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S Mookerjea

Publications and source records attributed to S Mookerjea.

At least 37 records · Page 2Linked to original sources
PubMed1988

Differential effect of inflammation and dexamethasone on dolichol and dolichol phosphate synthesis.

Inflammation and glucocorticoids stimulate hepatic glycoprotein synthesis, resulting in an increased secretion of serum glycoproteins. We now present evidence that the synthesis of dolichol and dolichol phosphate from mevalonate is increased in hepatocytes from inflamed rats. Also, in inflamed rats, the levels of dolichol and dolichol phosphate are increased in liver homogenates and microsomes. Dexamethasone treatment of the cells, however, does not increase the synthesis of dolichol and dolichol phosphate from mevalonate. The results suggest that the inflammation-induced dolichol-linked saccharide and glycoprotein synthesis is possibly mediated through an increase in the level of dolichol and dolichol phosphate in the liver. Since dexamethasone treatment does not increase the synthesis of dolichol and dolichol phosphate, its action on glycoprotein synthesis appears to be different and to affect the induction of enzymes in mannosyl phosphoryl dolichol- and dolichol-linked oligosaccharide synthesis.

Animals↗
PubMed1987

A study on the selective binding of apoprotein B- and E-containing human plasma lipoproteins to immobilized rat serum phosphorylcholine-binding protein.

Rat serum phosphorylcholine-binding protein (PCBP), a member of the pentraxin family of proteins, was previously shown to bind multilamellar liposomes prepared with egg phosphatidylcholine and lysophosphatidylcholine. The results suggested that the phosphorylcholine groups on the surface of liposomes play an important role in the binding process (Nagpurkar, A., Saxena, U., and Mookerjea, S. (1983) J. Biol Chem. 258, 10518-10523). A study on the binding of human plasma lipoproteins to PCBP immobilized on Sepharose has now been initiated. Very low density lipoproteins were partially bound to a Sepharose-PCBP column, and the bound fraction contained higher concentrations of apoprotein B and E. All the low density lipoproteins applied were bound to the column. In the case of high density lipoproteins, only a small fraction was retained on the column (based on protein analysis), and that bound fraction contained all the apoprotein E and Lp(a) lipoprotein. The binding of very low, low, and high density lipoproteins to Sepharose-PCBP was Ca2+-dependent, and the bound lipoproteins were quantitatively eluted by a phosphorylcholine gradient. Apoprotein B and E were also bound when whole human plasma was applied to Sepharose-PCBP. The effect of selective modification of lysine residues by acetoacetylation and of arginine residues by cyclohexanedione on the binding of low density lipoproteins to Sepharose-PCBP was examined. Modification of arginyl residues resulted in marked reduction of binding, whereas modification of lysine had no effect. Removal of sialic acid from PCBP also had no effect on the binding of low density lipoproteins to immobilized-desialylated PCBP column. The preferential binding of apoprotein B- and E-containing lipoproteins to Sepharose-PCBP indicates a possible physiological role of PCBP and other similar circulating phosphorylcholine-binding proteins of the pentraxin family in lipoprotein metabolism.

Animals↗
PubMed1987

Characterization of serum, liver, and intestinal sialyltransferases from rats treated with colchicine.

A modified high pressure liquid chromatographic method using lactose (Gal beta 1----4Glc) as an exogenous acceptor has been used to characterize the sialyltransferases known to increase in the serum of colchicine-treated rats. The results show a 10-fold increase of Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase (alpha 2----6 ST), whereas the Gal beta 1----3GlcNAc alpha 2----3 sialyltransferase showed only 1.6-fold increase in the serum after 17 h of colchicine treatment. The sialyltransferase activity in serum using exogenous desialylated, alpha 1-acid glycoprotein as acceptor also showed an eightfold increase. In liver homogenate and Golgi membrane, the sialyltransferase activity when assayed with desialylated alpha 1-acid glycoprotein as acceptor showed a slight decrease after 4 h, but returned to normal level after 17 h. A similar trend was seen when the two transferases were assayed with lactose as acceptor. The antiserum to rat alpha 2----6 ST inhibited the sialyltransferase activity in serum, liver, and jejunal incubation medium. Jejunal sections from rats treated with colchicine for 4 h in presence of heated serum showed a decrease of sialyltransferase, with consequent increase of the alpha 2----6 ST enzyme activity in the medium. This result suggests that intestinal tissue could be a source of increased serum enzyme activity in colchicine treatment.

Animals↗
PubMed1987

The role of the female Syrian hamster protein on the interaction between serum lipoproteins and heparin.

We have previously shown the effect of phosphorylcholine-binding proteins from rat (PCBP) and rabbit (CRP) on the precipitation of serum lipoproteins by heparin in presence of Ca2+. The present paper describes the effect of a phosphorylcholine-binding protein from the female Syrian hamster (FP) on the lipoprotein precipitation reaction. The precipitation of lipoproteins by heparin was lower in assays using female hamster serum in which FP is a prominent protein, compared with assays with male serum in which FP is present in very low concentration. Depletion of FP from female serum resulted in increased lipoprotein precipitation. The addition of purified FP to assays using human very low density lipoprotein (VLDL) inhibited the precipitation reaction. The precipitation of lipoproteins was also examined using serum from male hamsters treated with diethylstilbestrol and female hamsters treated with testosterone, treatments known to modulate the levels of FP. Results indicate an inverse relationship between serum FP levels from normal and hormone-treated hamsters and the precipitation of lipoproteins from their serum. The partially desialylated FP when added to precipitation assays using human VLDL resulted in reduced inhibition of VLDL precipitation.

Animals↗
PubMed1986

Inhibition of the binding of low density lipoproteins to liver membrane receptors by rat serum phosphorylcholine binding protein.

Rat serum phosphorylcholine binding protein (PCBP) is characterized by its Ca2+ dependent property to bind phosphorylcholine ligand. PCBP immobilized on sepharose has been shown to selectively bind human plasma apo B and E containing lipoproteins. The present report describes an inhibitory effect of PCBP on the binding of human 125I-LDL to LDL receptors on estradiol treated rat liver membranes. Pre-incubation of liver membranes with PCBP did not affect the binding of 125I-LDL to the membranes. Gel filtration analysis of the incubation products from the LDL-receptor assay showed a concentration dependent binding of 125I-PCBP to LDL. The inhibitory effect of PCBP is likely due to the formation of LDL-PCBP complex and not due to the binding of PCBP to the LDL receptor site.

Animals↗
PubMed1986

Recovery of dolichyl diphosphate oligosaccharide in methanolic aqueous phase prepared from rat liver microsomal fractions.

The synthesis of dolichyl diphosphate oligosaccharide was studied by incubating rat liver microsomes (microsomal fractions) with GDP-[14C]mannose, UDP-glucose, UDP-N-acetylglucosamine and [3H]dolichol phosphate. The labelled products obtained by the first step of extraction of the microsomes in methanolic aqueous phase (MAP fraction in chloroform/methanol/water; 3:2:1, by vol.) and in CMW fraction (chloroform/methanol/water; 10:10:3, by vol.) obtained by extraction of the interphase after the first step of extraction were analysed on a DEAE-cellulose column. With the progress of incubation, the radioactivity in unchanged GDP-mannose decreased, whereas the labelled dol-P-P-oligo in the MAP fraction increased about 5-6-fold. The lipid oligosaccharide in this fraction accounted for about 50-60% of the GDP-mannose used, whereas the recovery of the labelled lipid oligosaccharide in the CMW fraction was about 10%. The lipid oligosaccharide from both reactions after mild acid hydrolysis were analysed by gel filtration on Bio-Gel P-4. The oligosaccharide from the MAP fraction gave a peak of higher Mr distinctly separate from the lower-Mr peak obtained from the CMW fraction. Microsomes incubated with labelled lipid oligosaccharide from the MAP fraction showed incorporation of the label into endogenous protein.

Animals↗
PubMed1986

Activity and secretion of sialyltransferase in primary monolayer cultures of rat hepatocytes cultured with and without dexamethasone.

Monolayers of hepatocytes attached on collagen-coated dishes were cultured for 20-24 h and were found suitable to study the activity and secretion of CMP-N-acetylneuraminate:asialo-alpha 1-acid glycoprotein sialyltransferase. A progressive increase of sialyltransferase activity in the culture medium was observed during incubation of the hepatocytes. After 24 h 34-48% of the total sialyltransferase activity of the hepatocyte incubation system was present in the medium. The enzyme activity present in the medium was soluble in nature and could not be stimulated by Triton X-100. The secretion of the enzyme was stimulated about twofold by dexamethasone. The activity of sialyltransferase in the hepatocytes was also increased by dexamethasone. The Km of either hepatocyte or medium sialyltransferase for CMP-sialic acid was only slightly changed by dexamethasone, whereas the Vmax was increased about twofold. The secretion of sialyltransferase could be inhibited partially by the anti-microtubular agent colchicine. The dexamethasone-induced increase of the sialyltransferase activity in cells and media could be eliminated by inclusion of alpha-amanitin in the culture media at 0 h. The inhibiting effect of alpha-amanitin was only partially expressed when the drug was added 4 h after the addition of dexamethasone to the media. The results suggest that isolated rat hepatocytes actively secrete sialyltransferase and that the increase in the sialyltransferase activity in cells and media owing to the synthetic glucocorticosteroid dexamethasone results from increased synthesis of the enzyme molecule. It is supposed that in the intact rat the increased levels of the enzyme activity in serum observed in inflammation may originate from an induction of the synthesis of sialyltransferase in the hepatocytes of rat liver by the increased levels of circulating corticosteroids.

Amanitins↗
PubMed1985

Effect of dexamethasone on the synthesis of dolichol-linked saccharides and glycoproteins in hepatocytes prepared from control and inflamed rats.

Hepatocytes were prepared from control and inflamed rats. The incorporation of [14C]mannose into protein was increased in inflamed compared with control hepatocytes. The incorporation of [14C]mannose into protein was also increased when the hepatocytes were cultured in presence of dexamethasone (1 microM), either from control or inflamed rats. At the same time the incorporation of [14C]mannose into dolichol phosphate mannose and dolichol-linked oligosaccharide was increased due to inflammation. The presence of dexamethasone in the hepatocyte culture caused an increased formation of these two products; in particular its effect on oligosaccharide lipid formation was very pronounced. The ratios of activities of formation of [14C]mannose-labelled oligosaccharide lipid in inflamed over control hepatocytes gradually decrease when increasing amounts of exogenous dolichol phosphate was added in cell homogenate assay mixture. These results suggest that the increase of oligosaccharide lipid formation in inflammation could be due to a higher concentration of endogenous dolichol phosphate, as was shown for dolichol phosphate mannose formation in inflammation [Sarkar & Mookerjea (1984) Biochem. J. 219, 429-436]. In contrast, the ratio of activities of [14C]mannose-labelled oligosaccharide lipid between dexamethasone-treated and untreated hepatocytes shows only a slight increase when increasing concentrations of exogenous dolichol phosphate were added to the assays. This suggests that the stimulation of dolichol pyrophosphate oligosaccharide synthesis observed in dexamethasone treatment is probably due to the higher level of enzymes involved in oligosaccharide synthesis rather than higher level of endogenous dolichol phosphate in these cells.

Animals↗
PubMed1985

Contrasting effect of phosphorylcholine-binding protein from rat and rabbit on heparin-lipoprotein interaction: a role of sialic acid.

The effect of rat phosphorylcholine-binding protein (PCBP) and rabbit C-reactive protein (CRP) on the serum lipoprotein-heparin-Ca2+ precipitation reaction has been examined. Rabbit CRP, a nonglycosylated acute-phase protein, has been isolated from the serum after turpentine-induced inflammation following an isolation procedure similar to that used for rat PCBP. The mobilities of rabbit CRP and rat PCBP on polyacrylamide gel electrophoresis and immunoelectrophoresis have been found to be different. Rabbit CRP, even though a P-choline binding protein, was found to have no inhibitory effect on the serum lipoprotein-heparin-Ca2+ precipitation reaction. Desialylated rat PCBP obtained by enzymatic desialylation of rat PCBP no longer inhibited the precipitation reaction, even though it retained its P-choline binding property and immunochemical identity with rat PCBP. The results suggest that the binding of rat PCBP and rabbit CRP to the P-choline moiety is not a sufficient requirement, and it now appears that the sialic acid moiety must also be present on these proteins to observe the inhibition of lipoprotein precipitation. This requirement is fulfilled by rat PCBP, but not by rabbit CRP which is nonglycosylated.

Animals↗
PubMed1984

Synthesis and secretion of serum phosphorylcholine-binding protein by rat hepatocytes.

The incorporation of [1-14C]glucosamine into rat serum phosphorylcholine-binding protein in an isolated rat hepatocyte system was used to demonstrate the synthesis and secretion of this protein by the liver. The hepatocytes after incubating with colchicine resulted in an increased intracellular accumulation of phosphorylcholine-binding protein and less of the synthesized phosphorylcholine-binding protein was secreted into the medium. The synthesis of phosphorylcholine-binding protein was found to be significantly impaired when the hepatocytes were incubated with tunicamycin. The radiolabelled phosphorylcholine-binding protein co-eluted with exogenous phosphorylcholine-binding protein as a homogeneous peak by affinity chromatography. The identity of the radiolabelled phosphorylcholine-binding protein was further established by quantitative immunoprecipitation, polyacrylamide gel electrophoresis and isoelectric focusing.

Animals↗
PubMed1984

Increase of sialyltransferase activity in the serum and liver of inflamed rats.

Induction of inflammation by turpentine injection caused 1.5-2-fold increase of both sialyl- and galactosyltransferase activity in liver homogenates. The effect was apparent after 12 h of turpentine treatment. Serum sialyltransferase activity started to increase in the inflamed rats after 18 h, reaching a maximum of 4-fold at 48 h. In contrast, galactosyltransferase activity in serum showed no significant increase. The coordinated and temporal increase of sialyltransferase activity in liver and serum suggest involvement of a specific mechanism for the preferential release of this enzyme into serum.

Animals↗
PubMed1984

Characterization and partial purification of two enzymes transferring N-acetylglucosamine to dolichyl monophosphate and ribonuclease A.

Two N-acetylglucosamine (GlcNAc) transferases which catalyze the incorporation of GlcNAc into GlcNAc-P-P-dolichol (dolichol enzyme) and into bovine pancreatic ribonuclease A (RNAseA enzyme) were solubilized from the rat liver microsomes in a non-ionic detergent, Triton X-100. Both enzyme activities were adsorbed on activated CH-Sepharose 4B, and could be eluted with a linear KCl gradient. Two enzyme activities were separated by this column with the dolichol enzyme eluting before the RNAseA enzyme. A 49-fold and 136-fold purification was achieved for the dolichol and the RNAseA enzyme, respectively. The addition of exogeneous dolichyl phosphate resulted in a 3-5-fold stimulation of the purified dolichol enzyme, but did not affect the purified RNAseA enzyme. The addition of RNAseA stimulated only the RNAseA enzyme. Whereas, tunicamycin could inhibit only the dolichol enzyme. The purified dolichol enzyme had a Km of 14 X 10(-6) M for UDP-GlcNAc and the reaction was saturated with about 0.25 M dolichyl phosphate. The purified RNAseA enzyme had a Km of 4.55 X 10(-6) M for UDP-GlcNAc and was saturated with about 0.36 mM RNAseA. The pH optima and the metal ion requirement for the two enzymes were different. These results suggest that because of the different properties of these two enzymes they may have distinct functions regarding the core glycosylation of N-linked glycoproteins. It is well established that the dolichol enzyme catalyzes the formation of the first dolichol-linked intermediate GlcNAc-P-P-dolichol, whereas according to the present finding, the RNAseA enzyme may catalyze the transfer of GlcNAc directly from UDP-GlcNAc into acceptor protein.

Animals↗
PubMed1984

Effect of dexamethasone on mannolipid synthesis by hepatocytes prepared from control and inflamed rats.

Hepatocytes were prepared from control and inflamed rats. Mannose incorporation into dolichol monophosphate mannose in homogenate and microsomal fraction of the hepatocytes was increased 2-fold over the controls 24 h after induction of inflammation by turpentine injection. Incubation of hepatocytes from both control and inflamed rats with 0.1-10 microM-dexamethasone produced a 1.5-fold increase of dolichol phosphate mannose formation, whereas, 100 microM-dexamethasone decreased its formation. The increase in the ratio of dolichol phosphate mannose formation in inflamed over controls was virtually eliminated when the cell homogenate assay mixtures included 30 nmol of exogenous dolichol phosphate. This supports the earlier suggestion that the increase in the enzyme activity in inflammation could be due to higher concentrations of endogenous dolichol phosphate [ Coolbear & Mookerjea (1981) J. Biol. Chem. 256, 4529-4535]. In contrast, the increase in the ratio of dolichol phosphate mannose formation between dexamethasone-treated and untreated hepatocytes remained unchanged when increasing concentrations of exogenous dolichol phosphate were added to the assays. This suggests that the increase in glycosylation of dolichol phosphate in dexamethasone-treated hepatocytes is probably due to the increased mannosyltransferase activity, rather than due to higher concentrations of endogenous dolichol phosphate in these cells.

Animals↗
PubMed1983

Interaction of rat serum phosphorylcholine-binding protein with phospholipid-containing liposomes.

Rat serum phosphorylcholine-binding protein (PCBP) has been shown to inhibit the Ca2+-modulated heparin-lipoprotein precipitation reaction. This effect of PCBP on the reaction is prevented by phosphorylcholine (P-choline). A stoichiometric relationship between the serum very low density lipoproteins and PCBP was also evident in the heparin-very low density lipoprotein precipitation reaction (Nagpurkar, A., and Mookerjea, S. (1981) J. Biol. Chem. 256, 7440-7448). A study on the binding of PCBP to artificial liposomes was initiated to understand the mechanism of interaction between PCBP and phospholipids in soluble lipoproteins. Radioiodinated PCBP was incubated with multilamellar liposomes prepared with egg phosphatidylcholine (PC) and lysophosphatidylcholine, and it was found that the binding of PCBP to multilamellar liposomes was Ca2+-dependent and required the incorporation of about 25% lysophosphatidylcholine into the liposomes. Furthermore, the binding could be inhibited by addition of P-choline. The optimum concentrations of Ca2+ and liposomes as well as time and temperature required for binding were established. Analysis of Scatchard binding data yielded an association constant K alpha of 1.8 X 10(6) M-1 and a total binding capacity of 0.96 nmol/mumol of phospholipid. Substitution of P-choline head groups by phosphorylethanolamine and phosphorylserine on the PC of liposomes reduced the binding considerably, whereas the substitution of fatty acyl moieties on the PC of liposomes was without any effect. Bovine serum albumin was required in the assay to prevent artifactual binding of 125I-PCBP to the assay tubes. The results suggest that the P-choline groups on the surface of liposomes play an important role in the binding to PCBP and this may provide a possible explanation of the effect of PCBP on the Ca2+-dependent heparin-lipoprotein precipitation reaction.

Animals↗
PubMed1983

Key role of dolichol phosphate in glycoprotein biosynthesis.

Studies on the developmental changes in oviducts of hormone-treated chicks and embryos of sea urchins have indicated that the level of dolichol phosphate in the tissues may serve as a control for asparagine-linked glycoprotein biosynthesis. Acute-phase reactant glycoprotein biosynthesis is greatly increased in inflamed rats given a single injection of turpentine. As most of the serum glycoproteins are synthesized via the dolichol pathway, the rate of synthesis of mannosyl and glucosyl dolichol monophosphate and of glucosyl dolichol pyrophosphoryl oligosaccharide, CTP-dependent dolichol phosphokinase, and the level of dolichol phosphate were measured in the livers of inflamed rats. The activities of the glycosyltransferases were increased at least twofold as a result of inflammation. It was also observed that dexamethasone treatment reversed the inflammation-induced increase of mannosyl and glucosyl transfer to dolichol monophosphate. The endogenous level of dolichol phosphate and dolichol kinase activity were increased in microsomes 24 h after inflammation. With exogenous dolichol added to the microsome assay, increased kinase activity was observed as early as 6 h after turpentine injection. The increase of dolichol phosphate in inflammation is most likely due to both greater availability of dolichol and an increase in the level of CTP-dependent dolichol kinase. Studies with purified subcellular fractions showed that dolichol kinase activity is primarily localized in the rough endoplasmic reticular fraction. Since this is the major site of dolichol-phosphate-linked N-glycosylation reactions, a key role of dolichol phosphokinase activity in rough microsomes to initiate the first steps of N-glycoprotein synthesis seems plausible.

Animals↗
PubMed1981

A novel phosphorylcholine-binding protein from rat serum and its effect on heparin-lipoprotein complex formation in the presence of calcium.

An adsorbent was synthesized by attaching 4-aminophenylphosphorylcholine to cyanogen bromide-activated Sepharose. A phosphorylcholine (P-choline)-binding protein from rat serum was adsorbed on this affinity column which was eluted by 4 mM P-choline. The protein separated into two bands of Mr = 47,000 and 24,000 on sodium dodecyl sulfate-polyacrylamide gradient gels and contained 18% carbohydrate. A serum protein factor, precipitable by 30-50% (NH4)2SO4, was previously shown to inhibit Ca2+-heparin-rat serum very low density lipoprotein (VLDL) precipitation reaction, whereas P-choline counteracted the action of this protein (Mookerjea, S. (1978) Can. J. Biochem. 56, 746-752). It is now demonstrated that purified P-choline-binding protein prevents Ca2+-heparin-chylomicron or VLDL complex formation and P-choline reverses the effect of this protein. Antibody to P-choline-binding protein raised in rabbits produced a single precipitin line against the pure antigen. The antiserum, however, did not react against rat serum chylomicron, VLDL, low density lipoproteins, or high density lipoprotein. Human serum appears to lack P-choline-binding protein, since (a) the affinity column did not adsorb any such protein, (b) P-choline had no effect on the Ca2+-heparin-serum lipoprotein precipitation reaction, and (c) an immunodiffusion test against the antiserum was negative. However, when P-choline-binding protein was added to human serum, the lipoprotein precipitation was inhibited, and P-choline counteracted the effect of this protein. Preliminary experiments suggested a stoichiometric interaction between P-choline-binding protein and VLDL. Hydrophilic P-choline groups exposed on the surface of VLDL may possibly interact with the P-choline-binding protein and thereby affect the precipitation of lipoproteins by heparin and Ca2+.

Amino Acids↗
PubMed1981

Effects of turpentine-induced inflammation on the synthesis of dolichol-linked intermediates of N-glycosylation and the phosphorylation of dolichol by CTP-dependent dolichol kinase.

Inflammation was induced in rats by the subcutaneous injection of turpentine. Microsomes were prepared from the livers between 2 and 72 h after injection. Mannose and glucose incorporation into mannosyl and glucosyl dolichyl monophosphate was increased 2-fold over saline-injected controls 24 h after induction of inflammation. Synthesis of glycosylated dolichyl pyrophosphoryl oligosaccharides was also increased compared to controls. Extraction and assay of dolichol monophosphate from inflamed and control rat liver microsomes indicated that the endogenous levels of the lipid were elevated in the inflamed state. CTP-dependent phosphorylation of endogenous dolichol was also found to increase in microsomes from inflamed rats 24 h after injection of turpentine. When exogenous dolichol was added to the microsomal system an increase in phosphorylation was observed as early as 6 h after turpentine injection. Furthermore, the increase appeared to be biphasic, there being two peaks of elevated activity at 12 and 36-48 h after induction of inflammation. The earlier peak was the greater of the two. The results suggest that the increase in glycosylation of dolichol derivatives was due to greater amounts of endogenous dolichol monophosphate. The increase in dolichol monophosphate was itself due to greater availability of dolichol and an increase in the levels of CTP-dependent dolichol kinase.

Animals↗