Sialyltransferase activity in regenerating rat liver

Liver microsomal fractions catalyse the transfer of sialic acid from CMP-N-acetyl-neuraminic acid to various exogenous acceptors such as desialylated fetuin, desialylated human Tamm–Horsfall glycoprotein and desialylated bovine submaxillary-gland mucin. An increase in the rate of incorporation of sialic acid into desialylated glycoproteins was found after a lag period (7h) in regenerating liver. The increase was maximum 24h after partial hepatectomy for all acceptors tested. At later times after operation the sialyltransferase activity remained high only for desialylated fetuin. No soluble factors from liver or serum of partially hepatectomized animals influenced the activity of the sialyltransferases bound to the microsomal fraction. The sensitivity of sialyltransferases to activation by Triton X-100, added to the incubation medium, was unchanged in the microsomal preparation from animals 24h after sham operation or partial hepatectomy. The full activity of sialyltransferases towards the various desialylated acceptors showed some differences. Human Tamm–Horsfall glycoprotein was a good acceptor of sialic acid only when desialylated by mild acid hydrolysis. After this treatment, but not after enzymic hydrolysis, a decrease in molecular weight of human Tamm–Horsfall glycoprotein was observed. Further, the sialyltransferase activity as a function of incubation temperature gave different curves according to the acceptor used. The relationship between the biosynthesis of glycoproteins by regenerating liver and the sialyltransferase activity of microsomal fraction after partial hepatectomy is discussed.     

Characterization and solubilization of membrane bound diacylglycerol lipases from bovine brain

Bovine brain contains two diacylglycerol lipases. One is localized in purified microsomes and the other is found in the plasma membrane fraction. The microsomal enzyme is markedly stimulated by the non-ionic detergent, Triton X-100, and Ca2+, whereas the plasma membrane diacylglycerol lipase is strongly inhibited by Triton X-100 and Ca2+ has no effect on its enzymic activity. Both enzymes were solubilized using 0.25% Triton X-100. The solubilized enzymes followed Michaelis-Menten kinetics. The apparent Km values for microsomal and plasma membrane enzymes are 30.5 and 12.0 microM respectively. Both lipases are strongly inhibited by RHC 80267, with Ki values for microsomal and plasma membrane diacylglycerol lipases of 70 and 43 microM, respectively. The retention of microsomal diacylglycerol lipase on a concanavalin A-Sepharose column and its elution by methyl alpha-D-mannoside indicates the glycoprotein nature of this enzyme.     

Human lung adenocarcinoma alpha1,3/4-L-fucosyltransferase displays two molecular forms, high substrate affinity for clustered sialyl LacNAc type 1 units as well as mucin core 2 sialyl LacNAc type 2 unit and novel alpha1,2-L-fucosylating activity.

Human lung tumor alpha1,3/4-L-fucosyltransferase (FT) was purified (2000-fold, 29% recovery) from 290 g of tissue by including a chromatography step on Affinity Gel-GDP. Two molecular forms (FTA, larger size carrying 15% alpha1,4-FT activity; FTB, the major form with 85% activity) were separated by further fractionation on a Sephacryl S-100 HR column. A difference in the electrophoretic mobilities of these two activities was also found on native polyacrylamide gel electrophoresis (PAGE). Both forms were devoid of typical alpha1,2-fucosylating activity but were associated with the novel alpha1,2-fucosylating ability of converting the Lewis a determinant to Lewis b. Based on percentage activity toward 2-O-MeGalbeta1,3GlcNAcbeta-O-Bn, both forms exhibited the same extent of activity toward various acceptors, which included sulfated, sialylated, or methylated LacNAc type 1 or type 2 as well as mucin core 2 acceptors. However, FTA and FTB exhibited a difference in their ability to act on mucin core 2 3'-sialyl LacNAc (activities 24.2% and 40.8%, respectively, as compared to 2-O-MeGalbeta1,3GlcNAcbeta-O-Bn). The unsubstituted LacNAc type 1 acceptors were 15-20 times as active as the corresponding LacNAc type 2 acceptors. The 3-O-substitution on the beta1,4-linked Gal (methyl, sulfate, or sialyl) in mucin core 2 acceptors increased the efficiency of these acceptors five- to eightfold. The most efficient acceptor for FTA and FTB was 3-O-sulfoGalbeta1,3GlcNAcbeta-O-Al (K(m) 100 and 47 microM, respectively). The K(m) (mM) values for 2-O-methyl Galbeta1,3GlcNAcbeta-O-Bn and 3-O-sialyl Galbeta1,3GlcNAcbeta-O-Bn were 0.40 and 2.5 (FTA) and 0.16 and 0.67 (FTB), respectively. The 35-kDa glycoprotein ancrod (from Malayan pit viper venom) containing 36% complex N-glycans with the antennae NeuAcalpha2,3Galbeta1,3GlcNAcbeta- acted as the best macromolecular acceptor substrate (K(m): 45 microM), as examined with FTB. On desialylation the acceptor efficiency dropped to approximately 50% (K(m) for asialo ancrod: 167 microM). Sialylglycoproteins, such as carcinoembryonic antigen, fetuin, and bovine alpha(1)-acid glycoprotein, were better acceptors than asialo fetuin. On the contrary, fetuin triantennary glycopeptide containing predominantly NeuAcalpha2,3Galbeta1,4GlcNAcbeta- was only 55% active as compared to the asialo glycopeptide (K(m): 1.43 and 0.63 mM, respectively). Thus, the human lung tumor alpha1,3/4-L-FT has the potential to generate clustered sialyl Lewis a and Lewis b determinants in N-glycans and sialyl Lewis x determinant in mucin core 2 structures.     

Ganglioside biosynthesis in rat liver. Characterization of UDPgalactose--glucosylceramide galactosyltransferase and UDPgalactose-GM2 galactosyltransferase

The conditions for the quantitative determination of UDP-Gal:glucosylceramide galactosyltransferase and of UDP-Gal:GM2 galactosyltransferase in Golgi-enriched preparations of rat liver were optimized. Triton X-100 was the detergent routinely used as octyl glucoside acted as a galactose acceptor forming octyl lactoside. Manganese ions were required for full activity, but Co2+ and Mg2+ could substitute to some extent. The nucleotide pyrophosphatase activity of the Golgi preparations which interfered with the GL2-synthase assay was inhibited by addition of 20 mM IMP; the latter is without appreciable effect on the rate of GL2 synthesis. Apparent Km values for UDP-Gal were 130 microM and 140 microM with Gl2-synthase and Gm1-synthase, respectively. That for glucosylceramide was 80 microM with GL2-synthase; for GM2 it was 10 microM with GM1-synthase. Competition experiments with variable concentrations of the lipid acceptors showed that the two synthase activities are independent catalytic entities. The specific activity of GM1-synthase exceeds that of GL2-synthase by a factor of ca. 25 under the optimized conditions used here.     

Partial characterization of microsomal sialyltransferase from chicken liver and hepatoma Mc-29: II. Measurement of enzyme activities utilizing microsomal glycoproteins as exogenous acceptors

Microsomal sialyltransferase was assayed in chicken liver and hepatoma Mc-29 utilizing liver and hepatoma microsomal glycoprotein fractions, treated with Triton X-100, as exogenous acceptors. In a homologous assay system containing enzyme and acceptor from one and the same tissue no quantitative dependence of enzyme activity was revealed with increasing amount of the acceptor. In mixed experiments in which liver enzyme activity was tested towards hepatoma acceptor glycoproteins, a gradual drop in sialyltransferase activity occurred with increasing quantities of the acceptor. This effect seems to be a consequence of the presence of some inhibitor in the microsomal fractions from the hepatoma cells.     

Purification to homogeneity of a beta-galactoside alpha2 leads to 3 sialyltransferase and partial purification of an alpha-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase from porcine submaxillary glands.

Two different sialyltransferases (EC 2.4.99.1) have been resolved from Triton X-100 extracts of porcine submaxillary glands by affinity chromatography on CDP-hexanolamine agarose. The predominant sialyltransferase of this tissue, a CMP-N-acetylneuraminate: alpha-D-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase, has been obtained in a partially purified and stable form. A less abundant but highly active enzyme, a CMP-N-acetylneuraminate: beta-D-galactoside alpha2 leads to 3 sialyltransferase, was purified over 90,000-fold to homogeneity. Chromatography of the latter enzyme on Sephadex G-200 separated two noninterconverting forms, designated A and B, with Stokes radii of 51 A and 31 A, respectively. Both forms have equal specific activity toward lactose and contain a single polypeptide with a molecular weight of about 50,000 as estimated by gel electrophoresis. Form A appears to bind 1.18 g of Triton X-100 per g of protein, or nearly an entire detergent micelle per polypeptide, while Form B binds little or no detergent. The enzymatic properties of both forms are similar (Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) J. Biol. Chem. 254, 4444-4451) supporting the conclusion that Form A may represent the native sialyltransferase with an intact membrane-binding site, and Form B may be a large proteolytic fragment of Form A.     

Sialyltransferase of bovine serum: age- and hormone-related changes

Sialyltransferase activity of bovine serum with the acceptor asialofetuin exhibits a pH optimum at 6.0-6.5, no divalent cation dependence, and a Km for CMP-sialic acid of 0.05 mM. Although a 2-fold increase in sialyltransferase activity with the acceptor asialofetuin is observed in serum samples from 2-day-old vs 20-day-old calves, the relative activity towards other glycoprotein acceptors is not different between the groups. With the acceptor lactose, the major product (greater than 95%) for all samples is 3'-sialyllactose, suggesting that the elevated levels of sialyltransferase in 2-day-old calves are due to Gal-R (alpha 2-3) sialyltransferase.     

Studies on glycosyltransferases in fusion-defective conA-resistant L6 rat myoblast cell lines

1. Sialyl- and galactosyl-transferase activities were determined in wild type and conA-resistant L6 rat myoblasts with substrates derived from fetuin, alpha 1-acid glycoprotein and bovine submaxillary mucin; fetuin was the best acceptor for both enzyme activities, whereas the mucin did not act as an acceptor. 2. The optimum pH for sialyltransferase was 6.6 in both cell lines. 3. The optimum pH for galactosyltransferase in the wild type cell line was 6.2 which was slightly higher than the value of 5.8 found for the conA-resistant cells. 4. Values for Km for both enzyme activities increased five to ten-fold in the variant cell line with both acceptors. 5. The main sialyltransferase activity was the Gal beta 1----4GlcNAc alpha 2----3sialyltransferase for N-linked chains. The galactosyltransferase was most likely the enzyme that is responsible for the synthesis of the Gal beta 1----4GlcNAc structure.     

Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase

The detergent-induced amplification of lucigenin-dependent chemiluminescence of O2-, generated by xanthine oxidase or microsomal NADPH oxidase was studied. An assay system is described which is at least 10 times more sensitive than normal lucigenin-dependent chemiluminescence due to the amplification by high concentrations of octylphenylpolyethylene glycol (Triton X-100). Compared to the superoxide dismutase-sensitive reduction of acetylated cytochrome c, a 3750-fold lower amount of microsomal protein was necessary to produce an O2- signal 10-fold above the background. In contrast to cytochrome c reduction, detergent-amplified chemiluminescence of lucigenin was completely inhibited by superoxide dismutase and therefore more selective for O2-. The membrane-bound and Triton X-100-solubilized NADPH oxidase from microsomes of macrophages was activated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and inhibited by Ca2+ and sodium dodecyl sulfate. The membrane-bound enzyme showed a Km value of 1.35 microM, which decreased to 0.95 microM after the addition of 12% (g/g) Triton X-100. The Km and Vmax values of soluble xanthine oxidase were not influenced by Triton X-100, indicating that the enzyme activities were not impaired by the high concentrations of detergent.     

Glycoprotein biosynthesis in calf kidney. Glycoprotein sialyltransferase activities towards serum glycoproteins and calf Tamm-Horsfall glycoprotein.

CMP-AcNeu:glycoprotein sialyltransltransltransltransltransferase of calf kidney cortex was characterized using serum glycoproteins and Tamm-Horsfall glycoprotein, obtained from calf urine, as acceptors. Native calf Tamm-Horsfall glycoprotein showed the best acceptor properties, followed by desialylated calf fetuin and desialylated human alpha 1-acid glycoprotein exhibiting V values of, respectively, 114, 63 and 41 nmol/h per g wet wt. of kidney cortex and Km values of 0.12, 0.16 and 0.26 mM glycoprotein acceptor. Desialylated ovine submaxillary mucine appeared to be a very poor acceptor. Tamm-Horsfall glycoprotein sialyltransferase could be distinguished from serum glycoprotein sialyltransferase by competition studies. In addition the two glycoprotein sialyltransferase activities showed different distributions over the three regions of the calf kidney: the ratios of the Tamm-Horsfall to serum glycoprotein sialyltransferase activities decreased from 3.3 in the cortex to 0.8 and 0.4 in the medulla and the papilla, respectively. It was concluded that in calf kidney at least two different sialyltransferases exist. The high cortical Tamm-Horsfall glycoprotein sialyltransferases activity corresponds markedly to the origin of the urinary Tamm-Horsfall glycoprotein, namely the distal part of the kidney tubule. Inactivation of glycoprotein sialyltransferase activity by preincubation at various temperatures and during storage at 0 degree C, could be reduced by the addition of CMP-AcNeu. The possible relevance towards the in vivo sialylation of this finding is discussed.     

Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes

Some properties of two distinct rat brain sialyltransferases, acting on fetuin and asialofetuin, respectively, were investigated. These two membrane-bound enzymes were both strongly inhibited by charged phospholipids. Neutral phospholipids were without effect except lysophosphatidylcholine (lysoPC) which modulated these two enzymes in a different way. At 5 mM lysoPC, the fetuin sialyltransferase was solubilized and highly activated while the asialofetuin sialyltransferase was inhibited. Preincubation of brain microsomes with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), known as a specific anion inhibitor and a non-penetrating probe, led to a moderate inhibition of the asialofetuin sialyltransferase just as in the case of the ovomucoid galactosyltransferase (used here as a marker for the luminal side of the Golgi membrane); under similar conditions, the fetuin sialyltransferase was strongly inhibited. In the presence of Triton X-100, which induced a disruption of membranes, all three enzymes were strongly inhibited by DIDS. Trypsin action on intact membranes showed that asialofetuin sialyltransferase, galactosyltransferase and fetuin sialyltransferase were all slightly inhibited. After membrane disruption by Triton X-100, the first two enzymes were completely inactivated by trypsin while the fetuin sialyltransferase was quite insensitive to trypsin treatment. From these data, we suggest that the fetuin sialyltransferase, accessible to DIDS, is an external enzyme, oriented closely towards the cytoplasmic side of the brain microsomal vesicles (endoplasmic and Golgi membranes), whereas the asialofetuin sialyltransferase is an internal enzyme, oriented in a similar manner to the galactosyltransferase. Moreover, the anion site (nucleotide sugar binding site) of the fetuin sialyltransferase must be different from its active site, as this enzyme, when solubilized, is strongly inhibited by DIDS while no degradation is observed in the presence of trypsin.     

Effect of ethanol on mucus glycoprotein fatty acyltransferase from gastric mucosa

The enzyme activity that catalyzes the transfer of palmitic acid from palmitoyl coenzyme A to the deacylated intact or deglycosylated gastric mucus glycoprotein was demonstrated in the detergent extracts of the microsomal fraction of antral and body mucosa of the rat stomach. Both types of mucosa exhibited similar acyltransferase activities and acceptor specificities. A 10-14% decrease in the fatty acyltransferase activity was observed with the reduced and S-carboxymethylated mucus glycoprotein, but the proteolytically degraded glycoprotein showed no acceptor capacity. This indicated that the acylation of mucus glycoprotein with fatty acids occurs at its nonglycosylated polypeptide regions and that some of the fatty acids may be linked via thiol esters. Optimum enzyme activity was obtained at pH 7.4 with the detergent Triton X-100, NaF, and dithiothreitol. The apparent Km values for the intact and deglycosylated mucus glycoproteins were 0.45 and 0.89 microM, respectively. The acyltransferase activity of the microsomal enzyme was inhibited by ethanol. With both intact and deglycosylated glycoprotein substrates, the rate of inhibition was proportional to the ethanol concentration up to 0.4 M and was of the competitive type. The K1 values were 0.80 microM for the intact mucus glycoprotein and 1.82 microM for the deglycosylated glycoprotein. Preincubation of the microsomal enzyme with low concentrations of ethanol (up to 0.5 M) did not seem to exert any additional deterrent effect on acyltransferase activity. Higher concentrations of ethanol (1.0 M and above), however, caused substantial reduction in the transferase activity due to denaturation of the enzyme.     

Platelet membrane phosphatidylinositol kinase activity. Triton X-100 effects provide evidence for intramicellar reaction.

Phosphatidylinositol (PI) kinase activity of platelet membranes was solubilized and partially purified by anion-exchange chromatography to measure the initial enzymatic rates. Kinetic studies were performed in the presence of Triton X-100 to obtain mixed micelles. The partially purified enzyme exhibited a Michaelian behaviour towards ATP, with a Km of 58 microM. The enzymatic rates were dependent upon Triton concentrations. Upon increasing its concentration, this detergent exhibited an activating effect followed by an inhibitory one. The optimal micellar Triton concentration was proportionnal to the PI concentration used in the assay. Conversely, the behaviour of the enzyme towards PI was dependent upon the Triton concentration. However, when PI concentration was expressed as its surfacic concentration within the micelles, the activity became independent of the detergent concentration, and a Km value of 0.09 mol/mol was estimated. Therefore, in vitro phosphorylation of phosphatidylinositol by PI kinase is rate-limited by an intramicellar reaction, and provides a study model for the in vivo reaction.     

Glycosylation of kappa-casein. I. Localization and characterization of sialyltransferase in bovine mammary gland.

A sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) which attaches N-acetylneuraminic acid to the terminal end of the carbohydrate chain of kappa-casein was found to be concentrated in Golgi apparatus-enriched fractions of bovine mammary gland. Maximum sialyltransferase activity was obtained at pH 5.5 and 37 degrees C in the presence of 1 mM dithiothreitol and Triton X-100. A Km of 0.19 mg asialo-kappa-casein/ml (0.01 mM) was obtained for the sialyltransferase. Native kappa-casein also served as acceptor for N-acetylneuraminic acid transferase of Golgi apparatus-enriched fractions although at a slower rate than did asialo-kappa-casein. The sialyltransferase has a divalent cation requirement for maximum activity which was best satisfied by the presence of 10 mM Mn2+.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.     

Detergents inhibit exocytosis in PC 12 cells: evidence for an effect on ion fluxes

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+-stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X-100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 microM and 10 microM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4). The inhibitory effect of 30 microM Triton X-100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X-100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X-100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X-100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13-14 degrees C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished. In the presence of 30 microM Triton X-100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50-60%. These data suggest that the site of action of Triton X-100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.     

Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases

Three sialyltransferase activities involved in ganglioside biosynthesis were studied in Golgi-enriched preparations of rat liver: the formation of GM3, GD3 and GD1a. The conditions for the quantitative assays of these enzymatic reactions were standardized and optimized, with Triton X-100 being used as detergent. The apparent Km values of each sialyltransferase for N-acetyl-2-(5'-cytidylyl)neuraminic acid (1.5 mM with GM3 synthase, 0.2 mM with GD3 synthase, and 0.5 mM with GD1a synthase) and the respective glycolipid substrates (0.08 mM for lactosylceramide, 0.1 mM for GM3, and 0.5 mM for GM1) were determined. Competition experiments showed that the three sialyltransferase activities are three individual catalytic entities. Moreover, evidence was found that product inhibition may play a role in the regulation of the activity of sialyltransferases.     

NADH dehydrogenase from bovine neutrophil membranes. Purification and properties

A membrane-associated NADH dehydrogenase from beef neutrophils was purified to homogeneity, using detergent (cholate plus Triton X-100) extraction and chromatography on DEAE-Sepharose CL-6B, agarose-hexane-NAD, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 17,500, but the enzyme was highly aggregated (Mr greater than 450,000) in nondenaturing gels containing 0.1% Triton X-100. The protein band in nondenaturing gels was also stained for activity using NADH and nitro blue tetrazolium. The enzyme showed greatest electron acceptor activity with ferricyanide (100%), followed by cytochrome c (3.5%), dichloroindophenol (2.7%), and cytochrome b5 (0.34%). No activity was seen with oxygen. The Km values for NADH and ferricyanide were 18 and 9.5 microM, respectively, and NAD+ was a weak competitive inhibitor (Ki = 118 microM). No activity was seen with NADPH. No effects were seen with mitochondrial respiratory inhibitors such as azide, cyanide, or rotenone, but p-chloromercuribenzoate was strongly inhibitory and N-ethylmaleimide was weakly inhibitory. No free flavin was detectable in enzyme preparations. Based upon kinetic, physical, and inhibition properties, this NADH dehydrogenase differs from those previously described in microsomes and erythrocyte plasma membrane.     

Ganglioside GD3 biosynthesis in normal and mutant mouse embryos

CMP-sialic acid:GM3 sialyltransferase (GD3 synthase; EC 2.4.99.8) was characterized in a membrane-enriched preparation (P2 pellet) from mouse embryos at embryonic day 12 (E-12). Gangliosides GD3 and GM3 were the major radiolabeled products of the reaction. Optimum GD3 synthase activity was obtained at pH 6.0 using 0.1% detergent Triton CF-54. The Km values for GM3 and CMP-sialic acid were 55 and 80 microM, respectively. The Vmax value was calculated as 622 pmol/mg protein/hr. Ganglioside GD3, as end product, induced a two-step reduction of enzyme activity in the range of concentrations from 0 to 34 microM (40%) and from 150 to 300 microM (65%). The rate of GD3 formation was similar in whole embryos and in embryo head and body regions. GD3 synthase activity in tw1/tw1 mutant mouse embryos, which express defects in neuronal differentiation, was only 40% of that in normal wild-type (+/+) embryos. Enzyme activity in heterozygous (+/twl) embryos was similar to that in +/+ embryos. These findings suggest that the reduced GD3 synthase activity in the mutants might arise as a consequence of failed nervous system development and might reflect a secondary rather than a primary effect of the mutation.     

D-aspartate oxidase in mammalian brain and choroid plexus

Abstract— Synaptosomes from guinea-pig cerebral cortex contain a fetuin: sialyl glyco-protein: glycosyl transferase; evidence is presented which indicates that both a sialyl transferase; evidence is presented which indicates that both a sialyl transferase and endogenous acceptors were located in the synaptosome ‘ghost’ fractions. Following solubilization of synaptosomes with Triton X-100 and the use of fetuin minus NANA as acceptor, 25 per cent of the transferase was recovered after centrifugation and column chromatography on Sephadex G-100 and G-200 with a 64·0-fold purification. The enzyme had a pH optimum of 6·3, required no divalent metal cation for activity, and exhibited high activity with either fetuin minus sialic acid, prothrombin minus sialic acid, Tamm-Horsfall glycoprotein minus sialic acid, or orosomucoid minus sialic acid as acceptor; neither BSM nor PSM minus NANA functioned as an effective acceptor. The fetuin:sialyl transferase using fetuin minus sialic acid and CMP-sialic acid as substrates a and b, respectively, gave the following kinetic constants when using the Cleland bisubstrate model: K_a= 35μM; K_b= 3 μM; K_ia, = 25 μM; K_ib= 25μM; and V₁= 92 pmoles. min^?1.mg^?1 of protein. The following divalent cations inhibited the reaction: Ba²⁺ > Hg²⁺ > Pb²⁺ > Cu²⁺.