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J B Findlay

Publications and source records attributed to J B Findlay.

At least 19 recordsLinked to original sources
PubMed1996

Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase.

The organotin complex dibutyltin-3-hydroxyflavone bromide [Bu2Sn(of)Br] has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H(+)-ATPase. Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein. Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid. The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide. Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity. Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid. The fluorescent enhancement of affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity. Interaction of the probe with mutant proteolipid was unaffected by DCCD. These data suggest an overlap in the binding sites of organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the facuolar H(+)-ATPase.

Adenosine Triphosphate↗
PubMed1996

Membrane receptor for odour-binding proteins.

Specific binding of 125I-labelled bovine odour-binding protein (OBP) to isolated membranes from nasal mucosa was demonstrated. The interaction reached equilibrium within 30 min at 37 degrees C and was reversible. A Scatchard analysis of the equilibrium binding revealed a single population of binding sites, with the calculated equilibrium dissociation constant and maximum number of binding sites being 2.25 +/- 0.5 microM and 18.5 +/- 2 pmol/mg of membrane protein respectively (n = 2). Receptor activity was decreased on digestion by trypsin, proteinase K or endoglycosidase H, was heat labile and was sensitive to thiol-group-specific reagents. With the exception of rat and mouse major urinary proteins, which exhibit a high degree of structural similarity with OBP and bind similar ligands, other members of the lipocalin family, such as retinol-binding protein and beta-lactoglobulin, failed to inhibit the binding of 125I-labelled OBP to its receptor. The receptor seems not to be restricted to olfactory tissues, as it was detected in a variety of other tissues. This suggests that OBP is unlikely to play a role only in olfactory signal transduction. It might have a much broader role within the body; possibilities include a role in detoxification or signalling.

Animals↗
PubMed1995

The first putative transmembrane helix of the 16 kDa proteolipid lines a pore in the Vo sector of the vacuolar H(+)-ATPase.

The 16 kDa proteolipid is the major component of the vacuolar H(+)-ATPase membrane sector, responsible for proton translocation. Expression of a related proteolipid from the arythropod Nephrops norvegicus in a Saccharomyces strain in which the VMA3 gene for the endogenous proteolipid has been disrupted results in restored vacuolar H(+)-ATPase function. We have used this complementation system, coupled to cysteine substitution mutagenesis and protein chemistry, to investigate structural features of the proteolipid. Consecutive cysteines were introduced individually into putative transmembrane segment 1 of the proteolipid, and at selected sites in extramembranous regions and in segment 3 and 4. Analysis of restored vacuolar H(+)-ATPase function showed that segment 1 residues sensitive to mutation to cysteine were clustered on a single face, but only if the segment was helical. Only residues insensitive to mutation could be covalently modified by the cysteine-specific reagent fluorescein 5-maleimide. A cysteine introduced into segment 3 was the only residue accessible to a relatively hydrophobic reagent, suggesting accessibility to the lipid phase. Analysis of disulphide bond formation between introduced cysteines indicates that the first transmembrane alpha-helices of each monomer are adjacent to each other at the centre of the proteolipid multimeric complex. The data are consistent with a model in which the fluorescein maleimide-accessible face of helix I lines a pore at the centre of a hexameric complex formed by the proteolipid, with the mutationally sensitive face oriented into the protein core. The implications for ion-transport function in this family of proteins are discussed in the context of this structural model.

Animals↗
PubMed1995

A beta-subclass phosphatidylinositol-specific phospholipase C from squid (Loligo forbesi) photoreceptors exhibiting a truncated C-terminus.

A PCR-based strategy has been used to isolate a full length cDNA encoding a phosphatidylinositol-specific phospholipase C from a sized cDNA squid (Loligo forbesi) retinal library. The predicted protein sequence contains 875 amino acids, with calculated M(r) 98,181, and has marked similarity with PLC beta-isoforms, including conservation of the 'X' and 'Y' regions. It is unique in having a major C-terminal truncation. A major protein of apparent M(r) 120,000 estimated by SDS-PAGE has been isolated from squid photoreceptors and identified by partial protein sequence analysis to correspond to the protein sequence predicted from the cDNA clone. This protein has been shown to hydrolyse phosphatidylinositol 4,5-bisphosphate. It is not yet clear whether this represents the major light-activated PLC in squid vision.

Amino Acid Sequence↗
PubMed1995

Lipid-protein interactions and assembly of the 16-kDa channel polypeptide from Nephrops norvegicus. Studies with spin-label electron spin resonance spectroscopy and electron microscopy.

The assembly of 16-kDa polypeptide channel units in membranes from the hepatopancreas of Nephrops norvegicus has been studied both by electron microscopy and by the lipid--protein interactions reported with spin-labeled lipids. Membranes prepared by extraction with N-lauroylsarcosine and Triton X-100 have a low lipid/protein ratio (ca. 4-6.5 phospholipids and 1 cholesterol per 16-kDa monomer), and those prepared by alkaline extraction have a higher lipid/protein ratio (ca. 12-16 phospholipids and 3.5-4 cholesterols per 16-kDa monomer). In the membranes extracted with detergents, the protein is assembled in membrane sheets as hexagonally packed hexameric complexes, whereas the alkali-extracted preparations consist of closed vesicles in which the channel complexes are near randomly distributed. The electron spin resonance (ESR) spectra from lipids spin-labeled at the C-14 position of the (sn-2) chain show lower mobility for the membranes extracted with N-lauroylsarcosine than for the alkaline-extracted membranes. At higher temperatures, the ESR spectra reveal a population of lipids whose mobility is restricted by direct interaction with the intramembranous sections of the channel assemblies. The population of protein-associated spin-labeled phosphatidylcholine in the alkali-extracted membranes corresponds to 4-5 phospholipid molecules plus 1 cholesterol molecule per 16-kDa polypeptide monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Integration of a K+ channel-associated peptide in a lipid bilayer: conformation, lipid-protein interactions, and rotational diffusion.

The 26-residue peptide of sequence KEALYILMVLGFFGFFTLGIMLSYIR, which contains the single putative transmembrane domain of a small protein that is associated with slow voltage-gated K+ channels, has been incorporated in bilayers of dimyristoylphosphatidylcholine by dialysis from 2-chloroethanol to form complexes of homogeneous lipid/peptide ratio. Fourier transform infrared spectroscopy indicates that the peptide is integrated in the lipid bilayer wholly in a beta-sheet conformation. The electron spin resonance spectra of spin-labeled lipids in the lipid/peptide complexes contain a component corresponding to lipids whose chains are motionally restricted in a manner similar to those of lipids at the hydrophobic surface of integral transmembrane proteins. From the dependence of the lipid spin label spectra on the lipid/peptide ratio of the complexes, it is found that ca. 2.5 lipids per peptide monomer, independent of the species of spin-labeled lipid, are motionally restricted by direct interaction with the peptide in the bilayer. This value would be consistent with, e.g., a beta-barrel structure for the peptide in which the beta-strands either are strongly tilted or have a reverse turn at their center. A preferential selectivity of interaction with the peptide is observed for the negatively charged spin-labeled lipids phosphatidic acid, stearic acid, and phosphatidylserine, which indicates close proximity of the positively charged residues at the peptide termini to the lipid headgroups. The saturation-transfer electron spin resonance spectra of the peptide spin-labeled at a cysteine residue replacing Leu18 evidence rather slow rotational diffusion in the lipid complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Structural heterogeneity of Pseudomonas aeruginosa bacterioferritin.

The subunit composition, amino acid sequence and haem-binding characteristics of bacterioferritin (BFR) from Pseudomonas aeruginosa have been studied. Unlike other BFRs, P. aeruginosa BFR was found to contain two subunit types, designated alpha and beta, which differed considerably in their amino acid sequences. The N-terminal 69 and 55 amino acids of the alpha and beta subunits respectively were determined. The alpha subunit differed most from other BFRs. The two subunits were present in variable proportions in different preparations. The maximum stoichiometry of haem binding was found to be sample-dependent and to be different from the previously reported one per subunit [Kadir and Moore (1990) FEBS Lett. 271, 141-143]. This previous haem-binding study was shown to have been carried out with damaged protein, which contained both normal alpha and beta subunits and shorter versions of these that appeared to have been produced by cleavage of the normal subunits. The possibility that aging processes degrade ferritins and affect their haem-binding characteristics is discussed.

Amino Acid Sequence↗
PubMed1994

The ligand binding site of the neurokinin 2 receptor. Site-directed mutagenesis and identification of neurokinin A binding residues in the human neurokinin 2 receptor.

Thirteen residues in the human neurokinin 2 (NK2) receptor were identified as potential ligand-binding residues by molecular modeling and amino acid sequence analysis. Site-directed mutagenesis was used to alter these residues in order to ascertain their importance in binding neurokinin A (NKA), the physiological peptide ligand for the NK2 receptor, and the non-peptide NK2 receptor selective antagonist SR48968. Four sites appear to be critical for NKA binding (Gln109, His198, Ile202, and Gly273). The mutant receptors Gln109-->His, Ile202-->Val, Gly273-->Pro, and Gly273-->Thr maintain their affinity for SR48968, despite being unable to bind the peptide ligand. His198-->Ala and His198-->Leu no longer bind NKA or SR48968. We have also identified a residue (Leu292) which appears to play a minor role in the binding of substance P (SP) and neurokinin B (NKB) to the NK2 receptor. The mutant receptor Leu292-->Ser binds NKB and SP with approximately a 5-fold greater affinity in comparison with the wild type receptor while the affinity of NKA remains unaffected. The results suggest that intramembranous residues, as well as residues which lie close to the extracellular side of transmembrane helices 3, 5, and 6, form part of the NK2 receptor binding site. Binding of SP and NKB to the NK2 receptor may also be influenced by residues near the extracellular side of helix 7. These results suggest that some regions of the binding site for NKA in the NK2 receptor are not used for binding SP in the NK1 receptor. However, it also seems that the NKA binding site includes regions that are also used by other G-protein-coupled receptors such as rhodopsin and the beta 2-adrenergic receptors.

Amino Acid Sequence↗
PubMed1994

Changes in activation gating of IsK potassium currents brought about by mutations in the transmembrane sequence.

Expression of the rat kidney IsK protein in Xenopus oocytes produces slowly-activating potassium channel currents. We have investigated the relationship between structure and function of the single putative membrane-spanning domain using site-directed mutagenesis. Six mutants were constructed in which consecutive individual amino acids (53 to 58) of the transmembrane region were substituted by cysteine. Expression of four of these mutants in Xenopus oocytes resulted in currents which were similar to wild-type. However, for one mutant (position 55) activation curves were shifted in a hyperpolarising direction and for another mutant (position 58) activation curve were shifted in a depolarising direction. This suggests that the hydrophobic phenylalanine residues at positions 55 and 58 may play a critical role in IsK activation gating. This spacing of functional amino acids at every third residue may indicate an alpha-helical conformation for the membrane-spanning domain of IsK. Furthermore, these results also indicate that one face of the helix may represent a region of subunit association.

Amino Acid Sequence↗
PubMed1994

Solubilization and purification of the retinol-binding protein receptor from human placental membranes.

The membrane receptor for retinol-binding protein (RBP) has been solubilized from human placental brush-border membranes with octyl-beta-glucoside, Nonidet P-40 and CHAPS. A method, based on the preferential precipitation of 125I-RBP-receptor complex with poly(ethylene glycol) 8000, was developed in order to measure the RBP-binding activity in the detergent extracts. The receptor was fairly stable (4 degrees C, 7 days) in octyl-beta-glucoside and Nonidet P-40, but quickly lost activity in CHAPS. The detergent-solubilized form retained all the properties characteristic of the membrane-bound protein, except for a small decrease in affinity for RBP (3- and 7-fold in Nonidet P-40 and octyl-beta-glucoside respectively). The receptor was isolated using recombinant RBP coupled to Reacti-Gel 6X affinity matrix. The purified material contained major and minor protein species of 63 and 55 kDa respectively on SDS/PAGE.

Binding Sites↗
PubMed1994

Structure-function studies on human retinol-binding protein using site-directed mutagenesis.

Retinol-binding protein (RBP) transports vitamin A in the plasma. It consists of eight anti-parallel beta-strands (A to H) that fold to form an orthogonal barrel. The loops connecting the strands A and B, C and D, and E and F form the entrance to the binding site in the barrel. The retinol molecule is found deep inside this barrel. Apart from its specific interaction with retinol, RBP is involved in two other molecular-recognition properties, that is it binds to transthyretin (TTR), another serum protein, and to a cell-surface receptor. Using site-directed mutagenesis, specific changes were made to the loop regions of human RBP and the resultant mutant proteins were tested for their ability to bind to retinol, to TTR and to the RBP receptor. While all the variants retained their ability to bind retinol, that in which residues 92 to 98 of the loop E-F were deleted completely lost its ability to interact with TTR, but retained some binding activity for the receptor. In contrast, the double mutant in which leucine residues at positions 63 and 64 of the loop C-D were changed to arginine and serine respectively partially retained its TTR-binding ability, but completely lost its affinity for the RBP receptor. Mutation of Leu-35 of loop A-B to valine revealed no apparent effect on any of the binding activities of RBP. However, substitution of leucine for proline at position 35 markedly reduced the affinity of the protein for TTR, but showed no apparent change in its receptor-binding activity. These results demonstrate that RBP interacts with both TTR and the receptor via loops C-D and E-F. The binding sites, however, are overlapping rather than identical. RBP also appears to make an additional contact with TTR via its loop A-B. A further implication of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. This observation is consistent with our previously proposed mechanism for delivery of retinol to target tissues [Sivaprasadarao and Findlay (1988) Biochem. J. 255, 571-579], according to which retinol delivery involves specific binding of RBP to the cell-surface receptor, an interaction that triggers release of retinol from RBP to the bound cell rather than internalization of retinol-RBP complex.

Base Sequence↗
PubMed1994

Functional properties of a hybrid vacuolar H(+)-ATPase in Saccharomyces cells expressing the Nephrops 16-kDa proteolipid.

The hydrophobic 16-kDa polypeptide which forms gap-junction-like structures in the crustacean Nephrops norvegicus is a member of a highly conserved family of proteolipids involved in a variety of membrane transport functions in eukaryotic cells. This family also includes the product of the Saccharomyces cerevisiae VMA3 gene which encodes an integral membrane component of the vacuolar membrane H(+)-ATPase. The cDNA for the Nephrops proteolipid complements a mutation in the yeast VMA3 gene, resulting in assembly of a hybrid H(+)-ATPase comprising yeast catalytic subunits and Nephrops integral membrane components. The hybrid vacuolar ATPase was capable of ATP hydrolysis which was coupled to proton translocation and showed inhibitor binding and enzymological properties similar to those of wild-type V-ATPases (Km for ATP, 0.4 mM), suggesting that both yeast and crustacean proteolipids share conserved structure at regions of protein interaction. To facilitate isolation of the Nephrops proteolipid by affinity chromatography on a Ni(2+)-binding support, six C-terminal histidine residues were added to the proteolipid. This modification did not prohibit assembly into the hybrid H(+)-ATPase, although the resultant enzyme did have a markedly elevated Km (1.8 mM). The membrane-bound Vo sector of the ATPase was isolated by the affinity-chromatography procedure and reconstituted into synthetic vesicles. This complex was found to be impermeable to small cations in the absence of catalytic ATPase subunits either in situ in the vacuolar membrane or in the reconstituted system. The functional significance of this impermeability and the structure/function relationships between proteolipids from different sources are discussed.

Adenosine Triphosphate↗
PubMed1994

Primary structure of a beta subunit of alpha-dendrotoxin-sensitive K+ channels from bovine brain.

Voltage-dependent cation channels are large heterooligomeric proteins. Heterologous expression of cDNAs encoding the alpha subunits alone of K+, Na+, or Ca2+ channels produces functional multimeric proteins; however, coexpression of those for the latter two with their auxiliary proteins causes dramatic changes in the resultant membrane currents. Fast-activating, voltage-sensitive K+ channels from brain contain four alpha and beta subunits, tightly associated in a 400-kDa complex; although molecular details of the alpha-subunit proteins have been determined, little is known about the beta-subunit constituent. Proteolytic fragments of a beta subunit from bovine alpha-dendrotoxin-sensitive neuronal K+ channels yielded nine different sequences. In the polymerase chain reaction, primers corresponding to two of these peptides amplified a 329-base-pair fragment in a lambda gt10 cDNA library from bovine brain; a full-length clone subsequently isolated encodes a protein of 367 amino acids (M(r) approximately 40,983). It shows no significant homology with any known protein. Unlike the channels' alpha subunits, the hydropathy profile of this sequence failed to reveal transmembrane domains. Several consensus phosphorylation motifs are apparent and, accordingly, the beta subunit could be phosphorylated in the intact K+ channels. These results, including the absence of a leader sequence and N-glycosylation, are consistent with the beta subunit being firmly associated on the inside of the membrane with alpha subunits, as speculated in a simplified model of these authentic K+ channels. Importantly, this first primary structure of a K(+)-channel beta subunit indicates that none of the cloned auxiliary proteins of voltage-dependent cation channels, unlike their alpha subunits, belong to a super-family of genes.

Amino Acid Sequence↗
PubMed1994

Molecular cloning, sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum.

Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16,485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the substrates of greatest affinity.

Amino Acid Sequence↗
PubMed1994

Modulation by protein kinase A of a cloned rat brain potassium channel expressed in Xenopus oocytes.

A potassium channel from rat brain was expressed in Xenopus oocytes in order to study modulation of channel function by phosphorylation via protein kinase A. Application of 8-Br-cAMP to oocytes expressing the drk1 channel (with the first 139 amino acids of the N terminus deleted, delta Ndrk1) caused a voltage-independent elevation of current amplitude, which was not seen for endogenous currents or for wild-type full-length drk1 channel. This effect on delta Ndrk1 was blocked by pre-injection of oocytes with Walsh-peptide protein kinase A inhibitor, suggesting mediation via protein kinase A. The protein kinase inhibitor also reduced both delta Ndrk1 and full-length drk1 currents. Substitution of the serine residues by alanine at one or both of the two consensus protein kinase A phosphorylation sites on the C terminus (residues 440 and 492) of delta Ndrk1 resulted in a loss of function of the expressed channels. These results indicate that phosphorylation via protein kinase A modulates drk1 channel function and that both consensus phosphorylation sites seems to be essential for channels to function.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Fingerprinting G-protein-coupled receptors.

Recently we reported the design of a discriminating fingerprint for rhodopsin-like G-protein-coupled receptors (GPCRs). The fingerprint encodes the seven putative membrane-spanning motifs and was potently diagnostic of all GPCRs (52 in all) in version 8.1 of the OWL composite sequence database, readily distinguishing them from all other integral membrane proteins. With a 3-fold increase in the size of OWL, the fingerprint has been updated and now finds 332 receptors that match all the motifs. The situation, however, has grown in complexity: 61 sequences make imperfect matches with the fingerprint, yielding a total of 393 'hits'. The bulk of the partial hits are olfactory receptors: these appear to fall into discrete subfamilies in which one or more of the transmembrane motifs are either poorly matched or are not matched at all. These results are supported by preliminary phylogenetic analyses, which show the olfactory and various other partial matches clustering away from the main body of true hits. The approach has provided a powerful diagnostic tool for identifying GPCRs, and results are consistent with previous observations that the pheromone, cAMP and secretin-like receptors belong to separate families--these bear their own unique sequence fingerprints by which they may be distinguished from the rhodopsin-like superfamily.

Amino Acid Sequence↗