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Biomedical subjects

J P Earnest

Publications and source records attributed to J P Earnest.

9 recordsLinked to original sources
PubMed1995

Dystrophin-related protein in the platelet membrane skeleton. Integrin-induced change in detergent-insolubility and cleavage by calpain in aggregating platelets.

The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 x g) but the membrane skeleton requires 100,000 x g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin alpha IIb beta 3 induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following alpha IIb beta 3-ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate an approximately 140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.

Amino Acid Sequence↗
PubMed1989

Structure, oligosaccharide structures, and posttranslationally modified sites of the nicotinic acetylcholine receptor.

Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californica contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor. Asparagine-143 of the alpha subunit (in consensus numbering) is shown to be glycosylated with high-mannose oligosaccharide. Asparagine-453 of the gamma subunit is not glycosylated, a fact that bears on the question of the orientations of putative transmembranous helices M3, MA, and M4. The structures of the six major acetylcholine receptor oligosaccharides are determined: the major components (70%) are of the high-mannose type, with bi-, tri-, and tetraantennary complex oligosaccharides making up approximately equal to 22 mol% of the total carbohydrate. This application of a multichannel array detector mass spectrometer provided a breakthrough in sensitivity that allowed us to identify the site of attachment of, and the sequence of, oligosaccharides on a 300-kDa membrane protein from only 5 pmol of the isolated oligosaccharide.

Amino Acid Sequence↗
PubMed1988

Reconstitution of the nicotinic acetylcholine receptor using a lipid substitution technique.

The nicotinic acetylcholine receptor was purified by affinity chromatography in the presence of dioleoylphosphatidylcholine (DOPC). A method for replacing the DOPC with other lipids was developed by using detergent solubilization with a large excess of the new lipid followed by sucrose density gradient centrifugation in detergent-free buffers to separate receptor-lipid complexes from excess lipid and detergent. Homogenous complexes of defined lipid composition could be easily prepared and the efficiency of substitution was independent of lipid type. However, the functional properties of the resulting lipid complexes depended on the lipid composition.

Animals↗
PubMed1988

A minimum number of lipids are required to support the functional properties of the nicotinic acetylcholine receptor.

The detergent sodium cholate was used to both solubilize and partially delipidate the nicotinic acetylcholine receptor from Torpedo californica. Using both native membranes and reconstituted membranes, it is shown that the detergent to lipid molar ratio is the most important parameter in determining the effect of the detergent on the functional properties of the receptor. Receptor-lipid complexes were quantitatively separated from detergent and excess lipids by centrifugation through detergent-free sucrose gradients. The lipid to protein molar ratio of the complexes could be precisely controlled by adjusting the cholate and lipid concentrations of the starting membranes. Analyses of both ion influx activity and ligand binding revealed that a minimum of 45 lipids per receptor was required for stabilization of the receptor in a fully functional state. Progressive irreversible inactivation occurred as the lipid to protein mole ratio was decreased below 45, and complete inactivation occurred below a ratio of 20. The results are consistent with a functional requirement for a single shell of lipids around the perimeter of the receptor.

Allosteric Regulation↗
PubMed1988

Cesium ion liquid secondary ion mass spectrometry of membrane-bound glycoproteins: structural and topological considerations of acetylcholine receptor from Torpedo californica.

We report mass mapping of a large (270 kD) multisubunit membrane bound glycoprotein, nicotinic acetylcholine receptor from Torpedo californica, using enzymic digests of the affinity purified whole receptor and cesium ion liquid secondary ion mass spectrometry. Peptides, glycopeptides and derivatized N-linked oligosaccharides were isolated by HPLC and identified by LSIMS. We have shown that mass spectrometric sensitivity is improved a hundred-fold through use of computer-controlled mass window stepping of an electro-optical multichannel array detection system on a LSIMS double focusing mass spectrometer. This new method permitted determination of the complete fragmentation pattern of Man8N2-ABEE using only 5 picomoles of sample.

Amino Acid Sequence↗
PubMed1986

Binding of local anesthetics to reconstituted acetylcholine receptors: effect of protein surface potential.

Nicotinic acetylcholine receptor isolated from Torpedo californica electric organ is reconstituted into lipid bilayers of zwitterionic dioleoylphosphatidylcholine. These membranes are labeled with a spin-labeled quaternary amine local anesthetic (C6SLMeI), which has been shown previously to be a noncompetitive blocker of acetylcholine receptor-ion channel function in the micromolar concentration range. The electron spin resonance spectral component corresponding to protein-immobilized anesthetic spin-label can be resolved from the composite data spectrum by using spectral subtraction of lipid components. This protein-immobilized component is shown to represent C6SLMeI bound to a finite number of sites on the receptor. We demonstrate that C6SLMeI binds to the receptor as a function of the surface potential on the protein and suggest that the acetylcholine receptor reconstituted into zwitterionic phospholipid, which has no surface potential of its own, provides an excellent model system with which to study effects of protein surface charge. We hypothesize that the primary pathway of interaction of C6SLMeI with the acetylcholine receptor is via the aqueous medium.

Animals↗
PubMed1984

Multiple binding sites for local anesthetics on reconstituted acetylcholine receptor membranes.

Using electron spin resonance spectroscopy and a spin-labeled analog of a tertiary amine local anesthetic, we have identified several populations of the local anesthetic within reconstituted lipid membranes containing purified acetylcholine receptors. These populations represent the local anesthetic interacting with membrane lipid and with the acetylcholine receptor. The data also suggest the existence of at least two classes of binding sites for the local anesthetic on the acetylcholine receptor.

Anesthetics, Local↗