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J B Findlay

Publications and source records attributed to J B Findlay.

At least 127 records · Page 7Linked to original sources
PubMed1986

Amino acid composition of the 9 kDa phosphoprotein of pea thylakoids.

The 9 kDa phosphoprotein of pea thylakoids was isolated by electroelution from SDS-polyacrylamide gels and its amino acid composition determined. The result is at variance with the amino acid compositions predicted from published nucleotide sequences of the genes for apocytochrome b-559 and for CFo subunit III. The amino acid composition of the 9 kDa phosphoprotein resembles that of the 25 kDa light-harvesting chlorophyll a/b protein (LHC-II). We propose that the 9 kDa polypeptide is a chlorophyll-binding protein of photosystem II, that it functions as a link in excitation energy transfer between LHC-II and the reaction centre, and that its phosphorylation regulates excitation energy distribution by means of mutual electrostatic repulsion between itself and phosphorylated LHC-II.

Amino Acids↗
PubMed1986

Identification of the sites in opsin modified by photoactivated azido[125I]iodobenzene.

Opsin labelled with photoactivated 1-azido-4-[125I]iodobenzene was proteolysed in situ with Staphylococcus aureus V8 proteinase to yield two radioactive membrane-bound fragments. These were separated, cleaved with CNBr and the resultant peptides sequenced in order to locate the radiolabelled residues. In the whole molecule, there was clear evidence for modification of at least 20 sites, identified as derivatives of cysteine, tryptophan, tyrosine, histidine and lysine residues. The probe primary reacted, therefore, with nucleophilic substituents. The positions of the modified sites relative to the confines of the phospholipid bilayer were consistent with all other studies on the disposition of the polypeptide chain. The location of these sites substantiated an earlier suggestion that not all the transmembrane segments should be regarded as continuous regular alpha-helices.

Amino Acid Sequence↗
PubMed1986

Modification of ovine opsin with the photosensitive hydrophobic probe 1-azido-4-[125I]iodobenzene. Labelling of the chromophore-attachment domain.

The hydrophobic photosensitive probe 1-azido-4-[125I]iodobenzene (AIB) partitioned preferentially into photoreceptor disc membranes and, upon u.v. irradiation, became covalently bound to opsin and phospholipid. The labelling of both protein and phospholipid was linearly related to AIB concentration. The amount of probe incorporated into protein was not significantly different when membranes were irradiated at -100 degrees, 4 degrees or 25 degrees C, but irreversible aggregation of monomeric opsin was dramatically reduced by performing the photolysis at -100 degrees C. Labelling of opsin after irradiation at -100 degrees or 4 degrees was not significantly reduced by the presence of lysine in the aqueous buffer, indicating that significant amounts of reactive species did not enter the aqueous phase. The incorporation into phospholipid, unlike that into opsin, decreased as the temperature of irradiation increased. Some labelling of opsin occurred on incubation with pre-photoactivated AIB, indicating that reaction may also occur with reactive species of longer lifetimes than the nitrene. Proteolysis of labelled opsin with Staphylococcus aureus V8 proteinase yielded two radiolabelled membrane-bound fragments. The location of the modified sites (cysteine, tryptophan, tyrosine, lysine and histidine residues: all nucleophiles) in the smaller fragment was entirely consistent with putative models for the protein derived from other studies.

Affinity Labels↗
PubMed1986

Purification of vitamin D binding protein from human plasma using high performance liquid chromatography.

Vitamin D binding protein/group-specific component was purified from human plasma by chromatographic techniques utilising high performance liquid chromatography and by traditional low pressure chromatographic techniques alone. Use of high performance liquid chromatography considerably reduced the time taken to prepare pure vitamin D binding protein and increased the yield to 16% compared with 2.8% using the traditional methods. The vitamin D binding protein prepared by high performance liquid chromatography was shown to be highly pure by amino acid sequence, SDS gel electrophoresis and by antibody production. The amino acid sequence was confirmed and extended. The affinity constants of the high pressure liquid chromatography purified vitamin D binding protein for 25 hydroxycholecalciferol (25 OHD3) and 1,25 dihydroxycholecalciferol 1,25(OH)2D3 were 1.9 X 10(7) mol/l and 2.6 X 10(6) mol/l, respectively.

Amino Acid Sequence↗
PubMed1986

Posttranslational processing of concanavalin A precursors in jackbean cotyledons.

Metabolic labeling of immature jackbean cotyledons with 14C-amino acids was used to determine the processing steps involved in the assembly of concanavalin A. Pulse-chase experiments and analyses of immunoprecipitated lectin forms indicated a complex series of events involving seven distinct species. The structural relatedness of all of the intermediate species was confirmed by two-dimensional mapping of 125I-tryptic peptides. An initial glycosylated precursor was deglycosylated and cleaved into smaller polypeptides, which subsequently reannealed over a period of 10-27 h. NH2-terminal sequencing of the abundant precursors confirmed that the intact subunit of concanavalin A was formed by the reannealing of two fragments, since the alignment of residues 1-118 and 119-237 was reversed in the final form of the lectin identified in the chase and the precursor first labeled. When the tissue was pulse-chased in the presence of monensin, processing of the glycosylated precursor was inhibited. The weak bases NH4Cl and chloroquine were without effect. Immunocytochemical studies showed that monensin treatment caused the accumulation of immunoreactive material at the cell surface and indicated that the ionophore had induced the secretion of a component normally destined for deposition within the protein bodies. Consideration of the tertiary structure of the glycosylated precursor and mature lectin showed that the entire series of processing events could occur without significant refolding of the initial translational product. Proteolytic events included removal of a peptide from the surface of the precursor molecule that connected the NH2- and COOH-termini of the mature protein. This processing activated the carbohydrate-binding activity of the lectin. The chase data suggest the occurrence of a simultaneous cleavage and formation of a peptide bond, raising the possibility that annealment of the fragments to give rise to the mature subunit involves a transpeptidation event rather than cleavage and subsequent religation.

Amino Acid Sequence↗
PubMed1985

Comparison of water exposed area of cholera toxin when free in solution and bound to liposomes containing the ganglioside GM1.

Membrane impermeable diazocoupling reagents were used for studying the water exposure of subunits (alpha, beta, gamma) of cholera toxin (CT) when bound to liposomes containing the ganglioside GM1 (Lip-GM1). The interaction between CT with Lip-GM1 shielded the binding region in particular, since a maximum of one amino acid residue on each beta subunit was modifiable. When CT was labeled free in solution five residues of each beta subunit can be coupled, but it produced loss of binding ability. New area of beta subunit was exposed to reagents after having removed alpha subunit. This labeling may serve as a tool to assess the topology of CT upon binding with Lip-GM1.

Chemical Phenomena↗
PubMed1984

Phosphorylation of ovine rhodopsin. Identification of the phosphorylated sites.

Light-dependent phosphorylation of sheep opsin was obtained in purified discs to which was added a partially purified preparation of rhodopsin kinase. A maximum ratio of 1.8 mol of phosphate/mol of rhodopsin bleached was obtained. Perturbing the lipid bilayer did not alter the phosphorylation ratio. Dephosphorylation in both segments and discs was only achieved when the supernatant fraction from a retina homogenate was added. Complete dephosphorylation required the presence of the detergent dodecyltrimethylammonium bromide in the incubation medium. Treatment of phosphorylated disc membranes with Staphylococcal aureus V8 proteinase generated two membrane-bound fragments, only one of which (V8-S, Mr 12 000) was labelled, together with a soluble seven-residue peptide that contained [32P]phosphoserine. Peptide sequencing, together with subdigestion procedures, localized the phosphorylation sites to serine residues at positions 334, 338 and 343 in the whole sequence and threonine residues at positions 335 and 336.

Amino Acid Sequence↗
PubMed1984

Labelling of the cytoplasmic domains of ovine rhodopsin with hydrophilic chemical probes.

The disposition of polypeptide chain of ovine rhodopsin in the photoreceptor disc membrane was investigated by using two hydrophilic reagents, 3,5-di-[125I]iodo-4-diazobenzenesulphonate [( 125I]DDISA) and [14C]succinic anhydride. Both reagents were used to modify rhodopsin in intact disc membranes under conditions where no loss of A500 occurred. Reaction of [125I]DDISA with rhodopsin approached completion after 30 min. Binding was saturated at a 75-fold molar excess of reagent, which gave binding ratios of up to 2 mol/mol of rhodopsin. Proteolysis of rhodopsin, using Staphylococcus aureus V8 proteinase, yielded two membrane-bound fragments, both of which contained bound radioactive probe. Subsequent CNBr cleavage of these fragments produced five radiolabelled peptides which corresponded to the C-terminal region and cytoplasmic loops of rhodopsin. Similar studies with [14C]-succinic anhydride also gave binding ratios of up to 2 mol/mol of rhodopsin. Sequencing of the [14C]succinylated peptides identified the location of the reactive sites as lysine residues 66, 67, 141, 245, 248, 311, 325 and 339 in the polypeptide chain. Non-permeability of both probes was demonstrated by the absence of any radioactivity associated with the intradiscal N-terminal glycopeptide. Sonication of membranes in the presence of [125I]DDISA led to the incorporation of label in this peptide.

Amino Acid Sequence↗
PubMed1984

Sequence variability in the retinal-attachment domain of mammalian rhodopsins.

Ovine rhodopsin was regenerated with 11-cis-[15-3H]retinal and cleaved in situ by Staphylococcus aureus V8 proteinase to give two membrane-bound fragments of Mr 27 000 (V8-L) and 12 000 (V8-S). After purification of the proteolysed complex by affinity chromatography with concanavalin A-Sepharose 4B, [3H]retinal was covalently linked to the protein by reduction with borohydride. The purified [3H]-retinyl V8-S fragment was cleaved with CNBr and trifluoroacetic acid, the resulting peptides resolved by gel filtration and the [3H]retinyl peptide sequenced. The protocol developed for the isolation and sequencing of this region of the ovine protein was applied directly, and reproducibly, to bleached and unregenerated porcine and equine opsins. Comparisons of the primary structures of the fragments reveals marked variation in the sequence immediately after the lysine residue shown in the ovine protein to be the attachment point for the aldehyde group of the chromophore. Mutable positions are localized in regions previously predicted as adopting nonregular or distorted conformations and hint at structural arrangements that may provide a better understanding of the spectral and functional properties of the visual pigment.

Amino Acid Sequence↗
PubMed1984

The structure of mammalian rod opsins.

Ovine rhodopsin is organised in disc membranes as a monomer. The determination of its amino acid sequence has permitted the utilisation of structure prediction programmes which indicate the probable disposition of the polypeptide chain in the bilayer. This putative model is consistent with labelling data using the chemical probes, [14C]succinic anhydride, [125I]diazodiido sulphanilic acid and [125I]iodophenyl azide, and with the cleavage points for several proteases. More surprisingly the predicted structure points to the occurrence of breaks/distortions in the transmembrane helical segments. These distorted regions may be of primary functional importance to the protein and at least one is associated with the attachment point of the chromophore. This particular part of the structure is also identified as a "mutational hot spot", for bovine, equine, ovine and porcine opsins exhibit different sequences (but conserved molecular volumes) in the four residues following the retinyllysine. In an otherwise highly conserved protein with no obvious functional differences between the four species, the high substitution rate in this region is unexplained.

Amino Acid Sequence↗
PubMed1983

Investigation of the organisation of the major proteins in bovine myelin membranes. Use of chemical probes and bifunctional crosslinking reagents.

Bovine myelin was incubated with a variety of bifunctional reagents and chemical probes. The use of a photosensitive hydrophobic compound, 1-azido-[125I]iodobenzene, led to the suggestion that the proteolipid protein is deeply intercalated into the hydrophobic milieu of the membrane, but did not support the contention that regions of the basic protein behave in a similar fashion. Crosslinking studies indicated that both polypeptides may be present in the membrane as homodimers and these dimers may be part of much larger assemblies. These results give rise to a somewhat different model for the structural organisation of myelin to that proposed earlier.

Animals↗
PubMed1983

Isolation and characterization of the CNBr peptides from the proteolytically derived N-terminal fragment of ovine opsin.

Ovine rhodopsin may be cleaved in situ by Staphylococcus aureus V8 proteinase into two membrane-bound fragments designated V8-L (27 000 mol.wt.) and V8-S (12 000 mol.wt.). After purification of the proteolysed complex by affinity chromatography in detergent using concanavalin A immobilized on Sepharose 4B, the two polypeptide fragments may be separated by gel-permeation chromatography on Sephadex LH-60. Digestion of the N-terminal-derived V8-L fragment with CNBr in 70% (v/v) trifluoroacetic acid resulted in a peptide mixture that could be fractionated by procedures involving gel-permeation chromatography in organic and aqueous solvents and the use of differential solubility. The complete or partial sequences of all ten peptides are reported.

Amino Acid Sequence↗
PubMed1982

Characterization and partial sequence of di-iodosulphophenyl isothiocyanate-binding peptide from human erythrocyte anion-transport protein.

We investigated the presumed anion-binding domain of the anion-transport protein from human erythrocyte membranes, using 2,6-di-iodo-4-sulphophenyl isothiocyanate, an inhibitor of anion transport. The 125I-labelled reagent binds covalently to the protein with a half-maximal inhibitory concentration of 86 microM. Treatment of unsealed erythrocyte 'ghosts' with chymotrypsin yielded a membrane-bound fragment (mol.wt. 14 500 +/- 1000) that contained all the protein-bound radioactivity. The binding of the inhibitor to this peptide gave a pattern very similar to that obtained for the effect of the compound on phosphate transport into erythrocytes. The peptide is therefore presumed to be intimately involved in the mediation of anion exchange. Cleavage of the 14 500-mol.wt. transmembrane fragment with CNBr resulted in the production of two peptides with apparent molecular weights of 8800 and 4700. The 4700-mol.wt. peptide is the N-terminal portion of the 14 500-mol.wt. peptide. The attachment site for 2,6-di-iodo-4-sulphophenyl isothiocyanate is situated near the C-terminal of the 8800-mol.wt. peptide. This locates the inhibitor-binding site near the chymotrypsin cleavage point at the extracellular surface of the membrane. A partial sequence (residues 1--38) of the 8800-mol.wt. peptide was obtained.

Affinity Labels↗
PubMed1982

The effects of general anaesthetics on glucose and phosphate transport across the membrane of the human erythrocyte.

A study has been carried out into the effects of clinically important general anaesthetics, althesin, thiopentone and propanidid, on the transport of glucose and phosphate across the membrane of the human erythrocyte. In general these three substances all inhibit both transport processes but with characteristic inhibition profiles and varying degrees of efficacy. Glucose transport was more sensitive to the hydrophobic steroids and phosphate transport to propanidid. Some hydrophobic agents, e.g., iodobenzene and its azide, were not inhibitory. Removal of cholesterol to some extent augmented the inhibitory effects of most of these compounds (not propanidid). It is argued that these effects are due to the penetration of the anaesthetics into the lipid bilayer and either subsequent disruption of the lipid annuli surrounding the integral membrane proteins and/or direct anaesthetic-protein interaction.

Alfaxalone Alfadolone Mixture↗
PubMed1980

The isolation of human-erythrocyte band-3 polypeptide labelled with a photosensitive hydrophobic probe.

To investigate the intramembranous domains of the major band-3 polypeptide, human erythrocyte membranes were labelled with 1-azido-4-[125I]iodobenzene. The anion-exchange protein has been isolated by a new procedure that decreases possible contamination by other integral membrane proteins of similar molecular weight. After chemical modification, the protein was subjected to proteolytic cleavage by several proteinases to yield a distinct pattern of peptides, a few of which contained radioactivity.

Anion Exchange Protein 1, Erythrocyte↗