Chemical characteristics and immuno-modulating activities of exo-biopolymers produced by Grifola frondosa during submerged fermentation process

The immuno-modulating activities and chemical characteristics of exo-biopolymer (EX-GF) produced by a submerged mycelial culture of Grifola frondosa were studied. The EX-GF was fractionated into EX-GF-Fr.I, II, and III by Sephadex G-100 gel chromatography. Anti-complementary activity of EX-GF-Fr.III was highest (71.1%) among them, and its activation system occurred through both classical and alternative pathways, where the classical pathway found to be major one. Lysosomal enzyme activity and nitric oxide production ability of macrophage were also found to be mediated by EX-GF-Fr.III. The molecular weight of the EX-GF-Fr.I, II, and III was estimated to be about 163, 40, and 2.8 kDa, respectively. Total sugar and protein contents of the three fractions were 80.3, 61.9 and 89.3%, and 17.3, 35.2, and 10.7%, respectively. The sugar and amino acid compositions of the EX-GF-Fr.I, II, and III were also analyzed in detail.     

Purification and immobilization of dextranase

An enzymic characteristic of Novo dextranase was presented. In addition to a high dextranolytic activity (7,200 U/ml), the crude enzyme also contained small amounts of protease, glucoamylase, polygalacturonase, carboxymethylcellulase, laminarinase and chitinase. A highly purified dextranase was then simply separated from a commercial preparation by column chromatographies on DEAE-Sepharose, CM-Sepharose, and by chromatofocussing on Polybuffer Exchanger PBE-94. The enzyme was recovered with an over 200-fold increase in specific activity and a yield of 84%. The final preparation was homogeneous, as observed during high performance liquid chromatography (HPLC). Size-exclusion HPLC indicated that dextranase had a molecular mass of 35 kDa and its isoelectric point, established by chromatofocussing, was 4.85. Analysis of the dextran break-down products indicated that purified dextranase represents an endolytic mode of action, and isomaltose and isomaltotriose were identified as the main reducing sugars of dextran hydrolysis. The enzyme was then covalently coupled to the silanized porous glass beads modified by glutaraldehyde (Carrier I) or carbodiimide (Carrier II). It was shown that immobilization of dextranase gave optimum pH and temperature ranges from 5.4 to 5.7 and from 50°C to 60°C, respectively. The affinity of the enzyme to the substrate decreased by a factor of more than 13 for dextranase immobilized on Carrier I and increased slightly (about 1.4-times) for the enzyme bound to Carrier II.     

Properties of two sugar phosphate phosphatases from Streptococcus bovis and their potential involvement in inducer expulsion.

Streptococcus bovis possesses two sugar phosphate phosphatases (Pases). Pase I is a soluble enzyme that is inhibited by the membrane fractions from lactose-grown cells and is insensitive to activation by S46D HPr, an analog of HPr(ser-P) of the sugar phosphotransferase system. Pase II is a membrane-associated enzyme that can be activated 10-fold by S46D HPr, and it appears to play a role in inducer expulsion.     

Immune-stimulating properties of polysaccharides from Phellodendri cortex (Hwangbek)

Heteropolysaccarides were isolated from the Korean medicinal plant, Phellodendri cortex (Hwangbek), by hot water and alkali extractions. The extracted polysaccharides were fractionated into eight fractions and they are mainly composed of D-N-acetylglucosamine, D-galactose, D-mannose, and D-glucose. Among the polysaccharide fractions, Fr.-2 showed a potent B-lymphocyte-stimulating activity in a system using polyclonal antibody forming cells in C57BL/6XC3H mice at dosages of 2–10 mg. On the basis of their solubility in aqueous ethanol, four fractions of Fr.-2-1 to Fr.-2-4 were further obtained from the Fr.-2, and Fr.-2-3 was divided into Fr.-2-3-1, 2, 3, and 4 by DEAE cellulose chromatography. The main activity was found in Fr.-2-3-2, which contained 100% (w/w) of carbohydrates and further purified to Fr.-2-3-2-2 by gel filtration chromatography using TSK Gel HW50S. Fr.-2-3-2-2, having a molecular weight of about 230 kDa, showed the highest B-cell-stimulating activity and the half-maximal concentration for B-lymphocyte-stimulating activity was ca. 2.2 µg/ml.     

A potent inhibitor of platelet activating factor from the saliva of the leech Hirudo medicinalis.

Leech saliva is shown to contain protein platelet aggregation inhibitors and a range of selective low molecular weight (LMW) aggregation inhibitors. Gel filtration on Bio-Gel P-2 (cut-off kDa) yields a protein fraction (Fr. I) and three LMW fractions. Fr. I inhibits aggregation induced by collagen, ADP, epinephrine and arachidonic acid. Of all the fractions, only one, Fr. II (LMW) specifically inhibits aggregation induced by platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine). Fr. II also inhibits thrombin-induced platelet aggregation. Fr. III inhibits aggregation induced by ADP, epinephrine and arachidonic acid, and Fr. IV only that induced by arachidonic acid. Fr. II also inhibits PAF- and thrombin-induced thromboxane generation in platelets, but does not inhibit arachidonic acid-induced thromboxane generation. Efforts to separate the anti-PAF from the anti-thrombin activity have been unsuccessful. The inhibition may therefore be due to a single inhibitor, though it may also be due to several inhibitors. Fr. II also inhibits superoxide anion production in formyl Met-Leu-Phe (fMLP)- and ionophore 23187- stimulated neutrophils. This may be due to the inhibition of the effects of PAF generated within the cell. Preliminary results suggest that the Fr. II inhibitor(s) is (are) amphipathic. The interaction of platelets with PAF and their interaction with the inhibitor(s) are mutually exclusive, and the inhibition may be competitive.     

Multiple proteases from Streptomyces moderatus: I. Isolation and purification of five extracellular proteases

The presence of multiple proteases in the culture filtrate of Streptomyces moderatus was detected. After preliminary purification by ammonium sulfate precipitation and decolorization using DEAE-cellulose, the fractionation of various proteases was carried out using CM-trisacryl cation-exchange chromatography. By this procedure, four different protease fractions (Fr.) were separated (Fr. I, II, III, and IV). The first fraction was further separated into two different proteolytically active fractions (Fr. Ia and Fr. Ib) by DEAE-trisacryl anion-exchange chromatography. Fraction Ia was purified further by affinity chromatography on N-carbobenzoxy-d-phenylalanyl triethylenetetramine-Sepharose 4B. The second fraction (Fr. Ib) was purified by gel filtration on Ultrogel AcA 44. For the purification of the other protease fractions (Fr. II, III, and IV) single-step affinity chromatography methods were employed. Protease fractions II and III were purified by ϵ-aminocaproyl-4-(4-aminophenylazo)phenylarsonic acid Sepharose 4B and protease fraction IV was purified on ϵ-aminocaproyl trialanine-Sepharose 4B. All five proteases purified were found to be apparently homogeneous by gel electrophoretic methods.     

Purification and characterization of an extracellular polygalacturonase from Lactobacillus plantarum strain BA 11

Lactobacillus plantarum produced extracellular polygalacturonase in a medium containing 1.5% low methyl-pectin (w/v) and 0.5% glucose (w/v) as inducers. The enzyme was purified (approximately 70-fold) by ammonium sulphate fractionation, Sephadex G-100 gel filtration and DEAE-cellulose ion exchange chromatography. Two peaks (PG I and PG II) of enzymic activity were obtained from the DEAE-cellulose column. The molecular mass of PG I was similar to that of PG II (32 000 Da). The K _m values of PG I and PG II for sodium polypectate were calculated to be 1.63 mg/ml and 1.78 mg/ml respectively. Their isoelectric points were about pH 5.5. The pH optimum was 4.5, while the optimum temperature was 35°C for both PG I and PG II. The two purified enzymes had similar endo modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction and reducing group release.     

A secretin releasing peptide exists in dog pancreatic juice

Canine pancreatic juice has been shown to stimulate exocrine pancreatic secretion in the dog. In the present study we investigated whether there is a secretin-releasing peptide in canine pancreatic juice. Pancreatic juice was collected from the dogs with Thomas gastric and duodenal cannulas while pancreatic secretion was stimulated by intravenous administration of secretin at 0.5 microg/kg/h and CCK-8 at 0.2 microg/kg/h, respectively. The pancreatic juice was separated into three different molecular weight (MW) fractions (Fr) by ultrafiltration (Fr 1; MW > 10,000, Fr 2; MW=10,000-4,000 and Fr 3; MW < 4,000), respectively. All the fractions were bioassayed in anesthetized rats. Fraction 3 dose-dependently and significantly stimulated pancreatic juice flow volume from 78.0% to 99.4% (p<0.05) and bicarbonate output from 128.9% to 202.1% (p<0.01), respectively. Plasma secretin concentration also increased from 1.2 +/- 0.5 pM to 5.0 +/- 0.8 pM and 6.0 +/- 1.0 pM (p<0.05). None of these fractions increased pancreatic protein secretion or plasma CCK level. The stimulatory effect of Fraction 3 on pancreatic secretion and the release of secretin was completely abolished by treatment with trypsin (1 mg/ml for 60 min at 37 degrees C) but not by heating (100 degrees C, 10 min). Intravenous injection of a rabbit anti-secretin serum, which rendered plasma secretin almost undetectable in rat plasma, also abolished Fr 3-stimulated pancreatic secretion of fluid and bicarbonate secretion. These observations suggest that a secretin-releasing peptide exists in the canine pancreatic juice. It is trypsin-sensitive and heat-resistant. This peptide may play a significant physiological role on the release of secretin and regulation of exocrine pancreatic secretion.     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis.

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxic and suppressor activity of human peripheral blood lymphocytes stimulated by interleukin-2 and phytohemagglutinin before and after fractionation in the percoll gradient

The cultivation of peripheral blood lymphocytes (PBL) obtained from patients with colorectal or bladder carcinoma and melanoma and from healthy donors in the presence of interleukin-2 (IL-2) and PHA resulted in the induction of cytotoxic activity against autologous and/or allogeneic tumour cells in 12 out of 13 patients and in 10 out of 10 donors. A higher level of cytolytic activity was achieved when PBL were separated by means of Percoll density gradient (1.077; 1.067 and 1.056 g/ml) centrifugation and the cells of fraction II (1.077-1.067 g/ml) were employed in the experiment, the level of cytotoxicity being elevated in all cases (1.7-fold elevation in donors and 2-fold elevation in patients on the average). The addition of fraction I (1.067-1.056 g/ml) to fraction II prevented (PHA + IL-2)-mediated induction of cytotoxic activity in all the patients, but in 4 out of 10 donors, i.e. cells of fraction I expressed a suppressor activity. The immunofluorescent analysis has shown that fraction II was enriched by T cells (92%) and depleted of monocytes (7%), as compared to unseparated PBL (66% and 27%, respectively). On the contrary, fraction I was characterized by a decreased T cell ratio (36%) and an increased monocyte level (up to 69%).     

Chemical and biological characterization of a polysaccharide biological response modifier from Aloe vera L. var. chinensis (Haw.) Berg

Three purified polysaccharide fractions designated as PAC-I, PAC-II, and PAC-III were prepared from Aloe vera L. var. chinensis (Haw.) Berg. by membrane fractionation and gel filtration HPLC. The polysaccharide fractions had molecular weights of 10,000 kDa, 1300 kDa, and 470 kDa, respectively. The major sugar residue in the polysaccharide fractions is mannose, which was found to be 91.5% in PAC-I, 87.9% in PAC-II, and 53.7% in PAC-III. The protein contents in the polysaccharide fractions was undetectable. NMR study of PAC-I and PAC-II demonstrated the polysaccharides shared the same structure. The main skeletons of PAC-I and PAC-II are beta-(1-->4)-D linked mannose with acetylation at C-6 of manopyranosyl. The polysaccharide fractions stimulated peritoneal macrophages, splenic T and B cell proliferation, and activated these cells to secrete TNF-alpha, IL-1 beta, INF-gamma, IL-2, and IL-6. The polysaccharides were nontoxic and exhibited potent indirect antitumor response in murine model. PAC-I, which had the highest mannose content and molecular weight, was found to be the most potent biological response modifier of the three fractions. Our results suggested that the potency of aloe polysaccharide fraction increases as mannose content and molecular weight of the polysaccharide fraction increase.     

Purification by sucrose density gradient zonal centrifugation and affinity column chromatography of antigenic substances from the livers of mice infected with Tyzzer's disease

Antigenic substances from livers of mice infected with Tyzzer's disease were purified by means of sucrose density gradient zonal centrifugation and affinity column chromatography using antiserum and checking antigenicity with the complement fixation test. Fractions obtained from zonal centrifugation fell into three main groups with different molecular weights, two of which (Fr. I and Fr. II) positively reacted with antiserum in the complement fixation tests. Both fractions were further purified by affinity column chromatography. The molecular weights of the main antigenic substances derived from Fr. I and Fr. II were determined to be about 52 000 and 66 000, respectively, by means of SDS-PAGE.     

Role of endo-1,4-beta-glucanases from neisseria sicca SB in synergistic degradation of cellulose acetate

An enzyme hydrolyzing beta-1,4 bonds in cellulose acetate was purified 10.5-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which assimilate cellulose acetate as the sole carbon and energy source. The enzyme was an endo-1,4-beta-glucanase, to judge from the substrate specificity and hydrolysis products of cellooligosaccharides, we named it endo-1,4-beta-glucanase I (EG I). Its molecular mass was 50 kDa, 9 kDa larger than EG II from this strain, and its isoelectric point was 5.0. Results of N-terminal and inner-peptide sequences of both enzymes, and a similarity search, suggested that EG I contained a carbohydrate-binding module at the N-terminus and that EG II lacked this module. The pH and temperature optima of EG I were 5.0-6.0 and 45 degrees C. It hydrolyzed water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for these compounds were 0.296% and 1.29 micromol min(-1) mg(-1), and 0.448% and 13.6 micromol min(-1) mg(-1), respectively. Both glucanases and cellulose acetate esterase from this strain degraded water-insoluble cellulose acetate synergistically.     

Effect of SH-reagents on aldehyde dehydrogenase activity of the rat liver

SH-reagents: tetraethylthiuram disulphide (TETD), 5,5'-dithiobisnitrobenzoic acid (DTNB), p-chloromercurybenzoate (p-ChMB), N-ethylmaleimide (NEM) were studied for their effect on the aldehyde dehydrogenase activity of mitochondrion (isoenzymes I and II) and microsome (isoenzyme II) fractions of the rat liver. TETD is established to inhibit isoenzyme I and isoenzyme II activity of mitochondrial aldehyde dehydrogenase by 100 and 50%, respectively, and the microsomal enzyme activity by 20%. DTNB and NEM inhibit 30-50% of the activity in two isoforms of mitochondrial aldehyde dehydrogenase having no effect on the enzymic activity in microsomes; p-ChMB inhibits completely the activity of the enzyme under study both in the mitochondrial and microsomal fractions. A conclusion is drawn that SH-groups are very essential for manifestation of the catalytic activity in the NAD+-dependent aldehyde dehydrogenase from mitochondrial and microsomal fractions.     

Pectinase production by Neurospora crassa: purification and biochemical characterization of extracellular polygalacturonase activity.

The production of pectinase was studied in Neurospora crassa, using the hyperproducer mutant exo-1, which synthesized and secreted five to six times more enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were induced by pectin, and this induction was glucose-repressible. Polygalacturonase was induced by galactose four times more efficiently than by pectin; in contrast the activity of lyases was not affected by galactose. The inducing effect of galactose on polygalacturonase was not glucose-repressible. Extracellular pectinases were separated by ion exchange chromatography. Pectate and pectin lyases eluted into three main fractions containing both activities; polygalacturonase eluted as a single, symmetrical peak, apparently free of other protein contaminants, and was purified 56-fold. The purified polygalacturonase was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular mass 36.6-37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of the rate for polypectate. Km and Vmax for polypectate hydrolysis were 5.0 mg ml-1 and 357 mumol min-1 (mg protein)-1, respectively. Temperature and pH optima were 45 degrees C and 6.0, respectively. The purified polygalacturonase reduced the viscosity of a sodium polypectate solution by 50% with an increase of 7% in reducing sugar groups. The products of hydrolysis at initial reaction times consisted of oligogalacturonates without detectable monomer. Thus, the purified Neurospora crassa enzyme was classified as an endopolygalacturonase [poly(1,4-alpha-D-galacturonide) glycanohydrolase; EC 3.2.1.15].     

A comparison of the properties of 5'-nucleotidase purified from the cytosolic and synaptic plasma membrane fractions of rat forebrain.

1. 5'-Nucleotidase was purified 1247-fold from the post-microsomal supernatant (I) and 3862-fold from the synaptic plasma membrane (II) of rat brain homogenates. 2. The apparent molecular masses of I and II were 131 and 72 kDa respectively by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and 268 and 286 kDa respectively by Sephacryl S-300 chromatography. 3. The activities of both I and II were strongly inhibited by concanavalin A but were affected differently by digestion with glycosidases. for II, these were 0.083 and 0.056 mM respectively. 5. Activities of both I and II were strongly inhibited by ATP and ADP.     

The properties of alpha-galactosidase remaining in kidney and liver of patients with Fabry's disease

α-Galactosidase activity is diminished in the kidney and liver of patients with Fabry's disease. Less than 2% of the normal activity was found in their kidney, while more than 20% of the normal activity was retained in their liver. The residual enzyme in these two organs showed a single component of pI 4.5 with activity toward 4-methylumbelliferyl α-galactoside on isoelectric focusing. This component seemed to correspond to Fr. II of normal liver or kidney. Ceramide trihexosidase activity was observed as a single component in the same fractions as the α-galactosidase activity for the synthetic substrate.In normal liver, 4-methylumbelliferyl α-galactoside hydrolase was separated into four components with pI's of 4.9, 4.5, 4.2 and 3.9 by isoelectric focusing. Fr. II with pI 4.5 differed from Fr. I in its heat stability and inhibition by myoinositol. In spite of some dissimilarities in their properties, the ratios of enzyme activities for ceramide trihexoside and 4-methylumbelliferyl α-galactoside were similar in all the components of both normal liver and kidney.     

Methyl viologen hydrogenase II, a new member of the hydrogenase family from Methanobacterium thermoautotrophicum delta H.

Two methyl viologen hydrogenase (MVH) enzymes from Methanobacterium thermoautotrophicum delta H have been separated (resolution, Rs at 1.0) on a Mono Q column after chromatography on DEAE-Sephacel and Superose 6 Prep Grade. The newly discovered MVH (MVH II) was eluted at 0.5 M NaCl with a linear gradient of 0.45 to 0.65 M NaCl (100 ml). The previously described MVH (MVH I) eluted in a NaCl gradient at 0.56 M. The specific activities of MVH I and MVH II were 184.8 and 61.3 U/mg of protein, respectively, when enzyme activity was compared at pH 7.5, the optimal pH for MVH II. Gel electrophoresis in nondenaturing systems indicated that MVH I and MVH II had a similar molecular mass of 145 kDa. Denatured MVH II showed four protein bands (alpha, 50 kDa; beta, 44 kDa; gamma, 36 kDa; delta, 15 kDa), similar to MVH I. The N-terminal amino acid sequences of the alpha, gamma, and delta subunits of MVH II were identical with the sequences of the equivalent subunits of MVH I. However, the N-terminal amino acid sequence of the beta subunit of MVH II was totally different from the sequence of the beta subunit of MVH I. Both MVH I and MVH II had the same optimal temperature of 60 degrees C for maximum activity. The pH optima of MVH I and MVH II were 9.0 and 7.5, respectively. Most of the divalent metal ions tested significantly inhibited MVH I activity, but MVH II activity was only partially inhibited by some divalent cations. Both hydrogenases were shown to be stable for over 8 days at --20 degrees C under anaerobic conditions. When exposed to air, 90% of MVH I activity was lost within 2 min; however, MVH II lost only 50% of its activity in 3 h.     

Solubilization and characterization of the initial enzymes of the dolichol pathway from yeast

Preparation and purification of substrate amounts of radioactive as well as non-radioactive dolichyl diphosphate N-acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I-III). The test conditions are described in detail. All three enzymes were solubilized from yeast membranes with detergents. Enzyme II and III were purified to give a purification factor of 35-fold and 70-fold, respectively. The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+. Enzyme II was stimulated almost to the same extent also by Ca2+. The Km values for UDP-N-acetylglucosamine for enzyme I and II were 15 and 10 muM, respectively, and for GDP-mannose (enzyme III) 7 muM. The apparent Km values for the lipophilic acceptor was 180 muM for enzyme I (dolichyl phosphate), 40 muM for enzyme II (dolichyl diphosphate N-acetylglucosamine) and 17 muM for enzyme III (dolichyl diphosphate chitobiose). The corresponding V values were approximately 1, 10, and 50 nmol X h-1 X mg protein-1. All reactions were inhibited by nucleoside diphosphates.