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P Whiting

Publications and source records attributed to P Whiting.

At least 73 records · Page 4Linked to original sources
PubMed1991

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure.

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.

Animals↗
PubMed1991

Expression of angiotensin converting enzyme mRNA in rat brain.

Using molecular genetic approaches, two forms of angiotensin-converting enzyme (ACE) have so far been identified; a pulmonary form and a testicular form. This study utilized both northern blot analysis and in-situ hybridization to examine the expression of ACE mRNA in the rat brain. Northern blot analysis using oligonucleotide probes specific to the testicular or pulmonary forms of ACE demonstrated expression of a 4-5kb mRNA doublet in the rat brain, identical in size to pulmonary ACE mRNA. No expression of the testicular form of ACE mRNA was detected in the brain. In situ hybridization demonstrated that ACE mRNA was expressed in the choroid plexus, caudate putamen and cerebellum. A weak signal was also observed in the hippocampus. This distribution correlated well with the expression of ACE mapped by radioligand binding and autoradiography.

Amino Acid Sequence↗
PubMed1991

GABAA receptor subtypes immunopurified from rat brain with alpha subunit-specific antibodies have unique pharmacological properties.

The unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor were expressed in bacterial and used to produce subunit-specific polyclonal antisera. Antibodies immobilized on protein A-Sepharose were used to isolate naturally occurring alpha-specific populations of GABAA receptors from rat brain that retained the ability to bind [3H]muscimol, [3H]flunitrazepam, [3H]Ro15-1788, and [125I]iodo-clonazepam with high affinity. Pharmacological characterization of these subtypes revealed marked differences between the isolated receptor populations and was generally in agreement with the reported pharmacological profiles of GABAA receptors in cells transiently transfected with alpha 1 beta 1 gamma 2, alpha 2 beta 1 gamma 2, alpha 3 beta 1 gamma 2, and alpha 5 beta 1 gamma 2 combinations of subunits. Additional subtypes were also identified that bind [3H]muscimol but do not bind benzodiazepines with high affinity. The majority of GABAA receptor oligomers contains only a single type of alpha subunit, and we conclude that alpha 1, alpha 2, alpha 3, and alpha 5 subunits exist in vivo in combination with the beta subunit and gamma 2 subunit.

Animals↗
PubMed1991

Structural and pharmacological characterization of the major brain nicotinic acetylcholine receptor subtype stably expressed in mouse fibroblasts.

Previously, we purified the predominant subtype of brain nicotinic acetylcholine receptor (AChR), analyzed its structure, and found that it was composed of two kinds of subunit, with sequences encoded by cDNAs termed alpha 4 and beta 2. Here we express these cDNAs from chicken brain in stably transfected fibroblasts. We demonstrate by synthesis that these cDNAs encode subunit polypeptides of the expected sizes, which coassemble to form receptor macromolecules having the same size as native AChRs. Additionally, we demonstrate that the expressed AChRs exhibit the ligand-binding pharmacology of native brain AChRs and function as acetylcholine-gated ion channels.

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PubMed1990

Limited proteolysis and 'in vitro' mutagenesis of bovine brain inositol monophosphatase identifies an N-terminal region important for activity.

Bovine brain inositol monophosphatase is rapidly cleaved by endoprotease lys-C at a single site in the absence of SDS. Further sites are revealed only after prolonged incubation with high concentrations of protease. The initial cleavage occurs near one end of the enzyme, generating an N-terminally-derived 36-residue peptide, which is blocked, and a large 28 kDa fragment bearing a free N-terminus. The start sequence of this fragment was found to be Xaa-Ser-Pro-Ala-Asp-Leu-Val, consistent with the cDNA sequence, and Lys-36-Ser-37 was identified as the cleavage site. The activity of the cleaved enzyme was markedly decreased to 3% of that of the native enzyme, although its dimeric structure was preserved. The 36-residue peptide was not covalently associated with the large fragment after proteolytic cleavage, although the possibility of non-covalent association could not be excluded. Finally, the epitope for the inhibitory monoclonal antibody G-2A4 [Gee, Howell, Ryan & Ragan (1989) Biochem J. 264. 793-798] was found to lie proximal to the endoprotease lys-C cleavage site. In vitro mutagenesis further mapped the epitope for monoclonal antibody G-2A4 to residues around Cys-8 of the enzyme. These results suggest that the N-terminal region of the enzyme is important for activity.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of bovine brain inositol monophosphatase.

Inositol monophosphatase is a key enzyme of the inositol phosphate second messenger signaling pathway. It is responsible for the provision of inositol required for synthesis of phosphatidylinositol and polyphosphoinositides and has been implicated as the pharmacological target for lithium action in brain. Using oligonucleotide probes based on partial amino acid sequence data for the bovine brain enzyme, several overlapping cDNA clones of 2-3 kilobases in length have been isolated. All contain an open reading frame encoding a 277-amino acid protein. No significant sequence homology was found with any known protein. The open reading frame was inserted into a bacterial expression vector in order to confirm the presumed identity of the protein. The expressed protein reacted with an anti-inositol monophosphatase monoclonal antibody. In addition, the protein was enzymically active and indistinguishable from the bovine brain enzyme with respect to Km values for substrate and Li+ sensitivity of inositol 1-phosphate hydrolysis.

Amino Acid Sequence↗
PubMed1990

Structural and functional heterogeneity of nicotinic receptors.

Three gene families of the ligand-gated ion channel gene superfamily encode proteins which bind cholinergic ligands: (1) nicotinic acetylcholine receptors (AChRs) from skeletal muscle, (2) AChRs from neurons, and (3) neuronal alpha-bungarotoxin-binding proteins (alpha BgtBPs). AChRs from muscles and nerves function as ACh-gated cation channels, but alpha BgtBPs do not appear to function in this way. A family of neuronal AChR subtypes has been characterized using monoclonal antibodies and cDNA probes. Neuronal AChRs exhibit sequence homologies with muscle AChRs, but differ in subunit composition, pharmacological and electrophysiological properties, and, in some cases, apparent functional roles. The genes that encode the subunits of the various purified AChR subtypes have been determined in several cases. Histological localization of AChR subunit mRNAs by in situ hybridization and of subunit proteins by immunohistochemistry is being conducted with increasing resolution. The subunit structure of alpha BgtBP is uncertain, but cDNAs have been identified for two subunits. Sequences of these cDNAs reveal that alpha BgtBPs are members of the ligand-gated ion channel gene family, and suggest that they could function as gated cation channels. Biochemical and molecular genetic approaches to studies of neuronal AChRs and related proteins are merging to provide a detailed description of a complex family of AChRs widely dispersed throughout the nervous system, which are probably important to many activities of the nervous system, but whose functional roles are not yet well characterized.

Animals↗
PubMed1990

Brain alpha-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily.

alpha-Bungarotoxin (alpha Bgt) is a potent, high-affinity antagonist for nicotinic acetylcholine receptors (AChRs) from muscle, but not for AChRs from neurons. Both muscle and neuronal AChRs are thought to be formed from multiple homologous subunits aligned around a central cation channel whose opening is regulated by ACh binding. In contrast, the exact structure and function of high-affinity alpha Bgt binding proteins (alpha BgtBPs) found in avian and mammalian neurons remain unknown. Here we show that cDNA clones encoding alpha BgtBP alpha 1 and alpha 2 subunits define alpha BgtBPs as members of a gene family within the ligand-gated ion channel gene superfamily, but distinct from the gene families of AChRs from muscles and nerves. Subunit-specific monoclonal antibodies raised against bacterially expressed alpha BgtBP alpha 1 and alpha 2 subunit fragments reveal the existence of at least two different alpha BgtBP subtypes in embryonic day 18 chicken brains. More than 75% of all alpha BgtBPs have the alpha 1 subunit, but no alpha 2 subunit, and a minor alpha BgtBP subtype (approximately 15%) has both the alpha 1 and alpha 2 subunits.

Amino Acid Sequence↗
PubMed1990

Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of gamma 2 phosphorylation site.

Diversity of gamma-aminobutyrate type A (GABAA) receptors has recently been proposed to be achieved by assembly of receptor subtypes from a multitude of subunits (alpha 1-6, beta 1-3, gamma 1-2, and delta) encoded by different genes. Here we report a further mechanism for creating GABAA receptor diversity: alternative RNA splicing. Two forms of bovine gamma 2 subunit cDNA were isolated (gamma 2S and gamma 2L) that differed by the presence or absence of a 24-base-pair (8-amino acid) insertion in the cytoplasmic domain between the third and fourth putative membrane-spanning regions. Polymerase chain reaction from RNA demonstrated that the two forms of gamma 2 subunit are expressed in bovine, human, and rat brain. Sequencing of genomic DNA clones encoding the gamma 2 subunit demonstrated that the 24-base-pair insert is organized as a separate exon. Analysis of the sequence of the 8-amino acid insert revealed that it contains a protein kinase C consensus phosphorylation site. Expression of the large cytoplasmic loop domains of gamma 2S and gamma 2L in Escherichia coli, followed by phosphorylation of the recombinant proteins by protein kinase C, demonstrated that gamma 2L, but not gamma 2S, could be phosphorylated. Thus the two forms of gamma 2 subunit differ by the presence or absence of a protein kinase C phosphorylation site. This mechanism for creating GABAA receptor diversity may allow differential regulation of the function of receptor subtypes.

Amino Acid Sequence↗
PubMed1989

Antisera against an acetylcholine receptor alpha 3 fusion protein bind to ganglionic but not to brain nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptor (AChR) subtypes have been defined pharmacologically, immunologically, and by DNA cloning, but the correlations between these approaches are incomplete. Vertebrate neuronal AChRs that have been isolated are composed of structural subunits and ACh-binding subunits. A single kind of subunit can be used in more than one AChR subtype. Monoclonal antibody (mAb) 35 binds to structural subunits of subtypes of AChRs from both chicken brain and ganglia. By using antisera to a unique sequence of alpha 3 ACh-binding subunits expressed in bacteria, we show that ganglionic AChRs contain alpha 3 ACh-binding subunits, whereas the brain AChR subtype that binds mAb 35 does not. Subunit-specific antisera raised against recombinant proteins should be a valuable approach for identifying the subunit composition of receptors in multigene, multisubunit families.

Amino Acid Sequence↗
PubMed1989

A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671.

The human neuromedulloblastoma cell line TE671 is shown by single-channel recordings to express nicotinic acetylcholine receptors (AChRs) that are blocked by alpha-bungarotoxin (alpha Bgt). These AChRs do not react with antisera to the alpha Bgt-binding protein of brain or with monoclonal antibodies (mAbs) to brain nicotinic AChRs that do not bind alpha Bgt. TE671 AChRs do react with autoantibodies to muscle AChRs from myasthenia gravis patients and with mAbs to muscle AChRs, including mAbs specific for extrajunctional AChRs. AChRs. AChRs purified from TE671 cells are composed of 4 kinds of subunits corresponding to those of muscle AChR. Sequences of cDNAs for the ACh-binding alpha subunit and the delta subunit of this AChR further identify it as muscle AChR. Expression of TE671 AChR can be up-regulated by nicotine and dexamethasone, and down-regulated by forskolin.

Amino Acid Sequence↗
PubMed1988

Autoradiographic localization of nicotinic acetylcholine receptors in the brain of the zebra finch (Poephila guttata).

We have localized nicotinic acetylcholine receptors in the zebra finch brain by using three 125I-labelled ligands: alpha bungarotoxin and two monoclonal antibodies to neuronal nicotinic receptors (MAb 35 of Tzardos et al., J. Biol. Chem., 250: 8635-8645, '81; and MAb 270 of Whiting and Lindstrom: J. Neurosci. 6: 3061-3069, '86). Unfixed brains from intact adult male and female zebra finches were prepared for in vitro autoradiography. Low-resolution film autoradiograms and high-resolution emulsion autoradiograms were prepared for each of the three ligands. The major brain structures that bind all three of the ligands are hippocampus; hyperstriatum dorsalis; hyperstriatum ventralis; nucleus lentiformis mesencephali; nucleus pretectalis, some layers of the optic tectum; nucleus mesencephalicus lateralis; pars dorsalis; locus ceruleus; and all cranial motor nuclei except nucleus nervi hypoglossi. The major structures labelled only by [125I]-alpha bungarotoxin binding included hyperstriatum accessorium and the nuclei: preopticus medialis, medialis hypothalami posterioris, semilunaris, olivarius inferior, and the periventricular organ. Of the song control nuclei, nucleus magnocellularis of the anterior neostriatum; hyperstriatum ventralis, pars caudalis; nucleus intercollicularis; and nucleus hypoglossus were labelled. The binding patterns of the two antibodies were similar to one another but not identical. Both labelled nucleus spiriformis lateralis and nucleus geniculatus lateralis, pars ventralis especially heavily and also labelled the nucleus habenula medialis; nucleus subpretectalis; nucleus isthmi, pars magnocellularis; nucleus reticularis gigantocellularis; nucleus reticularis lateralis; nucleus tractus solitarii; nucleus vestibularis dorsolateralis; nucleus vestibularis lateralis; nucleus descendens nervi trigemini; and the deep cerebellar nuclei. Lobus parolfactorius and nucleus vestibularis medialis were labelled by only MAb 270, whereas only MAb 35 labelled nucleus laminaris and the medial and lateral pontine nuclei. These data extend previous reports of cholinergic participation in the song system (Ryan and Arnold: J. Comp. Neurol. 202: 211-219, '81) to suggest that the zebra finch song system may contain several closely related nicotinic receptors. In several brain nuclei it appeared that certain anatomical portions of a nucleus or a certain class of neurons were specifically labelled. Furthermore, in certain cases, the labelling appeared to be clustered around Nissl-stained cell nuclei, thus suggesting that the receptors are concentrated on or in somata.

Animals↗
PubMed1988

Formation of apolar ecdysteroid conjugates by ovaries of the house cricket Acheta domesticus in vitro.

The newly laid eggs of the house cricket Acheta domesticus contain apolar ecdysteroid conjugates, which we have hypothesized to be ecdysone long-chain fatty acyl esters [Whiting & Dinan (1988) J. Insect Physiol., in the press]. The ovaries of mature adult female A. domesticus in vitro convert [3H]ecdysone into apolar conjugates identical with those found in newly laid eggs. Comparison of the radioactive metabolites produced on incubation of [3H]ecdysone with various organs of adult female A. domesticus in vitro indicate that the fat-body is the major producer of polar ecdysteroid metabolites at this stage of development, whereas the ovaries are the major site of production of apolar metabolites. Apolar metabolites are also produced to a lesser extent by the crop, gut sections and the fat-body. Hydrolysis of radioactive metabolites produced by the ovaries with Helix enzymes releases only [3H]ecdysone, and thus ecdysone is not metabolized before conjugation by the ovaries. Formation of chemical derivatives (acetonide and acetates) of these 3H-labelled apolar conjugates strongly indicates that the position of conjugation is through the hydroxy group at C-22 of ecdysone. Extensive chromatographic analysis of the 3H-labelled apolar metabolites produced by the ovaries by t.l.c. and h.p.l.c. and comparison with authenticated reference compounds have conclusively demonstrated that the conjugates consist of ecdysone esterified at C-22 to a mixture of common long-chain fatty acids. The major fatty acyl esters have been identified and their percentage contribution to the mixture determined: laurate (0.5%), myristate (2.8%), palmitate (25.8%), stearate (8.4%), arachidate (1.0%), oleate (15.7%), linoleate (38.8%) and linolenate (2.1%). In addition there are three minor unidentified peaks, one of which has been tentatively identified as ecdysone 22-palmitoleate (2.6%). Comparison of this percentage composition with the previously published fatty acid composition of A. domesticus haemolymph [Wang & Patton (1969) J. Insect Physiol. 15, 851-860] reveals remarkable similarities, indicating that the acyl transferase(s) forming the conjugates have a broad specificity with regard to the fatty acyl substrate.

Animals↗
PubMed1988

cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors (AChRs) immunoaffinity-purified from brains are composed of only two kinds of subunits rather than the four kinds present in muscle-type AChRs. Here we report the N-terminal protein sequences of the structural subunits of AChRs from rat and chicken brains and the cloning of full-length cDNAs for the chicken brain AChR structural subunit. Previously, the N-terminal amino acid sequence of the ACh-binding subunit of AChR immunoaffinity-purified from rat brain was shown to correspond to the cDNA alpha 4. Thus, cDNA sequences are now known for both of the subunits that form one AChR subtype in vivo.

Amino Acid Sequence↗
PubMed1988

Action of antibodies directed against the acetylcholine receptor on channel function at mouse and rat motor end-plates.

1. The acute effects of antibodies (both polyclonal and monoclonal) raised against the acetylcholine receptor were studied at mouse and rat end-plates. Isolated muscles were incubated in solutions containing antibody for 2 1/4 to 3 1/2 h. Intracellular microelectrode techniques were then used to record miniature end-plate potentials (MEPPs) and voltage noise. 2. Most antibody preparations investigated did not reduce MEPP amplitudes as compared with controls. One monoclonal (C7) and one polyclonal (J) preparation irreversibly reduced MEPP amplitudes. Both preparations caused reductions in acetylcholine-induced depolarization and associated channel opening frequency (from voltage noise analysis). Single-channel depolarization was not altered by these antibodies. 3. On the basis of these and previous results, four antibody binding regions on the receptor surface were distinguished according to whether channel function and/or alpha-bungarotoxin binding were affected. Although most antibody preparations did not affect channel function, monoclonal antibody C7 appeared to alter function by acting on the channel itself so as to prevent channel opening.

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