Inhibitor Of Protein Glycosylation Research Articles

Cellular mechanisms of callose deposition in response to fungal infection or chemical damage

Cellular mechanisms of callose deposition induced in cowpea (Vigna unguiculata) leaves by the living cowpea rust fungus (Uromyces vignae), boric acid, or the fungus killed by polyoxin D, were investigated by the use of chemical inhibitors. Effects of the inhibitors were observed in both a resistant and a susceptible cowpea cultivar. The effect of inhibitors differed depending on the type of callose-inducing stimulus and cultivar used. Inhibitors of transcription (actinomycin D) and protein synthesis (blasticidin S, cycloheximide) lowered the incidence of fungus-induced callose deposits in both cultivars. Inhibitors of protein synthesis also reduced deposits induced by boric acid or fungal death. Callose deposition induced by the living fungus in the resistant cultivar was reduced by inhibitors of protein glycosylation (tunicamycin, deoxynojirimycin) and microfilament function (cytochalasins B and E), but these inhibitors had no effect on callose deposition in the susceptible cultivar or on chemical or fungal death-induced deposition in either cultivar. No reduction in callose deposits was observed in plants treated with inhibitors of Golgi-associated vesicle transfer (brefeldin A, monensin) or microtubule polymerization (colchicine, oryzalin). The results suggest that the cellular processes involved in callose deposition differ with differing triggering stimuli and that callose deposition triggered by the living fungus in a resistant host cultivar is not a typical wound or damage response. Keywords: callose, infection, inhibitors, resistance, rust fungi.

Effect of Tunicamycin on Growth and Mineralization of Balanus amphitrite amphitrite Darwin (Cirripedia)

ABSTRACT The effects of the protein glycosylation inhibitor tunicamycin (TM) on skeletal growth, the rate of formation and morphology of the mineral growth increments, and the fine structure of the scutal plates of the barnacle Balanus amphitrite amphitrite Darwin were examined. If glycoproteins are important in control of crystal form and mineral microstructure, then by using TM to reduce the amount of glycoprotein synthesized by the mineralizing cells, one might expect to observe changes in fine structure by electron microscopy. Accordingly, 15-day-old barnacles, that had been grown on plastic coverslips, were exposed to various concentrations of TM for 10 days and the effects then analyzed by scanning electron microscopy. TM at 0.1 µg/ml inhibited shell growth. At 0.01 µg/ml TM, shell growth was retarded in one experiment but not in another. The normal rate of formation of a mineral growth increment, as calculated in two experiments, was 3.6 and 2.8 days, respectively, at 24° ± 1°C. This rate was reduced significantly by 0.01 µg/ml and 0.1 µg/ml TM; 1.0 µg/ml TM completely blocked increment formation. The bases of setae on the scuta increased in size, and the number of setae also increased in barnacles treated with 0.1 µg/ml TM for 10 days. However, other portions of the scuta revealed no characteristic differences in crystal microstructure.

Up-regulation of functional voltage-dependent sodium channels by cyclic AMP-dependent protein kinase in adrenal medulla

Treatment of cultured bovine adrenal chromaffin cells with dbcAMP increased [ 3H]STX binding with an EC 50 of 126 μM and a half-effective time of 12 h; dbcAMP (1 mM × 18 h) raised the B max approximately 1.5-fold without altering the K d value. Forskolin (0.1 mM) or IBMX (0.3 mM) also increased [ 3H]STX binding, while dbcAMP had no effect. Effects of dbcAMP and forskolin were abolished by H-89, an inhibitor of cAMP-dependent protein kinase. Cycloheximide (10 μg/ml) and actinomycin D (10 μg/ml), inhibitors of protein synthesis, nullified the stimulatory effect of dbcAMP, whereas tunicamycin, an inhibitor of protein glycosylation, had no effect. Treatment with dbcAMP augmented veratridine-induced 22Na influx, 45Ca influx via voltage-dependent Ca channels and catecholamine secretion, while the same treatment did not alter 45Ca influx and catecholamine secretion caused by high K (a direct activation of voltage-dependent Ca channels) [25]. Na influx via single Na channel calculated from 22Na influx and [ 3H]STX binding was quantitatively similar between non-treated and dbcAMP-treated cells. Brevetoxin allosterically enhanced veratridine-induced 22Na influx approximately 3-fold in dbcAMP-treated cells as in non-treated cells. These results suggest that cAMP-dependent protein kinase is involved in the modulation of Na channel expression in adrenal medulla.

Selectin ligands on human melanoma cells.

Twelve established human melanoma lines were screened for surface expression of the carbohydrate antigens Lewisa (Lea), sialyl Lewisa (SLea), dimeric sialyl Lewisa (diSLea), sialyl LewisX (SLex) and dimeric sialyl LewisX (diSLeX). None of the lines expressed SLex, but 11/12 were positive for diSLeX and 7/12 were positive for SLea. Although both diSLeX and SLea have been reported to bind to E-selectin, none of the melanoma lines exhibited E-selectin-dependent adhesion to activated human umbilical vein endothelial cells (HUVECs). Three melanoma lines infected with a retroviral vector carrying the cDNA for the human Lewis fucosyltransferase (FucT-III) subsequently expressed SLeX at their cell surface and exhibited E-selectin-dependent adhesion to activated HUVECs. Treatment of these transduced cells with inhibitors of O-linked or N-linked protein glycosylation significantly inhibited E-selectin-mediated adhesion, though fluorescence-activated cell sorter analysis indicated no decrease in cell surface expression of SLeX, Slea or diSLeX. This suggests that the majority of SLeX/SLea-type glycans endogenously produced by human melanoma cells are not protein-associated and do not mediate E-selectin-dependent adhesion. These results support the hypothesis that E-selectin-dependent adhesion requires presentation of SLeX-type moieties on appropriate glycoproteins.

Nitrogen dioxide-induced expression of a 78 kDa protein in pulmonary artery endothelial cells

Exposure to nitrogen dioxide (NO 2) activates signal transduction in cultured pulmonary artery endothelial cells (PAEC). We examined whether NO 2-induced activation of signal transduction results in increased expression of proteins in PAEC. Exposure to 5 ppm NO 2 for 4, 12, and 24 h had no significant effect on total protein synthesis. However, two-dimensional gel electrophoresis of [ 35s]-methionine-labeled PAEC exposed to NO 2 for 24 h, but not 4 and 12 h, demonstrated increased synthesis of several proteins including a two- to five-fold increase of some proteins with molecular masses of 47, 64, 78, and 105 kDa compared to controls. N-terminal amino acid sequencing and immunodetection analysis identified the 78 kDa protein as 78 kDa glucose- regulated protein (GRP-78). Induction of GRP-78 by NO 2 exposure was regulated at the transcriptional level, and the induction required de novo protein synthesis. Exposure to NO 2 for 24 h also significantly ( p < .05) decreased glycosylation of proteins in PAEC. Exposure of cell monolayers to tunicamycin, an inhibitor of protein glycosylation, mimicked the effect of NO 2 exposure on expression of GRP-78. Increased expression of GRP-78 was also detected when cell monolayers were exposed to the calcium ionophore A 23187, to 2-deoxyglucose, or to glucose-free medium, which are also known to cause perturbations in protein glycosylation. These results demonstrate that exposure to NO 2 increases expression of a number of proteins including GRP-78 in PAEC. Increased expression of GRP-78 in NO 2-exposed cells appears to be associated with inhibition of glycosylation or through coordinated alterations in metabolic events that lead to inhibition of protein glycosylation.

Design and Evaluation of Potent Inhibitors of Asparagine-Linked Protein Glycosylation

Design and Evaluation of Potent Inhibitors of Asparagine-Linked Protein Glycosylation

Effects of high glucose concentrations on human mesangial cell proliferation.

High concentrations of glucose in vitro inhibit cell proliferation and stimulate matrix protein synthesis. These studies sought to characterize the relationship between the effects of glucose on cell proliferation and matrix synthesis and to assess the mechanism(s) responsible for these cellular effects of glucose. The initial experiments showed that high glucose levels stimulate fibronectin (FN) synthesis by human mesangial cells (HMC) but only in those cultures in which cell proliferation was inhibited by glucose. To assess whether this relationship was due to an effect of glucose on the capacity of HMC to respond to cytokines, the responses of HMC to serum or cytokines were measured in the presence of different glucose concentrations. High concentrations of glucose inhibited (3H)thymidine incorporation in response to serum and platelet-derived growth factor. Under the conditions of these experiments, transforming growth factor-beta (TGF-beta) also stimulated thymidine incorporation by HMC, and high glucose concentrations inhibited thymidine incorporation in response to TGF-beta. In contrast, high concentrations of glucose did not inhibit the stimulation of FN synthesis caused by platelet-derived growth factor, serum, or TGF-beta. The antiproliferative effects of high glucose levels were first observed after 48 h of incubation and were reversible after the withdrawal of high glucose from the media. The following evidence suggest that the effects of glucose may be mediated via protein kinase C (PKC): (1) incubation with high glucose concentrations caused an increase in HMC PKC levels; (2) PKC activation with phorbol esters inhibited HMC proliferation; and (3) depletion or inhibition of PKC stimulated HMC proliferation and prevented the antiproliferative effects of glucose. In contrast to these findings, inhibitors of protein glycosylation and myo-inositol supplementation of culture media did not prevent the antiproliferative effects of glucose. In conclusion, high glucose concentrations acutely and reversibly inhibit HMC proliferation, perhaps by a PKC-dependent mechanism. Because PKC can also stimulate FN synthesis, glucose-induced changes in PKC may explain the relationship between the effects of high glucose concentrations on cell proliferation and FN synthesis.

Effect of inhibition of protein glycosylation on auxin-induced growth and the occurrence of osmiophilic particles in maize (Zea maysL.) coleoptiles

The dependence of auxin (IAA)-induced elongation growth on protein glycosylation was investigated in abraded maize (Zea mays L.) coleoptile segments, employing 2-deoxy-D-glucose (DOG) and tunicamycin (TUM) as inhibitors of protein glycosylation. TUM had no detectable effect on growth at 100 μg ml -1 . DOG impaired growth at concentrations larger than 1 mM. Total inhibition of growth occurred at a concentration of 20 mM. Similar effects were observed with mannose and glucosamine. The effect on wall-synthetic processes in the growth-limiting epidermis was analysed by tracer incorporation studies. Within 30 min hemicellulose and cellulose synthesis, measured as 3 H-glucose incorporation, was not affected by DOG, indicating that inhibition of growth is not causally related to synthesis of both wall components. In contrast, protein synthesis and secretion into the walls, measured as incorporation of 3 H-leucine into the TCA-precipitable protoplasmic and wall-bound protein, was rapidly inhibited by DOG. Concomitant with the effect on growth, DOG as well as mannose inhibited the occurrence of osmiophilic particles (OPs) which specifically occur at the growth-limiting epidermis during IAA-induced growth. The results provide evidence that IAA-induced wall loosening underlying elongation growth is dependent on O-glycosylation of proteins and their subsequent secretion into the epidermal walls. It appears that interference with these processes is responsible for inhibition of lAA-induced growth by hexoses acting as anti-glucose antimetabolites.

Characterization of the epitope recognized by a mAb that reacts differentially with murine suppressor T cells.

Although reliable antibodies are available that distinguish human suppressor T (Ts) cells from CTL and other T cells, few are available for murine Ts cells. We have developed a mAb (984D4.6.5) that, in the presence of complement, depletes alloantigen-specific Ts cells but not CTL. This antibody recognizes activated Ts cells but not their precursors. In these studies, flow cytometric analysis demonstrates that 984D4.6.5 reacts with several Ts cell hybridomas, cloned Ts cell lines and WEHI-3 (a myelomonocytic tumor cell line). Reactivity was not detected with BW5147, Th cell hybridomas, cloned Th cells, CTL lines and hybridomas, B cell lines, thymocytes, splenocytes, bone marrow cells nor a variety of tumor cells. Among 984D4.6.5 positive lines, expression is heterogeneous and the number of cells expressing high levels of the epitope is increased when the hybridomas are maintained at a relatively high cell density. Neuriminidase and pronase deplete the epitope recognized by mAb 984D4.6.5. Protein synthesis and glycosylation inhibitors also reduce expression of this epitope. These observations suggest that the epitope recognized by 984D4.6.5 is a carbohydrate linked to a polypeptide. This antibody was tested by ELISA for binding to a large panel of carbohydrates and glycolipids coupled to BSA. The only one that bound 984D4.6.5 was LS tetrasaccharide c (NeuNAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), an O-linked carbohydrate. Comparative analysis shows that both the sequence and the linkage of these sugars are essential to the reactivity with the 984D4.6.5 antibody. This epitope is expressed by a glycoprotein of approximately 200 kDa, as shown by Western blots. The identity of this glycoprotein remains to be determined, but indirect evidence suggests that it is not CD45.

Tunicamycin potently inhibits tumor necrosis factor-induced hepatocyte apoptosis

The protein glycosylation inhibitor tunicamycin protected male BALB/c mice from tumor necrosis factor α-induced liver failure. Tunicamycin also inhibited tumor necrosis factor-induced cell death in primary hepatocyte cultures with a median inhibitory concentration of 8 nM, but not in the tumor cell line WEHI 164 clone 13. Hepatocyte death in our culture system was characterized by DNA fragmentation and apoptotic changes. These two characteristic signs of programmed cell death were also inhibited by tunicamycin treatment. These data suggest that protein glycosylation is an early and causal event of tumor necrosis factor (TNF)-induced parenchymal cell death in the liver.

Further analysis of the upregulation of bradykinin B 1 receptors in isolated rabbit aorta by using metabolic inhibitors

Kinins exert a contractile effect that develops as a function of the in vitro incubation time with isolated rabbit aorta. This response is mediated via receptors of the bradykinin B 1 type and interleukin-1 amplifies this upregulation process. Tissues continuosly treated with the protein synthesis inhibitor cycloheximide (71 μM) or with the protein trafficking inhibitor, brefeldin A (18 μM), failed to develop a contractile response to the bradykinin B 1 receptor agonist, des-Arg 9-bradykinin (1.7 μM) (72−100% inhibition of kinin response recorded at 3 or 6 h), whether or not they were exposed to interleukin-1β (290 pM). The protein glycosylation inhibitor tunicamycin exerted a selective and significant, but partial (50−76%), inhibition of des-Arg 9-bradykinin- induced responses. The biochemical effect of the metabolic inhibitors on the tissue has been validated in assays involving incorporation of [ 3H]leucine and of [ 3H]mannose into protein or glycoprotein fractions, respectively. The modulatory effects of metabolic inhibitors on the responses to kinins of the isolated rabbit aorta support the idea that a de novo formation of membrane bradykinin B 1 receptors is the molecular basis of both the spontaneous and the interleukin-1-stimulated upregulation phenomenon.

Novel inhibitory action of tunicamycin homologues suggests a role for dynamic protein fatty acylation in growth cone-mediated neurite extension.

In neuronal growth cones, the advancing tips of elongating axons and dendrites, specific protein substrates appear to undergo cycles of posttranslational modification by covalent attachment and removal of long-chain fatty acids. We show here that ongoing fatty acylation can be inhibited selectively by long-chain homologues of the antibiotic tunicamycin, a known inhibitor of N-linked glycosylation. Tunicamycin directly inhibits transfer of palmitate to protein in a cell-free system, indicating that tunicamycin inhibition of protein palmitoylation reflects an action of the drug separate from its previously established effects on glycosylation. Tunicamycin treatment of differentiated PC12 cells or dissociated rat sensory neurons, under conditions in which protein palmitoylation is inhibited, produces a prompt cessation of neurite elongation and induces a collapse of neuronal growth cones. These growth cone responses are rapidly reversed by washout of the antibiotic, even in the absence of protein synthesis, or by addition of serum. Two additional lines of evidence suggest that the effects of tunicamycin on growth cones arise from its ability to inhibit protein long-chain acylation, rather than its previously established effects on protein glycosylation and synthesis. (a) The abilities of different tunicamycin homologues to induce growth cone collapse very systematically with the length of the fatty acyl side-chain of tunicamycin, in a manner predicted and observed for the inhibition of protein palmitoylation. Homologues with fatty acyl moieties shorter than palmitic acid (16 hydrocarbons), including potent inhibitors of glycosylation, are poor inhibitors of growth cone function. (b) The tunicamycin-induced impairment of growth cone function can be reversed by the addition of excess exogenous fatty acid, which reverses the inhibition of protein palmitoylation but has no effect on the inhibition of protein glycosylation. These results suggest an important role for dynamic protein acylation in growth cone-mediated extension of neuronal processes.

Expression and Localization of Plant Protein Disulfide Isomerase.

A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules.

INHIBITORY EFFECTS OF CONCANAVALIN A AND TUNICAMYCIN ON SEXUAL ATTACHMENT OF ALEXANDRIUM CATENELLA (DINOPHYCEAE)1

ABSTRACTThe effect of concanavalin A (a lectin) and tunicamycin (an inhibitor of protein glycosylation) on sexual attachment of gamete pairs of the dinoflagellate Alexandrium catenella Whedon &amp; Kofoid was studied. Concanavalin A inhibited sexual attachment at a concentration of 0.005%, and this inhibition was released by the addition of glucose or mannose at 10 mM. Mating type plus cells of A. catenella treated with tunicamycin for 12 h were not capable of sexual attachment. These results are the first to suggest the existence of a cell–cell recognition system in sexual attachment in A. catenella.

Similar responses to pharmacological agents of 1,2‐OAG–induced compaction‐like adhesion of two‐cell mouse embryo to physiological compaction

A comparison was made of responses to pharmacological agents between cell adhesion induced by an activator of Ca(2+)-phospholipid-dependent protein kinase (PKC) and physiological compaction in mouse embryos. An activator of PKC, 1-oleoyl-2-acetyl-sn-glycerol (1,2-OAG) induced the compaction-like adhesion of cells in two-cell embryos within 5-10 min and the adhesion lasted during the course of treatment for 1 h. W-7 and W-5 (calmodulin antagonists) and cytochalasin B and cytochalasin D (inhibitors of the polymerization of microfilaments) each completely interfered with the 1,2-OAG-induced adhesion of cells. Two-cell embryos having once shown evidence of cell adhesion in response to 1,2-OAG were decompacted when they were transferred to a medium that contained 1,2-OAG and any one of the agents described above. Colchicine and colcemid (inhibitors of the polymerization of microtubules) and tunicamycin (an inhibitor of N-linked protein glycosylation) each had no effect on the 1,2-OAG-induced adhesion of cells. In Ca(2+)-free medium, treatment with 1,2-OAG failed to induce any cell adhesion. These results are very similar to those reported for physiological compaction at the late eight-cell stage. Thus, the compaction-like adhesion of cells in mouse embryos at the two-cell stage appears to be a calmodulin-dependent process, requiring assembled microfilaments and extracellular Ca2+ ions but not microtubules or N-linked glycoproteins as is the case for the physiological compaction.

Simultaneous determination of sugar incorporation into glycosphingolipids and glycoproteins

We assessed inhibitors of glycosylation by simultaneous determination of [ 14C]Galincorporated into glycosphingolipids and glycoproteins as well as of [ 3H]Leu incorporated into proteins of intact cells. After metabolic labeling in 96-well plates in the presence or absence of a test substance, cells were collected on glass-fiber filters. The lipid components were extracted from the filter and radioactivities of both extract and filter determined. The reliability of the procedure was tested with different drugs. Using the glucocerebroside synthetase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP; 5 μ m), glycolipid biosynthesis was shown to be reduced to 50% in the murine T-cell EL-4 6.1 line, whereas glycosylation of proteins was not disturbed. With 0.5 μ m tunicamycin, the glycosylation of proteins was 50% of that in the control. The procedure was also able to detect various specific effects: the inhibition of protein glycosylation with d-glucosamine and castanospermine, the inhibition of glycosphingolipid biosynthesis with l-cycloserine, and a slight enhancement of glycosphingolipid biosynthesis with conduritol B epoxide and castanospermine. Within a series of N-acyl homologs of PDMP the inhibitory potency increased with chain length. In contrast, these homologs were equipotent by enzymatic in vitro assays.

Disruptions in intracellular membrane trafficking and structure preclude the glucocorticoid-dependent maturation of mouse mammary tumor virus proteins in rat hepatoma cells.

We have previously shown that glucocorticoids regulate the trafficking and processing of mouse mammary tumor virus (MMTV) proteins in viral-infected M1.54 rat hepatoma cells. To examine the role of intracellular membrane integrity on MMTV protein maturation, brefeldin A (BFA) was utilized to disrupt membrane flow between the endoplasmic reticulum and Golgi. Immunoprecipitation and immunofluorescence microscopy revealed that in the presence of dexamethasone, BFA inhibited the proteolytic processing, cell surface delivery, and externalization of MMTV glycoproteins. Glycosidase digestion and inhibitors of protein glycosylation confirmed that the observed differences in apparent sizes of MMTV glycoprotein products are due to BFA-induced changes in oligosaccharide processing. BFA treatment inhibited the proteolytic processing of the MMTV phosphoprotein precursor, which normally associates with the cytoplasmic face of intracellular membranes. Similarities in salt extraction efficiency revealed that BFA did not affect the membrane affinity of the uncleaved phosphorylated precursor. In a complementary approach, proteolytic processing of the phosphorylated polyprotein did not occur in glucocorticoid-treated HTC cells transfected with a mutant MMTV provirus encoding a normal phosphorylated precursor, but which express a truncated MMTV glycoprotein missing its transmembrane domain and cytoplasmic tail. These results suggest that the MMTV glycoproteins and phosphoproteins may interact at a late step in the transport pathway in a manner required for their mutual processing in response to glucocorticoids and establishes the importance of functional interactions with intracellular membranes for maturation of the cytoplasmic MMTV phosphoproteins.

Adaptive regulation of Na+‐dependent phosphate transport in the bovine renal epithelial cell line NBL‐1

1. Na(+)-dependent phosphate transport activity from bovine brush border membrane vesicles (BBMV) was reconstituted into artificial phospholipid membranes. It was shown, using lectin affinity chromatography, that transport activity was mediated by a glycoprotein. 2. The bovine kidney epithelial cell line NBL-1 expresses a Na(+)-dependent phosphate transporter which shows adaptive regulation in response to various media phosphate concentrations. Cells incubated in low-phosphate medium and medium containing 10 mM phosphate have Vmax values of 6.34 +/- 0.18 and 2.95 +/- 0.1 nmol.mg-1.3 min-1 and Km values of 17.1 +/- 3.0 and 36.3 +/- 7.14 microM for phosphate transport, respectively. Thus there is a significant (P less than 0.001) decrease in the Km and increase in the Vmax of transport when cells are transferred from 10 mM phosphate medium to low-phosphate medium. 3. Adaptation of phosphate transport in NBL-1 cells to low-phosphate medium was abolished by tunicamycin, an inhibitor of protein glycosylation, indicating that the transporter in these cells, like that in bovine BBMV, is a glycoprotein. 4. Cells were adapted to low-phosphate medium in the presence of [3H]mannose. Under these conditions a single membrane protein of molecular mass 55 kDa was labelled in the absence but not the presence of tunicamycin. Little labelling occurred if the cells were maintained in high-phosphate medium. 5. On the basis of these observations, it is proposed that the phosphate transporter of NBL-1 cells is a 55-kDa glycoprotein. Phosphate is accumulated across the brush border membrane of renal proximal tubule cells via a Na+/phosphate cotransport system. Na(+)-dependent phosphate transport has been extensively studied in isolated brush border membrane vesicles from rabbit and rat kidney [1-3]. The symport of phosphate and Na+ is electroneutral with a stoichiometry of 2Na+/phosphate at physiological pH values.

Nascent synthesis and secretion of cellobiase inNeocallimastix frontalis EB188

De novo synthesis and the secretion of cellobiase fromNeocallimastix frontalis EB188 were studied. Cellobiase was secreted rapidly in cellulose switch cultures. Chemical inhibition of protein synthesis and radiotracer studies suggested secretion was dependent on nascent protein synthesis. Inhibitors of protein glycosylation also inhibited secretion. An 85,000 dalton protein (and several others) represented the principal differences in de novo synthesis between cellulose- and glucose-switched cultures. Concentrations of the 85,000 daltons protein increased with culture incubation time and ultimately accounted for 7% of the total protein secreted. This protein was purified by gel electrophoresis separations and was determined to be a cellobiase. Secretion of this cellobiase was correlated with its radiolabeling. The possibility of cellobiase induction representing a specialized gene control system inNeocallimastix frontalis EB188 is discussed.

Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of protein glycosylation inhibitors: measurement of protein molecular weights and isoelectric focusing values

Protein and cellulase secreted in the presence of glycosylation inhibitors by Neocallimastix frontalis EB188 were studied using gel electrophoresis. Tunicamycin and 2-deoxy-D-glucose added to established cultures inhibited the production and secretion of proteins and cellulases. Schiff reagent staining of proteins after denaturing polyacrylamide gel electrophoresis confirmed the presence of extracellular glycoproteins. Intracellular or extracellular cellulases from cultures treated with inhibitors possessed distinct isoelectric focusing values and native gel Rf values. In N. frontalis EB188, glycosylation of protein occurred and was important for the production, secretion and activity of cellulases.