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Biomedical subjects

S Kornfeld

Publications and source records attributed to S Kornfeld.

At least 163 records · Page 9Linked to original sources
PubMed1979

Structural studies of the major high mannose oligosaccharide units from Chinese hamster ovary cell glycoproteins.

The major high mannose-type glycopeptides present in Chinese hamster ovary cells have the compositions (Man)9(GlcNAc)2-Asn, (Man)8(GlcNAc)2-Asn, and (Man)6(GlcNAc)2-Asn. The structures of these glycopeptides were determined by the combination of methylation analysis, acetolysis, Smith periodate degradation, and alpha- and beta-mannosidase digestion. Their complete structures are: manalpha1 leads to 2Manaalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn, Manalpha1 leads to 2Manalpha1 leads to 6(Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn, and Manalpha1 leads to 6(Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn. These structures are compared with the structures of the peptide-bound oligosaccharide intermediates that are processed to form complex-type oligosaccharides. From these results, it is proposed that the high mannose-type oligosaccharides are a product of "incomplete" processing of the protein-bound oligosaccharide along the same pathway which leads ultimately to the formation of a complex-type oligosaccharide.

Animals↗
PubMed1979

Vertebrate lectins, Comparison of properties of beta-galactoside-binding lectins from tissues of calf and chicken.

Beta-galactoside-binding lectins were isolated from various calf tissues and from chicken hearts by affinity chromatography on asialofetuin-Sepharose, and were compared with respect to biochemical characteristics, binding properties, antigenic cross-reactivity, and cellular localization. The lectins are all thiol group-requiring, divalent cation-independent dimers, of apparent monomer mol wt 12,000 (calf lectins) or 13,000 (chicken lectin), and acidic pI. The calf lectins appear essentially identical by dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, amino acid composition, and radioimmunoassay, while the chicken lectin is distinctly different by these criteria. However, all of the lectins competed for the same binding sites on rabbit erythrocytes, and could be inhibited by the same saccharide haptens (notably lactose and thiodigalactoside). Immuno-fluorescence studies on several cultured cell lines revealed that the bovine and chicken lectins had primarily an intracellular cytoplasmic localization. The beta-galactoside-binding lectins of vertebrates appear to be species-specific rather than tissue-specific.

Amino Acids↗
PubMed1979

Restricted replication of two alphaviruses in ricin-resistant mouse L cells with altered glycosyltransferase activities.

Two mouse L cell variant lines (CL 3 and CL 6) selected for resistance to the toxic plant lectin ricin were restricted in their ability to replicate the two alphaviruses Sindbis virus and Semliki Forest virus. CL 3 cells have been shown to exhibit increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, whereas CL 6 cells have been shown to contain decreased UDPgalactose:glycoprotein galactosyltransferase and UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activities. The adsorption of Sindbis virus to CL 6 cells was considerably reduced, suggesting that the loss or inaccessibility of the receptors for Sindbis virus accounted for a major defect in virus production in these cells. In contrast, CL 3 synthesized Sindbis viral RNA and proteins but were unable to convert the precursor glycoprotein PE2 to the structural protein E2. The cleavage of PE2 to E2 was also blocked in both CL 3 and CL 6 cells infected with Semliki Forest virus.

Galactosyltransferases↗
PubMed1978

The synthesis of complex-type oligosaccharides. I. Structure of the lipid-linked oligosaccharide precursor of the complex-type oligosaccharides of the vesicular stomatitis virus G protein.

The synthesis of the complex-type oligosaccharide unit of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosaccharide from a lipid carrier to the nascent polypeptide. Following transfer the oligosaccharide is "processed" by removal of glucose and mannose residues and the sugars that constitute the outer branches of the complex-type oligosaccharide are added. The structure of the oligosaccharide moiety of the lipid-linked precursor has been elucidated in order to further define the steps involved in processing. Since it was not feasible to obtain adequate amounts of material for standard structural studies, most of the structural studies were performed on radiolabeled material, with radioactivity incorporated differentially into glucose, mannose, and N-acetylglucosamine. Based on endo-beta-N-acetylglucosaminidase CII digestion, alpha-mannosidase digestion, acetolysis, Smith periodate degradation, methylation analysis, and periodate oxidation, we propose the following structure for the oligosaccharide moiety of the lipid-linked oligosaccharide.

Carbohydrates↗
PubMed1978

Structure of the altered oligosaccharide present in glycoproteins from a clone of Chinese hamster ovary cells deficient in N-acetylglucosaminyltransferase activity.

Clone 15B cells, derived from Chinese hamster ovary cells and deficient in a specific UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity, synthesize glycoproteins with altered oligosaccharide units. Glycopeptides prepared from these glycoproteins contain large quantities of a glycopeptide with the composition (Man)5(GlcNAc)2-Asn whereas parent cells have only small amounts of this glycopeptide. The structure of the glycopeptide was determined by the combination of methylation analysis, acetolysis, Smith periodate degradation, and alpha- and beta-mannosidase digestion. Its complete structure is Manalpha 1 leads to 6[Manalpha1 leads to 3]-Manalpha1 leads to 6[Manalpha 1 leads to 3]-Manbeta1 leads to 4 GlcNAcbeta1 leads to 4 GlcNAc leads to Asn-peptide. The structures of two other glycopeptides found in smaller quantities in clone 15B but not detected in the parent cells were determined and are Manalpha 1 leads to 6 [Manalpha 1 leads to 3]-Manalpha1 leads to 6Manbeta 1 leads to 4 GlcNAcbeta 1 leads to 4GlcNAc-Asn-peptide and Manalpha 1 leads to 3 Manalpha 1 leads to 6[Manalpha 1 leads to 3] Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAc-Asn-peptide. It is proposed that the (Man)5(GlcNAc)2-Asn unit is the physiologic acceptor for the particular N-acetylglucosaminyltransferase which is deficient in clone 15B cells and that this reaction is necessary for complex oligosacchari-e biosynthesis.

Clone Cells↗
PubMed1978

Synthesis and infectivity of vesicular stomatitis virus containing nonglycosylated G protein.

The replication of vesicular stomatitis virus (VSV) is inhibited by tunicamycin (TM), an antibiotic that blocks the formation of N-acetylglucosaminelipid intermediates. We had shown previously that the viral glycoprotein (G) synthesized in cells treated with TM is not glycosylated and is not found on the outer surface of the cell plasma membrane. In this report, we shown that cells exposed to TM produce a low yield of infectious particles. The yield is increased when the temperature during infection is lowered from 37 to 30 degrees C. At 30 degrees C in the presence of TM, both wild-type VSV and the temperature-sensitive mutant ts045 produce particles that do not bind to concanavalin A Sepharose and contain only the nonglycosylated form of G. These particles have a specific infectivity (pfu/cpm) comparable to that of VSV containing glycosylated G.

Anti-Bacterial Agents↗
PubMed1978

Isolation and metastatic properties of detachment variants of B16 melanoma cells.

Variant clones of B16 melanoma cells have been isolated that detached from plastic tissue culture dishes either more or less readily than the parent line upon treatment with EDTA or ethyleneglycol-bis(b-amino ethyl ether)N,N'-tetraacetic acid. Cells that detached more readily were spindlier in culture than parent cells, covered less surface area per cell, and formed fewer pulmonary tumors when injected iv into C57BL/6J mice. Cells that detached less readily were flatter in culture and formed more pulmonary tumors when injected iv than the parent cells. After detachment with EDTA, the cells reattached to culture dishes at the same rate.

Animals↗
PubMed1978

Effect of tunicamycin on IgM, IgA, and IgG secretion by mouse plasmacytoma cells.

Tunicamycin, an antibiotic that prevents glycosylation of glycoproteins by blocking the formation of N-acetylglucosamine-lipid intermediates, was used to study the importance of glycosylation for the secretion of immunoglobulins by mouse plasmacytoma lines that produce immunoglobulins of different classes. Biosynthetically labeled secreted and intracellular immunoglobulins were measured by immunoprecipitation assays. Tunicamycin, at a concentration of 0.5 mug/ml produced an 81% inhibition of IgM secretion by MOPC 104E plasma cells without significantly affecting the initial rate of synthesis of intracellular IgM. No increase in the intracellular degradation of nonglycosylated IgM could be demonstrated. Tunicamycin also produced a 64% average inhibition of IgA secretion by several mouse IgA-secreting plasmacytoma lines. In contrast, despite inhibiting the incorporation of D-[14C] glucosamine into newly synthesized IgG, tunicamycin only produced a 28% average inhibition of IgG secretion, which was only slightly more than the nonspecific inhibition of secretion of the normally nonglycosylated lambda2 light chains by variant MOPC 315 plasmacytomas. These data indicate that the extent of inhibition of immunoglobulin secretion produced by tunicamycin depends on the immunoglobulin class produced by the plasma cell.

Animals↗
PubMed1977

Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus.

Tunicamycin, an antibiotic which prevents the glycosylation of newly synthesized proteins, inhibits the replication of both vesicular stomatitis virus and Sindbis virus. In tunicamycin-treated infected cells, all of the viral proteins are synthesized but the glycoproteins are devoid of carbohydrate. The nonglycosylated glycoproteins could not be detected on the outside of the plasma membrane by lactoperoxidase labeling, indirect immunofluorescence staining, or chymotrypsin treatment of intact cells, whereas the glycosylated glycoproteins were readily detected by all three methods. These results indicate that the bulk of the nonglycosylated glycoproteins are unable to undergo the normal migration to the cell surface. In contrast to the normal glycosylated viral glycoproteins, the nonglycosylated glycoproteins were insoluble in nonionic detergents such as Triton X-100. The nonglycosylated glycoprotein of vesicular stomatitis virus could be solubilized using a combination of 6 M guanidine hydrochloride and 0.2% Triton X-100, but precipitated when the 6 M guanidine was removed by dialysis. These results suggest that the lack of carbohydrate alters the properties of the glycoproteins, which may explain their impaired mobility through the intracellular membranous system.

Anti-Bacterial Agents↗
PubMed1977

Studies of the mechanism of tunicamycin in hibition of IgA and IgE secretion by plasma cells.

Tunicamycin, an antibiotic which blocks the formation of N-acetylglucosamine-lipid intermediates, thereby preventing glycosylation of glycoproteins, inhibits the secretion of IgA and IgE by MOPC 315 mouse plasma cells and IR162 rat plasma cells, respectively. At 0.5 microng of tunicamycin per ml, D-[14C]glucosamine incorporation into newly synthesized immunoglobulin was inhibited greater than 90% while the overall rate of protein synthesized was much less inhibited (40% in the case of MOPC 315 cells and 13% in the case of IR162 cells). This dose of tunicamycin produced an 85% inhibition of IgA secretion by the MOPC 315 cells and a complete inhibition of intact IgE secretion by the IR162 plasma cells. In contrast, tunicamycin had little effect on the secretion of normally nonglycosylated lambda light chains or on cell-free protein synthesis, demonstrating that tunicamycin is not a general inhibitor of protein synthesis or a non-specific inhibitor of protein secretion. No enhancement of intracellular degradation of nonglycosylated immunoglobulin could be demonstrated. Electron microscopy of tunicamycin-treated MOPC 315 cells revealed marked dilatations of the rough endoplasmic reticulum, and direct immunofluorescence indicated that the dilated rought endoplasmic reticulum contained IgA. These data indicate that glycosylation of newly synthesized IgA and IgE may be necessary for normal secretion to occur.

Acetylglucosamine↗
PubMed1977

Alpha-ketoglutarate dehydrogenase complex of Acetobacter xylinum. Purification and regulatory properties.

The alpha-ketoglutarate dehydrogenase complex of Acetobacter xylinum was purified to homogeneity. It consists of three main polypeptide chains with a total molecular weight of about 2.4 X 10(6). It catalyzes the overall Mg2+ and thiamin pyrophosphate-dependent, NAD+- and CoA-linked oxidative decarboxylation of alpha-ketoglutarate, as well as the partial reactions characteristic of the three enzyme components described for the complex from other sources. Initial velocity studies revealed marked positive cooperativity for the substrate alpha-ketoglutarate (Hill coefficient (nH) = 2.0; concentration of ligand at half-maximum effect (S0.5) = 8 mM). The sigmoidal [alpha-ketoglutarate]-velocity relationship became hyperbolic upon addition of AMP or 3-acetylpyridine adenine dinucleotide (AcPyAD) or in the presence of high concentrations of NAD. S0.5 (alpha-ketoglutarate) decreased to 1 mM, but Vmax was unchanged. Saturation curves for NAD and AMP are sigmoidal (nH = 2) at low alpha-ketoglutarate concentrations and become hyperbolic at high alpha-ketoglutarate concentrations. As judged by S0.5, the relative efficiency of the allosteric effectors is AcPyAD greater than AMP greater than alpha-ketoglutarate- greater than NAD+. Half-maximal changes in nH, S0.5, and activation by AMP occur at a pH significantly different from that of half-maximal activity. A model for the allosteric behavior of the complex is proposed in which the first enzyme component of the complex (E1) is the site for the allosteric interactions and AMP is the primary positive modifier, whereas NAD and AcPyAD act as AMP analogues. The overall reaction is competitively inhibited by NADH with respect to NAD (K1 = 20 micronM) and by succinyl-CoA with respect of CoA (K1 = 3 micronM). The properties of the alpha-ketoglutarate dehydrogenase complex of A. xylinum appear to provide for appropriate partitioning of alpha-ketoglutarate carbon between competing pathways in response to the energy state of the cells.

Adenosine Monophosphate↗
PubMed1977

Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose.

Several clones of Chinese hamster ovary cells have been selected for their resistance to the toxic effects of wheat germ agglutinin. The clones do not bind wheat germ agglutinin as well as parent cells and are 5- to 250-fold more resistant to the toxic effects of the lectin. Of three clones studied in detail, all exhibit a decrease in wheat germ agglutinin binding affinity. Two have normal numbers of wheat germ agglutinin binding sites, while one (Clone 13) has a 65% decrease in binding sites. Crude membrane preparations of the clones have a decrease in sialic acid content relative to parent cells, and Clone 13 membranes are also deficient in galactose, while the mannose and hexosamine contents of all three clones are normal. The membrane sugar deficiencies affect both glycoproteins and glycolipids. Sialyl-lactosylceramide is the major glycolipid in parent cells, while Clones 1 and 1021 have lactosylceramide and Clone 13 has glucosylceramide as the predominant glycolipid. Labeling experiments with N-[G-3H]acetylmannosamine suggest that Clone 1021 cells have a block in the transfer of sialic acid from CMP-sialic acid to glycoprotein and glycolipid acceptors. Yet CMP-sialic acid:glycoprotein sialyl-transferase activity in cell lysates of Clone 1021 cells is 80% of normal. While CMP-sialic acid:lactosylceramide sialyl-transferase activity is only 25% of normal, it can be restored to normal or elevated levels by sodium butyrate induction without an associated increase in cellular sialyl-lactosylceramide content. Similarly, the galactose-deficient Clone 13 can synthesize UDP-galactose and has normal levels of UDP-galactose:glycoprotein galactosyltransferase and UDP-galactose:glucosylceramide galactosyltransferase when assayed in vitro. The glycosyltransferases of both these clones can utilize their own glycoproteins as sugar acceptors in in vitro assays. These data suggest that the variant cells fail to carry out specific glycosyltransferase reactions in vivo despite the fact that they possess the appropriate nucleotide sugars, glycoprotein and glycolipid acceptors, and glycosyltransferases.

Carbohydrates↗
PubMed1977

Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses.

Tunicamycin (TM), an antibiotic that inhibits the formation of N-acetylglucosamine-lipid intermediates, thereby preventing the glycosylation of newly synthesized glycoproteins, inhibits the growth of Sindbis virus and vesicular stomatitis virus in BHK cells. At 0.5 mug of TM per ml, the replication of both viruses is inhibited 99.9%. Noninfectious particles were not detected. All the viral proteins were synthesized in the presence of TM, but the glycoproteins were selectively altered in that they migrated faster than normal viral glycoproteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting defective glycosylation. Within 1 h after TM addition, [14C]glucosamine incorporation into glycoproteins was inhibited 20%, whereas [35S]methionine incorporation was unaffected. By 2 to 3 h after TM addition, glucosamine incorporation had fallen to 15% of control value, with methionine incorporation being 60% of normal. TM did not affect the growth of the nomenveloped encephalomyocarditis virus in BHK cells, demonstrating that TM is not a general inhibitor of protein synthesis. These data demonstrate that TM specifically inhibits the glycosylation of viral glycoproteins and that glycosylation may be essential for the normal assembly of enveloped viral particles.

Anti-Bacterial Agents↗