A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions

Previous results suggested that F2A8, a glycosylation mutant of Chinese hamster ovary cells, had a lower amount of dolichyl phosphate available for asparagine-linked glycosylation reactions relative to parental cells. The steady-state amounts and identities of polyisoprenoid lipids were determined by incubating F2A8, its parental cell line B4-2-1, and wild-type Chinese hamster ovary cells for 24 h with [2-3H]mevalonate. The neutral lipids, ubiquinone, cholesterol, and cholesteryl esters, which were the most highly labeled from [3H]mevalonate, were labeled equally in all three cell types. In wild-type and B4-2-1 cells, mevalonate incorporation into the anionic glycosylated and phosphorylated derivatives of dolichol was 10-fold higher than into the neutral free dolichol and dolichyl esters. In contrast, in F2A8 cells, label accumulated in neutral polyisoprenol lipids, so that the ratio of neutral to anionic lipids was 1:1 rather than 1:10. In wild-type and B4-2-1 cells, the polyisoprenoid found as free alcohol and in phosphorylated and glycosylated forms was shown by high pressure liquid chromatography using a silica column to be primarily dolichol, a polyisoprenol that has a saturated terminal isoprene unit. In contrast, in F2A8 cells the polyisoprenoid found primarily as the free alcohol and in phosphorylated and glycosylated forms appeared to be completely unsaturated polyprenol. The distribution of chain lengths of the labeled polyisoprenols of F2A8, B4-2-1, and wild-type cells was the same as determined by high pressure liquid chromatography using a reverse-phase column, with the predominant chain length being 19 isoprene units. These results combined with our previous studies on the phenotype of the F2A8 mutant indicate that the unsaturated polyprenyl phosphate derivatives do not function as well as dolichyl phosphate derivatives in cellular glycosylation reactions.     

Effects of dibutyryl cyclic AMP on the syntheses of dolichol-linked saccharides and glycoproteins in cultured hepatoma cells. Correlation with the effect on the adhesiveness of the cells.

When the hepatoma cells (AH 70Btc, Clone 10-5) were cultured in the presence of 1 mM-dibutyryl cyclic AMP for 2 days, the incorporation of [14C]glucosamine into protein was increased over 2-fold. At the same time, dibutyryl cyclic AMP increased the incorporation of [14C]glucosamine into dolichol-linked N-acetylglucosamine and NN'-diacetylchitobiose about 1.5-fold and into dolichol-linked oligosaccharides about 3-fold. Analysis of cellular glycoproteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction showed that dibutyryl cyclic AMP specifically enhanced the glycosylation of a fibronectin-like glycoprotein with an apparent mol.wt. of 220 000 and two other high-molecular-weight glycoproteins (apparent mol.wts. 270 000 and 185 000). Increased glycosylation of the glycoproteins with mol.wts. of 220 000 and 185 000 was shown to be linked to increased synthesis of the polypeptide portion. In addition to the above effects, dibutyryl cyclic AMP enhanced the adhesiveness of AH 70Btc cells to glass surfaces. Both the effects on the glycosylation pathway and on adhesiveness of cells were reversed by further treatment of the cells with 1 microgram of tunicamycin/ml. The results indicated that dibutyryl cyclic AMP increased the synthesis of dolichol-linked oligosaccharides and N-glycosylation of proteins in AH 70Btc cells. The enhancement of adhesiveness may be mediated by the increased synthesis of dolichol-linked oligosaccharides and also may be related to the increased synthesis of fibronectin.     

Glucoamylase overexpression and secretion in Aspergillus niger: analysis of glycosylation

We have studied the effects of overexpression and secretion of a homologous model glycoprotein, glucoamylase (GAM-1), on glycosylation in a single gene copy wild-type parent and multiple gene copy transformants of Aspergillus niger. In batch culture the B36 strain, which possess 80 additional copies of the GAM glaA gene, secreted about 5-8-fold more protein and GAM-1 than the parent strain (N402). A comparison of the glycosylation of GAM-1 secreted by the parent strain with that secreted by the multiple copy and hyper-secreting B36 strain showed that both the N-linked and O-linked glycan composition was very similar. Short oligomannose N-linked glycans were found (Man(7-8)GlcNAc(2)). O-Linked glycans were comprised of short (1-3 residues) oligosaccharide chains of mannose and galactose. Evidence is presented that this galactose is present in the novel galactofuranose conformation. This glycan composition of GAM-1 differed from that of a commercially available (A. niger) GAM source. Microsomes prepared from the mycelium showed a 2-3-fold co-ordinated increase in the activity of the dolichol phosphate:glycosyltransferases. Similar results were obtained from strains B1 (20 copies of glaA) and N402 when grown at a low dilution rate in a chemostat, although both the levels of GAM secretion and the activities of the dolichol phosphate:glycosyltransferases were lower than found in batch culture. These data suggest that A. niger is capable of secreting large amounts of a single glycoprotein combined with higher activity levels of the dolichol phosphate:glycosyltransferases without an increase in the heterogeneity of the glycan structures. Thus, from a biotechnological viewpoint, protein glycosylation may not be a bottleneck to enhanced glycoprotein production using A. niger.     

Bioactivity of amino terminal fragments of parathyroid hormone and parathyroid hormone related peptide in rat kidney slices: no effect of dietary phosphate deprivation

Humoral hypercalcemia of malignancy has been associated with the production of a recently cloned peptide human parathyroid hormone related protein (hPTHRP). One of the markers of this disease is an increased urinary excretion of cyclic AMP. The postreceptor mechanism of action and physiological role of hPTHRP remain obscure. To study the activity of hPTHRP 1-34 compared to rat and human parathyroid hormone (PTH) 1-34 we incubated these peptides with rat kidney slices and measured the cyclic AMP generated in the supernatant. hPTHRP 1-34 was equipotent with human PTH 1-34 but both were 5 times less active than rat PTH 1-34. Previous studies have suggested that a low dietary phosphate intake results in renal resistance to the phosphaturic action of PTH perhaps mediated by reduced adenylate cyclase activation by PTH. To determine whether, during dietary phosphate restriction, hPTHRP 1-34 has actions different from hPTH 1-34 we studied their effects following dietary phosphate deprivation. Dietary phosphate restriction had no significant effect on the cyclic AMP generating activity of any of the peptides. We conclude that hPTHRP 1-34 may be operating through similar mechanisms as human PTH 1-34 and that the previously observed effects of dietary phosphate deprivation on PTH mediated cyclic AMP generation in a broken cell preparation do not occur in intact cell preparations.     

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO2 fixation in the presence of dihydroxyacetone phosphate.

1. Dihydroxyacetone phosphate in concentrations greater than or equal to 2.5 mM completely inhibits CO2-dependent O2 evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of Pi, but not by addition of 3-phosphoglycerate. In the absence of Pi, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the 14C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70-95%. Based on these results the mechanism of the inhibition of O2 evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts. 2. Although O2 evolution is completely suppressed by dihydroxyacetone phosphate, CO2 fixation takes place in air with rates of up to 65 mu mol-mg1 chlorophyll-h1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation. 3. Under anaerobic conditions, the rates of CO2 fixation in presence of dihydroxyacetone phosphate are low (2.5-7 mumol-mg1 chlorophyll-h1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2-10(-7) M) reaching values of up to 60 mumol-mg1 chlorophyll-h1. As under these conditions the ATP necessary for CO2 fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO2 fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded t-at only under anaerobic conditions an "overreduction" of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5-10(-7) M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO2 fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO2 fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation. 4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO2 fixation in presence of dihydroxyacetone phoshate by dibromothymoquinone under anaerobic conditions indicated that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.     

New aspects of formation of 1,2-cyclic phosphates by phospholipase C-delta1

Phosphoinositide-specific phospholipase C-delta1 (PI-PLC-delta1) cleaves phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2), 1), 5-phosphate (PI-5-P, 2) and 4-phosphate (PI-4-P, 3) to form the mixture of the corresponding 4,5-, 5- and 4-phosphorylated inositol 1,2-cyclic phosphate (IcP) and 1-phosphate (IP) (4-6 and 7-9, respectively). In this work, we have studied the rates of the cleavage and the ratios of the cyclic-to-acyclic phosphate products under various pH and Ca(2+) concentration conditions using 31P NMR to monitor the reactions. In agreement with the previous report (Kim et al. Biochim. Biophys. Acta 1989, 163, 177), our results indicate that the IcP/IP ratios strongly depend on the reaction conditions, with the cyclic phosphate products formed predominantly at low pH (pH 5.0) and high calcium concentration (5 mM). Surprisingly, however, we have found that at pH 8.0 and 5 mM Ca(2+), PI-5-P rather than PI-4,5-P(2) is the most preferred substrate with the highest V(max). The cleavage of PI-5-P generated also more cyclic phosphate product than the other two substrates. In addition, we have studied the analogous reaction of phosphorothioate analogues of 1 with the sulfur placed in the nonbridging (10) or bridging (13) positions. We have found that the phosphorothioate analogue 10 produced exclusively the cyclic product 11, whereas the analogue 13 afforded exlusively the acyclic product 7. These results are discussed in terms of the mechanism of PI-PLC, where the cyclic product is formed by 'leaking' from the active site before its subsequent hydrolysis. The potential significance of the cyclic products in the signaling pathways is also discussed.     

Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.     

Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment

A coupled cell-free translation/glycosylation system, prepared from Spodoptera frugiperda insect cells, was established and optimized for protein production and glycosylation efficiency. Both translation and glycosylation were stimulated by addition of Mg2+, K+, ATP, GTP, creatine kinase and creatine phosphate, suggesting that glycoprotein productivity is largely determined by translation efficiency. However, high concentrations of creatine phosphate significantly inhibited translation. Spermidine stimulated both translation and glycosylation, but glycosylation required higher concentrations of spermidine than translation. Furthermore, extracts prepared at a nitrogen pressure of 10 kg/cm2 with the Mini-Bomb cell disruption chamber had the highest glycoprotein productivity; and extracts prepared at the higher nitrogen pressure of 15 kg/cm2 retained glycosylation ability. While extracts prepared with the Potter-Elvehjem homogenizer could mediate translation, no glycosylation was achieved. This indicated that the posttranslational machinery might survive disruption by high pressure, but not by physical shearing force. This insect cell-free system was able to synthesize approximately 25 microg of glycosylated gp120/ml of reaction mixture.     

Kinetic analysis of the formation of inositol 1:2-cyclic phosphate in carbachol-stimulated pancreatic minilobules. Half is formed by direct phosphodiesteratic cleavage of phosphatidylinositol

The issue as to whether there is direct phosphodiesteratic cleavage of phosphatidylinositol (PI), in addition to that of phosphatidylinositol 4,5-bisphosphate (PIP2), on agonist stimulation of cells has been controversial. In an attempt to resolve this issue, we have studied the kinetics of the formation and breakdown of the cyclic inositol phosphates. This approach is fairly straightforward, since the turnover of the cyclic inositol phosphates is very slow as compared to that of the noncyclic inositol phosphates and proceeds from inositol 1:2-cyclic 4,5-trisphosphate to inositol 1:2-cyclic phosphate (I(c1:2)P) directly by dephosphorylation without any branching pathways, in contrast to the multiple branchpoints of the noncyclic inositol phosphate pathway. Mouse pancreatic minilobules were prelabeled with [3H]inositol for 30 min, followed by washing to remove free inositol. They were then stimulated with carbachol for 30 min. The inositol cyclic polyphosphates reached steady state at 10-15 min, and I(c1:2)P reached steady state at 25 min. We blocked the action of carbachol by addition of an excess of atropine at 30 min, and the rate of disappearance of the three cyclic inositol phosphates was measured. From these data, the contribution of the inositol cyclic polyphosphate pathway to I(c1:2)P was calculated, which was 40-50% of total I(c1:2)P formation. Thus, 40-50% of the I(c1:2)P formed must have been derived from direct phosphodiesteratic cleavage of PI. This approach should prove useful in measuring the relative contributions of PI hydrolysis and PI phosphorylation (phosphatidylinositol 4,5-bisphosphate hydrolysis) in the overall PI cascade.     

Phosphorylation of cardiac troponin by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

The purpose of this investigation was to characterize the phosphorylation of bovine cardiac troponin by cyclic AMP-dependent protein kinase. The purified troponin-tropomyosin complex from beef heart contained 0.78 +/- 0.15 mol of phosphate per mol of protein. Analysis of the isolated protein components indicated that the endogenous phosphate was predominately in the inhibitory subunit (TN-I) and the tropomyosin-binding subunit (TN-T) of troponin. When cardiac troponin or the troponin-tropomyosin complex was incubated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, the rate of phosphorylation was stimulated by cyclic AMP and inhibited by the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase. The 32P was incorporated specifically into the TN-I subunit with a maximal incorporation of 1 mol of phosphate per mol of protein. The maximal amount of phosphate incorporated did not vary significantly between troponin preparations that contained low or high amounts of endogenous phosphate. The Vmax of the initial rates of phosphorylation with troponin or troponin-tropomyosin as substrates was 3.5-fold greater than the value obtained with unfractionated histones. The rate or extent of phosphorylation was not altered by actin in the presence or absence of Ca2+. The maximal rate of phosphorylation occurred between pH 8.5 and 9.0. At pH 6.0 and 7.0 the maximal rates of phosphorylation were 13 and 45% of that observed at pH 8.5, respectively. These results indicate that cyclic AMP formation in cardiac muscle may be associated with the rapid and specific phosphorylation of the TN-I subunit of troponin. The presence of endogenous phosphate in TN-T and TN-I suggests that kinases other than cyclic AMP-dependent protein kinase may also phosphorylate troponin in vivo.     

Oxyntomodulin and related peptides control somatostatin secretion in RIN T3 cells.

We studied the effects of oxyntomodulin (OXM), of its C-terminal (19-37) fragment (OXM (19-37)) and of glucagon (GLU) on somatostatin release, cyclic AMP accumulation and inositol phosphate turnover in somatostatin-secreting RIN T3 cells in culture. Rapid changes in cellular free Ca2+ were also measured using fura-2. Carbachol was used as a control test agent for the parameters involving the inositol phosphate/Ca2+ cascade. OXM, GLU and OXM (19-37) were all able to stimulate somatostatin release with relative ED50 of approx. 1, 22 and 45, respectively. OXM and GLU stimulated cyclic AMP levels with relative ED50 of approx. 1 and 30, respectively, whereas OXM (19-37) was totally ineffective on this parameter. In contrast to carbachol, none of the peptides significantly modified the inositol phosphate turnover or induced rapid changes in cellular free Ca2+. We conclude that the RIN T3 cells contain a receptor-cyclic AMP system similar to that found in gastric mucosa and that this system is linked to somatostatin release. Another receptor-second messenger mechanism linked to somatostatin release is triggered by the (19-37) fragment. This mechanism is not the inositol phosphate/Ca2+ cascade triggered in the same cells by cholinergic agents.     

Inhibition of beta-lactamases by monocyclic acyl phosph(on)ates

The cyclic acyl phosph(on)ates, 1-hydroxy-5-phenyl-2,6-dioxaphosphorinone(3)-1-oxide, its 4-phenyl isomer, and the phosphonate (2-oxo) analogue of the latter inhibited typical class A (TEM-2) and class C (Enterobacter cloacae P99) beta-lactamases in a time-dependent fashion. No enzyme-catalyzed turnover was detected in any case. The interactions occurring were interpreted in terms of the reaction scheme E + I left arrow over right arrow EI left arrow over right arrow EI', where EI is a reversibly formed noncovalent complex, and EI' is a covalent complex. Reactions of the cyclic phosphates with the P99 beta-lactamase were effectively irreversible, while that of the 4-phenyl cyclic phosphate with the TEM beta-lactamase was slowly reversible. The 4-phenyl cyclic phosphate was generally the most effective inhibitor, both kinetically and thermodynamically, with second-order rate constants of inactivation of both enzymes around 10(4) s(-1) M(-1). This compound also bound noncovalently to both enzymes, with dissociation constants of 25 microM from the P99 enzyme and 100 microM from the TEM. It is unusual to find an inhibitor equally effective against the TEM and P99 enzymes; the beta-lactamase inhibitors currently employed in medical practice (e.g., clavulanic acid) are significantly more effective against class A enzymes. The results of lysinoalanine analysis after hydroxide treatment of the inhibited enzymes and of a (31)P nuclear magnetic resonance spectrum of one such complex were interpreted as favoring a mechanism of inactivation by enzyme acylation rather than phosphylation. Molecular modeling of the enzyme complexes of the 4-phenyl phosphate revealed bound conformations where recyclization and thus reactivation of the enzyme would be difficult. The compounds studied were turned over slowly or not at all by acetylcholinesterase and phosphodiesterase I.     

A Protein Kinase Activity Is Associated with and Specifically Phosphorylates the Neural Cell Adhesion Molecule LI

The neural cell adhesion molecule L1 is a phosphorylated integral membrane glycoprotein that is recovered from adult mouse brain by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, 80, and 50 kilodaltons (L1-200, L1-180, L1-140, L1-80, and L1-50, respectively). In the present study, we show that two kinase activities are associated with immunopurified L1: One specifically phosphorylates L1-200 and L1-80 but not L1-180, L1-140, or L1-50. This pattern of phosphorylation corresponds to the one described for L1 after metabolic phosphate incorporation into cultures of cerebellar cells. In both cases, serine is the main amino acid that is labeled by radioactive phosphate. The kinase activity is not activated by Ca2+, calmodulin, phosphatidylserine, diolein, cyclic AMP, or cyclic GMP, a result suggesting that the enzyme is distinct from Ca2+/calmodulin-dependent kinases, from protein kinase C, or from cyclic AMP/cyclic GMP-dependent kinases and may belong to the independent kinase group. The other kinase phosphorylates only casein but not L1, utilizes GTP as well as ATP, and is strongly inhibited by heparin. Because the primary structure of the L1 protein does not contain consensus sequences characteristic for known kinases, we believe that the catalytic activities detectable in immunopurified L1 are due to kinases that are strongly enough associated with L1 to withstand the stringent purification procedures.     

Mevalonate-regulated mechanisms in cell growth control: role of dolichyl phosphate in expression of the insulin-like growth factor-1 receptor (IGF-1R) in comparison to Ras prenylation and expression of c-myc

One or more mevalonate derivatives of non-sterol type have beenproposed to be of indispensable importance for cell growth.Conceivable mevalonate-dependent mechanisms involved in growthcontrol are farnesylation of Ras proteins, regulation of c-mycexpression, and N-linked glycosylation of the IGF-1 receptor.The latter mechanism might be rate-limited by dolichyl phosphate,which acts as a donor of oligosaccharides in glycoprotein synthesisin the endoplasmic reticulum. In order to study the significancefor cell proliferation of the three aforementioned mevalonate-dependentprocessings, their inhibitory response due to mevalonate deprivationwas explored and compared with the effect on DNA synthesis inthe malignant melanoma cell line SK-MEL-2. We found that mevalonatedepletion due to treatment with 3 µM lovastatin for 24h, which efficiently growth-arrested the cells, hardly at allaffected the expression of c-myc, and although Ras prenylationwas inhibited by 50%, the most pronounced effect of lovastatinwas seen on N-linked glycosylation of IGF-1 receptors, whichwas inhibited by more than 95%. The order and magnitude of thedecreased IGF-1 receptor glycosylation, which was followed bya decreased expression of IGF-1 receptors at the cell membrane,correlated well with the inhibition of DNA synthesis. We investigatedwhether dolichol, and in particular dolichyl phosphate, throughits participation in N-linked glycosylation, act as regulatorsof IGF-1 receptor expression. First, we could confirm that exogenousdolichol became phosphorylated and in this form took part inthe glycosylation processing. Secondly, we showed that dolichylphosphate, in a dose-dependent manner, could increase the numberof IGF-1 receptors at the cell membrane, simultaneously as DNAsynthesis was stimulated. Taken together, our results providedirect evidence for an important role of dolichyl phosphateas a regulator of cell growth through limiting N-linked glycosylationof the IGF-1 receptor. dolichyl phosphate IGF-1 receptor c-myc N-linked glycosylation Ras     

Substrate specificity of N-acetylglucosamine 1-phosphate transferase activity in Chinese hamster ovary cells.

The assembly pathway of the oligosaccharide chains of asparagine-linked glycoproteins in mammalian cells begins with the formation of GlcNAc-PP-dolichol in a reaction catalysed by the enzyme N-acetylglucosamine 1-phosphate transferase. We have investigated the efficiency of two lipid substrates for the transferase activity in an in vitro assay using Chinese hamster ovary (CHO) cell membranes as an enzyme source. Experiments were carried out with varying concentrations of dolichyl phosphate or its precursor, polyprenyl phosphate. We determined that enzyme activity was optimal at pH 9, where the enzyme exhibited a 3-fold higher Vmax and a 2-fold lower Km for the dolichol substrate. At pH 7.4, the Km and Vmax differences between the two lipids were 10-fold. Under all assay conditions tested, we found that GlcNAc-PP-lipid was the only product formed. We conclude from these results that dolichyl phosphate rather than polyprenyl phosphate is the preferred substrate for the transferase enzyme in CHO cells. This observation is significant in light of the fact that we have previously isolated CHO glycosylation mutants which fail to convert polyprenol into dolichol, and hence utilize polyprenyl derivatives for glycosylation reactions. Thus, these results contribute to our understanding of the glycosylation defects in the mutant cell lines.     

Cyclic AMP mediates beta-adrenergic-induced increases in N-linked protein glycosylation in rat parotid acinar cells.

beta-Adrenergic stimulation of rat parotid cells by isoprenaline (isoproterenol) results in 2-3-fold increases in [3H]mannose incorporation into N-linked oligosaccharides. This occurs without perceptible lag and is linear with time for 60 min after agonist addition. Concomitantly, isoprenaline markedly increases cellular cyclic AMP. Examination of individual proteins by sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis reveals that glycosylation changes are primarily associated with four secretory proteins, of approx. Mr 17000, 32000, 38000 and 220000. Beta-Adrenoreceptor activation additionally elicits a slight increase in parotid protein synthesis. The greatest increase in [14C]leucine incorporation is that into another secretory protein (Mr approx. 24000). Exposure of cells to dibutyryl cyclic AMP yields results comparable with those after isoprenaline treatment. Forskolin, which increases parotid-cell cyclic AMP, also causes similar effects. Conversely, dibutyryl cyclic GMP shows no such response. The data are consistent with the notion that beta-adrenergic stimulation of N-linked protein glycosylation in rat parotid cells is mediated by cyclic AMP.     

Degradation of the cyclic AMP antagonist prostaglandylinositol cyclic phosphate (cyclic PIP) by dephosphorylation

The cAMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), is synthesized from prostaglandin E and activated inositol phosphate. From various tissues only that amount of cyclic PIP can be isolated that constitutes the difference between synthesis and degradation. In order to overcome this drawback, the cyclic PIP degrading enzyme or enzymes had to be characterized prior to searching for inhibitors. Cyclic PIP degrading activities have been found in all rat tissues tested, and are lowest in brain (380 pmol x min(-1) x g(-1) wet weight) and highest in liver (1460 pmol x min(-1) x g(-1) wet weight). They are associated primarily with particulate structures of the cells, but not with the plasma membrane. There appear to be at least two different enzymatic activities involved in the degradation of cyclic PIP, because there are two pH-optima, one between pH 7 and 8 and another between pH 4 and 5. It is assumed that these activities are located in microsomes and lysosomes. Because prostaglandylinositol is the final product obtained in the degradation of cyclic PIP, a phosphodiesterase and a phosphatase should be involved, which could not yet be identified individually. Like alkaline phosphatase, cyclic PIP-degrading enzymes require Mg2+ and they are inhibited by heavy metal ions such as mercuric and copper chloride, by sodium fluoride and interestingly, by prostaglandins.     

The study of the reaction of terminated oligomerization in the synthesis of oligo-(β1-6)-glucosamines

The applicability of terminated oligomerization to the synthesis of oligo-(β1-6)-glucosamines, fragments of the intercellular polysaccharide adhesin of staphylococci, was studied. The reactions of terminated oligomerization were carried out with mono-, di-, and trisaccharide monomers and N-protected aminopropanol; and spacered mono-and disaccharides as terminating molecules were also attempted. The primary formation of cyclic products of monomer intramolecular glycosylation was observed in almost all the reactions. Only the experiments with the monomer based on the disaccharide bromide under the conditions of the Helferich reaction led to reduced yields (30%) of the cyclic products. However, even in this case, the desired terminated oligosaccharides were generated in approximately 10% yield and mainly were the products of single glycosylation of the terminator by the monomer. These experiments allow the conclusion that, under the examined conditions, the reaction of terminated oligomerization could not result in the synthesis of oligoglucosamines with a high molecular mass.     

The study of the reaction of terminated oligomerization in the synthesis of oligo-(beta1-6)-glucosamines

The applicability of terminated oligomerization to the synthesis of oligo-(beta1-6)-glycosamines, fragments of the intercellular polysaccharide adhesin of staphylococci, was studied. The reactions of terminated oligomerization were carried out with mono-, di-, and trisaccharide monomers and N-protected aminopropanol; and spacered mono- and disaccharides as terminating molecules were also attempted. The primary formation of cyclic products of monomer intramolecular glycosylation was observed in almost all the reactions. Only the experiments with the monomer based on the disaccharide bromide under the conditions of the Helferich reaction led to reduced yields (30%) of the cyclic products. However, even in this case, the desired terminated oligosaccharides were generated in approximately 10% yield and mainly were the products of single glycosylation of the terminator by the monomer. These experiments allow the conclusion that, under the examined conditions, the reaction of terminated oligomerization could not result in the synthesis of oligoglucosamines with a high molecular mass.     

Stereoselective Synthesis of 2-Deoxy-2-Fluoroarabinofuranosyl-α-1-Phosphate and Its Application to the Synthesis of 2′-Deoxy-2′-Fluoroarabinofuranosyl Purine Nucleosides by a Chemo-Enzymatic Method

Stereoselective introduction of a phosphate moiety into 2-deoxy-2-fluoroarabinofuranose derivatives at the anomeric position was investigated by two methods. One involved a stereoselective hydrolysis of 1-bromo-derivative, and the consecutive phosphorylation of 2-deoxy-2-fluoro-α-D-arabinofuranose via a phosphoramidite derivative. The other method involved stereoselective α-phosphorylation of the 1-bromo-derivative at the 1-position. The resulting α-1-phosphate was utilized to prepare 2′-deoxy-2′-fluoroarabinofuranosyl purine nucleosides by an enzymatic glycosylation reaction. This chemo-enzymatic method will be applicable to the synthesis of some 2′F-araNs, and three important 2′F-araNs were actually obtained in 30–40% yields from 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinose with high purity.