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J B Findlay

Publications and source records attributed to J B Findlay.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1994

Evidence that the 16 kDa proteolipid (subunit c) of the vacuolar H(+)-ATPase and ductin from gap junctions are the same polypeptide in Drosophila and Manduca: molecular cloning of the Vha16k gene from Drosophila.

The 16 kDa proteolipid (subunit c) of the eukaryotic vacuolar H(+)-ATPase (V-ATPase) is closely related to the ductin polypeptide that forms the connexon channel of gap junctions in the crustacean Nephrops norvegicus. Here we show that the major protein component of Manduca sexta gap junction preparations is a 16 kDa polypeptide whose N-terminal sequence is homologous to ductin and is identical to the deduced sequence of a previously cloned cDNA from Manduca (Dow et al., Gene, 122, 355-360, 1992). We also show that a Drosophila melanogaster cDNA, highly homologous to the Manduca cDNA, can rescue Saccharomyces cerevisiae, defective in V-ATPase function, in which the corresponding yeast gene, VMA3, has been inactivated. Evidence is presented for a single genetic locus (Vha16) in Drosophila, which in adults at least contains a single transcriptional unit. Taken together, the data suggest that in Drosophila and Manduca, the same polypeptide is both the proteolipid subunit c component of the V-ATPase and the ductin component of gap junctions. The intron/exon structure of the Drosophila Vha16 is identical to that of a human Vha16 gene, and is consistent with an ancient duplication of an 8 kDa domain. A pilot study for gene inactivation shows that transposable P-elements can be easily inserted into the Drosophila ductin Vha16 gene. Although without phenotypic consequences, these can serve as a starting point for generation of null alleles.

Amino Acid Sequence↗
PubMed1994

The evolution and structure of aminergic G protein-coupled receptors.

An analysis of aligned sequences of the seven transmembrane segments of 59 aminergic G protein-coupled receptors (GPCRs) shows that their classification into subclasses based upon the nature of their ligands is over-simplified. Sequences similarity dendrograms indicate that the evolution of these receptors occurs at two sites (the G protein-coupling regions and the ligand binding site) and this results in convergent as well as divergent evolution. Amino acid substitution patterns at each position in the alignment discriminate between the internal residues and those that face the lipid and the periodicity in these patterns matches that of the classical alpha-helix. The point at which the fifth transmembrane helix protrudes into the cytoplasm and the point at which the seventh transmembrane helix protrudes on the extracellular side of the bilayer are detected. The positions of charged residues on the external face of the remaining helices are used to determine the point at which they enter and leave the bilayer. These data are used to construct three-dimensional models of GPCRs, which are based on the recently determined arrangement of the helices of bovine rhodopsin rather than on the structure of bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1993

Expression of functional human retinol-binding protein in Escherichia coli using a secretion vector.

In order to express human serum retinol-binding protein (sRBP) in Escherichia coli in a form that is structurally indistinguishable from the native protein, we placed the coding sequence of the RBP cDNA next to that of the outer membrane protein A (OmpA) signal sequence in the secretion vector, pIN-III-OmpA1. However, this construct did not generate detectable expression of RBP in E. coli. When the DNA fragment consisting of the ribosome-binding site and the OmpA-RBP fusion sequence was subcloned downstream to the T7 promoter of pKS-Bluescript, however, the resultant construct (pOmp-RBP2) gave low but detectable secretion of RBP into the periplasm. Deletion of the 3' untranslated region of the RBP cDNA (pOmp-RBP3) further improved the expression (by approx. 20-fold). After charging with retinol, the secreted RBP was purified from the periplasm on a transthyretin-affinity resin. The purified protein exhibited all the three molecular recognition properties characteristic of sRBP, i.e. it interacted with retinol, transthyretin and its cell-surface receptor. Comparison of the receptor binding properties of the recombinant RBP (rRBP) with those of the serum protein revealed that while the affinity of rRBP is similar to sRBP (50 +/- 20 nM), the Bmax of the rRBP is about 6-8-fold higher. This indicates that a major proportion of RBP, isolated from serum, is incapable of interacting with the receptor.

Autoradiography↗
PubMed1993

The molecular cloning of the squid (Loligo forbesi) visual Gq-alpha subunit and its expression in Saccharomyces cerevisiae.

The sequence of the alpha-subunit of the major G-protein from the squid (Loligo forbesi) retina was predicted from its cDNA to be a member of the Gq subclass. The abundance of the squid Gq-alpha in the squid photoreceptor membranes suggests that the protein functions in phototransduction; the sequence of this G-protein is consistent with it mediating the light-dependent activation of a phospholipase C. The squid G-alpha was expressed in the yeast Saccharomyces cerevisiae, where it was unable to replace the function of GPA1, the yeast G-alpha homologue that regulates the mating response, suggesting that Gq-alpha was unable to interact with the endogenous G-beta gamma (STE4-STE18).

Amino Acid Sequence↗
PubMed1993

Rhodopsin mobility, structure, and lipid-protein interaction in squid photoreceptor membranes.

Treatment of outer segment membranes from Loligo forbesi with endoprotease-V8 from Staphylococcus aureus results in cleavage of the C-terminal extension of the squid rhodopsin, with accompanying reduction of the apparent molecular weight from 47,000 to 36,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Negative-stain electron microscopy of the intact membranes shows that small clusters of the rhodopsin C-termini form structures extending from the membrane surface and that these are absent after protease treatment. Fourier transform infrared spectra of the amide I band of the protein indicate that removal of the C-terminal extension increases the relative alpha-helical content of squid rhodopsin to a level comparable to that for bovine rhodopsin in disk membranes, and to an extent which suggests that the alpha-helical structure lies mainly in the M(r) 36,000 (transmembrane) section of the protein. Saturation-transfer electron spin resonance (ESR) spectroscopy of the spin-labeled protein reveals that the rotational diffusion of squid rhodopsin in outer segment membranes that have been extensively washed with urea to remove peripheral proteins is much slower than that of bovine rhodopsin in rod outer segment disk membranes. This reduction in rotational mobility is also found with purified squid rhodopsin reconstituted in egg phosphatidylcholine and in urea-washed outer segment membranes which have been treated with endoprotease-V8 to remove the C-terminal extension of squid rhodopsin. In the latter case, the saturation-transfer ESR spectra are virtually identical to those of the non-proteolyzed membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1993

Evidence for a common structure for a class of membrane channels.

Electron microscopic analysis of gap-junction-like structures isolated from an anthropod (Nephrops norvegicus) and composed of a 16-kDa polypeptide, show the functional unit to be a star-shaped hexamer of protein arranged around a central channel which runs perpendicular to the plane of the membrane. Estimations of the molecular volume carried out on an averaged projection are consistent with a subunit mass of 16-18 kDa. Fourier transform infrared spectroscopy indicates a high alpha-helical content for the protein, supporting secondary-structure predictions of four transmembrane alpha helices/monomer. The averaged projection shows a close resemblance to a hexamer of the 16-kDa protein built on the basis of a four alpha-helical bundle [Finbow, M. E., Eliopoulos, E. E., Jackson, P. J., Keen, J. N., Meagher, L., Thompson, P., Jones, P. C. & Findlay, J. B. C. (1992) Protein Eng. 5, 7-15]. The reconstructed image is also similar to that obtained for gap-junction-like channels isolated from a related arthropod [Homarus americanus; Sikerwar, S. S., Downing, K. H. & Glaeser, R. M. (1991) J. Struct. Biol. 106, 255-263] whose protein content was unknown but which we demonstrate may be composed of a related 16-kDa protein. Previous studies have shown a high sequence identity of the Nephrops 16-kDa protein with the 16-kDa proteolipid subunit c of the vascular H(+)-ATPase, both of which in turn bear similarity to the 8-kDa proteolipid subunit of the F1F0-ATP synthase. Expression of cDNA coding for the Nephrops 16-kDa protein in Saccharomyces cerevisiae, in which the endogenous gene coding for the V-ATPase proteolipid has been inactivated, restores V-ATPase activity and cell growth.

Amino Acid Sequence↗
PubMed1993

Design of a discriminating fingerprint for G-protein-coupled receptors.

A systematic method for designing discriminating protein sequence fingerprints is described. The approach used is iterative, and diagnostic performance is evaluated in terms of the relative abilities of sequences to match with individual elements of the fingerprint. The method allows complete protein folds to be characterized in terms of a number of separate 'features', without the requirement to define specific intervals between them, and is described here with reference to the derivation of a fingerprint for G-protein-coupled receptors: this comprises the seven hydrophobic regions shown by protein chemistry approaches to be membrane-spanning. The fingerprint is potently diagnostic of all sequences of this type in the database in which it was derived (the OWL composite sequence database, version 8.1), and has continued to perform well on subsequent database updates, identifying 240 receptors in OWL17.0. Results are compared with a commonly used pattern template for this class of receptors. The investigation suggests that discriminating power is improved in the fingerprint approach because the recognition of individual features is made mutually conditional. Furthermore, by avoiding the definition of predetermined feature separations, members of protein families possessing all or only part of the fingerprint may be identified.

Algorithms↗
PubMed1993

Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction.

An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.

Automation↗
PubMed1992

Pheromone binding to two rodent urinary proteins revealed by X-ray crystallography.

The principal protein excreted in male rat urine, urinary alpha 2-globulin and the homologous mouse protein, major urinary protein, have been well characterized, although their functions remain unclear. Male rat urine affects the behaviour and sexual response of female rats, leading to the proposal that rodent urinary proteins are responsible for binding pheromones and their subsequent release from drying urine. Urinary alpha 2-globulin is also involved in hyaline droplet nephropathy, an important toxicological syndrome in male rats resulting from exposure to a number of industrial chemicals and characterized by the accumulation of liganded urinary alpha 2-globulin in lysosomes in the kidney, followed by the induction of renal cancer. We now report the three-dimensional structures of mouse major urinary protein (at 2.4 A resolution) and rat urinary alpha 2-globulin (at 2.8 A resolution). The results corroborate the role of these proteins in pheromone transport and elaborate the structural basis of ligand binding.

Alpha-Globulins↗
PubMed1992

The gamma-subunit of the principal G-protein from squid (Loligo forbesi) photoreceptors contains a novel N-terminal sequence.

The squid (Loligo forbesi) visual system presents as accessible a system for study of G-protein mediated signal transduction as the vertebrate rod outer segment with the added advantage that the major G-protein is a member of the Gq-class. Here the cDNA clone encoding the gamma-subunit of this G-protein is reported, thereby completing the molecular cloning of the heterotrimeric G-protein. The deduced protein structure of G-gamma has relatively little sequence identity with known mammalian counterparts particularly in comparison with the relatively high degree found for both the alpha- and beta-subunits of this protein. In particular, the N-terminus of the squid visual G-gamma contains a repetitive, highly charged region, rich in lysine and glutamate, that has no parallel in other G-proteins. The amino acid sequence of a number of peptides derived by chemical cleavage of G-gamma accounted for much of the protein sequence predicted from the cDNA, including the unusual N-terminal region.

Amino Acid Sequence↗
PubMed1992

Homology of a vesicular amine transporter to a gene conferring resistance to 1-methyl-4-phenylpyridinium.

The vesicular amine transporter (VAT) catalyzes transport and storage of catechol and indolamines into subcellular organelles in a wide variety of cells. It plays a central role in neurotransmission and is the primary target for several pharmacological agents. One of the drugs, reserpine, binds very tightly to the transporter and remains bound even after solubilization, a finding that has proven useful for purification of the transporter from bovine adrenal medulla in a fully functional state. The sequences of 26 N-terminal amino acids and of an additional 7-amino acid internal peptide are presented. Antibodies against a synthetic peptide based on the above sequences immunoprecipitate the transporter, confirming the conclusion that the peptide sequence is derived from bovine VAT. To our knowledge, documentation of sequences of vesicular neurotransmitter transporters has not been presented previously. In addition, the sequences obtained are highly homologous to the predicted sequence of a protein from PC12 cells that confers to Chinese hamster ovary cells resistance to 1-methyl-4-phenylpyridinium (MPP+), an agent that causes parkinsonism in model systems, confirming the hypothesis that the protein conferring resistance to MPP+ is a VAT.

1-Methyl-4-phenylpyridinium↗
PubMed1992

A protein homologous to glyceraldehyde-3-phosphate dehydrogenase is induced in the cell wall of a flocculent Kluyveromyces marxianus.

A protein with an apparent molecular weight of 37,000 (p37) is present in very large amounts in the cell wall of Kluyveromyces marxianus, after the induction of flocculation of the yeast. This protein was isolated by preparative gel electrophoresis and its purity checked by SDS-PAGE and reverse-phase HPLC. SDS-PAGE, endoglycosidase-H treatment and peptide sequencing indicated that p37 is a glycoprotein with a high identity to cytosolic glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae. Polyclonal antibodies were used for Western blot analysis and immunocytochemistry, which showed that p37 is present in the cell wall of non-flocculent K. marxianus and, therefore, is a constitutive protein of the cell wall.

Amino Acid Sequence↗