Further Evidence for an Intrinsic Neuraminidase in CNS Myelin

An intrinsic neuraminidase activity in rat brain CNS myelin has been demonstrated and compared with the neuraminidase activity in rat brain microsomes. With use of ganglioside GM3 as a substrate, the myelin-associated neuraminidase exhibited a shallow pH curve with an optimum at pH 4.8 whereas the microsomal activity had a marked optimum at pH 4-4.3. Neuraminidase activity in both fractions was optimized in 0.3% Triton CF-54 but activation was much greater in the microsomes. When the neuraminidase activities were examined at 60 degrees C, the myelin neuraminidase activity was more than sevenfold of that observed at 37 degrees C and was linear for at least 2 h; the microsomal activity increased only fivefold initially and exhibited a continual loss in activity. Addition of excess microsomes to the total homogenate prior to myelin isolation resulted in no change in myelin neuraminidase activity. When the two membrane fractions were examined at equivalent protein concentrations in the presence of additional cations or EDTA (1 mM), similar but not identical effects on neuraminidase activity were seen. The microsomal neuraminidase was considerably more susceptible to inhibition by divalent copper ion. Activity in both fractions was markedly inhibited by Hg2+ and Ag+ whereas EDTA had no effect on either activity. The myelin-associated neuraminidase activity was the highest in cerebral hemispheres, followed by brainstem, cerebellum, and spinal cord and was extremely low in sciatic nerve. In fact, the myelin neuraminidase activity was higher than the microsomal enzyme activity in the cerebral hemispheres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of ortho- and paramyxovirus neuraminidase on gangliosides. Hydrolysis of ganglioside GM1 by Sendai virus neuraminidase

The action of neuraminidase of influenza A virus, Sendai virus and Newcastle disease virus particles on bovine brain ganglioside GM1 and the properties of Sendai virus neuraminidase for GM1 were studied. With Sendai virus, GM1 was hydrolyzed to asialo-GM1 (GA1) and N-acetylneuraminic acid even in the absence of surfactant or other additives, while the hydrolysis of GM1 by Newcastle disease virus or influenza A virus was very low or undetectable under the same conditions. The formation of GA1 by Sendai virus neuraminidase was confirmed by thin-layer chromatography and immunodiffusion test using anti-GA1 antiserum. The apparent Km of Sendai virus neuraminidase for GM1 hydrolysis was found to be 2.67 x 10(-4) M and the optimum pH was 5.6. GM3, GM2 and oligosaccharide of GM1 were hydrolyzed more effectively than GM1 in the absence of surfactant (GM3 greater than GM2 greater than oligosaccharide of GM1 greater than GM1). The hydrolysis of GM1 by the Sendai virus enzyme was stimulated by the addition of sodium cholate or sodium taurocholate, but was inhibited by divalent cations (10 mM), Ca2+, Mg2+, ZN2+, Fe2+ and CU2+. In the absence of the surfactant, Sendai virus neuraminidase hydrolyzed GM1 more efficiently than Arthobacter ureafaciens neuraminidase which has been reported recently as being an adequate enzyme to hydrolyze ganglioside GM1 as a substrate.     

Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity.

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.     

Characterization of a CMPNeuAc: lactosylceramide alpha 2----3sialyltransferase from rainbow trout hepatoma (RTH-149) cells

1. The rainbow trout (Oncorhynchus mykiss) CMPNeuAc:lactosylceramide alpha 2----3sialytransferase enzyme from RTH-149 cells has been characterized. 2. Transfer of sialic acid to lactosylceramide was optimal at a pH of 5.9, temperature of 25 degrees C, and in the pressure of 0.3% CF-54, 10 mM Mn2+, 0.1 M sodium cacodylate, and 2 mM ATP. 3. Golgi-rich membrane fractions of RTH-149 cells were found to be enriched in sialidase activity and as such the addition of 40 microM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was necessary to assay alpha 2----3sialyltransferase activity optimally. 4. Apparent Km for donor (CMPNeuAc) and acceptor (lactosylceramide) were found to be 243 microM and 34 microM, respectively. 5. The alpha 2----3sialyltransferase characterized was found to be primarily specific for lactosylceramide though minor activity with other glycolipid acceptors was observed. 6. The presence of another sialyltransferase with differing substrate specificity was noted. 7. Properties of this enzyme, compared to analogous mammalian enzymes, are discussed.     

Macular cherry-red spots and myoclonus with dementia: coexistent neuraminidase and beta-galactosidase deficiencies.

A patient was previously characterized as having a variant form of G_M1 gangliosidosis based on severe deficiencies in β-galactosidase activity in both leukocytes and fibroblasts using 4-methylumbelliferyl-β-D-galactoside and G_M1 ganglioside. Reexamination of her cultured fibroblasts revealed a severe deficiency in neuraminidase activity using neuramin lactose, fetuin and 2-(3′-methoxyphenyl)-N-acetyl-D-neuraminic acid as substrates, but normal neuraminidase activity using G_M3 ganglioside as a substrate. The presence of normal levels of β-galactosidase activity in leukocytes from the mother of the patient indicates that the β-galactosidase deficiency is not the primary enzyme defect in this type of patient.     

Characterization of influenza virus neuraminidase with hemagglutinin activity and its comparison with that of viral neuraminidase

The neuraminidase associated with the bifunctional protein, hemagglutinin-neuraminidase, of influenza virus has been characterized. The enzyme has a pH optimum of 4.5, does not require Ca2+ and is inactivated (98%) by incubation at 50 degrees C. The enzyme has a Km of 2.00 X 10(-3) M and 0.06 X 10(-3) M with the substrates 2-(3-methoxyphenyl)-N-acetylneuraminic acid and fetuin, respectively. The Ki is 400 X 10(-6) with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. The incorporation of labeled cysteine, valine and leucine in the hemagglutinin-neuraminidase protein is different from that of viral neuraminidase. A comparison of the properties of the neuraminidase associated with protein hemagglutinin-neuraminidase with that of viral neuraminidase or sialidase showed that the former is biochemically different and an antigenically distinct enzyme. The unique feature of the new enzyme is that it has the hemagglutinin activity as well. The two biological activities could not be separated from each other in all systems used. Apparently, protein hemagglutinin-neuraminidase is genetically transferable and it is detectable in a laboratory recombinant virus E-2971 (H3 Aichi X N7). These results suggest that protein hemagglutinin-neuraminidase is a unique surface protein of the influenza virus A/Aichi/2/68 (H3N2).     

The influence of lysosomes on glycogen metabolism.

Treatment of rabbit spermatozoa with 50mM-MgCl2 removes the plasma and the outer acrosomal membranes. Subsequent treatment with the detergents Hyamine 2389 and Triton X-100 solubilizes spermatozoal neuraminidase bound to the inner acrosomal membrane. The enzyme was further purified by DEAE-cellulose, Sephadex G-150 and Bio-Gel P-300 column chromato. The enzyme showed a single major band, with the possibility of some minor contaminants, on disc-gel electrophoresis. It had a specific activity of 0.37 micronmal of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2 leads to 6'-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72 X 10(-6)M with Cowper's-gland mucin, 1.17 X 10(-5)M with fetuin and 8.8 X 10(-4)M with sialyl-lactose as a substrates. The Vmax. was 0.112 micronmol/min per mg with the Cowper's-gland mucin, 0.071 micronmol/min per mg with fetuin and 0.033 micronmol/min per mg with sialyl-lactose as substrate. The enzyme hydrolysed sheep submaxillary-gland mucin as readily as the Cowper's-gland mucin. The optimum of enzyme activity was at pH 5.0 on the Cowper's-gland mucin and at pH4.3 on sialyl-lactose. The enzyme activity was unaffected by 20mM-Na+ and-K+, but was inhibited by 20mM-Ca2+,-Mn2+,-Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at --20 degrees C.     

Characterization of sialidase from bloodstream forms of Trypanosoma vivax

Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.     

Neuraminidase Activities in Oligodendroglial Cells of the Rat Brain

Neuraminidase activities in oligodendroglial cells were characterized using rats of different ages. Rat oligodendroglial cells had intrinsic neuraminidase activities directed toward GM3 and N-acetylneuramin(2-3)lactitol (NL). Developmental profiles of the neuraminidase activities toward the two substrates in oligodendroglial cells were different from each other. The neuraminidase activity toward GM3 increased rapidly with the onset of active myelination and, after 26 days of development, reached the adult level which was about 18 times higher than that in myelin. At the adult age, oligodendroglial cells had the highest neuraminidase activity toward GM3 among the individual brain cell types examined. The activity of NL-neuraminidase showed a less remarkable developmental profile, with a peak value at 26 days. The UDP-galactose:ceramide galactosyltransferase activity in oligodendroglial cells increased during the period of active myelination and, afterward, returned to the basal level. The enrichment and unique developmental profile in oligodendroglial cells of the neuraminidase activity toward GM3 suggest that this enzyme may play an important role in the formation and maintenance of the myelin sheath.     

Subcellular Fractionation of Rat Sciatic Nerve and Specific Localization of Ganglioside LM1 in Rat Nerve Myelin

Subcellular fractionation of rat sciatic nerve was developed to determine the specific localization of gangliosides in the nerve membrane fractions. Myelin, microsomal, and a plasma membrane-like fraction were isolated and purified by sucrose density gradient centrifugation. These subfractions were characterized by electron microscopy, marker enzyme assays, and their protein and lipid profile. In rat sciatic nerve myelin, 90 mol% of the total gangliosides were monosialogangliosides. LM1 (sialosyl-lactoneotetraosylceramide) (61 mol%) and GM3 (21%) were the major gangliosides of the rat nerve myelin. Two other neolacto series of gangliosides, viz., sialosyl-lactoneonorhexaosylceramide and sialosyl-lactoneooctaosylceramide, were also localized mostly in the myelin fraction. GM1 was only a minor (less than 2%) ganglioside in myelin. The ganglioside patterns of the microsomal and plasma membrane-like fractions were similar with minor quantitative differences and were entirely different from that of myelin. Monosialogangliosides were approximately 70-75 mol% of the total in these fractions. The major gangliosides of the microsomal and plasma membrane-like fractions were GM3 (approximately 40%) and GM1 (approximately 20%). LM1 in these fractions was minimal (less than approximately 5%). Significant amounts of GM3 with N-glycolylneuraminic acid (approximately 10%) and GM1b (4-14%) were also identified in the microsomal and plasma membrane-like fractions but not in myelin. These and the higher lactoneo series of gangliosides have not been previously reported to be present in the rat nervous system. Almost exclusive localization of LM1 in myelin in rat peripheral nervous system is consistent with our previous observation that deposition of LM1 in the nerve with age was very similar to that of myelin marker lipids cerebrosides and sulfatides.     

Role of Myelin-Associated Neuraminidase in the Ganglioside Metabolism of Rat Brain Myelin

The role of myelin-associated neuraminidase in ganglioside metabolism was examined using rats of ages ranging from 17 to 97 days. The neuraminidase activity directed toward the ganglioside GM3 in the total myelin fraction was high during the period of active myelination and, thereafter, decreased rapidly to the adult level. The ganglioside composition became simpler during development with an increasing amount of GM1 and decreasing percentages of di- and polysialogangliosides. The decrease in the proportion of GD1a was most prominent, whereas relative amounts of GD1b and GT1b increased transiently before reducing to the adult levels. The heavy myelin subfraction contained higher percentages of di- and polysialo-species compared to the light myelin fraction at young and adult ages. The in vitro incubation of myelin of young rats under an optimal condition for neuraminidase action produced a profile of ganglioside changes similar to that observed in in vivo development. These results strongly suggest that myelin-associated neuraminidase may play a pivotal role in the developmental changes in the ganglioside composition of rat brain myelin.     

Properties of a protein activator of glycosphingolipid hydrolysis isolated from the liver of a patient with GM1 gangliosidosis,Type 1

The heat stable protein activator of GM1 ganglioside hydrolysis was isolated from the liver of a patient with GM1 gangliosidosis, Type 1. It was found to be present at a level about 35 times that found in a liver sample from an age matched control. This activator protein was demonstrated to stimulate the hydrolysis of GM1 ganglioside and GA1 (asialo-GM1 ganglioside) in the presence of purified GM1 ganglioside β-galactosidase without the need for bile salt detergents. It could not stimulate the hydrolysis of two other galactosphingolipids, galactosylceramide and lactosylceramide, in the presence of the same enzyme. Lactosylceramide was a good substrate for this enzyme when sodium glycodeoxycholate was included in the assay. This activator protein had two isoelectric points pH 4.1 and 4.6, and it had an apparent molecular weight of 27,000 by gel filtration.     

The influence of ganglioside insertion into brain membranes on the rate of ganglioside degradation by membrane-bound sialidase

Microsomal membranes isolated from calf brain contain a sialidase which cleaves ganglioside substrates naturally occurring within these membranes as well as exogenously added [3H]ganglioside GD1a. Micelles of [3H]ganglioside GD1a bind to the microsomal membranes in two steps. The first step, called adsorption, is fast and reversible by treatment with trypsin; the second step, called uptake, is slower and not reversible. The product of the enzymic degradation, [3H]ganglioside GM1, is exclusively located in the ganglioside pool taken up by the sialidase-bearing membranes, and not in the trypsin-releasable pool. Electron spin resonance (ESR) studies using a spin-labelled analogue of ganglioside GD1a indicate that the ganglioside uptake by microsomal membranes is accompanied by the disappearance of the micellar structure and by the 'dilution' of the probe molecules with membrane lipids. These findings suggest that exogenously added ganglioside substrate inserts into the microsomal membrane before it is recognized as substrate by the membrane-bound sialidase. Therefore, the influence of pH, ionic strength and membrane-fluidizing agents on the degradation rate measured with exogenous ganglioside GD1a does not only reflect kinetic parameters of the enzymic reaction itself but also the velocity of ganglioside insertion. Increasing ionic strength reduces the degradation rate. The acceleration of insertion with falling pH values shifts the measured pH optimum of the ganglioside degradation to lower values (pH 3.6) and masks the substantial residual sialidase activity at pH 5-7. The membrane-fluidizing alcohol n-hexanol greatly accelerates ganglioside insertion as well as ganglioside degradation. The latter was clearly demonstrated by studying the hydrolysis of endogenous ganglioside substrates, and is due to a decrease of the apparent Km value and an increase in the Vmax value. The Vmax value was also enhanced by freezing and thawing of the microsomal membranes.     

Purification and characterization of GM1 ganglioside beta-galactosidase from normal feline liver and brain.

GM1 ganglioside beta-galactosidase (GM1-beta-galactosidase) was purified from normal cat brain and liver by a combination of classical and affinity procedures. The final preparation of brain GM1-beta-galactosidase was enriched over 2000-fold with a 36% yield. However, the product was shown to contain several components by disc gel electrophoresis. GM1-beta-galactosidase was also purified from liver with greater than a 30 000-fold enrichment and 40% yield. The liver enzyme was judged homogeneous by disc gel electrophoresis at pH 4.3, 8.1, and 8.9 and by gel chromatography. Both liver and brain GM1-beta-galactosidase(s) eluted as sharp symmetrical peaks from Sephadex G-200 with molecular weights of 250 000 +/- 50 000. The apparent Km determined for 4-methylumbelliferyl beta-D-galactopyranoside (4-MU-Gal) using partially purified brain GM1-beta-galactosidase was 1.73 X 10(-4) M. Liver GM1-beta-galactosidase gave a Km with 4-MU-Gal of 3.25 X 10(-4) M and for [3H]GM1 ganglioside a Km of 4.51 X 10(-4) M was calculated. The pH optima of brain and liver GM1-beta-galactosidase using 4-MU-Gal was 3.8-4.5. By contrast, liver GM1-beta-galactosidase gave a sharp activity peak at pH 4.2 with [3H]GM1 ganglioside. Inhibition by mercuric chloride and sensitivity to hydrogen peroxide and persulfate suggest the involvement of a sulfhydryl in catalysis.     

Characterization of human placental neuraminidases. Stability, substrate specificity and molecular weight.

1. At least two components of neuraminidase can be distinguished on the basis of thermolability and sedimentability by using the artificial fluorogenic substrate 4-methylumbelliferyl N-acetyl-alpha-D-neuraminate. 2. In crude homogenates, thermodenaturation at 25 degrees C showed a biphasic curve corresponding to component A (half-life, 21 min) and B (half-life, 85 min). The two components were partially resolved by centrifugation. A being soluble and B sedimentable. Both had similar pH-activity curves (pH optimum, 4.4), Km values (A, 0.10 mM; B, 0.06 mM) and molecular weight as determined by radiation inactivation (A, 67000; B, 63000). 3. The soluble A form was still aggregated or bound to membranous debris since almost all neuraminidase activity was eluted near or at the void volume of a Sephacryl S-300 column. 4. Both soluble and sedimentable fractions of placenta hydrolysed the GD1A ganglioside and N-acetyl-neuraminyl-D-lactose linearly for 12 h but no fetuin hydrolysis was detected. 5. The neuraminidase activity with the artificial fluorogenic substrate was inhibited by N-acetylneuraminyl-D-lactose but not by the GD1A ganglioside. These preliminary results suggest that there exist two closely related enzymes hydrolysing both the artificial substrate and N-acetylneuraminyl-D-lactose and a third one hydrolysing the GD1A ganglioside exclusively.     

Effect of bile salts on the hydrolysis of gangliosides, glycoproteins and neuraminyl-lactose by the neuraminidase of Clostridium perfringens.

Studies were done on the effect of bile salts on the rates of hydrolysis of the N-acetylneuraminyl linkages of several sialic acid-containing compounds by the neuraminidase of Clostridium perfringens. When GM3-ganglioside, two glycolipids (glycophorin and orosomucoid) and neuraminyl-lactose were used as substrates, hydrolysis was obtained even in the absence of bile salts, but addition of this detergent, below its critical micellar concentration, increased the reaction rates; above the critical micellar concentration of the detergent rates decreased again. When a second ganglioside, GM1, was used as substrate, the requirement for bile salts was absolute; hydrolysis was not observed at all without this detergent. With increasing concentrations of bile salt and in the presence of high concentrations of enzyme, rates of hydrolysis increased, reaching maximal values at fixed ratios of bile salt to GM1-ganglioside. Physical measurements showed that mixtures of bile salt and GM1-ganglioside form mixed micelles that have a higher critical micellar concentration, a lower molecular weight and greater axial ratio than the corresponding micelles of pure GM1-ganglioside.     

Neuraminidase in Calf Retinal Outer Segment Membranes

Abstract: An enzyme catalyzing the hydrolysis of sialic acid ( N -acetylneuraminic acid: NeuNAc)-containing glycoconjugates has been found in bovine retinal rod outer segment (ROS) membranes. The enzymatic activity is optimal at pH 4.0 and is stimulated by 0.15% Triton X-100. Total activity was determined by the release of NeuNAc from endogenous and exogenous substrates (GDla). The ROS enzyme preferentially hydrolyses the ROS gangliosides, possibly because they are more accessible than the glycoproteins as substrates for the neuraminidase. Release of NeuNAc from gangliosides leads to important changes in the ganglioside patterns; whereas the amounts of GM1 increased throughout the incubation, the levels of polysialogangliosides GTlb and GD3 diminished owing to their rapid hydrolysis. The finding that gangliosides are hydrolysed more extensively than glycoproteins suggests that endogenous ROS gangliosides may be the principal source of metabolically available sialic acid in ROS. It was also observed that the activity of ROS neuraminidase is not affected by illumination of the membranes.     

Characterization of neuraminidase activity of cultured human fibroblasts.

Investigations have been carried out to establish the enzymatic properties and specificities of the neuraminidase of cultured human fibroblasts. Homogenates of these cells cleaved the actylated derivative of neuraminic acid from fetuin, N-acetylneuraminyllactose and 2-(3' methoxyphenyl)-N-acetyl-alpha-neuraminic acid. Maximum activity occurred between pH 4.2 and 4.6 in sodium acetate buffer. The Km values were 3.6 . 10(-4) M, 3.0 . 10(-3) M and 1.1 . 10(-3) M, respectively, against fetuin, N-acetylneuraminyllactose and 2-(3'methoxyphenyl)-N-acetyl-alpha-neuraminic acid. Against the first two substrates, the rate of hydrolysis fell below the expected value as the cell homogenate was diluted with water or 10 mM NaCl. Dilution with 8 mg/ml bovine serum albumin prevented the deviation and yielded the expected linear decrease. After the first 2-h incubation, the rate of hydrolysis decreased from the initial linear rate. The enzyme(s) was partially or completely inactivated by sonication at 20 kHz, freeze-thaw treatment, incubation at 52 degrees C or storage for 48 h at -70 degrees C. Suspension of the fibroblasts in water for 10 min at room temperature, followed by homogenization with a tissue grinder, yielded preparations that were suitable for the assay of the neuraminidase activity.     

Purification and properties of GM2 synthase, UDP-N-acetylgalactosamine: GM3 beta-N-acetylgalactosaminyltransferase from rat liver

A UDP-N-acetylgalactosamine:ganglioside GM3 beta-N-acetylgalactosaminyltransferase which catalyzes the conversion of ganglioside GM3 to GM2 has been purified over 6300-fold from a Triton X-100 extract of rat liver particulate fractions by hydrophobic chromatography and affinity chromatography on GM3-acid-Sepharose. The purified enzyme has two identical subunits of 64,000 daltons. The enzyme has a pH optimum of pH 6.7-6.9 and requires divalent cations such as Mn2+ and Ni2+. In studies on substrate specificity GM3 containing N-acetylneuraminic acid (GM3(NeuAc] and GM3 containing N-glycolylneuraminic acid were both good acceptors for the purified enzyme. The plots of the activity of transferase as a function of GM3(NeuAc) showed sigmoidal relationships. The oligosaccharide of GM3, sialyllactose, was also a good acceptor, which indicates that the preferred acceptor substrate has the possible structure NeuAc alpha 2- or NeuGc alpha 2-3 Gal beta 1-4Glc-OR.     

Studies of the catalytic mechanism of microsomal UDP-glucuronyltransferase. Alpha-glucuronidase activity and its stimulation by phospholipids

The rate at which a specific, purified form of microsomal UDP-glucuronyltransferase (designated as the GT2P type of this enzyme) catalyzes the hydrolysis of UDP-glucuronic acid was measured with pure, delipidated enzyme and enzyme reconstituted with different lysophosphatidylcholines. This activity of the GT2P type of UDP-glucuronyltransferase is referred to as alpha-glucuronidase activity. For delipidated enzyme, the rate of hydrolysis of UDP-glucuronic acid catalyzed by GT2P extrapolated to infinite concentrations of UDP-glucuronic acid was 1 X 10(-9) mol/min/mg of protein. This compares with a rate of glucuronidation of p-nitrophenol of 96 X 10(-9) mol/min/mg of enzyme, for delipidated enzyme. Addition of oleoyl- or myristoyllysophosphatidylcholine to GT2P did not affect the alpha-glucuronidase activity significantly. This activity was stimulated, however, in the presence of compounds that bind at the aglycone site but that do not undergo glucuronidation. alpha-Glucuronidase activity extrapolated to infinite concentration of UDP-glucuronic acid was 4.0 X 10(-9) mol/min/mg for delipidated enzyme assayed in the presence of less than saturating concentrations of p-nitrophenyl phenyl ether. Moreover, when the aglycone site of GT2P was occupied by ethers, the alpha-glucuronidase activity of this enzyme was enhanced by addition of phospholipids to delipidated enzyme. The extent of activation of the alpha-glucuronidase activity of GT2P, when the aglycone site was occupied, depended on the acyl chain of the lipid added to delipidated enzyme. These data indicate that the GT2P form of UDP-glucuronyltransferase catalyzes the hydrolysis of UDP-glucuronic acid at a significant rate and that lysophosphatidylcholines can influence this rate.