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J C Venter

Publications and source records attributed to J C Venter.

At least 109 records · Page 6Linked to original sources
PubMed1987

Structural analysis of purified beta-adrenergic receptors.

We have characterized the structure of purified beta-adrenergic receptors by a combination of photoaffinity labeling, high performance liquid chromatography (HPLC)-tryptic mapping, CNBr fragmentation, target size analysis, and electron microscopy of purified receptor molecules. Guinea pig lung beta-adrenergic receptors purified by affinity chromatography, ion exchange chromatography, and HPLC size exclusion chromatography or photoaffinity labeled with [125]-iodocyanopindolol diazirine displayed mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that corresponded to Mr = 68,000. Purified, radioiodinated guinea pig lung beta-receptors were subjected to complete trypsin digestion and subsequent reverse-phase HPLC analysis, which revealed nine peptides. Active site labeling and tryptic digestion of partially purified hamster lung beta-receptors produced one peptide, whereas CNBr digestion of the same material produced two labeled fragments, yielding information about the location of the active site within the primary sequence. Purified guinea pig lung receptors were examined with transmission electron microscopy. Electron micrographs revealed slightly asymmetric, rod-shaped structures with an average length of 13 nm and width of 3.4 nm. Many receptors were arranged as apparent dimeric structures. These findings confirm data obtained from target size analysis of guinea pig lung beta-receptors in situ which suggest that receptors may exist as oligomeric arrays in the native membrane. Taken together, these data provide information about putative functional domains of the beta-adrenergic receptor and its quaternary structure.

Amino Acid Sequence↗
PubMed1987

Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.

Amino Acid Sequence↗
PubMed1987

The structure and evolution of adrenergic and muscarinic cholinergic receptors.

We have studied the molecular structures of the neurotransmitter receptors of the autonomic nervous system (alpha- and beta-adrenergic and muscarinic cholinergic receptors) using biochemical and immunological techniques. Each of these receptors has been purified and their molecular characteristics including molecular mass, isoelectric point, membrane topology, and quaternary structure have been determined. Limited proteolysis has identified structural and functional domains while complete digestion and peptide mapping by high performance liquid chromatography (HPLC) have allowed comparison of the various receptors. Purification and characterization of these neurotransmitter receptors have revealed considerable structural and functional homology between these pharmacologically distinct classes of information transduction molecules.

Animals↗
PubMed1986

Target size of the adenosine Ri receptor.

The adenosine receptor of rat cerebral-cortical membranes was examined by radiation inactivation. In control membranes the receptor is distributed between high- and low-affinity states, that can be preferentially expressed by Mg2+ ions and guanine nucleotides respectively. Upon exposure to increasing doses of radiation, the high-affinity receptor decayed linearly as a function of radiation dose. This decay rate corresponded to a target size of 63,000 Da, when compared with the decay of the muscarinic cholinergic receptor that was also measured in these membranes.

Animals↗
PubMed1986

Alpha and beta adrenergic and muscarinic cholinergic receptor structure.

Purification and characterization of the neurotransmitter receptors of the autonomic nervous system have revealed considerable structural and functional homology between these pharmacologically distinct classes of information transduction molecules. Alpha 1- and alpha 2-adrenergic receptors are single polypeptides with molecular mass 85,000 Da and pI 4.6. Beta 1- and beta 2-adrenergic receptors are single polypeptides with molecular mass 68,000 Da and pI 5.0. Muscarinic cholinergic receptors from a variety of tissues and species are single polypeptides with molecular mass 80,000 Da and pI 4.2. Proteolytic digestion and analysis of affinity-labelled adrenergic and cholinergic receptors indicates a striking similarity in the number and sizes of peptides produced. Topographical analysis of the receptors has shown that they have a similar membrane orientation with more than half of the protein exposed to the extracellular environment. Peptide-mapping studies of soluble and membrane-bound receptors suggest that the ligand-binding domain of adrenergic and cholinergic receptors is localized near the end of the protein that is exposed to the extracellular environment. The marked similarity between alpha- and beta-adrenergic and muscarinic cholinergic receptor structure is perhaps not unexpected in light of the fact that these receptors interact with the same transmitters (in the case of alpha- and beta-adrenergic receptors) and/or with the same effector proteins in the membrane (stimulatory and inhibitory guanine nucleotide regulatory proteins, ion channels). Yet, depending on the tissue distribution of receptors and their effectors, this limited number of proteins can modulate dramatically different physiological effects. It may be that the differences in pharmacological specificity of ligand binding and the differences in receptor-effector interactions observed among adrenergic receptor subtypes and muscarinic cholinergic receptors are due to minor structural differences within the active sites of these proteins. Obviously, the real answers as to the extent of structural homology between the receptor classes will be derived from the amino acid sequencing of the purified proteins or from the cloning of the receptor genes and recently the genes coding for the beta-adrenergic receptor have been cloned and sequenced from human brain, hamster lung, and turkey erythrocytes. Comparison of the derived protein sequences reveals a high degree of structural homology between the avian and mammalian receptors with approximately 50% primary sequence identity and highly conserved secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

A sulfhydryl group of the canine cardiac beta-adrenergic receptor observed in the absence of hormone.

Canine cardiac beta-adrenergic receptors contain a free sulfhydryl group in the adrenergic ligand binding site. [125 I]-Iodohydroxybenzylpindolol [( 125 I]-IHYP) binding to cardiac beta-receptors was inhibited 80% by treatment with 1 mM p-chloromercuribenzoic acid (pCMB). Occupation of the beta-receptors by an antagonist prior to treatment with pCMB prevented this effect suggesting that a sulfhydryl group is present in or near the ligand binding site of the cardiac beta-receptor. In the presence of agonists, the sensitivity of cardiac beta-receptors to pCMB was increased. Incubation of isoproterenol-occupied cardiac beta-receptors, resulted in a 57% inhibition of [125 I]-IHYP binding measured after extensive washing to remove bound agonist. The ability of isoproterenol to increase the reactivity of cardiac beta-adrenergic receptors supports the hypothesis that agonists produce a conformational change upon binding.

Adrenergic beta-Agonists↗
PubMed1985

Molecular comparison of alpha 1- and alpha 2-adrenergic receptors suggests that these proteins are structurally related "isoreceptors".

The structures of human platelet alpha 2-adrenergic receptors and rat liver alpha 1-adrenergic receptors were compared by utilizing isoelectric focusing, NaDodSO4/PAGE, and monoclonal antibody crossreactivity. Digitonin-solubilized alpha 1- and alpha 2-adrenergic receptors have an identical isoelectric point of 4.6. Under reducing conditions in NaDodSO4/polyacrylamide gels, the alpha 1-adrenergic receptor has an apparent molecular mass of 85 kDa. Similarly, the alpha 2-adrenergic receptor, which had been affinity-labeled with [3H]phenoxybenzamine and partially purified by isoelectric focusing or photoaffinity-labeled with p-[3,5-3H]azidoclonidine, was also found to have an apparent molecular mass of 85 kDa. One hybridoma, developed from a fusion between SP2/O myeloma cells and splenic lymphocytes from BALB/c mice immunized with human platelet alpha 2-adrenergic receptors, secreted a monoclonal antibody (alpha 2-116p) against the ligand binding site of alpha 2-adrenergic but not alpha 1-adrenergic receptors. In contrast, three monoclonal antibodies raised against the alpha 1-receptor polypeptide backbone but not the ligand binding site were found to specifically immunoprecipitate human platelet alpha 2-adrenergic receptors. These data suggest that the alpha 1- and alpha 2-adrenergic receptors are "isoreceptors," sharing immunogenic and, by implication, structural determinants that most likely evolved as a result of gene duplication.

Adrenergic alpha-Agonists↗
PubMed1985

Radiation inactivation (target size analysis) of the gonadotropin-releasing hormone receptor: evidence for a high molecular weight complex.

In the present study we used radiation inactivation (target size analysis) to measure the functional mol wt of the GnRH receptor while it is still a component of the plasma membrane. This technique is based on the observation that an inverse relationship exists between the dose-dependent inactivation of a macromolecule by ionizing radiation and the size of that macromolecule. This method demonstrates a mol wt of 136,346 +/- 8,120 for the GnRH receptor. This estimate is approximately twice that obtained (60,000) by photoaffinity labeling with a radioactive GnRH analog followed by electrophoresis under denaturing conditions and, accordingly, presents the possibility that the functional receptor consists of a high mol wt complex in its native state. The present studies indicate that the GnRH receptor is either a single weight class of protein or several closely related weight classes, such as might occur due to protein glycosylation.

Animals↗
PubMed1984

Partial functional reconstitution of the cardiac muscarinic cholinergic receptor.

Digitonin-solubilized cardiac muscarinic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack high-affinity muscarinic receptors. The number of receptors reconstituted was proportional to the quantity of soluble receptors added to the reconstitution system. Specific [3H](-)-quinuclidinyl benzilate binding to the reconstituted receptor was found to be saturable with a Kd (dissociation constant) equal to 48 +/- 4 pM and a Bmax (maximal density of binding sites) equal to 50 +/- 5 fmol/mg of protein. Competitive binding studies indicated that the reconstituted receptors showed stereoselectivity and drug specificity consistent with a high-affinity muscarinic receptor. Agonist binding to the reconstituted receptor was decreased by the addition of guanyl-5'-yl imidodiphosphate. Sixty per cent of the reconstituted receptors were found to be integral membrane proteins. The molecular weight of the reconstituted receptor as determined by sodium dodecyl sulfate-gel electrophoresis was 76,000 +/- 2,000 and was identical to the molecular weight of the muscarinic receptor in the original cardiac membranes. The data indicate that a partially functional, intact muscarinic receptor was reconstituted into human erythrocyte acceptor membranes and that membrane constituents may be required to stabilize the receptor in a high-affinity state for antagonists.

Binding, Competitive↗
PubMed1984

Nitrendipine and isoproterenol induce phosphorylation of a 42,000 dalton protein that co-migrates with the affinity labeled calcium channel regulatory subunit.

Slow inward calcium channels in canine cardiac membranes were affinity labeled with the calcium channel analogue, [3H]o-NCS [2,6 dimethyl-3,5-dicarbomethoxy-4-(2- isothiocyanatophenyl )-1, 4-dihydropyridine], in the presence and absence of cold o-NCS or nicardipine. A major specifically labeled peak was identified with Mr 42,000 on NaDodSO4 polyacrylamide gels. In parallel experiments the effects of the calcium channel antagonist, nitrendipine and a variety of other chemical mediators were tested for their ability to stimulate protein phosphorylation in cardiac membranes. These data demonstrate that both nitrendipine and isoproterenol induce the phosphorylation of a 42,000 dalton protein via a kinase endogenous to the cardiac membranes and that the effects of isoproterenol are attenuated by carbachol.

Affinity Labels↗
PubMed1984

The differential loss of [3H]pirenzepine vs [3H] (-) quinuclidinylbenzilate binding to soluble rat brain muscarinic receptors indicates that pirenzepine binds to an allosteric state of the muscarinic receptor.

[3H]Pirenzepine [( 3H]PZ) and [3H] (-)Quinuclidinylbenzilate [( 3H] (-)QNB) specific binding to soluble rat brain muscarinic cholinergic receptors was assessed as a function of time subsequent to receptor solubilization. The soluble brain muscarinic receptor is stable at 4 degrees C when assayed by [3H] (-)QNB binding (t 1/2 = 80 hrs). In contrast the pirenzepine state of the receptor decays rapidly (t 1/2 = 3.0 hrs). Prior occupation of the receptor with [3H] (-)QNB or [3H]PZ increases the receptor stability by two to five fold (t 1/2 QNB greater than 1,000 hrs; t 1/2 PZ = 6.5 hrs). These data indicate that pirenzepine binds to an allosteric state of the muscarinic receptor and that caution should be employed in the assignment of receptor subtypes based solely upon the binding of ligands which recognize unique conformational states.

Allosteric Site↗
PubMed1984

Monoclonal antibodies detect the conservation of muscarinic cholinergic receptor structure from Drosophila to human brain and detect possible structural homology with alpha 1-adrenergic receptors.

Muscarinic cholinergic receptors isolated from Drosophila heads, rat and human brain, dog heart, and monkey ciliary muscle were examined for structural similarities/differences by utilizing isoelectric focusing, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and monoclonal antibody crossreactivity. Muscarinic receptors were affinity labeled with [3H]propylbenzilylcholine mustard and subjected to isoelectric focusing. Muscarinic receptors from each species focused with an isoelectric point of 5.9. The same proteins all migrated with an apparent molecular mass of 80,000 daltons on sodium dodecyl sulfate gels. Six hybridomas secreting monoclonal antibodies specific for muscarinic receptors were developed by using purified rat brain muscarinic receptors as the antigen. The six different monoclonal antibodies immunoprecipitated muscarinic receptors from all tissues and species tested, including human and Drosophila brains, with equal efficacy. These data indicate that muscarinic receptors are highly conserved over a considerable evolutionary period. One of the six muscarinic receptor monoclonal antibodies also immunoprecipitated rat liver alpha 1-adrenergic receptors. Furthermore, two out of five monoclonal antibodies raised against alpha 1-receptors immunoprecipitated muscarinic receptors. These data suggest that some degree of structural homology exists between muscarinic cholinergic receptors and alpha 1-adrenergic receptors.

Animals↗