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P H Atkinson

Publications and source records attributed to P H Atkinson.

At least 55 records · Page 3Linked to original sources
PubMed1981

Application of high-field proton magnetic resonance spectroscopy in the structural determination of membrane-derived Sindbis virus glycopeptides.

Sindbis virus membrane glycopeptides have ben purified in chemical quantities and their oligosaccharide structures analyzed by 1H NMR spectroscopy at 360 MHz. Interpretable spectra could be obtained with approximately 100 micrograms of oligosaccharide. Spectral analysis of the sialyl glycopeptides S1, S2, and S3 at high and low temperatures confirms their structures to be NANA alpha (2,3)Gal beta (1,4)-GlcNAc beta (1,2)Man alpha (1,6)-[NANA alpha (2,3)Gal beta (1,4)-GlcNAc beta (1,2)Man alpha (1,3)]-Man beta (1,4)GlcNAc beta (1,4)-[Fuc alpha-(1,6)]-GlcNAc beta 1-Asn. These are heterogeneous with respect to sialic acid (NANA). Spectra of two endo-beta-N-acetylglucosaminidase products of the S4 glycopeptides are reported. The interpretation of these spectra is consistent with Man5GlcNAc and Man7GlcNAc oligosaccharide structures. Their chemical shifts are essentially identical with those reported for ovalbumin glycopeptides of the same composition, with exception to the perturbations arising from their oligosaccharide nature.

Animals↗
PubMed1981

Determination of the Structure of four glycopeptides from hen ovalbumin using 360-MHz proton magnetic resonance spectroscopy.

Four glycopeptides have been purified by Dowex and Bio-Gel P2 chromatography from Pronase digests of hen ovalbumin. The high-resolution proton magnetic resonance spectra of these glycopeptides and various products of their enzymatic digestion have been obtained at 360 MHz. By use of information derived from the spectra of a number of model compounds, an unambiguous assignment of all C1-H and Man C2-H resonances in the spectra can be made. On this basis structures are proposed for the four glycopeptides which are identical with those structures previously deduced from destructive chemical methods.

Animals↗
PubMed1981

Demonstration of heterogeneity of chick ovalbumin glycopeptides using 360-MHz proton magnetic resonance spectroscopy.

Ovalbumin glycopeptides AC-C and AC-D at various stages of purification were studied by high-field proton magnetic resonance spectroscopy (1H NMR). In a homogeneous substance, the intensity of the various resonances appears in integral amounts, while subintegral intensities usually denote mixtures of structure. We show how 1H NMR can be used to nondestructively assay the purification of major components from mixtures. In glycopeptide AC-C we have spectroscopic evidence for the four different glycopeptide species, three of which have been described [Shepherd, V., & Montgomery, R. (1978) Carbohydr. Res. 61, 147; Tai, T., Yamashita, K., Ito, S., & Kobata, A. (1977) J. Biol. Chem. 252, 6687]. However, we did detect a fourth structure not previously reported. In glycopeptide AC-D, we have spectroscopic evidence for five different compounds, only two of which have been previously reported (Tai et al., 1977; Shepherd & Montgomery, 1978).

Animals↗
PubMed1980

Growth-dependent alterations in oligomannosyl glycopeptides expressed in Sindbis virus glycoproteins.

Sindbis virus was used to examine host cell growth-dependent expression of oligomannosyl glycopeptides within single species of viral glycoproteins. Four mannosyl oligosaccharides ranging from 5 to 8 mannose residues were separated on Bio-Gel P4 column chromatography. Virus derived from rapidly growing chicken embryo fibroblasts displayed greater quantities of larger sized oligomannosyl glycopeptides in intact virus and in purified Sindbis virus glycoproteins E1 and E2 compared with virus from nongrowing cells. The pattern of mannosyl oligosaccharides in the two glycoproteins differed remarkably. E1 and E2 contained predominantly Man5GlcNAc and Man7GlcNAc, respectively; in addition, E1 relative to E2 contained more complex-type glycopeptides than mannosyl-type glycopeptides. These data clearly demonstrate that host-dependent alterations in glycosylation are expressed in Sindbis virus and that differences in specific glycosylation patterns are obscured in oligosaccharides derived from mixtures of glycoproteins. It is apparent that processing of these cotranslated glycoproteins can yield different patterns of oligosaccharide structures.

Animals↗
PubMed1978

Gycoprotein and protein precursors to plasma membranes in vesicular stomatitis virus infected HeLa cells.

Vesicular stomatitis virus is known to mature at HeLa cell plasma membranes. To study the process, cells, infected with vesicular stomatitis virus, were fractionated after short term labeling studies (1 min pulse, 1 min chase) to determine the assembly kinetics of G protein and M protein into plasma membranes. Newly synthesized M protein was found released in the supernatant from which free polysomes were sedimented during sucrose gradient analysis of these polysomes. If this M protein is particle bound, it must have a density of less than 1.08 g/ml. About 40% of this M protein so labeled was not sedimentable at 165,000 X g for 16 h. This newly synthesized M protein had not yet assembled into plasma membrane and thus must represent an internal pool. This and previous studies show that it has a subsequent transit time to the plasma membrane of about 2 min. Once associated with plasma membranes, M protein decayed in an approximately logarithmic fashion indicating that newly synthesized M randomly mixes (and turns over) with preexisting M protein. G protein was particle bound in a 1 min pulse, 1 min chase, and was never found released in a soluble form. At the later time when fucose is added to G protein, the oligosaccharide moiety is near to complete, and on completion is about 2,000 in molecular weight. Evidence is presented showing that fucose is probably attached to the N-acetylglucosamine of the protein carbohydrate linkage. G protein to which fucose had just been added was located internally on a membranous fraction of density 1.14 g/ml in sucrose; its subsequent transit time from this pool (which in uninfected cells is between 1--2% of the total cell fucosyl glycoprotein) was about 15 min. Because their densities were different and their transit times were different, internal newly synthesized M and fucosyl G protein which assemble into plasma membranes were not on the same internal membranous component. Association of M protein with the plasma membranes may thus occur from a nonsedimentable soluble cytoplasmic pool by a process of direct adsorption.

Cell Membrane↗
PubMed1978

Further characterization of HeLa S3 plasma membrane ghosts.

A plasma membrane fraction of HeLa S3 cells, consisting of ghosts, is characterized more fully. A simple procedure is described which permits light and electron microscope study of the plasma membrane fraction through the entire depth of the final product pellet and through large areas parallel to the surface. Contamination by nuclei is 0.14%, too little for DNA detection by the diphenylamine reaction. Contamination by rough endoplasmic reticulum and ribosomes is small, a single ghost containing about 3% of the RNA in a single cell. Mitochondria were not encountered. Electron microscopy also shows (a) small vesicles associated with the outer surface of the ghosts, and (b) a filamentous web at the inner face of the ghost membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis shows that of the many Coomassie Blue-stained bands two were prominent. One, 43,000 daltons, co-migrated with purified rabbit muscle actin and constituted about 7.5% of the plasma membrane protein. The other major band, 34,000 daltons, was concentrated in the plasma membrane fraction. Two major glycoproteins detected by autoradiography of [14C]fucose-labeled glycoproteins on the gels, had apparent molecular weights of 35,000 daltons and 32,000 daltons. These major bands did not stain with Coomassie Blue. There were many other minor glycoprotein bands in the 200,000- to 80,000-dalton range. Ouabain-sensitive, Na+, K+-adenosine triphosphatase (ATPase) activity of the ghost fraction is purified 9.1 (+/- 2.2) times over the homogenate; recover of the activity is 12.0 (+/- 3.8%) of the homogenate. Enrichment and recovery of fucosylglycoprotein parallel those for ouabain-sensitive Na+, K+-ATPase activity. Fucosyl glycoprotein is recovered more than the enzyme activity in a smooth membrane vesicle fraction probably containing the bulk of plasma membrane not recovered as ghosts.

Actins↗
PubMed1977

Studies on mannose-containing glycopeptides from a normal and an SV40 transformed human cell.

The oligosaccharide moiety of cell-surface mannose-labelled glycopeptides from a normal (WI38) and an SV40 transformed cell (W118Va) have been investigated using specific glycosidases. Partially purified mannose-containing glycopeptides were separated into acidic and neutral species by high voltage paper electrophoresis. Endo-beta-N-acetylgucosaminidase D, in the presence of three exoglycosidases, released from the acidic glycopeptides of non-growing cells a product completely absent in growing cells. However, the acidic species from growing WI18 Va and WI38 were found to be similar in the products released by enzyme digestion. The neutral species from growing normal cells contained a proportion of the glycopeptides resistant to endoglycosidase D while those from the non-growing cells were almost free of these resistant species. The SV40 transformed cells were further enriched, when compared to normal cells (WI38), in these neutral resistant species. We suggest that the oligomannosyl core of the majority of the susceptible species contains three mannose residues while that of the resistant species contains between six and eight.

Acetylglucosaminidase↗
PubMed1977

Equilibration of fucosyl glycoprotein pools in HeLa cells.

The pool sizes, label equilibration times, and specific radioactivity relationships of fucosyl glycoproteins and precursors have been examined in exponentially growing HeLa S3 cells (generation time about 23 h) using a quantitative radioisotopic approach. The specific radioactivity of the precursor GDP-fucose (pool size 0.52 +/- 4% nmol/10(7) cells) equilibrates with radioactive fucose in the medium in about 1 h. 10(7) cells contain 5.3 +/- 16% nmol of glycoprotein fucose of which 96-98% resides in or on the cell surfaces and is equilibrated isotopically within 22 h of labeling; 2% or less is in an internal pool, some of which is precursor to plasma membranes and some of which is released as soluble glycoprotein directly to the medium without random mixing with the plasma membrane glycoprotein. Because we cannot rule out the presence of internal free fucose, 2% of the total glycoprotein fucose could be in the degradative pathway being recycled internally before release of free fucose. In rate terms and in a particular culture where 10(7) cells contained 4.4 nmol of glycoprotein fucose, a total of 11.1 nmol of glycoprotein fucose is synthesized per generation (9-11% of the total cell glycoprotein fucose/h). Of this, 2.5 nmol of glycoprotein fucose per generation is released directly into the growth medium without mixing with the plasma membrane glycoprotein fucose. The small internal pool feeding glycoprotein fucose to the plasma membrane does so at the rate of 8.6 nmol/10(7) cells per generation, 4.2 nmol per generation of which, after mixing with the plasma membrane glycoprotein fucose, is ultimately released into the growth medium, 75-80% as free frucose. This release process is independent of cell density and the presence of serum in the growth medium.

Cell Fractionation↗
PubMed1976

Characterization of mannose-labeled glycopeptides from human diploid cells and their growth-dependent alterations.

Mannose-labeled glycopeptides were prepared from human diploid fibroblasts harvested by brief pronase digestion. Combined use of endo-beta-N-acetylglucosaminidase H and D converted most of the mannose-label into arrays of oligosaccharides. They were separated by paper chromatography and were characterized by Sephadex G-25 column chromatography, by affinity column chromatography on concanavalin A-Sepharose, and by successive digestion with alpha-mannosidase and beta-mannosidase. The results indicated that mannose residues existed as clusters of various sizes, which we refer to as "oligomannosyl cores". The large oligomannosyl cores (approximately 7 to 8 mannosyl residues) were predominant in the glycopeptides from growing cells and were preferentially associated with neutral glycopeptides, similar to Unit A glycopeptides of thyroglobulin (mannose-N-acetylglucosamine unit). In glycopeptides from nongrowing cells, the ratio of the large oligomannosyl cores decreased, accompanying the increase of a small oligommanosyl core consisting of 3 mannosyl residues. The small core was preferentially associated with acidic glycopeptides.

Acetylglucosaminidase↗
PubMed1976

Oligosaccharide moieties of the glycoprotein of vesicular stomatitis virus.

Vesicular stomatitis virus contains a single structural glycoprotein whose carbohydrate sequences are probably specified by the host cell. The glycopeptides derived by Pronase digestion of the glycoprotein of vesicular stomatitis virus grown in HeLa cells have an average molecular weight of 1,800. There are multiple oligosaccharide chains on the vesicular stomatitis virus glycoprotein with protein-carbohydrate linkages that are cleaved only by strong alkali under reducing conditions, suggesting that they contain asparagine and N-acetylglucosamine. The oligosaccharide moieties, in addition, appear to be heterogeneous in sequence on the basis of their mobilities during electrophoresis and their sensitivities to cleavage by an endoglycosidase. The carbohydrate-peptide linkage region of the major class of oligosaccharides of the vesicular stomatitis virus glycoprotein has the proposed sequence: (see article).

Acetylglucosamine↗
PubMed1975

Fucosyl-glycoprotein and precursor polls in HeLa cells.

An enzymatic-radioactive isotope method has been developed for the direct quantitation of L-fucose in amounts as low at 0.5 plus or minus 0.05 nmol. Fucose kinase is used to transfer [32-P]phosphate from ATP to [3-H]fucose. The labeled enzymatic products are then separated electrophoretically and the amount and specific activity of the fucose are determined from the known specific activity of the phosphate donor. This assay has been used to measure the GDP-L-fucose and macromolecualar fucose in HeLa cells after extraction and purification of the sugar. It has been determined there are 0.5 nmol of GDP-L-fucose in 10-7 cells with a nine- to tenfold dilution of specific activity in converting L-[3-H] fucose to GDP-L-[3-H]fucose. After 2 to 3 days of labeling, the GDP-L-[3-H]fucose pool is essentially at equilibrium with the macromolecular pool, and hence it can be concluded that the dilution of label is due to a nine- to tenfold contribution to GDP-L-fucose from an endogenous source, as compared to exogenously supplied fucose. The fucosyl-glycoprotein pool has been shown to be much larger containing 6 to 8 nmol of fucose in 10-7 cells. It has further been shown that GDP-fucose is the only soluble fucose intermediate present in significant amount.

Cell Division↗
PubMed1975

Synthesis and assembly of HeLa cell plasma membrane glycoproteins and proteins.

At least 60 percent of the fucose residues in HeLa cell glycoprotein are nonreducing, terminal, and closely proximal to the protein carbohydrate linkage. As determined by pulse-labeling with (3H) fucose and sizing glycopeptides in Sephadex chromatography these residues are added near the time of completion of oligosaccharide chains. Glycoproteins, the large bulk if not the only macromolecules labeled with radioactive fucose in HeLa cells, were not soluble in ethanol or chloroform-methanol, 2:1, but were substanially solubilized by chloroform-methanol-water, 10:10:3. Folch extraction of labeled cells and analysis of the upper phase revealed little if any (3H) fucose-labeled glycosphingolipids. Studies on the distribution of radioactively labeled glycoprotein in various cell fractions show that in uniform labeling conditions fucosylated glycoproteins accumulate in the plasma membrane specifically. Pulse-chase and protein synthesis inhibitor studies show that there is an internal pool of completed fucosylated glycoprotein, taking not less than 12 min to deplete. From this pool newly synthesized glycoprotein moves to the plasma membrane with a transit time of 12 min and little was found soluble in the cell. By contrast, a pool of protein labeled with 14C-aminoacids and precursor to plasma membrane protein is small and depleted almost immediately. From this pool newly synthesized protein molecules move to the plasma membrane with a transit time of less than 2 min. It would appear that these two distinct molecular components of plasma membranes may be assembled into membranes sequentially or into the plasma membrane independently.

Amino Acids↗
PubMed1975

Growth-dependent alterations in oligomannosyl cores of glycopeptides.

Mannose-labeled glycopeptides from the surface of growing and nongrowing human diploid cells (KL-2) were separated into neutral glycopeptides and acidic glycopeptides by paper electrophoresis. Growth-dependent alterations occurred in oligomannosyl cores in the neutral glycopeptides; namely, the neutral glycopeptides from the surface of growing cells were more resistant to endo-beta-N-acetylglucosaminidase D but were more susceptible to alpha-mannosidase (EC 3.2.1.24; alpha-D-mannose mannohydrolase) than those derived from the surface of nongrowing cells. Another growth-dependent change was found when the endoglycosidase-resistant material from acidic glycopeptides was compared by paper electrophoresis at pH 1.9. The material from the surface of nongrowing cells contained a component that was absent or greatly reduced in growing cells.

Acetylglucosaminidase↗
PubMed1975

Role of carbohydrate in determining the immunochemical properties of the major glycoprotein (gp71) of Friend murine leukemia virus.

Treatment of Friend leukemia virus gp71 with protease-free glycosidase enzymes results in removal of the major portion of the carbohydrate without affecting the amount of protein present. The digested material migrates as protein of about 60,000 to 65,000 molecular weight on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Analyses of the serological properties of gp71 after enzyme treatment indicated that the type, group, and interspecies determinants were not destroyed. In contrast, treatment with proteolytic enzymes led to the complete destruction of the gp71 molecule, including the total elimination of its serological reactivity as measured by direct and competition radioimmunoassay and by a serum cytotoxicity assay. We conclude that the carbohydrate portion of gp71 is not of major significance in defining the antigenic determinants of this viral glycoprotein.

Carbohydrates↗