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Biomedical subjects

W L Roberts

Publications and source records attributed to W L Roberts.

At least 73 records · Page 4Linked to original sources
PubMed1987

Differences in the glycolipid membrane anchors of bovine and human erythrocyte acetylcholinesterases.

Acetylcholinesterases (AcChoEases; EC 3.1.1.7) from bovine (Ebo) and human (Ehu) erythrocytes were purified to apparent homogeneity by affinity chromatography. The hydrophobic portion of the glycolipid membrane anchor of each enzyme was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Several cleavage procedures demonstrated that this radiolabel was highly selective for the fatty acid portion of the anchor in both enzymes. The labeled enzymes were digested with phosphatidylinositol (PtdIns)-specific phospholipase C (EC 3.1.4.10), and label release was assessed by polyacrylamide gel electrophoresis. About 85% of the radiolabel was cleaved from Ebo AcChoEase, whereas only 5% was released from Ehu AcChoEase. This finding agrees with a report that Ebo AcChoEase was quantitatively released from intact erythrocytes by PtdIns-specific phospholipase C but Ehu AcChoEase was not [Low, M. G. & Finean, J. B. (1977) FEBS Lett. 82, 143-146]. The two AcChoEases contained comparable amounts of the anchor components ethanolamine, glucosamine, and myo-inositol, but qualitative and quantitative differences were found in the fatty acids. Thin-layer chromatography of radiolabeled fragments generated from Ebo and Ehu AcChoEases by nitrous acid deamination revealed a major difference in the membrane anchors of the two enzymes. The fragment released from Ebo AcChoEase by this procedure comigrated with PtdIns, whereas the corresponding fragment from Ehu AcChoEase had a mobility much greater than that of PtdIns even though it contained myo-inositol and fatty acids. These studies show that 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine is useful for analysis of lipid-containing compounds and indicate that, whereas Ebo AcChoEase contains PtdIns in its glycolipid anchor, Ehu AcChoEase has a different anchor structure, which is resistant to PtdIns-specific phospholipase C. This observation suggests the existence of a class of glycolipid-anchored membrane proteins resistant to this phospholipase.

Acetylcholinesterase↗
PubMed1986

Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid.

Membrane-associated decay accelerating factor (DAF) of human erythrocytes (Ehu) was analyzed for a C-terminal glycolipid anchoring structure. Automated amino acid analysis of DAF following reductive radiomethylation revealed ethanolamine and glucosamine residues in proportions identical with those present in the Ehu acetylcholinesterase (AChE) anchor. Cleavage of radiomethylated 70-kilodalton (kDa) DAF with papain released the labeled ethanolamine and glucosamine and generated 61- and 55-kDa DAF products that retained all labeled Lys and labeled N-terminal Asp. Incubation of intact Ehu with phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the anchors in trypanosome membrane form variant surface glycoproteins (mfVSGs) and murine thymocyte Thy-1 antigen, released 15% of the cell-associated DAF antigen. The released 67-kDa PI-PLC DAF derivative retained its ability to decay the classical C3 convertase C4b2a but was unable to membrane-incorporate and displayed physicochemical properties similar to urine DAF, a hydrophilic DAF form that can be isolated from urine. Nitrous acid deamination cleavage of Ehu DAF at glucosamine following labeling with the lipophilic photoreagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) released the [125I]TID label in a parallel fashion as from [125I]TID-labeled AChE. Biosynthetic labeling of HeLa cells with [3H]ethanolamine resulted in rapid 3H incorporation into both 48-kDa pro-DAF and 72-kDa mature epithelial cell DAF. Our findings indicate that DAF and AChE are anchored in Ehu by the same or a similar glycolipid structure and that, like VSGs, this structure is incorporated into DAF early in DAF biosynthesis prior to processing of pro-DAF in the Golgi.

CD55 Antigens↗
PubMed1986

Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase.

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.

Acetylcholinesterase↗
PubMed1986

Glycolipid membrane-binding domain of human erythrocyte acetylcholinesterase.

The membrane-binding domain of human erythrocyte acetylcholinesterase is a small hydrophobic structure at the COOH-terminus of the enzyme subunits. Papain digestion cleaves a COOH-terminal dipeptide linked to the hydrophobic structure with the sequence His-Gly-ethanolamine-Z, where the ethanolamine is in amide linkage to the glycine and Z is a partially characterized glycolipid. This glycolipid includes a second residue of ethanolamine and a residue of glucosamine, both of which have free primary amino groups accessible to radiomethylation. The glycolipid also contains a carbohydrate residue or residues that bind to concanavalin A and nearly stoichiometric amounts of both palmitate and C22 unsaturated fatty acids. Similarities in this membrane-binding structure to those reported for trypanosome variant surface glycoproteins and Thy-1 glycoprotein suggest an important new category of posttranslational modifications involving the attachment of COOH-terminal glycolipid.

Acetylcholinesterase↗
PubMed1985

Identification of covalently attached fatty acids in the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase.

Human erythrocyte acetylcholinesterase is an amphipathic enzyme whose hydrophobic membrane-binding domain can be selectively labeled with a lipophilic photoreagent and removed by digestion with papain. In this paper we demonstrate that methanolysis releases covalently bound fatty acids from the hydrophobic domain and thus confirm that this domain is a covalently linked glycolipid at the enzyme subunit C-terminus. About one mole of saturated and one mole of unsaturated fatty acids were released per mole of domain. Since the predominant unsaturated fatty acids (22:4 and 22:5) are minor components of the esterified fatty acid pool in human erythrocyte membranes, assembly of the glycolipid must involve a selected unsaturated fatty acid pool.

Acetylcholinesterase↗
PubMed1985

Education standards for critical care nursing: conceptual framework.

AACN exists to promote the health and welfare of mankind by advancing the science and art of critical care nursing. One of AACN's long-range goals is to promote educational standards for critical care nursing. As a prelude to dissemination of AACN's Education Standards for Critical Care Nursing, this article presented the conceptual framework used to develop this set of standards.

Critical Care↗
PubMed1978

The consistent acceleration of lifted objects: implications for kinesthetic illusions and the perception of weight.

In examining films of lifting movements in a study of the size-weight illusion (Davis & Roberts, 1976) a consistency was noted in the values obtained for the maximum accelerations of the objects lifted. While at first surprising, this finding can be embedded significantly in theories relating to kinesthetic illusions and the perception of weight and to theories on the control of general physical movement. This study was designed to confirm its existence. Twenty-four subjects were filmed lifting four objects differing in size, shape, substance, color, and weight. The film was analyzed frame-by-frame and the data were subjected to a two-way analysis of variance. Subjects, while differing from one another, were consistent in the maximum accelerations they applied to the three heaviest of the four objects. The accelerations of the lightest object differed significantly from the accelerations of the other three, but it seems likely that this was due to the experimental task itself.

Journal Article↗