Two epidermolytic toxins, produced by different strains of Staphylococcus aureus, split human skin at a site in the upper epidermis. Clinical effects are most common in infants, but adults are susceptible. Epidermolysis may also be observed in the mouse, in vivo and in vitro, and in a few other mammals. Recent in vitro experiments have demonstrated an inhibition by chelators and point to metal-ion, possibly Ca2+, involvement. The epidermolysis effect is insensitive to a wide range of other metabolic inhibitors. The toxin amino acid sequences are similar to that of staphylococcal proteinase, and new experiments by chemical modification and site-directed mutagenesis have shown that toxicity depends on ‘active serine’ residues of a catalytic triad similar to that found in serine proteases. Furthermore the toxins possess esterolytic activity, also dependent on the ‘active serine’ sites. However, the toxins have low or undetectable activity towards a range of peptide or protein substrates. In histological and related studies, the toxins bound selectively to an intracellular skin protein, profilaggrin, but there was no evidence that the toxin can enter intact epidermal cells. Therefore, although the circumstantial evidence that the toxins act by proteolysis is convincing, a specific skin proteolytic substrate for the toxin has not been identified.

1H n.m.r. was used to observe tryptophan formation from indole and L-serine, proton exchange at C-2 of L-tryptophan, and proton exchange at C-2 of L-serine, catalysed by yeast tryptophan synthase in the presence of 2H2O. Tryptophan synthesis took place with compulsory replacement of C-2 hydrogen by solvent hydrogen. The exponential decay rate (kobs) of the serine exchange reaction was insensitive to serine concentration in the range 2-20mM and was used to calculate kcat./Km values. However, kobs. was very sensitive to pH* values in the range 6.5-8.5 and the data require that the free enzyme is active in the base form resulting from two inseparable ionizations of pKa 7.3, and inactive after a third ionization controlled by a pKa of 7.5. Initial rates measured by u.v. absorbance and colorimetric procedures were used to calculate kinetic parameters of the tryptophan synthesis reaction. From pH 6.5 to 7, kcat./Km values for L-serine in the tryptophan synthesis and hydrogen exchange reactions were indistinguishable and increased rapidly under the control of two acid-base groups of pKa 6.7 and 7.2. Above pH 7, this equivalence breaks down because the exchange reaction alone is responsive to the third pKa value of the free enzyme. The pH dependence of the catalytic constant for tryptophan synthesis was qualitatively similar to that of the kobs. for serine exchange. A mechanism to explain the results is contrasted with recent proposals for the Escherichia coli system.

An epidermolytic toxin target was observed in keratohyalin granules of sectioned epidermis by a ‘direct’ fluorescence procedure using FTC-toxin, but not by an ‘indirect’ procedure using sequential reaction with toxin, anti-toxin and FTC-secondary antibody. The investigation of the two procedures was extended to keratinocytes. A dispase digestion procedure yielded three fractions which corresponded to basal, spinous and granular cells according to biochemical and morphological criteria. It was shown that the ‘direct’ and ‘indirect’ procedures both detected the toxin target in the keratohyalin granules of granular cells, but that the ‘indirect’ procedure was very insensitive. In control experiments, the profilaggrin of keratohyalin granules was detected readily in cells by a ‘direct’ procedure using FTC-antiprofilaggrin but only weakly by an ‘indirect’ double antibody procedure. Insensitivity to ‘indirect’ procedures thus appears to be a particular property of the keratohyalin granule site. It was shown that the toxin target was readily accessible in permeable (trypsin-isolated) granular cells but inaccessible in impermeable (dispase-isolated) cells.

Fluorescein-labelled epidermolytic toxin (FTC-toxin) ofStaphylococcus aureus and ferritin—toxin conjugate have been prepared and purified. FTC-toxin bound selectively to cryostat and resin-impregnated sections of neonatal mouse skin. Binding was localized at the keratohyalin granules and in the stratum corneum. In an epidermal cell (granular, spinous and basal) preparation, only keratohyalin granules of the granular cells bound FTC-toxin. Ferritin—toxin conjugate bound to skin sections at the same two sites as FTC-toxin and was competitive with the binding of free toxin. Keratohyalin granules in unstained sections had a novel ‘patched’ appearance under the electron microscope, and the ferritin—toxin conjugate bound preferentially to the electron-lucent areas. In the stratum corneum it was shown by quantitative estimation that the target density decreased as the surface of the tissue was approached.

Conference Article| April 01 1986 A search for the target site of epidermolytic toxin THOMAS P. SMITH; THOMAS P. SMITH 1Department of Biochemistry, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar CHRISTOPHER J. BAILEY CHRISTOPHER J. BAILEY 1Department of Biochemistry, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1986) 14 (2): 439–440. https://doi.org/10.1042/bst0140439 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation THOMAS P. SMITH, CHRISTOPHER J. BAILEY; A search for the target site of epidermolytic toxin. Biochem Soc Trans 1 April 1986; 14 (2): 439–440. doi: https://doi.org/10.1042/bst0140439 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: SDS, sodium dodecyl sulphate This content is only available as a PDF. © 1986 Biochemical Society1986 Article PDF first page preview Close Modal You do not currently have access to this content.

Book Review| June 01 1985 Electrophoresis ‘84 Electrophoresis ‘84. V. NEUHOFF. Verlag Chemie GmbHWeinheim 1984, pp. 522, DM 110 (softback) CHRISTOPHER J. BAILEY CHRISTOPHER J. BAILEY Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1985) 13 (3): 637. https://doi.org/10.1042/bst0130637a Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation CHRISTOPHER J. BAILEY; Electrophoresis ‘84. Biochem Soc Trans 1 June 1985; 13 (3): 637. doi: https://doi.org/10.1042/bst0130637a Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1985 Biochemical Society1985 Article PDF first page preview Close Modal You do not currently have access to this content.

It has been shown that yeast tryptophan synthase (L-serine hydro-lyase (adding indoleglycerol-phosphate) EC 4.2.1.20) catalyses tritium exchange reactions between protons on the alpha-carbon of L-serine of L-tryptophan, and water. The absolute rates of these reactions and indole-serine condensation (reaction B), all of which are pyridoxal phosphate-dependent, were measured. L-Serine exchange was resolved into two components, a high-affinity, slow, Michaelian reaction (KmS,H = 0.06 mM, kcats,H 3 X 10(-3) s-1) and a faster reaction (kcat greater than 2.5 S-1) which was not saturated even at 100 mM L-serine. Hydrogen exchange by tryptophan was a Michaelian process (KmT,H = 2.9 mM; kcatT,H = 0.6 s-1). Indole did not inhibit either exchange reaction. A plausible explanation of the results, that reaction B has a ping-pong mechanism with serine as first substrate and water and L-tryptophan as first and second products, respectively, was inadequate because of the observations that L-tryptophan is as first and second products, respectively, was inadequate because of the observations that L-tryptophan is synthesised with less than 1 mol of exchanged proton per mol amino acid, and that the ratio kcat/Km for serine changes between enzyme reactions. A branched modification with two enzyme-serine complexes, only one of which will exchange protons with water, will fit all the results.

Two serotypes of epidermolytic toxin were purified from culture filtrates of different strains of Staphylococcus aureus. The amino acid composition of the proteins is similar, each containing no cystine and one methionine, but type ii contains no tryptophan, whereas type i has 1 mol/mol protein. The molecular weights of type i and type ii toxins were 30,000 and 29,500, respectively, as found by SDS-polyacryamide gel electrophoresis and confirmed by studies of CNBr fragments and tryptic peptides. Dansylation gave a single different N-terminal amino acid for each toxin; the C-terminus of each is lysine. Peptide mapping of tryptic digests showed that very few peptides are common to the two amino acid sequences.

The amino acid sequence of plastocyanin from dock has been completed. It is a single polypeptide chain of 99 residues which is closely related to other plant plastocyanins. Compared to a preliminary sequence presented earlier, the completed sequence now shows two changes, at positions 53 and 92.

1.1. Enzymic tests, based on the effect of N-acetylglucosamine and on tryptic digestion, have been developed to distinguish hen- and goose-type lysozyme activities in egg whites.2.2. The lytic activity of the egg whites of 25 Anseriforme species has been measured and grouped into types by the enzymic tests.3.3. The simultaneous occurrence of both types of lysozyme in an egg was frequently observed; 6 species were mainly or completely goose-type; 3 species contained mainly hen-type enzyme.

Conference Article| December 01 1975 The Action of Hen and Goose Lysozymes on the Cell-Wall Peptidoglycan of Micrococcus lysodeikticus PETER GEOFFROY; PETER GEOFFROY 1Department of Biochemistry, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar CHRISTOPHER J. BAILEY CHRISTOPHER J. BAILEY 1Department of Biochemistry, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1975) 3 (6): 1212–1214. https://doi.org/10.1042/bst0031212 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation PETER GEOFFROY, CHRISTOPHER J. BAILEY; The Action of Hen and Goose Lysozymes on the Cell-Wall Peptidoglycan of Micrococcus lysodeikticus. Biochem Soc Trans 1 December 1975; 3 (6): 1212–1214. doi: https://doi.org/10.1042/bst0031212 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1975 Biochemical Society1975 Article PDF first page preview Close Modal You do not currently have access to this content.

AbstractAn automated chromatographic procedure for FDNBa‐available lysine determination is presented. “Available tyrosine” values can be obtained from the results and it is suggested that measurement of 100% tyrosine availability is a useful control for complete reaction of feedstuffs with FDNB. Application of the method to a range of feedstuffs confirmed the high availability of lysine in commercial feedstuffs. Using the tyrosine availability figures as a control, it is suggested that low values of lysine availability for fishmeal and meat and bone meal are an artifact but that grassmeal contains incompletely available lysine.

Conference Article| December 01 1974 Studies of the Amino Acid Sequence of Plastocyanin from Rumex obtusifolius (Broad-Leaved Dock) BARRY HASLETT; BARRY HASLETT 1Department of Botany, University of Durham, Durham DH1 3LE, U.K. Search for other works by this author on: This Site PubMed Google Scholar CHRISTOPHER J. BAILEY; CHRISTOPHER J. BAILEY 2Department of Biochemistry, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar JOHN A. M. RAMSHA; JOHN A. M. RAMSHA 1Department of Botany, University of Durham, Durham DH1 3LE, U.K. Search for other works by this author on: This Site PubMed Google Scholar MICHAEL D. SCAWEN; MICHAEL D. SCAWEN 1Department of Botany, University of Durham, Durham DH1 3LE, U.K. Search for other works by this author on: This Site PubMed Google Scholar DONALD BOULTER DONALD BOULTER 1Department of Botany, University of Durham, Durham DH1 3LE, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1974) 2 (6): 1329–1331. https://doi.org/10.1042/bst0021329 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation BARRY HASLETT, CHRISTOPHER J. BAILEY, JOHN A. M. RAMSHA, MICHAEL D. SCAWEN, DONALD BOULTER; Studies of the Amino Acid Sequence of Plastocyanin from Rumex obtusifolius (Broad-Leaved Dock). Biochem Soc Trans 1 December 1974; 2 (6): 1329–1331. doi: https://doi.org/10.1042/bst0021329 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1974 Biochemical Society1974 Article PDF first page preview Close Modal You do not currently have access to this content.

Conference Article| December 01 1974 The Purification and Structure of Tryptophan Synthetase from Neurospora crassa DAPHNE OWENS; DAPHNE OWENS 1Department of Biochemistry, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar CHRISTOPHER J. BAILEY CHRISTOPHER J. BAILEY 1Department of Biochemistry, Trinity College, Dublin 2, Ireland Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1974) 2 (6): 1331–1332. https://doi.org/10.1042/bst0021331 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation DAPHNE OWENS, CHRISTOPHER J. BAILEY; The Purification and Structure of Tryptophan Synthetase from Neurospora crassa. Biochem Soc Trans 1 December 1974; 2 (6): 1331–1332. doi: https://doi.org/10.1042/bst0021331 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1974 Biochemical Society1974 Article PDF first page preview Close Modal You do not currently have access to this content.

The amino acid sequence of plastocyanin from potato was determined. It consists of a single polypeptide chain of 99 residues, of molecular weight 10332. The sequence was determined by using a Beckman 890c sequencer and by dansyl–Edman analysis of peptides derived from purified CNBr fragments. The sequence shows considerable similarity with that of Chlorella fusca, and also with the C-terminal region of bacterial azurins.

The relationship between the electrophoretic mobility, the molecular weight and the charge of peptides at pH1.9 was re-investigated. It was shown that the relationship at pH1.9 is quantitatively similar to that which holds at pH6.5.

Four sub-units of vicilin were separated by sodium dodecyl sulphate gel electrophoresis and their molecular weights determined by calibration. The amino acid composition of vicilin was determined by ion-exchange chromatography and by other methods for half-cystine and tryptophan. Tryptic peptide maps and N-terminal analysis following cyanogen bromide cleavage, were used to deduce a chemical molecular weight of vicilin of 100–130 × 103. These structural results on vicilin are discussed in relationship to its biological role.

Legumin from broad bean seed has been carboxymethylated and analysed. The protein contained three N‐terminal residues, leucine, glycine and threonine. Three polypeptide chains were found by polyacrylamide gel electrophoresis of the dissociated protein; their molecular weights were 56000, 42000 and 23000. 140 peptides were found on fingerprints of tryptic digests of S‐carboxymethyl‐legumin. Of these 14 were radioactive in digests of [14C]carboxymethylated legumin, and 16 were radioactive in digests of legumin prepared from material labelled in vivo with [35S]sulphate. Three new end groups were found after cyanogen bromide cleavage corresponding to Met‐Val, Met‐Phe and Met‐Pro sequences. Taken in conjunction with the amino acid analyses these peptide mapping experiments suggest that the protein contains a total sequence weight of 115000, which is in agreement with the finding of three polypeptide chains whose weights total 121000.On the basis of dye binding studies and of the incorporation in vivo of uniformly 14C‐labelled amino acid mixture, it is suggested that the polypeptide chains of molecular weights 56000, 42000 and 23000 are present in molar ratios of 1:4:6 or 1:3:6, respectively. From consideration of experimental accuracy the 1:3:6 structure is preferred. The proposed 10 chains model for legumin has a molecular weight of 320000.

Cultured slices of cotyledon tissue from 60-day broad bean have been shown to synthesise globulin protein extensively. About 80% of the 3H-leucine incorporated into protein was incorporated into globulin protein; 35S-sulphate was also incorporated by the slices.