Robert G. L. Shorr

Characterization of a digitonin-solubilized bovine brain H3 histamine receptor coupled to a guanine nucleotide-binding protein.

Abstract: The H3 receptor is a high‐affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD= 4.6 nM; Bmax= 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15–30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD= 6.1 nM; Bmax= 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size‐exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide‐binding protein.

Kinetics of cysteine oxidation in immobilized pH gradient gels

Abstract It was recently found that, during the gel polymerization step, persulphate could oxidize the alkaline Immobiline species (acryloyl weak bases used for producting immobilized pH gradients, IPG) by forming N-oxides. When focusing proteins in alkaline pH ranges, free SH groups were oxidized to SS bonds, generating artefactual, higher pI bands. The kinetics of this phenomenon were investigated in a model system consisting in incubating free Cys with a crushed IPG gel auto-buffered at pH 9.0 under aerobic or anaerobic conditions. After appropriate incubation periods, any excess free SH was blocked with acrylonitrile. After dansylation, Cys and its oxidized derivatives were separated and quantified by high-performance liquid chromatography in a C18 column. Under anaerobic conditions, control Cys (incubated in the absence of IPG gel) showed 30% oxidation after a period of 12 h, whereas Cys incubated with a pH 9.0 crushed IPG gel was 100% oxidized to cystine. When the same experiment was repeated under aerobic conditions, the oxidation process was accelerated by a factor of 3–4. Successful focusing of alkaline proteins can still be achieved if (a) the IPG gel is washed in 100 mM ascorbic acid, prior to the run, for elimination of N-oxides from the matrix, (b) the IPG gel is run anaerobically and (c) the focusing time is kept to a minimum (e.g., 4 h vs. the standard overnight run).

Fast affinity chromatography using small particle silica-based packing materials

Summary Affinity chromatography is one of the most powerful techniques for the purification of biologically active proteins available (for review see [1]). The ability of this method to purify proteins is based on highly specific, selective or characteristic interactions with immobilized ligands. Several advantages over traditional soft gel affinity supports have been observed with the use of small particle silica based materials for high performance affinity chromatography. These include greatly improved mass transfer properties which allow separations that are not always practical in the low performance mode, greatly reduced equilibration and isolation times, high available ligand densities, small elution volumes, excellent recovery of very small quantities of protein and high dynamic capacities. The criteria for developing a general, derivatizable, high performance support for high performance affinity chromatography are discussed. The step-by-step examination of these criteria and experimental evidenc...

Human Liver Cytochrome P-450 Related to a Rat Acetone-Inducible, Nitrosamine-Metabolizing Cytochrome P-450: Identification and Isolation

A monoclonal antibody (MAb) to a rat acetone-inducible and nitrosamine-metabolizing form of microsomal cytochrome P-450, P-450ac, detected a related P-450 in human liver microsomes by both immunoblot and competitive radioimmunoassay. This MAb was also used to immunopurify microsomal cytochromes P-450 from both human liver and acetone-treated rats; these were electrophoretically homogeneous with apparent molecular weights of 56,200 and 53,000 daltons, respectively. The structures of the cytochromes P-450 were compared by peptide mapping and amino-terminal sequence analyses. They differed in their peptide maps but displayed amino-terminal sequence similarity in their first 19 residues. This report thus demonstrates the utility of MAbs to rat cytochromes P-450 for detection, identification and structural characterization of human P-450s.

Selective purification of cardiac myosin by a high-performance salicylate affinity column

The cardiac muscle proteins, myosin and actin, were purified in one step using a salicylate-silica affinity column. The affinity columns were prepared by coupling sodium salicylate via its hydroxyl group to an Altex Ultraffinity-EP column. Crude detergent extracts from guinea pig hearts were passed through the column and the myosin-actin complex was then eluted with excess free salicylate or high salt. The affinity of cardiac myosin for immobilized salicylate was unique as myosin heavy chain from guinea pig leg muscle detergent extracts could not be purified by this procedure. Commercially purified rabbit leg muscle myosin also appeared to have no interaction with the salicylate affinity column, suggesting that the column is specific for cardiac myosin.

Interactions of β-adrenergic receptors with a membrane protein other than the stimulatory guanine nucleotide-binding protein

Beta-adrenergic receptors on membranes prepared from rat lung, wild-type S49 lymphoma cells, and the adenylate cyclase-deficient variant of S49 lymphoma cells (cyc-) bind the agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) with high affinity and this binding of [3H]HBI can be inhibited by GTP. Membranes prepared from these tissues were incubated with the agonist [3H]HBI or the antagonist [125I]iodopindolol ([125I]IPIN), labeled receptors were solubilized with digitonin, and the apparent molecular sizes of the ligand-bound receptor complexes were determined by high-performance size-exclusion chromatography. Results with all three tissues demonstrated that receptors labeled with [125I]IPIN were retained by the size-exclusion columns longer than receptors labeled with [3H]HBI. Thus, the apparent molecular size of soluble beta-adrenergic receptors from rat lung, wild-type S49 cells, and cyc- S49 cells was larger when receptors were occupied with an agonist rather than an antagonist. The results suggest that receptors, including those on cyc- S49 cells, interact with a membrane protein, presumably a guanine nucleotide-binding protein. Since cyc- S49 cells do not contain a functional stimulatory guanine nucleotide-binding protein, but do contain a functional inhibitory guanine nucleotide-binding protein, an interaction between beta-adrenergic receptors and the inhibitory guanine nucleotide-binding protein may be responsible for the apparent increase in the molecular size of the receptor after occupation of the receptor with an agonist.

Evidence for tyrosine at the ligand binding center of beta-adrenergic receptors

Beta 1- and beta 2-adrenergic receptor radioligand antagonist binding activities in plasma membrane preparations from mammalian lung, as well as amphibian and avian red blood cells, have been shown to be inactivated by agents which specifically alkylate tyrosine. In membrane preparations, protection against inactivation was afforded by both agonists and antagonists. In soluble purified preparations, antagonists but not agonists protected against inactivation. These results suggest that tyrosine is located at or near the ligand binding site of both beta 1- and beta 2-adrenergic receptors.

Rapid Generation of Peptides for N-Terminal Sequence Analysis Using Immobilized Pronase

The primary structure of proteins can often be determined by successive enzymatic and/or chemical digestion procedures that generate peptides yielding overlapping sequence data. Such methodology is time-consuming, labor intensive and subject to sample loss due to handling. We have developed a fast and simple protein fragmentation strategy which will generate overlapping peptides and eliminates much sample handling.