography.

A simple method is described for the separation and quantification of the subunits of GSH transferases present in rat tissue extracts. This method, involving GSH-agarose affinity chromatography followed by reverse-phase h.p.l.c., is rapid and sufficiently sensitive to measure 5 micrograms of each subunit in a mixture. Examples are given of its application to extracts of rat kidney, adrenal, testicular interstitial cells and seminiferous tubules. The analysis of seminiferous tubules indicates that the technique may be of value for the identification of novel subunits. Preliminary separations of subunits from human GSH transferases are also described.

Animals↗
PubMed1987

Use of Sep-Pak cartridges for on-line preparative high-performance liquid chromatography.

Sep-Pak C18 cartridges have found wide application to the extraction, concentration, and fractionation of peptides. Their use has hitherto been limited to stepwise loading, washing, and eluting procedures. By the use of commercially available plastic column end-fittings it was possible to connect steel tubing to both ends of the cartridges and allow low-pressure operation in a high-performance liquid chromatography (HPLC) instrument. The cartridge was then able to function as a disposable, reversed-phase (RP) preparative column. Operation in a gradient mode gave several advantages over step-wise elution, including the direct UV absorption monitoring of the eluant. Gradient conditions could be optimised for sample recovery. The cartridge was compared with standard analytical RP-HPLC columns for the separation of both proteins and peptides. A test protein gave a single peak with good recovery. Experiments with peptide mixtures showed that the cartridge could resolve some components sufficiently to allow fractions of pure peptides to be collected.

Buffers↗
PubMed1987

Amino acid sequence of beta-galactoside-binding bovine heart lectin. Member of a novel class of vertebrate proteins.

A variety of animal tissues contain beta-galactoside-binding lectins with molecular masses in the range 13-17 kDa. There is evidence that these lectins may constitute a new protein family although their function in vivo is not yet clear. In this work the major part of the amino acid sequence of the 13 kDa lectin from bovine heart muscle has been determined. Comparison of this sequence with the cDNA-deduced sequence published for the chick embryo skin lectin showed 58% homology. Comparison of the bovine lectin sequence with partial sequences from two cDNA clones from a human hepatoma library and partial amino acid sequences of human lung lectin showed 70, 40 and 85% homology, respectively. The sequences of these vertebrate lectins are thus clearly related, supporting earlier results of immunological cross-reactivity within this group of proteins. Computer searching of protein sequence databases did not detect significant homologies between the bovine lectin sequence and other known proteins.

Amino Acid Sequence↗
PubMed1987

Direct analysis of plasma fibrinogen-derived fibrinopeptides by high-performance liquid chromatography: investigation of nine congenital fibrinogen abnormalities.

A simple method has been developed for the rapid analysis of fibrinopeptides contained on fibrinogen in small anticoagulated plasma samples. Following incubation with thrombin the plasma is diluted, boiled and then studied by high performance liquid chromatography (HPLC). The three forms of FPA (AP, A, AY) and two forms of FPB (B, des Arg B) can be identified and quantified in samples of less than 200 microliters. Additionally, the FPB peak height can be used to measure the plasma fibrinogen level. This method has been used to screen plasma samples with abnormal clotting times for possible congenital fibrinogen abnormalities. Results of the study of nine unrelated cases are presented. Four cases of congenital dysfibrinogenaemia were diagnosed directly from HPLC analysis alone. Fibrinogen Sheffield and Paris VI were identified as A alpha Arg 16----His substitutions and fibrinogens London VI and Madrid II were found to be heterozygous for an unknown substitution preventing thrombin cleavage at A alpha Arg 16. A case of dysfibrinogenaemia (fibrinogen Ashford) with a normal fibrinopeptide release stoichiometry was confirmed to have a primary polymerization abnormality using purified fibrin monomers. Similarly, a case of hypodysfibrinogenaemia (fibrinogen London V) had normal fibrinopeptides and a fibrin polymerization abnormality. In one case of hypofibrinogenaemia and two cases of afibrinogenaemia, no fibrinopeptide or functional abnormalities could be definitely established. This rapid and simple method of fibrinopeptide analysis is recommended for screening of plasma samples taken from patients suspected of having abnormalities of fibrinogen synthesis.

Blood Coagulation Disorders↗
PubMed1986

Direct analysis of plasma fibrinogen-derived fibrinopeptides by high performance liquid chromatography: investigation of A alpha-chain N-terminal heterogeneity.

Fibrinopeptide A (FPA), released from the fibrinogen A alpha-chain by thrombin, can be resolved by high-performance liquid chromatography (HPLC) into three forms, the intact peptide (A), a modified peptide phosphorylated at the serine in position 3 (AP), and an N-terminally degraded form (AY). A new method has been developed, using HPLC, that allows direct measurement of the proportions of AP, A, and AY released by thrombin from fibrinogen in plasma samples of 200 ul or less. The method was used to examine variations in the proportions of AP and AY expressed as a % of total FPA in a number of patient and control groups. The mean percentages of AP and AY of plasma fibrinogen were found to be 21.7 and 14.2%, respectively, in normal laboratory controls. In older, apparently normal, individuals these figures were 27.0 and 15.5%, respectively. Cord plasma exhibited very high AP and slightly reduced AY levels (41.6 and 12.4%, respectively) compared with normal adults. Patients with liver failure had low AP levels and high AY levels (11.6 and 21.1%, respectively). Patients recovering from major surgery or acute thrombotic stroke showed an acute-phase rise in fibrinogen level that was accompanied by an increase in AP and variable reduction in AY. Incubation of heparinized whole blood for 8 days in vitro demonstrated a gradual decrease in the proportion of AP and increase in AY of plasma fibrinogen. These results provide some support for the idea that an increased "aging" of fibrinogen in the circulation may result in a decrease in the AP content of fibrinogen accompanied by a more variable increase in AY.

Aged↗
PubMed1986

Direct analysis of plasma fibrinogen-derived fibrinopeptides by high-performance liquid chromatography.

The fibrinopeptides released from fibrinogen by thrombin can be conveniently quantitated by high-performance liquid chromatography (HPLC). This work describes a method that enables direct analysis of fibrinopeptides derived from fibrinogen contained in small amounts of plasma or whole blood without the necessity of fibrinogen purification. Aliquots of plasma or whole blood were treated with thrombin, diluted in buffer at pH 6.0, boiled and centrifuged. After filtration to 0.22 micron the supernatant was analysed directly by HPLC using a 3 microns C18 analytical column fitted with a pre-column. All the known normal fibrinopeptide peaks and their degradation products were clearly resolved from other plasma-derived peptides and the release pattern was identical to that observed with purified fibrinogen. Some samples contained interfering endogenous peptides which were removed by rapid gel-filtration of 200 microliters of plasma before thrombin treatment. The method was quantitative and the peak heights of fibrinopeptide B (FPB) could be used to accurately measure plasma fibrinogen concentrations directly in samples equivalent to 20 microliters of plasma. These fibrin determinations were unaffected by the type of anticoagulant used were performed on blood samples of both human and animal origin collected and/or stored under a variety of conditions. Variations of the method were specifically developed for different applications requiring quantitative fibrinopeptide analysis.

Animals↗
PubMed1985

Characterization of peptides cleaved by plasmin from the C-terminal polymerization domain of human fibrinogen.

The C-terminal region of the fibrinogen gamma chain is known to participate in several functional interactions including fibrin polymerization. This part of the molecule is retained on the gamma chain of fragment D (FgD) when fibrinogen is digested by plasmin in the presence of calcium to produce the fragment D-fragment E (FgD X FgE) complex but is lost if FgD is prepared in the absence of calcium. In an attempt to characterize the C-terminal polymerization domain we have used three techniques to examine this further degradation of FgD following the addition of EDTA and plasmin. Analysis of the digestion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a progressive cleavage of the gamma chain to two small remnants. The polymerization-inhibitory activity of the whole digest was studied using acid-solubilized fibrin. A progressive loss of inhibitory activity was associated with gamma chain shortening, reaching greater than a 120-fold reduction at the end of digestion. The cleavage of peptides was followed by reverse-phase high performance liquid chromatography and the release of a characteristic peptide triplet was associated with gamma chain cleavage. Manual sequencing, amino acid analysis, and fast atom bombardment mass spectrometry established the three peptides as gamma 303-356, 357-373, and 374-405. These peptides have sequences in common with those peptides recently reported by other investigators to be potent polymerization inhibitors. However, when a mixture of the three peptides was added in a 200-fold molar excess to polymerizing fibrin, no inhibitory activity could be demonstrated. It is concluded that the C-terminal polymerization domain of fibrinogen may be an extended region which includes the sequence gamma 303-405, when this is contiguous with the remainder of the gamma chain.

Amino Acid Sequence↗
PubMed1985

Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.

Blood Platelets↗
PubMed1985

Thrombin-induced fibrinopeptide release from a fibrinogen variant (fibrinogen Sydney I) with an Aalpha Arg-16----His substitution.

Fibrinogen, purified from a recently identified case of dysfibrinogenaemia, fibrinogen Sydney I, was shown by thrombin digestion, high-performance liquid chromatography (HPLC) and amino acid analysis to be a heterozygous case of an A alpha Arg-16----His substitution. Kinetic studies have been carried out on the thrombin-induced release of fibrinopeptide A (FPA), fibrinopeptide B (FPB) and the variant peptide [His16]FPA. When thrombin was added to fibrinogen Sydney I at a concentration of 0.2 U/ml release of FPA was rapid and there was a 79-fold reduced rate of release of [His16]FPA, but the rate of release of FPB was not appreciably reduced. In contrast, at lower thrombin concentrations the rate of FPB release was reduced in proportion to the rate of total FPA release, supporting the view that release of fibrinopeptides is a sequential process. The second-order kinetic constant kcat/Km for hydrolysis of the abnormal A alpha chain by thrombin was calculated from Lineweaver-Burk plots to be 16-30-fold less than that for the normal A alpha chain. Molecular modelling studies, using a refined model of the trypsin-pancreatic-trypsin-inhibitor complex have been used to suggest how the histidine at the P1 site can be accommodated within the enzyme hydrophobic active-site pocket.

Amino Acids↗
PubMed1983

Fibrinogen Manchester: identification of an abnormal fibrinopeptide A with a C-terminal arginine leads to histidine substitution.

Purified samples of fibrinogen Manchester, a congenital dysfibrinogenaemia with impaired fibrinopeptide A (FPA) release, were digested with thrombin. Amino acid sequencing of the fibrin showed that FPA had been completely released. High performance liquid chromatographic (HPLC) analysis of the clot supernatant showed the presence of a new peptide eluting ahead of the normal FPA. The amino acid composition and sequence of the new peptide established its identity as a variant of FPA containing histidine in position 16 instead of the usual arginine. The chromatograms from both siblings with the defect demonstrated that they were heterozygous for this clotting defect.

Amino Acid Sequence↗