Evidence for the Presence of CDP-Ethanolamine: 1,2-diacyl-sn-glycerol Ethanolaminephosphotransferase in Rat Central Nervous System Myelin

Highly purified rat brain myelin isolated by two different procedures showed appreciable activity for CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase (EC 2.7.8.1). Specific activity was close to that of total homogenate and approximately 12-16% that of brain microsomes. Three other lipid-synthesizing enzymes, cerebroside sulfotransferase, lactosylceramide sialyltransferase, and serine phospholipid exchange enzyme, were found to have less than 0.5% the specific activity in myelin compared with microsomes. Washing the myelin with buffered salt or taurocholate did not remove the phosphotransferase, but activity was lost from both myelin and microsomes by treatment with Triton X-100. It resembled the microsomal enzyme in having a pH optimum of 8.5 and a requirement for Mn2+ and detergent, but differed in showing no enhancement with EGTA. The diolein Km was similar for the two membranes (2.5-4 x 10(-4) M), but the CDP-ethanolamine Km was lower for myelin (3-4 x 10(-5) M) than for microsomes (11 - 13 x 10(-5 M). Evidence is reviewed that this enzyme is able to utilize substrate from the axon in situ.     

Cloning, sequence, and expression of a cDNA encoding hamster UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase

UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT) catalyzes the initial reaction required for synthesis of dolichol-P-P-oligosaccharides. We report here on the sequence and expression of a full-length cDNA clone encoding hamster GPT. The cDNA predicts a protein of 408 amino acid residues including 10 hydrophobic segments. Several portions of the hamster GPT sequence constituting one-third of the protein have 60% or greater identity with yeast GPT, and one-half of the conserved sequence falls within the hydrophobic segments. In addition, hamster GPT has two copies of a putative dolichol recognition sequence recently identified in three yeast enzymes that interact with dolichol. The protein lacks KDEL or DEKKMP-type carboxyl-terminal ER sorting sequences. When expressed in COS-1 cells, the cDNA causes a 5-7-fold increase of GPT activity in membrane fractions. The activity was completely inhibitable by tunicamycin, and the primary product was shown to be GlcNAc-pyrophosphoryldolichol. This cDNA represents the first enzyme of the dolichol-oligosaccharide biosynthetic pathway to be cloned from a vertebrate source and demonstrates structural homology between the enzymes of the yeast and mammalian pathways.     

Kinetic studies on microsomal glucose dehydrogenase in rat liver.

Glucose dehydrogenase from rat liver microsomes was found to react not only with glucose as a substrate but also with glucose 6-phosphate, 2-deoxyglucose 6-phosphate and galactose 6-phosphate. The relative maximum activity of this enzyme was 29% for glucose 6-phosphate, 99% for 2-deoxyglucose 6-phosphate, and 25% for galactose 6-phosphate, compared with 100% for glucose with NADP. The enzyme could utilize either NAD or NADP as a coenzyme. Using polyacrylamide gradient gel electrophoresis, we were able to detect several enzymatically active bands by incubation of the gels in a tetrazolium assay mixture. Each band had different Km values for the substrates (3.0 x 10(-5)M glucose 6-phosphate with NADP to 2.4M glucose with NAD) and for coenzymes (1.3 x 10(-6)M NAD with galactose 6-phosphate to 5.9 x 10(-5)M NAD with glucose). Though glucose 6-phosphate and galactose 6-phosphate reacted with glucose dehydrogenase, they inhibited the reaction of this enzyme only when either glucose or 2-deoxyglucose 6-phosphate was used as a substrate. The Ki values for glucose 6-phosphate with glucose as substrate were 4.0 x 10(-6)M with NAD, and 8.4 x 10(-6)M with NADP; for galactose 6-phosphate they were 6.7 x10(-6)M with NAD and 6.0 x 10(-6)M with NADP. The Ki values for glucose 6-phosphate with 2-deoxyglucose 6-phosphate as substrate were 6.3 x 10(-6)M with NAD and 8.9 x 10(-6)M with NADP; and for galactose 6-phosphate, 8.0 x 10(-6)M with NAD and 3.5 x 10(-6)M with NADP. Both NADH and NADPH inhibited glucose dehydrogenase when the corresponding oxidized coenzymes were used (Ki values: 8.0 x 10(-5)M by NADH and 9.1 x 10(-5)M by NADPH), while only NADPH inhibited cytoplasmic glucose 6-phosphate dehydrogenase (Ki: 2.4 x 10(-5)M). The results indicate that glucose dehydrogenase cannot directly oxidize glucose in vivo, but it might play a similar role to glucose 6-phosphate dehydrogenase. The differences in the kinetics of glucose dehydrogenase and glucose 6-phosphate dehydrogenase show that glucose 6-phosphate and galactose 6-phosphate could be metabolized in quite different ways in the microsomes and cytoplasm of rat liver.     

A phospholipid requirement for dolichol pyrophosphate N-acetylglucosamine synthesis in phospholipase A2-treated rat lung microsomes

The role of phospholipids in the activity of UDP-Glc-NAc:dolichol phosphate GlcNAc-1-phosphate transferase of rat lung microsomes has been investigated. Treatment of microsomes with phospholipase A2 in the presence of delipidated bovine serum albumin resulted in a time-dependent loss of 65 to 75% of the enzyme activity and approximately 30% of the phospholipids. Addition of phosphatidylglycerol to the enzyme assay system containing phospholipase A2-treated microsomes restored activity to that obtained with native microsomes and phosphatidylglycerol. Addition of phosphatidylinositol, phosphatidylcholine, or cardiolipin resulted in only partial restoration of activity, whereas phosphatidylserine and phosphatidylethanolamine were without effect. Triton X-100 was not by itself capable of restoring activity, but was required for the phospholipid effect. Measurements of the phospholipase A2 hydrolysis products released from the microsomes during digestion, and other control experiments of adding fatty acids and lysophospholipids to the enzyme assay system, indicated that the loss of UDP-GlcNAc:dolichol phosphate GlcNAc-1-phosphate transferase activity was not due to product inhibition.     

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.     

Selection of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosaminyltransferase activity

Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) have been isolated by a stepwise selection procedure with progressive increments of TM added to the medium. TM inhibits asparagine-linked glycoprotein biosynthesis by blocking the transfer of N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the lipid carrier. The TM-resistant cells exhibited a 200-fold increase in their LD50 for TM and were morphologically distinct from the parental cells. The rate of asparagine-linked glycoprotein biosynthesis was the same for wild-type and TM-resistant cells. Membrane preparations from TM-resistant cells cultured for 16 d in the absence of TM had a 15-fold increase in the specific activity of the UDP-N- acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase as compared to membranes of wild-type cells. The products of the in vitro assay were N-acetylglucosaminylpyrophosphoryl-lipid and N,N'-diacetylchitobiosylpyrophosphoryl-lipid for membranes from both TM- resistant and wild-type cells. The transferase activity present in membrane preparations from wild-type of TM-resistant cells was inhibited by comparable levels of TM. The data presented are consistent with overproduction of enzyme as the mechanism of resistance in these variant CHO cells.     

Biosynthesis of dolichyl diphosphate N-acetylglucosamine intermediates in Ascaridia galli microsomes.

1. N-acetylglucosamine-1-phosphate transferase was demonstrated in the microsomal fraction of Ascaridia galli. 2. The transferase reaction depends on exogenous dolichyl phosphate as lipid acceptor and was found to be inhibited by tunicamycin. 3. The enzyme activity was optimal in the presence of sodium deoxycholate as detergent and Mg cations after 10 min of incubation. 4. The product of the transferase reaction--dolichyl diphosphate N-acetylglucosamine was converted into lipid-disaccharide-dolichyl diphosphate N,N'-diacetylchitobiose. 5. The maximum level of the conversion was achieved at 5 mM concentration of unlabelled UDP-N-acetylglucosamine, while this conversion was negligible at lower UDP-N-acetylglucosamine concentrations (0.1 and 0.5 mM).     

Isolation of transketolase from rabbit liver and comparison of some of its kinetic properties with transketolase from other sources

1. Rabbit liver transketolase activity was purified 56-fold using the following steps: ammonium sulfate precipitation, chromatography on DEAE-Sephadex A-25, concentration through an Amicon ultrafiltration cell and rechromatography on DEAE-Sephadex A-25. 2. The enzyme showed an optimum PH for activity at 7.8-8.0. 3. The optimum temperature was around 40 degrees C and the activation energy calculated from the Arrhenius plot was found to be 11.4 kcal/mole. 4. The molecular weight of the enzyme, as determined by gel filtration, was found to be approximately 162,000, while the content of thiamin diphosphate was between 1.8 and 2 mumole per mole protein. 5. Addition of thiamin diphosphate and magnesium chloride did not influence the activity. 6. From the kinetic studies of the enzyme, the Km values for xylulose-5-phosphate, ribose-5-phosphate and fructose-6-phosphate were 3.8 x 10(-5) M, 9.5 x 10(-5) M and 1.1 x 10(-2) M, respectively.     

Properties of a dolichol phosphokinase activity associated with rat liver microsomes

Rat liver microsomes show a capacity to synthesize [1-3H]dolichyl phosphate from [1-3H]-dolichol. Formation of [1-3H]dolichyl phosphate increased continuously over 15 min although the reaction rate was never completely linear. Product formation was directly proportional to microsomal protein concentration between 1.1 mg/mL and the highest concentration tested, 5.5 mg/mL. The reaction rate was linear with respect to the dolichol content of the assay mixture to a saturation point (120 microM). An apparent Km of 50 microM was established for dolichol. The normal phosphate donor for the reaction is CTP and not ATP. The optimum concentration of CTP was 10 mM, and an apparent Km of 4 mM was calculated for this nucleoside triphosphate. The reaction was totally dependent on divalent metal ion, magnesium being more effective than calcium. The optimum concentration of magnesium ion and CTP were the same (10 mM), suggesting that MgCTP2- is utilized as the normal enzyme substrate. Activity measured in the absence of Triton X-100 was only 5% of the activity observed at the optimum (0.5% w/v) detergent concentration. The measurable levels of dolichol phosphokinase could be doubled by the inclusion of 10-15 mM NaF as phosphatase inhibitor. Optimal enzymatic activity was obtained between pH 7.0 and pH 7.5 and could be inhibited by EDTA. The sulfhydryl reagent DTT was slightly stimulatory while the product of the reaction, dolichyl phosphate, was noninhibitory at the highest concentration tested (13.8 microM). The second reaction product (CDP) inhibits the enzymatic phosphorylation of dolichol.     

Purification and properties of phosphoglycerate phosphomutase from spores and cells of Bacillus megaterium.

Phosphoglycerate phosphomutase has been purified to homogeneity from vegetative cells and germinated spores of Bacillus megaterium, and the spore and cell enzymes appear identical. The enzyme is a monomer of molecular weight 61,000. The compound 2,3-diphosphoglyceric acid is not required for activity, but the enzyme has an absolute and specific requirement for Mn2+. The enzyme is inhibited by ethylenediaminetetraacetate and sulfhydryl reagents, has a pH optimum of about 8.0, and has Km values for 3-phosphoglyceric acid and Mn2+ of 5 x 10(-4) and 4 x 10(-5) M, respectively.     

Assay and properties of N-acetylglucosamine-6-phosphate deacetylase from rat liver

A single-vial assay has been developed for N-acetylglucosamine-6-phosphate deacetylase, in which [3H]acetate released from 3H-acetyl-labeled substrate is measured in a biphasic liquid scintillation counting system after acidification of the reaction mixture. The deacetylase was partially purified from rat liver, and some of its properties were determined. Chromatography on a calibrated Sepharose CL-6B column indicated a molecular weight of 345,000. The Km for the substrate at pH 8.0 was 0.3 mM. Glucosamine 6-phosphate and glucose 6-phosphate inhibited the enzyme, whereas N-acetylgalactosamine, N-acetylglucosamine, N-acetylglucosamine 1-phosphate, and glucosamine 1-phosphate were without effect. The effects of several divalent cations were also examined. Under the conditions tested, Ca2+, Mg2+, and Ba2+ had essentially no effect, whereas Mn2+, Ni2+, and Cu2+ were inhibitory and Co2+ stimulated activity at low concentrations but inhibited above 5 mM. An increase in the ionic strength of the reaction mixture to 0.3 M decreased the activity by 40%.     

Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization.

Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme.     

Glucose dehydrogenase (hexose 6-phosphate dehydrogenase) and the microsomal electron transport system. Evidence supporting their possible functional relationship.

The ability of a microsomal enzyme, glucose dehydrogenase (hexose 6-phosphate dehydrogenease) to supply NADPH to the microsomal electron transport system, was investigated. Microsomes could perform oxidative demethylation of aminopyrine using microsomal glucose dehydrogenase in situ as an NADPH generator. This demethylation reaction had apparent Km values of 2.61 X 10(-5) M for NADP+, 4.93 X 10(-5) m for glucose 6-phosphate, and 2.14 X 10(-4) m for 2-deoxyglucose 6-phosphate, a synthetic substrate for glucose dehydrogenase. Phenobarbital treatment enhanced this demethylation activity more markedly than glucose dehydrogenase activity itself. Latent activity of glucose dehydrogenase in intact microsomes could be detected by using inhibitors of microsomal electron transport, i.e. carbon monoxide and p-chloromercuribenzoate (PCMB), and under anaerobic conditions. These observations indicate that in microsomes the NADPH generated by glucose dehydrogenase is immediately oxidized by NADPH-cytochrome c reductase, and that glucose dehydrogenase may be functioning to supply NADPH.     

Dog liver glucose-6-phosphate dehydrogenase: purification and kinetic properties

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme.Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway.     

Dolichol kinase activity in the developing rat testis

Dolichyl phosphate concentrations, a primary factor in regulating the rate of N-glycosidically linked glycoprotein synthesis, are dependent upon a cytidine triphosphate (CTP)-dependent dolichol kinase. This study examines dolichol kinase in rat testicular microsomes and defines assay conditions. As with dolichol kinases from other tissues, addition of 2-mercaptoethanol increased activity 60%. Inclusion of NaF, an inhibitor of testicular dolichyl phosphate phosphatase activity, also resulted in a 38% increase in activity. Triton X-100 was necessary for phosphorylation of both endogenous and exogenous dolichol; however, concentrations of detergent in excess of 0.25-0.35% were inhibitory. A 2- to 5-fold stimulation of kinase activity was obtained by addition of 50-100 microM exogenous dolichol. The high level of nucleoside triphosphatase activity in testicular microsomes mandated the inclusion of high levels of uridine triphosphate (UTP) to protect the [gamma-32 P] CTP. Increasing UTP concentrations up to 50 mM resulted in increased product formation. A clear requirement for divalent cations was observed; 5 mM ethylenediaminetetraacetate (EDTA) abolished activity. The following order of cation effectiveness was observed: Mn greater than or equal to Ca greater than Cd greater than Zn much greater than Mg. Ten mM optima were established for Ca2+ and Mn2+; the presence of UTP, however, results in significantly reduced concentrations of free Ca2+. Ion combination studies demonstrated interactive inhibitory effects between Ca2+ and other stimulatory divalent cations. Addition of 2 microM brain calmodulin, in the presence of 10 mM Ca2+, resulted in a 75-100% stimulation of activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of transketolase from human erythrocytes

Transketolase was isolated from human red blood cells with over 6,200 fold purification by a new method. The stepwise procedure for the isolation of the enzyme from erythrocyte hemolysate included the use of ethanol/chloroform precipitation, chromatography on hydroxyapatite and finally, affinity adsorption on carboxymethyl-cellulose. The molecular weight of erythrocyte transketolase, as determined by polyacrylamide gel electrophoresis, appeared to be about 140,000. The pH optimum for activity was between 7.6 and 7.8 and the optimum temperature for activity was 50 degrees C. The Km values for xylulose-5-phosphate, ribose-5-phosphate and fructose-6-phosphate were 2.0 x 10(-4) M, 3.2 x 10(-4) M and 2.0 x 10(-3) M, respectively.     

Purification and characterization of uridine and thymidine phosphorylase from Lactobacillus casei

Uridine and thymidine phosphorylases have been purified to homogeneity from crude extracts of Lactobacillus casei. Both enzymes had an apparent molecular mass of about 80 kDa. Uridine phosphorylase consisted of four identical subunits while thymidine phosphorylase was composed of two identical ones. The sequence of 23 amino-acid residues from its N-terminal end was analyzed. Uridine phosphorylase had a Km of 5.0 x 10(-3) M for uridine and 1.24 x 10(-1) M for phosphate, while thymidine phosphorylase had a Km of 1.32 x 10(-1) M for thymidine and 1.0 x 10(-1) M for phosphate. Uridine phosphorylase was equally active with uridine and 5-methyluridine, but had a low activity towards thymidine. Its activity was inhibited competitively by 3-O-methyl-alpha D-glucopyranoside, on the other hand thymidine phosphorylase activity was not affected by this compound. Thymidine phosphorylase showed specificity towards the deoxyribosyl moiety of the substrate. In addition, it required a nonsubstituted pyrimidine moiety or one which was substituted in position 5. The pattern of the double-reciprocal plots of the initial velocities vs. the concentrations of either one of the substrates, and the product inhibition kinetics, indicated that the catalytic mechanism of both enzymatic reactions is sequential rather than Ping-Pong and that the sequence of the addition of the substrates is random (rapid equilibrium). In the case of the uridine phosphorylase-catalyzed reaction, the products are also released randomly, while in the thymidine phosphorylase-catalyzed reaction deoxyribose 1-phosphate is released after thymine.     

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.     

Esterification of dolichol in rat liver

In the various subcellular fractions of rat liver 45-75% of the total dolichol was esterified with a fatty acid. The esterification reaction was localized exclusively in the microsomes, and the transferase activity is 3-fold higher in the cation-insensitive smooth microsomes than in other microsomal subfractions. Although fatty acyl-CoAs tested served as substrates, palmitoyl-CoA was the most rapidly utilized. None of the phosphatidylcholine or phosphatidylethanolamine species tested could be utilized to esterify dolichol with a fatty acid, indicating the absence of transacylation. alpha-Saturated dolichols were esterified at a higher rate than their alpha-unsaturated counterparts. Albumin and low concentrations of Triton X-100 activated the esterification reaction, which was not dependent on mono- or divalent cations, ATP, or CoA. The sensitivity of the transferase activity to trypsin indicates localization of the enzyme(s) involved on the outer surface of microsomes (i.e. the cytoplasmic surface of the endoplasmic reticulum), as is also the case for enzymes of dolichol biosynthesis. Transferase activity was detected in all tissues examined but at a much lower level than in liver and testis. The patterns of fatty acids in dolichol esters of different organelles exhibited some specificity. Labeling in vivo indicated that esterification of dolichol may play a role in targeting this lipid from the endoplasmic reticulum to lysosomes.     

Restoration by phospholipids of dolichol pyrophosphate N-acetylglucosamine synthesis in delipidated rat lung microsomes

The effects of phospholipids on the reaction catalyzed by UDP-GlcNAc:dolichol phosphate GlcNAc-1-phosphate transferase have been studied with delipidated rat lung microsomes. Deoxycholate-solubilized enzyme was depleted of measurable phospholipid by either gel filtration on Sephadex G-100 or affinity chromatography on pentyl-agarose. The latter procedure also removed nucleotide and sugar nucleotide hydrolases. Delipidated protein fractions were devoid of GlcNAc-1-phosphate transferase activity unless supplemented with phospholipids. Maximal recovery of enzyme activity was obtained with an approximate 1:1 weight ratio of phosphatidylglycerol:phosphatidylcholine, with the observed rate being synergistic as compared to rates observed for each individual phospholipid. Variable recoveries of enzyme activity were obtained with mixtures containing other acidic phospholipids and phosphatidylcholine. Enzyme activity in the fraction eluted from pentyl-agarose could be recovered, after removal of Triton X-100, with sedimented phospholipid vesicles. Significant stabilization of enzyme activity associated with the phospholipid vesicles was obtained by the inclusion of dolichol phosphate.