Regulation of fibroblast cyclooxygenase synthesis by interleukin-1

We have prepared polyclonal antiserum against sheep seminal vesicle prostaglandin H synthase (also termed cyclooxygenase) which cross-reacted with human cyclooxygenase, thereby enabling us to directly determine the synthetic rate of cyclooxygenase protein and its modulation by the monokine interleukin-1 (IL-1). Cultured human dermal fibroblast cells were labeled with [35S]methionine, and the membrane-bound cyclooxygenase was solubilized and immunoprecipitated 35S-labeled fibroblast cyclooxygenase migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular size of approximately 73,000 daltons, similar to that of native sheep cyclooxygenase and of cyclooxygenase covalently labeled by [3H]aspirin, i.e. [3H]acetylcyclooxygenase. Additional validation of the immunoprecipitated 35S-labeled cyclooxygenase band indicated that it was specifically displaced by unlabeled sheep cyclooxygenase. N-terminal amino acid radiosequence analysis of [3H]proline-labeled cyclooxygenase revealed [3H]proline residues in positions 3, 6, and 8, consistent with the previously reported N-terminal sequence of sheep cyclooxygenase. Endoglycosidase H treatment of 35S-labeled fibroblast cyclooxygenase caused a decline in apparent molecular size (due to removal of mannose residues) which was similar to that seen with the native sheep cyclooxygenase. [35S]Methionine pulse-chase experiments indicated a half-life of 1 h for fibroblast cyclooxygenase. The monokine interleukin-1 stimulated fibroblast cyclooxygenase synthesis in a time- and dose-dependent fashion; as little as 0.03 unit/ml of IL-1 produced significant stimulation of 35S-labeled cyclooxygenase synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with 0.3 unit/ml of IL-1 for 12-16 h. IL-1 treatment of cells yielded parallel dose-response curves for stimulation of prostaglandin E2 formation, increased cellular cyclooxygenase activity, and increased synthetic rate of newly formed cyclooxygenase, suggesting that the IL-1 effect is mediated mainly, if not solely, via induction of cyclooxygenase synthesis.     

The cell surface receptor for immunoglobulin E. Effect of tunicamycin on molecular properties of receptor from rat basophilic leukemia cells

Cell surface receptors for immunoglobulin E were isolated by repetitive affinity chromatography from rat basophilic leukemia cells biosynthetically labeled with L-[35S]methionine and D-[3H]mannose. Native immunoglobulin E receptor appeared as a very broad band in the 45,000 to 62,000 Mr region in sodium dodecyl sulfate polyacrylamide gels. However, from cells cultured in the presence of tunicamycin, a relatively narrow band with an apparent Mr of 38,000 was isolated. The 38,000 Mr band rebound to immunoglobulin E-Sepharose, was immunoprecipitated with antibodies to immunoglobulin E receptor, shared tryptic peptides with native receptor, and was labeled with L-[35S]methionine but not D-[3H]mannose, and thus appears to be immunoglobulin E receptor lacking N-linked oligosaccharides. It is demonstrated that N-linked oligosaccharides account for much of the apparent heterogeneity of native receptor in sodium dodecyl sulfate polyacrylamide gels and in two-dimensional gel electrophoresis. A receptor-associated protein with apparent Mr = 30,000, prominently labeled with L-[35S]methionine but not with D-[3H]mannose, did not have altered molecular properties when isolated from tunicamycin-cultured cells, and did not share tryptic peptides with receptor.     

Fast-Transported Glycoproteins and Nonglycosylated Proteins Contain Sulfate

35SO4-labeled fast-transported proteins of bullfrog dorsal root ganglion neurons were separated by two-dimensional gel electrophoresis, and their mobilities were compared to similar species labeled with [3H]mannose or [3H]fucose. Fluorography revealed regions of poorly resolved, high molecular weight material, likely to represent sulfated proteoglycans, as well as many well resolved spots that corresponded in mobility to individual [35S]methionine-labeled fast-transported proteins. The majority of these well resolved spots appeared as "families," previously identified as glycoproteins based on their labeling with sugars. Thus, sulfate can be a contributor to the carbohydrate side-chain charge that underlies microheterogeneity. The most heavily 35SO4-labeled species, however, corresponded to fast-transported proteins that were not labeled by either sugar. The relative acid labilities of 35SO4 associated with individual species cut from the gel confirmed the assignments of these spots as glycoproteins or nonglycoproteins. A group of spots intermediate in their acid lability was also detected, suggesting that some proteins may contain sulfate linked to carbohydrate as well as to amino acid residues.     

Mechanisms of intracellular protein catabolism. Intracellular fate of microinjected polypeptides translated in vitro.

Erythrocyte-mediated microinjection was used to introduce [35S]polypeptides translated in vitro into 3T3-L1 cells. Such [35S]polypeptides are not degraded after loading into erythrocytes and are stable for the first 2 h after microinjection into growing 3T3-L1 cells. Similarly, little or no degradation of microinjected [35S]polypeptides is observed in either growing or confluent 3T3-L1 cells over a 70 h period. Microinjection of reticulocyte lysate alone does not affect the rate of degradation of long-lived endogenous protein. Reductively [3H]methylated lysate haemoglobin is degraded after microinjection by a cytosolic mechanism. Microinjected 125I-labelled bovine serum albumin is rapidly degraded by a cytosolic mechanism at the same rate in the absence or presence of reticulocyte lysate. The data do not support the notion that the observed lack of degradation of microinjected [35S]polypeptides translated in vitro is due to the presence of proteolytic inhibitors in reticulocyte lysates which can inhibit the degradation of microinjected or cellular proteins.     

The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum

We used a conjugate of transferrin and horseradish peroxidase (Tf/HRP) to label the intracellular transferrin receptor route in the human hepatoma cell line HepG2. The recycling kinetics of [125I]Tf/HRP were similar to those of unmodified [125I]Tf, implying identical routes for both ligands. 3,3'Diaminobenzidine (DAB)-cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were incubated with both Tf/HRP and [125I]Tf, and caused two different effects: (a) the equilibrium density of [125I]Tf containing microsomes in a Percoll density gradient was increased, and (b) the amount of immunoprecipitable [125I]Tf from density-shifted lysed microsomes was only 20% of that of nonDAB treated microsomes. The whole biosynthetic route of alpha 1-antitrypsin (AT), a typical secretory glycoprotein in HepG2 cells, was labeled during a 60-min incubation with [35S]methionine. DAB cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were also incubated with Tf/HRP. DAB cytochemistry caused approximately 40% of microsome- associated "complex" glycosylated [35S]alpha 1-antitrypsin ([35S]c-AT) to shift in a Percoll density gradient. Only part of the density shifted [35S]c-AT could be recovered by immunoprecipitation. A maximum effect was measured already after 10 min of Tf/HRP uptake. The density distribution of the "high mannose" glycosylated form of 35S-alpha 1- anti-trypsin [( 35S]hm-AT) was not affected by Tf/HRP. If in addition to Tf/HRP also an excess of non-conjugated transferrin was present in the medium, [35S]c-AT was not accessible for Tf/HRP, showing the involvement of the transferrin receptor (TfR) in the process. Furthermore, we show that if Tf/HRP and [35S]c-AT were located in different vesicles, the density distribution of [35S]c-AT was not affected by DAB-cytochemistry. Pulse-labeling with [35S]methionine was used to show that [35S]c-AT became accessible to endocytosed Tf/HRP minutes after acquirement of the complex configuration. A common intracellular localization of endocytosed Tf/HRP and secretory protein could be confirmed by immuno-electron microscopy: cryosections labeled with anti-albumin (protein A-colloidal gold) as well as DAB reaction product showed double-labeling in the trans-Golgi reticulum.     

Effect of monensin on synthesis, post-translational processing, and secretion of dopamine beta-hydroxylase from PC12 pheochromocytoma cells

Monensin was used to ascertain the location in the biosynthetic pathway where the 77,000-Mr membrane-bound subunit form of dopamine beta-hydroxylase is post-translationally converted to the 73,000-Mr soluble form. Treatment with low concentrations of monensin (less than or equal to 50 nM) completely depleted the cells of the norepinephrine and dopamine, had a small effect on protein synthesis, and enhanced post-translational processing of only dopamine beta-hydroxylase which was previously synthesized and presumably packaged into neurosecretory vesicles. At these low concentrations, exit from the Golgi apparatus did not appear to be blocked since stimulated secretion of a group of high molecular weight [35S]methionine-labeled proteins was not inhibited. Treatment with higher concentrations of monensin (200 nM) prevented the secretion of the [35S] methionine-labeled proteins normally released with a secretagogue, and also prevented the secretion of [3H] mannose-labeled proteins including dopamine beta-hydroxylase. Surprisingly, a group of lower molecular weight [35S]methionine-labeled proteins was now released from monensin-treated cells. Treatment with high concentrations of monensin (greater than or equal to 200 nM) appeared to block the secretory pathway prior to the packaging step, probably in the Golgi apparatus. If the proteins were packaged prior to monensin treatment, they were released upon stimulation with secretagogues. Monensin treatment (200 nM) enabled the post-translational processing of newly synthesized dopamine beta-hydroxylase, from the 77,000-Mr to the 73,000-Mr subunit form, to go to completion. The susceptibility of this 73,000-Mr subunit form to endoglycosidase H digestion was unaltered, suggesting that dopamine beta-hydroxylase from monensin-treated cells may have the same high mannose oligosaccharide content as native dopamine beta-hydroxylase. These experiments indicate that the post-translational processing of dopamine beta-hydroxylase occurs in the Golgi apparatus and may continue in immature granules prior to their acidification.     

Immunopurification of the suppressor tRNA dependent rabbit beta-globin readthrough protein

In mammalian cells, the rabbit beta-globin readthrough protein is the only known example of a naturally occurring readthrough protein which does not involve a viral system. To provide an efficient means for its isolation, detection, and study, we elicited specific antibodies against this unique protein. The 22 amino acid peptide corresponding to the readthrough portion of this protein was synthesized, coupled to keyhole limpet hemocyanin, and injected into sheep. Specific antibodies to the peptide were produced as demonstrated by the enzyme-linked immunosorbent assay technique and by immunoblotting. The antibodies did not react with globin. The rabbit beta-globin readthrough protein was separated from globin and other reticulocyte proteins by polyacrylamide gel electrophoresis and visualized by silver staining or by labeling with [35S]methionine. Incorporation of [35S]methionine into the readthrough protein was significantly enhanced upon addition of an opal suppressor tRNA to reticulocyte lysates. Immunoblotting revealed that the readthrough protein also occurs in lysates without added suppressor tRNA. The antibodies were purified on an affi-gel column which had been coupled with the peptide antigen. The readthrough protein was then purified from reticulocytes by immunoaffinity chromatography and by high-performance liquid chromatography. The results provide conclusive evidence that the beta-globin readthrough protein is naturally occurring in rabbit reticulocytes.     

Rubella virus contains one capsid protein and three envelope glycoproteins, E1, E2a, and E2b.

We have analyzed the structure of rubella virus proteins labeled metabolically with [35S]methionine, [3H]mannose, and [3H]glucosamine or externally with [3H]borohydride after galactose oxidase treatment. Four structural proteins, with MrS of about 58,000 (E1), 47,000 (E2a), 42,000 (E2b), and 33,000 (C), were resolved on sodium dodecyl sulfate-polyacrylamide gels. Tryptic peptide maps obtained from [35S]methionine-labeled proteins indicated that E1 and C were unrelated to each other and to E2a and E2b, whereas the latter two gave similar, if not identical, maps. E1, E2a, and E2b were associated with the envelope and were located externally on the virus particle, whereas the C protein was associated with the RNA in the nucleocapsid. Solubilization of the virus with Triton X-100, followed by removal of the nucleocapsid and the detergent, resulted in the formation of soluble envelope protein complexes (rosettes) containing E1, E2a, and E2b. Although external labeling with [3H]borohydride and metabolic labeling with [3H]glucosamine suggested that all three proteins were glycosylated, only E1 and E2b were efficiently labeled with [3H]mannose. It is thus possible that the difference in migration between E2a and E2b is due to differences in glycosylation. Analysis by immunoprecipitation and sodium dodecyl sulfate-gel electrophoresis of intracellular [35S]methionine-labeled structural proteins synthesized in the presence and absence of tunicamycin supported the conclusion that E1 and E2 are glycoproteins. Unglycosylated E1 and E2 had an Mr of about 53,000 and 30,000, respectively.     

The hamster transferrin receptor contains Ser/Thr-linked oligosaccharides: use of a lectin-resistant CHO cell line to identify glycoproteins containing these linkages.

We recently reported that the human transferrin receptor (TfR) contains O-linked GalNAc residues [1]. To investigate whether this modification is shared by transferrin receptors in other mammals, we investigated the glycosylation of TfR in hamster cells. To facilitate our analysis the lectin-resistant Chinese hamster ovary (CHO) cell line Lec8 was used. These cells are unable to galactosylate glycoproteins, resulting in truncation of the Ser/Thr-linked oligosaccharides to a single residue of terminal alpha-linked GalNAc. This structure is bound with high affinity by the lectin Helix pomatia agglutinin (HPA). The TfR was affinity purified from Lec8 cells metabolically radiolabeled with [3H]glucosamine and the receptor was found to bind tightly to HPA-Sepharose. Treatment of the purified TfR with mild alkaline/borohydride released [3H]GalNAcitol, demonstrating the presence of O-linked GalNAc. We also found that many other unidentified [3H]glucosamine-labeled glycoproteins from Lec8 cells were bound by HPA-Sepharose. The bound and unbound glycoproteins were separated by SDS/PAGE and individual species were selected for treatment with mild base/borohydride. Treatment of glycoproteins bound by HPA, but not those unbound, resulted in the release of [3H]GalNAcitol. These studies demonstrate both that the hamster TfR contains O-linked oligosaccharides and that this approach may have general utility for identifying the presence of these oligosaccharides in other glycoproteins.     

The effect of castanospermine on the synthesis of synaptic glycoproteins by rat brain slices

Slices were prepared from rat forebrains and the incorporation of [³H]mannose and [³⁵S]methionine into proteins and glycoproteins determined. The incorporation of methionine continued to increase for up to 8 hours whereas mannose incorporation was maximal between 2 and 4 hours and declined thereafter. Glycopeptides prepared by pronase digestion of [³H]mannose-labeled glycoproteins were digested with endoglucosaminidase H (endo H) and analysed by gel filtration. The major endo H-sensitive oligosaccharide eluted in a position similar to standard Man₈GlcNAc. In the presence of castanospermine, which inhibits glucosidase I, the first enzymatic step in the processing of N-linked oligosaccharides, a new endo H-sensitive glycan similar in size to standard Glc₃Man₉GlcNAc₂ accumulated. Synaptic membranes (SMs) were isolated from slices which had been incubated with either [³H]mannose or [³⁵S]methionine in the presence and absence of castanospermine. In the presence of inhibitor the relative incorporation of [³H]mannose into high-mannose glycans of synaptic glycoproteins was increased. The incorporation of newly synthesized, [³⁵S] methioninelabeled, Con A-binding glycoproteins into SMs was not affected by the addition of inhibitor. Many of the glycoproteins synthesized in the presence of castanospermine exhibited a decreased electrophoretic mobility indicative of the presence of altered oligosaccharide chains. The results indicate that changes in oligosaccharide composition produced by castanospermine had little effect on the subsequent transport and incorporation of glycoproteins into synaptic membranes.To whom to address reprint requests.     

Lymphocyte function-associated antigen 1 (LFA-1) contains sulfated N-linked oligosaccharides

The murine lymphocyte function-associated antigen 1 (LFA-1) is a glycoprotein heterodimer consisting of an Mr 180,000 alpha-chain and an Mr 95,000 beta-chain. Although LFA-1 has been studied extensively in the past few years due to its involvement in various antigen-specific T lymphocyte responses, virtually nothing is known about its glycosylation. In this report, we have analyzed the oligosaccharide moieties of the murine LFA-1 molecule. Utilizing a T lymphoma cell line, EL-4, it was found that [35S] sulfate, [3H]glucosamine, [3H]mannose, and [3H]fucose were incorporated into both the alpha- and beta-chains of LFA-1. Isolated alpha- and beta-chains from anti-LFA-1 immunoprecipitates of [3H]glucosamine-labeled NP-40 lysates were subjected to tryptic-chymotryptic digestion, and the resulting glycopeptides were fractionated by reverse-phase high performance liquid chromatography. Five major [3H]glucosamine-labeled glycopeptides were generated by this procedure from each of the two polypeptide chains. Treatment of the individual glycopeptides with almond emulsin peptide:N-glycosidase or Endo F demonstrated that the [3H]glucosamine label existed almost entirely in N-linked oligosaccharide structures (Mr 5000 to 10,000). By using similar techniques, the majority of the [35S]sulfate moieties were also found covalently bound to N-linked oligosaccharides. In addition, both [35S]sulfate-labeled alpha- and beta-chains were susceptible to Keratanase and endo-beta-galactosidase digestions, indicating the presence of sulfated N-acetyllactosamine sequences. The expression of [35S]sulfate-labeled LFA-1 on various cell types was also examined. LFA-1 was found to be sulfated only on thymocytes and splenic T cells, but not on macrophages, splenic B, or bone marrow cells.     

Structural characterization of the glycinin precursors

Poly(A)-RNAs enriched for glycinin coding sequences were injected into frog oocytes and translated in the presence of either [3H]leucine or [3H]isoleucine. Sodium dodecyl sulfate electrophoresis indicated that radioactive proteins similar in size to the authentic acidic and basic polypeptide components of glycinin were not present among the glycinin-related proteins synthesized. Instead, high molecular weight precursors (Mr = 58,000-67,000) were immunoprecipitated. Unlike disulfide-linked native glycinin complexes which were cleaved by disulfide reduction, products purified from either rabbit reticulocyte lysate or oocyte translation systems were insensitive to reducing agents. The glycinin-related proteins synthesized in the oocyte were 1000 to 2000 daltons smaller than those synthesized in the reticulocyte lysate system. This result, which suggested that the oocyte system had removed NH2-terminal leader sequences of the preglycinin polypeptides, was confirmed by NH2-terminal sequence analysis of proteins synthesized in oocytes. Radioactive label was found exactly at the positions predicted by the NH2-terminal sequences of the acidic polypeptide component of native glycinin. Glycinin precursors, therefore, have an NH2-terminal leader sequence followed by the acidic peptide component and then the basic polypeptide component, joined in peptide linkage.     

A source of error in the chromatographic study of 35S-sulfate labeled mucous glycoproteins secreted by the gill epithelium of Mytilus edulis

HPLC combined with [35S]-sulfate/[3H]-glucosamine radiolabeling were employed to study the synthesis and secretion of mucous glycoproteins. The secreted radiolabeled glycoproteins were separated from the medium by precipitation with a mixture of trichloroacetic-phosphotungstic acids (TCA/PTA). The redissolved glycoproteins were chromatographed on an anion exchange protein column at varying pH of the mobile phase and fractions were collected for liquid scintillation counting. Varying the pH of the mobile phase from pH 3 to 7 resulted in a decrease of glycoprotein bound [35S] from 69.5 to 0.5% of the total recovered [35S]-sulfate with the remainder recovered as free [35S]-sulfate. The [3H]-labeled glycoprotein recovered under the uV peaks at this pH range was 99.5%. When high performance size exclusion chromatography was performed the change in mobile phase pH did not affect the 100% recovery of either [35S]-or [3H]-labels under the uV peaks. No free [35S]-sulfate was obtained when [35S]-labeled glycoproteins were separated from the medium using dialysis. These data suggest that the standard method of TCA/PTA precipitation of [35S]-labeled glycoproteins may cleave the [35S]-sulfate ester linkages to the oligosaccharide chains. The [35S]-sulfate may then rebind to the macromolecule by a relatively strong noncovalent bond. This may prove critical in anion exchange protein HPLC studies.     

The binding of Met-tRNAf to isolated 40-S ribosomal subunits and the formation of Met-tRNAf - 80-S-ribosome initiation complexes.

Different forms of 40-S ribosomal subunit, distinguishable by their buoyant densities on CsCl equilibrium density gradients, are formed when derived 40-S ribosomal subunits are incubated with partially purified reticulocyte ribosomal wash proteins. One of these subunits, the 1.37-g-cm-3 form is not present in the cell but the other two forms, the 1.40-g-cm-3 and 1.40-g-cm-3 subunits, are present in cell extracts. 35S label is bound to 1.37-g-cm-3 and 1.40-g-cm-s subunits when [35S]Met-tRANf, GTP and poly(A,U,G) are included in the incubations. The 35S-labelled 40-S subunits recovered, and the amount of 35S label bound to them, are changed if the [35S]Met-tRNAf-40-S-subunit-poly(A,U,G) complexes are first purified on sucrose gradients before analysing them on CsCl. The 1.37-g-cm-3 particle is no longer seen and the total quantity of 35S label on the 40-S subunits is 90% lower after sucrose gradient purification. Between 30% and 40% of the 40-S subunits bind [35S]Met-tRNAf when 1 mM GTP, an excess of ribosomal wash proteins and [35S]Met-tRNAf over derived 40-S subunits, and poly(A,U,G) or AUG is included in the incubations. The omission of poly(A,U,G) or AUG from the incubations substantially lowers the amount of subunit-bound 35S label ultimately recovered. With these incubations less than 10% of the 40-S subunits have bound [35S]Met-tRNAf. [35S]Met-tRNAf binding is affected by the nature of the RNA added. The addition of poly(U), rRNA and native 9-S golbin mRNA is without effect, whereas denatured globin mRNA is stimulatory. Maximum binding is obtained however with AUG. Poly(A,U,G) is less stimulatory than AUG but more stimulatory than denatured mRNA, suggesting that the number as well the accessibility of the AUG initiations condons determines the amount of 35S label bound. Similar results are obtained for the ribosomal-wash-dependent binding of [35S]Met-tRNAf to 80-S ribosomes. Contrary to the binding results, the ability of mRNA to stimulate protein synthesis is dependent on the integrity of the mRNA. Thus, native 9-S globin mRNA but not poly(A,U,G) stimulatex protein synthesis in the wheat germ system. HCHO-treated globin mRNA, although stimulatory, is 45% less effective than native mRNA. The addition of AUG, derived 60-S subunits and extra ribosomal wash is required for the formation of [35S]Met-tRNAf-80-S-ribosome complexes from sucrose-gradient-purified [35S]Met-tRNAf-40-S-subunit complexes. The 80-S ribosome complexes are able to form peptide bonds. Thus, if puromycin is added to the full incubations at zero time, no 35S label is present on the 80-S ribosome. 35S label is released as methionyl-puromycin. If the [35S]Met-tRNAf-40-S-subunit complexes are assembled with poly(A,U,G) or AUG in the incubations and then purified, only derived 60-S subunits are required to form [35S]Met-tRNAf-80-S-ribosome complexes. 35S label is not released from them when puromycin is added to the incubations unless extra ribosomal wash is also added.     

The efficiency of methionine incorporation from isoaccepting species of tRNAMet into rabbit globin in an homologous reticulocyte lysate system.

Rabbit globin alpha and beta chains were labeled with [3H]leucine, and with [35S] -methionine from reticulocyte tRNAMet isoacceptors using a rabbit reticulocyte cell-free synthesis system. [35S]Methionine from the three tRNAMet species isolated by RPC-5 chromatography was incorporated into internal positions of both alpha and beta globin. The initiator tRNA, tRNAIMet, exhibited very low efficiency for incorporating methionine internally, while tRNAIIMet was four times more efficient than tRNAIIIMet. Amino acid analysis of the tryptic peptides of the labeled globins revealed that all three isoacceptors incorporated methionine into the normal methionine peptides. Similar studies with Escherichia coli [35S]Met-tRNAfMet showed a 3-fold increase over the reticulocyte initiator tRNA in its capacity to incorporate methionine into the internal positions of rabbit globin.     

Glycosaminoglycan profiles in cloned granulated lymphocytes with natural killer function and in cultured mast cells: their potential use as biochemical markers

Proteoglycans from three cloned, granulated lymphocyte cell lines with natural killer (NK) function (NKB61A2, HY-3, H-1) and one mast cell line (PT-18) were labeled with [35S]sulfate. [35S]proteoglycans were extracted in 1 M NaCl with protease inhibitors to preserve their native structure and were separated from unincorporated [35S]sulfate by Sephadex G-25 chromatography. [35S]proteoglycans from all four cell lines were chromatographed over Sepharose 4B and were found to have a similar range of m.w. The [35S]glycosaminoglycans from each cell line were then separated from parent proteoglycans by treatment with 0.5 M NaOH. The [35S]glycosaminoglycans from the three lymphocyte cell lines exhibited a similar m.w. as assessed by Sepharose 4B gel filtration, whereas the [35S]glycosaminoglycans from the mast cell line chromatographed as a smaller m.w. molecule. [35S )glycosaminoglycan charge characteristics were evaluated with DEAE C1-6B ion exchange chromatography. The consistency of the elution patterns was determined by using [35S]glycosaminoglycans obtained from radiolabelings of each cell line separated by 6 mo in culture. Each NK lymphocyte cell line reproducibly produced two distinct [35S]glycosaminoglycan chains that eluted in two regions well before the commercial heparin marker. The proportions of each chain were dependent upon the specific cell line. The mast cell line produced a single [35S]glycosaminoglycan chain, which eluted overlapping the internal commercial heparin marker, consistent with its higher charge characteristics. [35S]glycosaminoglycans from all cell lines were identified as chondroitin sulfates with the use of specific polysaccharidases. The NK lymphocyte glycosaminoglycans contained chondroitin 4-sulfate disaccharides. The mast cell glycosaminoglycans contained oversulfated disaccharides and chondroitin 4-sulfate disaccharides. Thus, each granulated NK lymphocyte cell line produced chondroitin sulfate glycosaminoglycans that were characteristic of that cell line and of different composition and less charge than those produced by cultured mast cells. These findings demonstrate that glycosaminoglycan profiles are useful biochemical markers in the characterization of diverse granulated cell lines including NK lymphocytes and mast cells.     

Brefeldin A inhibits oligosaccharide processing of glycoproteins in mouse hypothyroid pituitary tissue at several subcellular sites

We have studied the effects of brefeldin A (BFA) and monensin on the processing of the oligosaccharides of thyrotropin (TSH), free alpha-subunits, and cellular glycoproteins of mouse pituitary tissue to clarify the subcellular sites of action of BFA. BFA was previously shown to inhibit the translocation of glycoproteins from the rough endoplasmic reticulum to the Golgi apparatus but action at other sites was possible. Pituitaries from hypothyroid mice were incubated with [35S]methionine, [3H]mannose, [3H]galactose, [3H]fucose, N-[3H]acetylmannosamine, or [35S]sulfate for 2 hr in the absence or presence of 5 micrograms of BFA/ml or 2 microM monensin. TSH and free alpha-subunits were immunoprecipitated from tissue lysates and analyzed by sodium dodecyl sulfate-gel electrophoresis. The tryptic glycopeptides of TSH were separated using high-performance liquid chromatography. Total glycoproteins in cell lysates were precipitated using trichloroacetic acid. Labeled oligosaccharides were released from the tryptic glycopeptides of TSH and cellular glycoproteins by endoglycosidase H and they were analyzed by paper chromatography. Compared with control incubations, BFA caused the intracellular accumulation of glycoproteins having less than expected amounts of Man9GlcNAc2 units, but with excess Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2 units. There was a lesser accumulation of glucose-containing oligosaccharides, especially Glc1Man9GlcNAc2. Monensin also caused the accumulation of certain high mannose species, but the pattern differed from that seen for BFA, since Man9GlcNAc2 units were preserved and there was less excess of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2 units. BFA did not block the initial attachment of oligosaccharides at any of the three Asn-glycosylation sites of TSH, but caused the accumulation of Man5-8GlcNAc2 units at each site. Both monensin and BFA inhibited fucosylation, sulfation, and sialylation more markedly than mannose incorporation. Thus, in addition to its previously described action of inhibiting rough endoplasmic reticulum to Golgi transport, BFA appears to partially inhibit the glucose-trimming enzymes as well as some Golgi enzymes.     

Separation and analysis of cell types involved in early stages of carrot somatic embryogenesis

The occurrence of the polarized synthesis of DNA in embryogenic cell clusters of carrot on the third and fourth days after transfer to an embryogenesis-inducing medium was observed by labeling with [³H]thymidine and autoradiography. The cells that were actively synthesizing DNA were separated from cells that were not synthesizing DNA by maceration of cell clusters into individual protoplasts and centrifugation in a Percoll density gradient. [³⁵S]Methionine-labeled proteins extracted from the two types of cell were analyzed by SDS-PAGE and fluorography. Three polypeptides (of 69, 98 and 108 kD, respectively) were found only in cells that were actively synthesizing DNA and could be candidates for markers of the polarity of DNA synthesis that is specific to embryogenesis.     

Initiation and translation in vitro of mRNA for MOPC 315 immunoglobulin heavy chain and characterization of translation product.

An initiation study of mineral oil-induced plasmacytoma (MOPC) 315 heavy chain immunoglobulin (H315) in vitro has been conducted using formyl-[35S]methionyl-tRNAfMet and a highly purified 18 S message from MOPC 315 solid tumor in a crude rabbit reticulocyte lysate system. The product was specifically precipitated by antibodies directed against MOPC 315 immunoglobulin and H315. The in vitro H315 products terminally labeled with formyl-[35S]methionine or internally labeled with [3H]leucine were electrophoretically identical with in vivo H315 on sodium dodecyl sulfate-polyacrylamide gels. All of the [35S]-methionine was incorporated at the NH2 terminus, not internally, since there is a near complete recovery of [35S]methionine following one cycle of Edman degradation. The NH2-terminal cyanogen bromide peptide, CN2, of in vivo and in vitro H315 co-migrated exactly on gel electrophoresis under conditions which completely resolved two proteins differing in size by only 14 amino acids. These data strongly suggest that there is no NH2-terminal precursor of H315 in this system. Cyanogen bromide peptide profiles of in vivo and in vitro H315 were chromatographically indistinguishable. Three peptides, CN1, CN2, and CN4, which represent approximately 85% of the total amino acids of H315 were isolated and further characterized by electrophoresis and paper chromatography. All were very similar to the corresponding peptides of authentic H315. We conclude that the fidelity of H315 translation is preserved in vitro.     

Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits

The biosynthesis and carbohydrate processing of the insulin receptor were studied in cultured human lymphocytes by means of metabolic and cell surface labeling, immunoprecipitation with anti-receptor autoantibodies, and analysis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. In addition to the two major subunits of Mr = 135,000 and Mr = 95,000, two higher molecular weight bands were detected of Mr = 210,000 and Mr = 190,000. The Mr = 210,000 band and the two major subunits were labeled by [3H]mannose, [3H]glucosamine, [3H]galactose, and [3H]fucose, and were bound by immobilized lentil, wheat germ, and ricin I lectins. On the other hand, the Mr = 190,000 band was labeled only by [3H]mannose and [3H]glucosamine and was bound only by lentil lectin. All four components could be labeled with [35S] methionine; however, in contrast with the other three polypeptides, the Mr = 190,000 band was not labeled by cell surface iodination with lactoperoxidase, suggesting that it is not exposed at the outer surface of the plasma membrane. Pulse-chase studies with [3H]mannose showed that the Mr = 190,000 was the earliest labeled component of the receptor; radioactivity in this band reached a maximum 1 h after the pulse, clearly preceded the appearance of the other components, and had a very brief half-life (t1/2 = 2.5 h). The Mr = 210,000, Mr = 135,000, and Mr = 95,000 bands were next in appearance and reached a maximum 6 h in the chase period. Monensin, an ionophore which interferes with maturation of some proteins, blocked both the disappearance of the Mr = 190,000 protein and the appearance of the Mr = 135,000 and Mr = 95,000 subunits. The mannose incorporated in the Mr = 190,000 component was fully sensitive to treatment with endoglycosidase H while that in the Mr = 210,000 band and the two major subunits was only partially sensitive. Tryptic fingerprints of the 125I-labeled Mr = 210,000 band suggested that this component contains peptides of both the Mr = 135,000 and Mr = 95,000 subunits. In conclusion, the Mr = 190,000 component appears to represent the high mannose precursor form of the insulin receptor that undergoes carbohydrate processing and proteolytic cleavage to generate the two major subunits. In addition, the Mr = 210,000 band is probably the fully glycosylated form of the precursor that escapes cleavage and is expressed in the plasma membrane.