Abstract: The last two years have seen a dramatic expansion in public cheminformatics, as exemplified by the approximate five-fold growth of PubChem from over 50 contributing data sources. Consequently, medicinal chemists who were hitherto limited to commercial databases now also have access to public sources that they can download and/or query directly over the Web. The range of public sources, particularly where they link out to structured bioinformatic and biological data, already offer utilities that have no commercial equivalent. This work reviews compound content comparisons between selected public and commercial databases that capture bioactive content. We focused particularly on those that specify relationships between compounds and their protein targets. Our stringent filtering produced lower unique compound numbers than those reported for individual databases and thereby facilitated standardised comparisons of content. The resultant matrix shows the pairwise comparison of each database and selected subsets. Overall, this showed an unexpected degree of non-overlap, thereby emphasising the complementarity gained from combining public and commercial sources. This conclusion is supported by a Venn-type analysis of GVKBIO, WOMBAT (both commercial) and PubChem (public). These databases show not only overlap but also unique bioactive content in each case because of their different strategies for source selection and data collection.
Abstract: Multiple alignments and phylogenetic tree constructions are established techniques for examining the evolutionary history of protease sequences in organisms such as humans, mice, fruitflies, nematode worms and yeast. They also facilitate the mapping of those conserved positions that are important for structure and catalytic function. However, the continued increase in completed or draft genomes offers new opportunities for examining protease evolution across a broader (e.g. more mammals) and deeper (e.g. more invertebrates) phylogenetic range. In addition, the improving annotation not only of proteases, but also of their substrates, interaction partners in proteolytic complexes and endogenous inhibitor proteins now means that aspects of co-evolution can be addressed. The increasing phylogenetic coverage is also important for resolving orthology issues that arise from protease gene duplication or loss in different lineages. Selected sequences will be used to exemplify the utility of Internet resources and present results for these types of analysis.
Abstract: Splice variants play an important role within the cell in both increasing the proteome diversity and in cellular function. Splice variants are also associated with disease states and may play a role in their etiology. Information about splice variants has, until now, mostly been derived from the primary transcript or through cellular studies. In this study information from the transcript and other studies is combined with tertiary structure information derived from homology models. Through this method we have determined that it is possible to effectively model splice variants. Forty models of splice variants for fourteen proteins were produced. Analysis of the models shows that deletions produce superior model validation values. Additions to sequences where there is little homology become increasingly difficult to model with increasing sequence length. Many of the splicing events are associated with post-translational modification either in the N-terminal region by changing the signal peptide or by affecting the number or availability of glycosylation sites. Often the alternative exon combinations are associated with loss or gain of whole structural units, as opposed to just changing small loop regions. Losing part of the secondary structure may destabilize neighboring parts of the same secondary structure. Detailed analysis is given of four biomedically relevant proteins (Beta-site Amyloid Precursor Protein Cleaving enzyme (BACE), Interleukin-4, Frataxin and Hereditary hemochromatosis protein) and their associated splice variant models. The visualization of these possible structures provides new insights about their functionality and the possible etiology of associated diseases.
Abstract: Since the identification of approximately 25,000 proteins from the draft human genome assembly in 2001, estimates of the total have oscillated between 30,000 and 70,000. The recently announced genome closure has not generated a consensus gene count despite this being a key parameter for many areas of biology including drug target discovery and characterization of the human proteome. Contrary to earlier predictions of constitutive under-detection for eukaryotic genes, the latest model organism updates have produced minor increases in the worm but fly and yeast gene numbers have decreased. The postdraft, precompletion interval has produced large increases in human transcript coverage, continuous improvements in genome assembly and refinements in automated genomic annotation. Notably these enhancements have resulted in an Ensembl human protein-coding gene number of 22,184, a decrease of 1862 since the first release. Longitudinal database surveys indicate that redundancy-reduced human mRNA and protein collections are flattening out at approximately 28,000, although Ensembl maps approximately 20,000 known sequences. Observations suggest high-throughput cloning projects are predominantly extending known genes or sampling new splice forms and novel protein discovery has slowed to a trickle. The hypothesis that substantial numbers of short proteins remain experimentally and computationally undetected in mammalian genomes is neither supported by sequence data nor by the extensive homology between mouse and human proteins. Aggregating the independent annotations for complete transcripts from seven completed human chromosomes extrapolates to approximately 25,000 genes. The inclusion of partial putative genes would increase this to above 30,000 but recent data suggest these represent predominantly nonprotein-coding transcripts. Mass spectrometry-based proteomics has already verified more than 10% of human genes but has not identified significant numbers of unpredicted proteins. The available data are thus converging to a basal protein-coding gene number well below 30,000, which could even be as low as 25,000.
Abstract: The discovery, design and evaluation of new medicines is critically dependent on the elucidation of protein mechanisms involved in human diseases. Since the proteome of a cell or tissue is not a simple reflection of its transcriptome, direct protein-based analysis is needed. Advances in proteomic technologies are improving the analysis of membrane proteins and signaling complexes with increased speed and molecular detail. Changes in protein isoforms due to post-translational modifications, such as phosphorylation induced by cell signaling events and alternative splice forms of receptors, may be mapped to an altered protein expression pattern in clinically relevant cell populations with a causative or diagnostic disease link. A CNS proteome database derived from primary human tissues may avoid ambiguities of experimental models. It will also accelerate the development of more specific diagnostic and prognostic disease markers as well as new selective therapeutics. Proteomics is also being applied to resolve in silico gene prediction uncertainties by direct open reading frame verification. These advances hold great promise for improvements in the understanding, diagnosis and therapy of central nervous system disorders.
Abstract: A proteomic study of rat urine was undertaken using two-dimensional gel electrophoresis, microbore high performance liquid chromatography, mass spectrometry and N-terminal sequencing. Five known urinary proteins were identified but two novel peptide fragments matched a large number of rat expressed sequence tags (ESTs) from a liver library. By combining protein chemical and nucleotide data, two 101-residue open reading frames with 90% amino acid identity were determined, rat urinary protein 1 (RUP-1) and RUP-2. The data established signal peptide removal and provided evidence for N-glycosylation. A third related sequence, rat spleen protein (RSP-1) was confirmed from EST searches. These three proteins have been submitted to SWISS-PROT as P81827, P81828 and Q9QXN2, respectively. A fourth novel homologue was found in porcine and bovine ESTs from embryo libraries. Alignment with known homologues showed conserved cysteine positions characteristic of a secreted subfamily of Ly-6 proteins. In two cases, antineoplastic urinary protein and caltrin, these homologues have unverified functional annotations. The RUP sequences showed high scoring matches to three unrelated rat mRNAs subsequently established to be chimeric. Two of these share extended sectional identity to RUP-1 but the third may represent another novel Ly-6 homologue. These chimeras have caused serious annotation errors in secondary databases.
Abstract: Cells precisely monitor the concentration and functionality of each protein for optimal performance. Protein quality control involves molecular chaperones, folding catalysts, and proteases that are often heat shock proteins. One quality control factor is HtrA, one of a new class of oligomeric serine proteases. The defining feature of the HtrA family is the combination of a catalytic domain with at least one C-terminal PDZ domain. Here, we discuss the properties and roles of this ATP-independent protease chaperone system in protein metabolism and cell fate.
Abstract: Of the approximately 400 known human proteases, approximately 14% are under investigation as drug targets. Although the total is certain to rise during the finishing phase of the human genome project, the initial annotation of the approximately 30,000 human proteome set includes approximately 500 proteases. Bioinformatic analysis can now be performed on complete human protease families and will soon include comparisons with mice and fish. New sequences will require evaluation of their function in normal physiology and human disease. By revealing details such as splice variants and population polymorphisms, genomic sequence information will have a central role in the validation of protease drug targets.
Abstract: Over 400 human proteases documented in secondary databases can already be delineated in genomic sequence. A Genome Ontology annotation of 30585 sequences in the provisional human proteome set recognises 498 proteases, i.e. 1.6%. Homology searches against finished sequence and comparisons between mouse and zebrafish are likely to increase this total. However, the data already indicate that the mechanistic class, sequence family and domain distribution of the genomic complement of proteases is unlikely to shift significantly from that already observed in the transcript data. Genomically derived novel sequences will require bioinformatic analysis and biochemical verification. The increasing availability of annotated genomic data will enable studies of splice variants, transcriptional control, polymorphisms, pseudogenes, inactive homologues and evolution. Comparative work on complete human protease families should produce a more integrated picture of their biochemistry and physiology. Genomic data will also lead to the identification of new protease involvement in disease processes and their evaluation as drug targets.
Abstract: Database searching with bacterial serine beta-lactamases identified mouse expressed sequence tags (ESTs) with significant similarity scores.The cloned mouse cDNA encodes a novel 551-amino-acid protein, LACTB, with a predicted amino-terminal transmembrane domain but no signal peptide. It contains an active site motif related to C-class beta-lactamases. Homologues were detected in sequence data from human, rat, cow, rabbit, pig, toad, zebrafish, and Caenorhabditis elegans, but not in Saccharomyces cerevisiae or Drosophila melanogaster. The genes were mapped to human chromosome 15q22.1 and mouse chromosome 9. Sequencing of a 14.7-kb fragment of mouse genomic DNA defined six exons. A virtual human cDNA and a 549-residue protein, predicted from unfinished genomic sequence, showed the same intron/exon structure. Northern blot analysis showed expression of the 2.3-kb mRNA predominantly in mouse liver and human skeletal muscle. This is the first reported vertebrate example of this microbial peptidase family.
Abstract: We have identified human and mouse cDNAs encoding a novel ubiquitin-specific protease designated USP23. Both cDNAs encode a 62-kDa protein containing the highly conserved His and Cys domains characteristic of the C19 cysteine protease family of ubiquitin-specific processing proteases (UCH-2). Human tissue Northern blots revealed USP23 to be ubiquitously expressed, whereas USP12, its closest human paralogue, displayed a more restricted expression pattern. The human USP23 gene mapped to chromosome 1q22.
Abstract: Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.
Abstract: Sequential proteolytic processing of the Amyloid Precursor Protein (APP) by beta- and gamma-secretases generates the 4-kDa amyloid (A beta) peptide, a key component of the amyloid plaques seen in Alzheimer's disease (AD). We and others have recently reported the identification and characterisation of an aspartic proteinase, Asp2 (BACE), as beta-secretase. Here we describe the characterization of a second highly related aspartic proteinase, Asp1 as a second beta-secretase candidate. Asp1 is expressed in brain as detected at the mRNA level and at the protein level. Transient expression of Asp1 in APP-expressing cells results in an increase in the level of beta-secretase-derived soluble APP and the corresponding carboxy-terminal fragment. Paradoxically there is a decrease in the level of soluble A beta secreted from the cells. Asp1 colocalizes with APP in the Golgi/endoplasmic reticulum compartments of cultured cells. Asp1, when expressed as an Fc fusion protein (Asp1-Fc), has the N-terminal sequence ALEP..., indicating that it has lost the prodomain. Asp1-Fc exhibits beta-secretase activity by cleaving both wild-type and Swedish variant (KM/NL) APP peptides at the beta-secretase site.
Abstract: The revealing of the entire complement of protease and protease inhibitor sequences by the Human Genome Project will be of great importance to both academic and pharmaceutical research. Although the finishing phase is not yet complete, a selection of secondary annotation sources and comparisons with completed model organism genomes already allow useful estimates to be made. Conservative extrapolation suggests a total of approximately 1.8% for human proteases. This is close to the figures for yeast (1.7%) and worm (1.8%) but lower than the fly (3.4%) which has a large trypsin-like protease content. Using estimates for the human proteome of between 40,000 and 60,000 genes would extrapolate to 700-1,100 proteases, compared with approximately 360 currently represented as GenBank mRNAs. Preliminary comparisons between domain annotations for predicted human gene products and completed proteins suggest the genomic protease family and mechanistic class distributions will broadly reflect those in the current transcript data. The protease:inhibitor ratio at the mRNA level is currently approximately 9:1, but genome annotation data indicate that inhibitory domains are more widespread than this ratio would indicate.
Abstract: The family and motif databases, PROSITE, PRINTS, Pfam and ProDom, have been integrated into a powerful resource for protein secondary annotation. As of June 2000, InterPro had processed 384 572 proteins in SWISS-PROT and TrEMBL. Because the contributing databases have different clustering principles and scoring sensitivities, the combined assignments compliment each other for grouping protein families and delineating domains. The graphic displays of all matches above the scoring thresholds enables judgements to be made on the concordances or differences between the assignments. The website links can be used to analyse novel sequences and for queries across the proteomes of 32 organisms, including the partial human set, by domain and/or protein family. An analysis of selected HtrA/DegQ proteases demonstrates the utility of this website for detailed comparative genomics. Further information on the project can be found at the European Bioinformatics Institute at http://www.ebi.ac.uk/interpro/
Abstract: A range of high-performance liquid chromatography (HPLC) columns with internal diameters of 0.25 to 1.8 mm have been constructed by securing glass or plastic tubing into standard HPLC fittings. These were packed with chromatographic materials chosen for operation at moderate pressures with high flow rates. These columns were shown to be effective in a conventional HPLC instrument for peptide and protein separations in reverse-phase mode and for proteins in ion-exchange and size-exclusion modes. The simple construction and low cost of these microbore columns allow them to be considered as disposable. Using only small amounts of any type of packing material, they have the flexibility to be adapted to a wide range of analytical and micropreparative separations.
Abstract: The Alzheimer's disease beta-amyloid peptide (Abeta) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of beta- and then gamma-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of beta-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal beta-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the beta-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Abeta. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.
Abstract: Using expressed sequence tag (EST) homology screening, a new human serine dependent phospholipase A2 (HSD-PLA2) was identified that has 40% amino acid identity with human low density lipoprotein-associated phospholipase A2 (LDL-PLA2). HSD-PLA2 has very recently been purified and cloned from brain tissue but named PAF-AH II. However, because the homology with LDL-PLA2 suggested a broader substrate specificity than simply platelet activating factor (PAF), we have further characterized this enzyme using baculovirus-expressed protein. The recombinant enzyme, which was purified 21-fold to homogeneity, had a molecular mass of 44kDa and possessed a specific activity of 35 micromol min-1 mg-1 when assayed against PAF. Activity could also be measured using 1-decanoyl-2-(4-nitrophenylglutaryl) phosphate (DNGP) as substrate. Like LDL-PLA2, HSD-PLA2 was able to hydrolyse oxidatively modified phosphatidylcholines when supplemented to human LDL prior to copper-stimulated oxidation. A GXSXG motif evident from sequence information and inhibition of its activity by 3,4, dichloroisocoumarin, diisopropyl fluorophosphate (DFP) and diethyl p-nitrophenyl phosphate (DENP) confirm that the enzyme is serine dependent. Moreover, sequence comparison indicates the HSD-PLA2 probable active site triad positions are shared with LDL-PLA2 and a C. elegans homologue, suggesting that these sequences comprise members of a new enzyme family. Although clearly structurally related with similar substrate specificities further work reported here shows HSD-PLA2 and LDL-PLA2 to be different with respect to chromosomal localization and tissue distribution.
Abstract: A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 micromol.min-1.mg-1 and 12 micromol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2.
Abstract: In this study we have explored the behaviour of peptides after capillary electrophoresis (CE) followed by elution under pressure. The use of D2O- rather than H2O-based buffer solutions appears to restrict the diffusion of peptides after CE, resulting in little loss of resolution when peptides are eluted by dynamic flow. In this paper we present results showing that a simple two-step process, involving CE at a low voltage, switching off the power supply, and connecting the fused capillary at the anode end to a syringe pump for dynamic flow, can retain separation characteristics and can be used for the isolation of picomole quantities of peptides for sequence determination.
Abstract: The capillary electrophoresis (CE) of peptide fragments from the tryptic digest of salmon calcitonin and elcatonin has been carried out in H2O- and D2O-based buffer solutions. Analysis in heavy water was found to be superior to that in H2O especially at a pH or pD of 7.93. From a single CE run on elcatonin digest we were also able to harvest three pure cleavage peptides in sufficient quantity to determine each amino acid residue by protein sequencing. The order of elution from CE agreed with that predicted on the basis of net charge calculated for each peptide.
Abstract: O6-alkylguanine-DNA-alkyltransferase (ATase) activity was increased in rat liver from 80 to 320 fmoles/mg total protein 48 h after administration of 2-acetylaminofluorene at 60 mg/kg body weight. This tissue was used as a source of ATase which was purified by ammonium sulphate precipitation and DNA-cellulose, molecular exclusion and ion exchange chromatography (IEC). IEC purified material showed a major 24 kDa band after polyacrylamide gel electrophoresis (PAGE) with silver staining. Fluorography of purified ATase following incubation with [3H]-methylated substrate DNA and PAGE showed a single band at 24 kDa suggesting that, as with bacterial ATases, the protein itself accepts the alkyl group from O6-alkylguanine in substrate DNA during the repair reaction. Further purification of the protein using reverse phase HPLC resulted in a single peak representing approximately 125,000 fold purification. This was subjected to amino-terminal sequencing and it was found that the protein was blocked at the amino-terminal end: it was cleaved using trypsin or cyanogen bromide and the amino acid sequence of several reverse phase HPLC purified fragments was determined.
Abstract: ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.
Abstract: A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.
Abstract: p-Cresol is a mechanism-based inhibitor of bovine dopamine beta-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate: oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) (DBH) which covalently modifies a tyrosine at position 216 during inactivation (DeWolf, W.E., Jr., Carr, S.A., Varrichio, A., Goodhart, P.J., Mentzer, M.A., Roberts, G.D., Southan, C., Dolle, R.E. and Kruse, L.I. (1988) Biochemistry 27, 9093-9101). Here we report the recovery and characterization of additional minor peptides that are produced during the inactivation of DBH with p-[3H]cresol. Sequence and structural analysis of these peptides indicates tyrosine 357 as a second, minor site of modification.
Abstract: 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.
Abstract: 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)
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: Fibrinogen Manchester is an abnormal fibrinogen with an impaired release of fibrinopeptide A (FPA) and a polymerization abnormality. In the accompanying article we have identified the amino acid substitution in fibrinogen Manchester as A alpha 16 Arg leads to His. When fibrinogen Manchester was digested with low thrombin concentrations approximately 40-50% of the total FPA content was release at a rate similar to FPA release from normal fibrinogen. The fibrin so formed exhibited an impaired polymerization of monomers. Digestion of fibrinogen Manchester with high concentrations of thrombin for prolonged times released the remaining FPA which had an abnormal retention time when studied by high performance liquid chromatography (HPLC). This fibrinopeptide has been shown previously to contain the A alpha 16 Arg leads to His substitution. fibrin resulting from this exhaustive digestion had normal polymerization of monomers. The normal and substituted FPAs were isolated by HPLC and compared in a double antibody competitive-binding assay for normal FPA. The immunological cross-reactivity of the abnormal peptide was reduced, so that approximately 5 times more abnormal peptide was required on a molar basis to displace labelled normal FPA. Normal intact fibrinogen was 10-fold less reactive (on a half molar basis) than free normal FPA and the crossreactivity of fibrinogen Manchester was measurably less than that of normal fibrinogen. It is concluded that immunological measurement alone of FPA released from abnormal fibrinogens may not give a complete description of the kinetics of peptide release if the amino acid substitution lies within the FPA sequence. The combination of radioimmunoassay and HPLC, however, provides a powerful analytical approach that should be useful in classifying and characterizing abnormal fibrinogens.
Abstract: 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.
