Protein-bound oligosaccharides of Semliki Forest virus

Semliki Forest virus was grown in BHK cells and labeled in vivo with radio-active monosaccharides. promnase digenst of the virus chromatographer on Bio-Gel P 6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson J. and Clamp J.R. (1971) Biochem. J. 123, 739–745) The former was labeled with [³H]fucose, [³H]galactose, [³H]mannose and [¹⁴C]glucosamine, the latter only with [³H]mannose and [¹⁴C]glucosamine. The three envelope glycoproteins E₁, E₂ and E₃ were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E₁ and E₃ revealed glycopeptides of A-type. E₂ revealed glycopeptides of B-type. E₂ yielded additionally a glycopeptide (M_r3100) which was heavily labeled from [³H]galactose, but only marginally from [¹⁴C]glucosamine, [³H]fucose and [³H]mannose. Wether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E₁ and 4000 in E₃; the B-type unit of E₂ had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggast that E₁ and E₃ contain on the average one A-type unit; E₂ probably contains one 3100 dalton unit plus one or two B-type units.     

The NH2-terminal amino acid sequence of human proparathyroid hormone by radioisotope microanalysis.

The amino terminal amino acid sequence of human proparathyroid hormone was determined by a highly sensitive radioisotope method. Fresh human parathyroid glands were incubated with one of several ³H-labeled amino acids after which human proparathyroid hormone labeled with [³H]lysine, [³H]serine, [³H]valine, [³H]arginine, or [³H]leucine was isolated. These specimens were subjected to several cycles of Edman degradation. An increase in the amount of radioactivity liberated at any cycle was taken as evidence that the amino acid at that cycle was the one with which the sample was labeled. By this approach, we found that the amino-terminal sequence of human proparathyroid hormone is lys¹-ser²-val³-lys⁴-lys⁵-arg⁶-ser⁷-val⁸-ser⁹ … leu¹³ … leu¹⁷ … lys¹⁹ … Based on these results, we conclude that the amino-terminal region of human proparathyroid hormone consists of a hexapeptide lys-ser-val-lys-lys-arg followed by the amino acid sequence of human parathyroid hormone.     

Biosynthesis and release of glycoproteins by human skin fibroblasts in culture

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-³H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated ³H was present as [³H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [³H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [³H]fucose-labelled material before and after trypsin digestion. 3. The [³H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [³H]fucose and ¹⁴C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [³⁵S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

The secretion of parathormone and glycosylated proteins by parathyroid cells in culture

The secretion of radioactive peptides by dispersed porcine parathyroid cells incubated with [³H]- or [¹⁴C]amino acids, [³H]glucosamine and [³H]mannose was analyzed. After incubation, the culture medium contained radioactive parathormone, as expected, and two radioactive glycopeptides: SP I and SP II. SP I appears to be identical with $\text{parathyroid}\text{secretory}\text{protein}$, heretofore not recognized as a glycoprotein. SP II has not been previously identified. SP I, but not SP II or parathormone, was adsorbed by Concanavalin A possibly reflecting a high mannose content of this molecule. Raising the concentration of calcium in the medium suppressed the secretion of radioactive parathormone and SP I in a similar fashion but did not affect the secretion of SP II. Our results suggest that SP I may play a fundamental role in parathyroid synthetic or secretory processes.     

Density labeling of proteins with 13C-labeled amino acids: no accumulation of an inactive alpha-amylase precursor in barley aleurone cells.

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus ¹³C-labeled amino acids and [³H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [³H]leucine was negligible in comparison to that of ¹³C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of ¹³C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.     

Prostaglandin E1 binding and adenylate cyclase activation in normal and transformed fibroblasts

The binding of [³H]prostaglandin E₁ to membranes of clones of normal rat kidney fibroblasts (NRK cells) has been measured. Cell lines that responded to prostaglandin E₁, such as NRK and NRK transformed with Schmitt-Ruppin strain of Rous sarcoma virus (SR-NRK cells), have a high affinity prostaglandin E₁ binding site. Murine-sarcoma-virus-transformed lines of NRK cells are unresponsive to prostaglandin E₁ and have reduced prostaglandin E₁ binding. Exposure of cells to prostaglandin E₁ results both in decreases prostaglandin E₁ responsiveness and reduced prostaglandin E₁ binding.Activation of adenylate cyclase is correlated to binding of prostaglandin E₁ to receptors in both NRK and SR-NRK cell membranes. Mathematical models suggest that GTP decreases the affinity of hormone for its receptor while increasing the catalytic efficiency of adenylate cyclase, and that aggregates of occupied receptors may play an important role in the activation of adenylate cyclase.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Prostaglandin E2, prostaglandin E1 and thromboxane B2 release from human monocytes treated with C3b or bacterial lipopolysaccharide

C3b or lipopolysaccharide treatment of human peripheral blood monocytes in culture stimulates an early release of thromboxane B₂ and a delayed release of prostaglandin E into culture supernatants. Immunoreactive thromboxane B₂ release is maximal from 2–8 h, whereas prostaglandin E release is maximal from 16–24 h after stimulation of monocytes in culture. We further examined this process by comparing the time course of labelled prostaglandin E₂, prostaglandin E₁ and thromboxane B₂ release from human monocytes which were pulse or continuously labelled with [³H]arachidonic acid and [¹⁴C]eicosatrienoic acid. The release of labelled eicosanoids was compared with the release of immunoreactive prostaglandin E and thromboxane B₂. The time course of prostaglandin E₂ release was virtually identical to the release of prostaglandin E₁ in all culture supernatants regardless of labelling conditions. However, release of immunoreactive prostaglandin E paralleled the release of labelled prostaglandin E₁ and E₂ only for continuously labelled cultures. The release of labelled prostaglandin E₁ and E₂ from pulse labelled cultures paralleled the release of thromboxane B₂ and not immunoreactive prostaglandin. In contrast, labelled and immunoreactive thromboxane B₂, quantitated in the same culture supernatants, demonstrated similar release patterns regardless of labelling conditions. These findings indicate that the differential pattern of prostaglandin E and thromboxane B₂ release from human monocytes is not related to a time-dependent shift in the release of prostaglandin E₁ relative to prostaglandin E₂. Because thromboxane B₂ and prostaglandin E₂ are produced through cyclooxygenase mediated conversion of arachidonic acid, these results further suggest that prostaglandin E₂ and thromboxane B₂ are independently metabolized in human monocyte populations.     

Parathyroid hormone is not involved in 1,25-dihydroxyvitamin D regulation of 25-hydroxyvitamin D metabolism in vivo

1,25-Dihydroxyvitamin D₃ administration to vitamin D-deficient rats suppresses accumulation of 1,25-dihydroxy-[3α-³H]vitamin D₃ and stimulates accumulation of 24,25-dihydroxy-[3α-³3H]vitamin D₃ from 25-hydroxy-[3α-³H]vitamin D₃ equally well in the presence and absence of parathyroid glands. These results demonstrate that this regulatory action is not mediated by the parathyroid glands and support conclusions from $\text{in}\text{vitro}$ studies that this represents a direct action of 1,25-dihydroxyvitamin D₃.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

Axonal transport of newly synthesized glycoproteins in a single identified neuron of Aplysia californica

Increasing amounts of glycoprotein synthesized from L-[³H]fucose injected into the cell body of R2, an identified Aplysia neuron, were found in the right pleuro-abdominal connective. Autoradiography revealed that the glycoproteins were localized in the axon of R2. Glycoproteins appearing in the axon presumably were synthesized in the cell body, since no significant incorporation was observed when [³H]fucose was injected directly into the axon. [³H]glycoproteins were detected in the connective after a delay of 1 h after intrasomatic injection. Thereafter, transport from the cell body was rapid, and by 10 h after injection, 45% of the total neuronal [³H]glycoprotein had appeared in the axon. By analysing the radioactivity in cell body and connective 4, 10, and 15 h after injection, we found that [³H]glycoproteins were transported selectively compared to nonmacromolecular material. Sequential sectioning of the connective revealed that [³H]glycoproteins were transported in discrete waves. The population of membrane-associated [³H]glycoproteins in the axon differed from that in the cell body. Two of the five somatic components appeared to be transported preferentially. In addition a new component appeared in the axon 10 h after injection.     

β2-Adrenergic regulation of prostaglandin D2 receptor in rabbit platelets

[³H]Prostaglandin D₂ binding to rabbit platelets was increased by about 150% in the presence of β-adrenoceptor agonist, isoproterenol. The isoproterenol-induced potentiation of the [³H]prostaglandin D₂ binding gave a bell-shaped dose-response relationship (maximum response at 3·10⁻⁸ M) in a stereospecific manner. Similar and moderate potentiation was obtained with terbutaline. On the other hand, β-adrenoceptor antagonists such as alprenolol, propranolol and butoxamine (β₂-specific) had no potentiating effect on [³H]prostaglandin D₂ binding; rather, they abolished the isoproterenol-induced increase of [³H]prostaglandin D₂ binding. The β₁-specific antagonist, metoprolol, did not have any effect. Rabbit platelets were found to possess one [³H]prostaglandin D₂ binding site (K_d = 6·10⁻⁷ M, B_max = 787 fmol/mg protein). In the presence of isoproterenol at 3·10⁻⁸ M, B_max was increased with unaltering K_d value. Isoproterenol did not increase [³H]prostaglandin E₁, [³H]prostaglandin E₂ and [³H]prostaglandin F_2α bindings to platelets. The potential effect of isoproterenol was mimicked by forskolin, theophylline, dibutyryl cyclic AMP, prostaglandin E₁ and prostaglandin I₂, but it was abolished by 2′, 5′-dideoxyadenosine, an inhibitor of adenylate cyclase, indicating that elevated level of cyclic AMP may be available for the induction of the increase of [³H]prostaglandin D₂ binding. Prostaglandin D₂-induced cyclic AMP synthesis and antiaggregation activity were also augmented in the presence of isoproterenol. These results suggest a β₂-adrenoceptor-mediated cyclic AMP-dependent mechanism for the regulation of prostaglandin D₂ receptor binding in rabbit platelets.     

Different secretory actions of pancreastatin in bovine and human parathyroid cells

Chromogranin A is an acidic protein that is costored and cosecreted with parathyroid hormone (PTH) from parathyroid cells. Pancreastatin (PST), is derived from chromogranin A, and inhibits secretion from several endocrine/neuroendocrine tissues. Effects of different pancreastatin peptides were investigated on dispersed cells from bovine and human parathyroid glands. Bovine PST(1–47) and bovine PST(32–47) inhibited PTH release from bovine cells in a dose-dependent manner. The former peptide was more potent and suppressed the secretion at 1–100 nM. This inhibition was evident in 0.5 and 1.25 mM, but not in 3.0 mM external Ca²⁺. Both peptides failed to alter the concentration of cytoplasmic Ca²⁺([Ca²⁺]_i) of bovine cells. Human PST(1–52) and PST(34–52) did not affect PTH release or [Ca²⁺]_i of parathyroid cells from patients with hyperparathyroidism, nor [Ca²⁺]_i of normal human parathyroid cells. Furthermore, bovine PST(1–47) and bovine PST(32–47) failed to alter the secretion of abnormal human parathyroid cells. The study indicates that PST exerts secretory inhibition on bovine but not human parathyroid cells, and that this action does not involve alterations of [Ca²⁺]_i.     

Synthesis of glycoproteins in a single identified neuron of Aplysia californica

Incorporation of L-[³H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [³H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [³H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [³H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [³H]fucose, gel profiles of particulate fractions labeled with [¹⁴C]valine were much more complex.     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly purified nuclei exhibited specific binding with ¹²⁵I-labeled human choriogonadotropin, [³H]prostaglandin E₁ and [³H]prostaglandin F_2α. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E₁) and 8.9% (prostaglandin F_2α) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F_2α binding site and 5′-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21–26% (prostaglandins) of original specific binding despite virtual disappearance of 5′-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound ¹²⁵I-labeled human choriogonadotropin and [³H]prostaglandin F_2α to the same extent or significantly more ([³H]prostaglandin E₁, P < 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Role of calcium ions in rapid effects of L-thyroxine on phosphoinositide metabolism in rat liver cells

The role of calcium ions in the L-thyroxine-induced initiation of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) and also the course of releasing individual fractions of inositol phosphates and diacylglycerides (DAG) were studied in liver cells during early stages of the hormone effect. L-Thyroxine stimulated a rapid hydrolysis in hepatocytes of PtdInsP₂ labeled with [¹⁴C]linoleic acid and [³H]inositol mediated by phosphoinositide-specific phospholipase C. This was associated with accumulation of [¹⁴C]DAG, total inositol phosphates, [³H]inositol 1,4,5-trisphosphate (Ins1,4,5P₃) and [³H]inositol 1,4-bisphosphate (Ins1,4P₂). Elimination of calcium ions from the incubation medium of hepatocytes did not abolish the effect of thyroxine on the accumulation of [¹⁴C]DAG and total [³H]inositol phosphates. Preincubation of liver cells with TMB-8 increased the stimulatory effect of L-thyroxine on the accumulation of [¹⁴C]DAG. During the incubation of hepatocytes in the presence of the hormone the content of ¹⁴C-labeled fatty acids did not change. The L-thyroxineinduced accumulation of [³H]Ins1,4,5P₃ and [³H]Ins1,4P₂ did not depend on the presence of calcium ions in the incubation medium of the cells.     

THE CELL-FREE SYNTHESIS OF ALKALOID-BINDING PROTEINS IN IMMATURE RAT BRAIN1

Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [¹⁴C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble ¹⁴C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of ¹⁴C-labeled amino acids. Criteria for the formation of vinblastine-binding, ¹⁴C-labeled proteins were: (1) aggregation of ¹⁴C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of ¹⁴C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [³H]colchicine-binding, ¹⁴C-labeled protein were based upon: (1) co-precipitation of the ³H-and ¹⁴C-labeled materials by vinblastine sulfate and (2) the coincidence of ³H- and ¹⁴C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, ¹⁴C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with ¹⁴C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, ¹⁴C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [³H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.     

O-Linked fucose in glycoproteins from Chinese hamster ovary cells

We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-³H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A)— Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[³H]fucitol quantitatively from [³H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [³H]fucose-labelledglycoproteins from Lec1 cells were separated by SDS—PAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [³H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDS—PAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[³H]fucitol. Trypsin treatment of [³H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked