Triton CF-54 Research Articles

Dimeric Galectin-1 Binds with High Affinity to α2,3-Sialylated and Non-sialylated Terminal N-Acetyllactosamine Units on Surface-bound Extended Glycans

Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an approximately 29.5-kDa noncovalent homodimer (dGal-1) that is widely expressed in many tissues. Here, we report that human recombinant dGal-1 bound preferentially and with high affinity (apparent K(d) approximately 2-4 microM) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Galbeta1-4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (-3Galbeta1-4GlcNAcbeta1-)(n)) sequences, complex-type biantennary N-glycans, or novel chitin-derived glycans modified to contain terminal LN. Although terminal Gal residues are important for dGal-1 recognition, dGal-1 bound similarly to alpha3-sialylated and alpha2-fucosylated terminal LN, but not to alpha6-sialylated and alpha3-fucosylated terminal LN. The binding specificity of human recombinant dGal-1 was similar to that observed with purified bovine heart-derived dGal-1. Unexpectedly, dGal-1 bound free ligands in solution with relatively low affinity and displayed no preference for extended glycans, indicating that dGal-1 preferentially recognizes extended glycans only when they are surface-bound, such as found on cell surfaces. Human dGal-1 also bound to both native and desialylated human promyelocytic HL-60 cells with similar affinity as observed for immobilized long chain PL. Binding to these cells was reduced upon treatment with endo-beta-galactosidase, which cleaves PL sequences, indicating that cell-surface PLs are ligands. To test the role of dimerization in dGal-1 binding, we examined the binding of a mutated form of dGal-1 that weakly dimerizes (monomeric Gal-1 (mGal-1)) and a covalently dimerized (chemically cross-linked) form of mGal-1 (cd-mGal-1). dGal-1 and cd-mGal-1 had similar affinities that were both approximately 3.5-fold higher for immobilized PL than observed for mGal-1, suggesting that dGal-1 acts as a dimer to cross-link terminal LN units on immobilized PL. These results indicate that dGal-1 functions as a dimer to recognize LN units on extended PLs on cell surfaces.

N-Acetylaspartate synthase is bimodally expressed in microsomes and mitochondria of brain

N-Acetylaspartate (NAA) is an abundant amino acid derivative of the central nervous system that is localized primarily in neurons and has found widespread use in clinical NMR spectroscopy (MRS) as a non-invasive indicator of neuronal survival and/or viability. Its function, although still obscure, is thought to reflect its unusual metabolic compartmentalization wherein NAA synthase occurs in the neuron and aspartoacylase, the hydrolytic enzyme that removes the acetyl moiety, occurs in myelin and glia. The NAA synthase enzyme, acetyl-CoA/ l-aspartate N-acetyltransferase (ANAT), was previously shown to function in mitochondria (MIT), although other subcellular fractions were apparently not examined. In this study we confirmed its presence in MIT but also found significant activity in rat brain microsomes (MIC). The reaction mixture, consisting of [ 14C]aspartate plus acetyl-CoA in Na-phosphate buffer (pH 7), gave rise to [ 14C]NAA that was separated and quantified by TLC. Reaction rates were 29.0±0.46 and 6.27±0.27 nmol/h/mg for MIC and MIT, respectively. K m values and pH optima were similar, and both fractions showed modest enhancement of ANAT activity with the detergents Triton CF-54 and CHAPS. Our tentative conclusion is that ANAT is bimodally targeted to MIT and a component of MIC—likely endoplasmic reticulum. ANAT activity increased in both MIC and MIT between 29 and 60 days of age but differed thereafter in that only MIT ANAT showed a decrease after 1 year.

An amino acid region at the N-terminus of rat hepatoma alpha1-->2 fucosyltransferase modulates enzyme activity and interaction with lipids: strong preference for glycosphingolipids containing terminal Galbeta1-->3GalNAc-structures.

A GDP-fucose:GM1 alpha1-->2 fucosyltransferase (FucT) is induced during early stages of chemical hepatocarcinogenesis in parenchymal cells of Fischer 344 rats fed a diet supplemented with 0.03% N-2-acetylaminofluorene (AAF). This enzyme is undetectable in normal rat liver tissues but is highly expressed in many rat hepatoma cell lines, including rat hepatoma H35 cells. Enzymatic properties and acceptor specificity of native rat hepatoma H35 cell alpha1-->2FucT, expressed recombinant full-length H35 cell alpha1-->2FucT, and a truncated form missing the first 27 amino acid residues from the N-terminus, comprising the cytoplasmic and transmembrane domains of the enzyme, were studied. The results indicate that the recombinant full-length enzyme has a specific activity over 80-fold higher than the truncated enzyme. Both the native and recombinant full-length enzymes display significant activity in the absence of detergent or phospholipid and optimal activity in the presence of Triton CF-54 detergent. The truncated enzyme is optimally activated by CHAPSO, showing little activity in its absence. These findings are in agreement with previous studies demonstrating a requirement of a lipidic environment for optimal activity with this enzyme and suggest that the N-terminal transmembrane domain is important either in the maintenance of an active conformation or in allowing efficient interaction with acceptor glycolipids. Both the full-length and truncated enzymes transfer fucose not only to GM1 and asialo-GM1 (Gg4) but also to galactosyl globoside (Gb5) as well. Weak or undetectable transfer to lacto- and neolacto-series acceptors was observed, demonstrating a strong preference for terminal Galbeta1-->3GalNAc- structures. The structures of two reaction products generated by expressed recombinant full-length alpha1-->2FucT, which are known to be important tumor-associated antigens (fucosyl-GM1 and fucosyl-Gb5), were unambiguously confirmed by 1H-NMR spectral analysis.

Extractions of polychlorinated biphenyl (PCB) compounds from surfactant suspension/soil extracts with dechlorination on-line.

Polychlorinated biphenyl (PCB) formulation (Aroclor 1242 or 1248)-surfactant (Brij 97 or Triton CF54) emulsions (0.1-1.0%, v/v) were extracted with supercritical carbon dioxide (scCO2). The declinations curve that resulted for each loading of PCB substrate was accurately modelled as a single-exponential decay, and the half-life [t1/2, 10.76 min +/- 2% (Brij 97) or 10.0 min +/- 14% (Triton CF54)] was independent of the level of loading. When the extraction was combined with on-line dechlorination, t1/2 was increased to 17.62 min +/- 6% or 16.46 min +/- 5%, respectively The dechlorination reactor contributed appreciably to the back-pressure of the system so that, under identical scCO2 head pressures, the flow rate at the exit of the system was reduced from 1500 mL min-1 (as decompressed gas) to 900 mL min-1. When corrected for the difference in flow rates, the declination curves in the absence and presence of the reactor became virtually identical. For a surfactant suspension from a soil that was field-burdened with only 6 ppm PCBs, extended operation (extraction-dechlorination) for 15 h, during which the substrate suspension was replaced every 30 min, caused no loss of the dechlorination efficiency; however, for 1% (v/v) Aroclor 1242 or 1248 in surfactant suspension, the reactor gradually lost reactivity over 5 h of continued operation. However, the reactivity could be restored virtually completely by purging the reactor column with scCO2 for 3 h or washing the column at ambient temperature with 30 mL of methanol-water (7 + 3, v/v). Mass spectrometry of the reaction products indicated that the principal products included biphenyl and toluene, but methylated benzenes and phenols were also present.

Development of an Enzyme-Linked Immunosorbent Assay-Based Method for Measuring Galactosyltransferase Activity Using a Synthetic Glycopolymer Acceptor Substrate

A lectin-assisted enzyme-linked immunosorbent assay (ELISA)-based method using a synthetic glycopolymer as an acceptor substrate was developed for measuring β1,4-galactosyltransferase (GalT) activity. A polyacrylamide derivative having a β-linkedN-acetylglucosamine (GlcNAcβ) moiety on each monomeric unit was synthesized chemically and immobilized on a polystyrene microtiter plate as an acceptor substrate for GalT. After the plate was incubated with bovine GalT, the enzyme reaction product, β-linked Gal residue on the polyacrylamide-bound GlcNAc residue, was detected by usingRicinus communisagglutinin 1 (RCA1), rabbit anti-RCA1 antibody, and a peroxidase-labeled anti-rabbit IgG. The lowest GalT concentration detectable by this method was about 0.5 mU/ml, which is comparable to those by the previously reported ELISA-based assays. The unique property of the glycopolymer, PAP(GlcNAcβ), of binding noncovalently but tightly to the polystyrene microtiter plate allowed the use of this acceptor substrate for the GalT activity measurement even in the presence of 1% Triton CF-54 and X-100. Our system was successfully applied to assess GalT activity in milk of various mammals.

Dependence of rat liver CMP- N-acetylneuraminate:GM 1 sialyltransferase (SAT IV) activity on the ceramide composition of GM 1 ganglioside

The dependence of CMP- N-acetylneuraminate:GM 1 sialyltransferase (SAT IV) activity of rat liver Golgi apparatus on GM 1 ganglioside ceramide composition was evaluated. SAT IV activity was assayed on GM 1 molecular species carrying homogeneous ceramide moieties containing long chain bases of different length (18 or 20 C atoms) unsaturated or not, linked to 14:0, 16:0, 18:0 or 22:0 fatty acids. The results obtained in the presence of the detergent Triton CF-54, when enzyme and substrate are presumably part of the same supramolecular structure, show that either the long chain base or the fatty acid composition can affect enzyme activity. This feature was not displayed when GM 1 was embedded in dipalmitoylphosphatidylcholine vesicles in the absence of detergent. Under the latter conditions, the enzyme was not sensitive to the lipid composition of GM 1 but to the ganglioside/phospholipid ratio in the vesicles. These results indicate for the first time that SAT IV is affected by the lipid composition of the substrate and strengthen the hypothesis that glycosyltransferases may contribute to control the cellular glycosphingolipid ceramide pattern.

Characterization of sialidase activity in mouse synaptic plasma membranes and its age‐related changes

Sialidase activity in synaptic plasma membranes (SPM) isolated from C57BL/6 mouse brain was examined using exogenous ganglioside substrates. The enzyme activity directed toward GM3 showed sharp pH dependency with optimal pH of 4.0, and was greatly enhanced by Triton CF-54, Nonidet P-40 or CHAPS. The apparent Km and Vmax values for enzyme activity in SPM were 11 microM and 164 pmol/mg protein/min, respectively. Examination of sialidase activities in subcellular fractions of brain tissues showed the enrichment of enzyme activity in SPM prepared from either young adult or senescent mice. Substrate specificity of SPM sialidase was compared with that of myelin sialidase using delipidated, solubilized enzyme preparations. The SPM sialidase hydrolyzed GD1a more effectively as compared with the myelin enzyme. While SPM sialidase could hydrolyze GM1, the hydrolytic rate by the SPM enzyme was significantly lower than that by the myelin enzyme. The sialidase activity in SPM decreased with increasing age; activity was highest between the ages of 4-7 months, decreased to a relatively constant level between 13-25 months, and reached its lowest level at 31 months. These results demonstrate that SPM contain a distinct sialidase activity which is regulated in an age-dependent manner.

Characterization of sialyltransferase-IV activity and its involvement in the c-pathway of brain ganglioside metabolism.

To characterize the sialyltransferase-IV activity in brain tissues, the activities of GM1b-, GD1a-, GT1b-, and GQ1c-synthases in adult cichlid fish and rat brains were examined using GA1, GM1, GD1b, or a cod brain ganglioside mixture as the substrate. The GD1a-synthase activity in the total membrane fraction from cichlid fish brain required divalent cations such as Mg2+ or Mn2+ and Triton CF-54 for its full activity. The Vmax value was 1,340 pmol/mg of protein/h at an optimal pH of 6.5, whereas the apparent Km values for CMP-sialic acid and GM1 were 172 and 78 microM, respectively. Cichlid fish and rat brains also contained GM1b-, GT1b-, and GQ1c-synthase activities. The ratio of GM1b-, GD1a-, and GT1b-synthase activities in fish brain was 1.00:0.89:1.13, respectively, and in rat brain 1.00:0.60:0.63. Incubation of fish brain membranes with a cod brain ganglioside mixture, which contains GT1c, and [3H]CMP-sialic acid produced radiolabeled GQ1c. It is interesting that the adult rat brain also contains an appreciable level of GQ1c-synthase activity despite its very low concentrations of c-series gangliosides. The GD1a- or GQ1c-synthase activity in fish and rat brain was inhibited specifically by coincubation with the glycolipids that serve as the substrates for other sialyltransferase-IV reactions. Thus, the GD1a-synthase activity was inhibited by GA1 and GD1b, but not by LacCer, GM3, or GD3. In a similar manner, the synthesis of GQ1c was suppressed by GA1, GM1, and GD1b, but not by LacCer, GM3, or GD3.(ABSTRACT TRUNCATED AT 250 WORDS)

A New Method for Detecting β1,4-Galactosyltransferase Activity in Sera of Cancer Patients

A new method for assaying the activity of the enzyme that catalyzes the formation of a cancer-associated glycolipid, paragloboside (nLc 4Cer), from lactotriaosyl-ceramide (Lc 3Cer) and UDP-galactose has been developed that is based on a time-resolved fluoroimmunoassay (TRFIA) with a Europium (Eu)-chelate-labeled antibody. The substrate, Lc 3Cer, immobilized on a microtiter plate, was incubated with UDP-galactose, MnCl 2, Triton CF-54, and the enzyme. The content of the incubation product, nLc 4Cer, was determined by the TRFIA with anti-nLc 4Cer monoclonal antibody H-11 as the first antibody and Eu-labeled anti-mouse IgM antibody as the second one. The lower limit of detection of nLc 4Cer was estimated to be 0.2 pmol. This method was used to detect the galactosyltransferase activity in sera from patients with colorectal cancer or benign colorectal adenomas and from healthy subjects of a reference sample group. The reference interval was 0-0.25 pmol/25 μl serum/2 h. Activity was significantly greater in patients with colorectal cancer than in those with colorectal benign adenoma ( P < 0.05) and the subjects of the reference sample group ( P < 0.01).

Purification and characterization of CMP-N-acetylneuraminic acid:lactosylceramide (alpha 2-3) sialyltransferase (GM3-synthase) from rat brain.

CMP-N-acetylneuraminic acid:lactosylceramide (alpha 2-3) sialyltransferase (GM3-synthase) was purified to homogeneity from a Triton CF-54 extract of young rat brain. The enzyme was separated by affinity chromatography on CDP-Sepharose column and resolved by linear NaCl gradient elution from the same adsorbent. Final purification of GM3-synthase was achieved by chromatography on a "lactosylceramide acid"-Sepharose column and specific elution with lactosylceramide. The enzyme activity was highest at pH 6.5 and required the presence of Triton CF-54 (0.15%) and Mn2+ (10 mM) for its full activity. The product of the reaction catalyzed by the enzyme was identified as GM3 based on its mobility on thin layer chromatographic plates using two different solvent systems. Comparison with several glycolipid substrates showed high specificity of GM3-synthase for lactosylceramide. The apparent Km value for lactosylceramide and CMP-N-acetylneuraminic acid were 80 and 210 microM, respectively. The apparent molecular mass of the enzyme determined on SDS-polyacrylamide gel electrophoresis was 76 kDa.

UDP-galactose:lactosylceramide ?-galactosyltransferase activity in human placenta

The activity of UDP-Gal:LacCer galactosyltransferase in human placenta was studied by using crude homogenate and Triton CF-54 extract as the source of enzyme. Transfer of galactose to lactosylceramide was optimal in the presence of 0.1% Triton CF-54 and Mn2+ at pH 6.3, and the reaction product was susceptible to alpha-galactosidase.

Purification and characterization of a novel sialidase found in procyclic culture forms of Trypanosoma brucei

A membrane-bound sialidase (EC 3.2.1.18) was found in procyclic trypomastigotes of Trypanosoma brucei. The mammalian stage bloodstream form, however, displayed no sialidase activity. This sialidase is an integral surface protein, linked to the membrane via a glycosylphosphatidylinositol anchor. After osmotic lysis and solubilization with Triton CF-54, the enzyme was purified 1900-fold by gel filtration and ion exchange chromatography. Its size, as determined by conventional and high-performance liquid gel chromatography, is 67 kDa. The sialidase is active over a broad pH and temperature range with optima at pH 6.9 and 35°C, respectively. No loss of activity is observed after 4 freeze-thaw cycles. T. brucei sialidase activity is inhibited by N-(4-nitrophenyl)oxamic acid and 2-deoxy-2,3-didehydro- N-acetylneuraminic acid, the latter, however, being less effective. N-Acetylneuraminic acid shows no inhibitory effect, whereas a variety of metal ions are potent inhibitors. The sialidase is activated by di- and tricarboxylic acids, but inhibited by chloride. Relative hydrolysis rates of various sialic acid-containing compounds reveal that de- O-acetylated bovine submandibular gland mucin is the preferred substrate and that α(2–3)-linkages are hydrolyzed faster than α(2–6)-linkages.

UDP-galactose:globotriaosylceramide alpha-galactosyltransferase activity in rat pheochromocytoma (PC12h) cells.

The activity of alpha-galactosyltransferase in cultured rat pheochromocytoma subcloned (PC12h) cells was examined using Gb3 as the acceptor for the galactose from UDP-galactose. The major reaction product was identified as gal alpha 1-3Gb3 based on its mobility on thin-layer chromatographic (TLC) plates and susceptibility to specific galactosidases. The enzyme activity in PC12h cells was the highest at pH 7.0 while the presence of Triton CF-54 (0.1%) and Mn2+ (5 mM) was required for its full activity. The apparent Km values for Gb3 and UDP-galactose were 57 and 17 microM, respectively. The enzyme activity in PC12h cells was compared with that in parent PC12 cells, in which gal alpha 1-3Gb3 is not expressed in an appreciable amount. In the enzyme reaction with exogenous Gb3, the enzyme activity in PC12h cells was about 1.5-fold higher than that in PC12 cells. In the absence of exogenous Gb3, this difference became even more pronounced; gal alpha 1-3Gb3 was generated from endogenous Gb3 at a much higher rate in PC12h cells than in PC12 cells. These findings suggest that the higher level of the alpha-galactosyltransferase activity in PC12h cells may, at least in part, be responsible for the accumulation of unique neutral glycosphingolipids having gal alpha 1-3 terminal residues in the cells.

Peroxidase-amplified assay of sialidase activity toward gangliosides

Sialidase assays were carried out with the substrate, ganglioside G D1a, coated onto enzyme immunoassay plate wells. Following the incubation of G D1a with sialidase from V. cholerae, the amount of ganglioside G M1 produced was measured as follows: cholera toxin B subunit conjugated to horseradish peroxidase was added to specifically bind to G M1, and then the amount of bound peroxidase was determined in a colorimetric enzymatic assay. In the absence of detergent, linearity for the detection of G M1 was 0 to 0.5 pmol per well, and the sensitivity for sialidase detection was about 3 fmol of product formed per minute. The addition of detergent (Triton CF-54) to the assay reduced the sensitivity and increased the amount of substrate required. Application of this assay for the detection of cell-derived neutral (pH 6.5) sialidase activities in the conditioned medium of human skin fibroblasts is described.

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.

Substrate specificity of α2↔3‐sialyltransferases in ganglioside biosynthesis of rat liver golgi*

The acceptor specificities of four sialytransferases (I, II, IV and V) involved in ganglioside biosynthesis were studied in Golgi vesicles derived from rat liver. The activities of these sialytransferases were strongly detergent-dependent. Competition experiments with different detergent concentrations using LacCer (Gal beta 1----4Glc beta 1----1Cer), GM1a [Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer] and GD1b [Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer] as substrates, and as mutual inhibitors for ganglioside sialyltransferase activity, suggested that sialyltransferase IV was able to catalyze the sialyltransfer in alpha 2----3 linkage to the galactose residues of LacCer as well as of GM1a and GD1b. The other three sialyltransferases (I, II and V) seemed to be quite specific for their respective glycolipid acceptors, LacCer, GM3 and GM1b, GD1a and GT1b. Furthermore the kinetic data showed that sialyltransferase I was inactive at higher detergent concentrations (greater than 75 micrograms Triton CF-54); under these conditions, formation of GM3 and GD1a was catalyzed only by sialyltransferase IV. These results have been integrated into a model for ganglioside biosynthesis and its regulation.

Biosynthesis in vitro of core lacto-series glycosphingolipids by N-acetyl- d-glucosaminyltransferases from human colon carcinoma cells, Colo 205

Two N-acetyl- d-glucosaminyltransferases have been detected in human colon carcinoma Colo 205 cells. These enzymes catalyze the biosynthesis in vitro of the core-glycolipid of Type 1 and Type 2 lacto-series antigens and of the polylactosamine-containing longer chain antigenic structures, respectively. The first enzyme, GlcNAcT-1, which catalyzes the formation of lactotriosylceramide [LcOse 3Cer, β- d-Glc pNAc-(1→3)-LcOse 2Cer, the core for all lacto-series Type 1 and Type 2 chains] from lactosylceramide [β- d-Gal p-(1→4)- d-Glc p-Cer, LcOse 2Cer] and UDP-GlcNAc shows optimum activity in the presence of nonionic detergent Triton CF-54. The other enzyme, GlcNAcT-2, which catalyzes the biosynthesis in vitro of iLcOse 5Cer [β- d-Glc pNAc-(1→3)-nLcOse 4Cer, the core for polylactosamine-containing antigens] from nLcOse 4Cer [β- d-Gal p-(1→4)-LcOse 3Cer] and UDP-GlcNAc, is optimally active with the zwitterionic detergent, Zwittergent 3–14, when membrane-bound. Both of these activities, however, can be extracted from the membrane by use of a nonionic detergent, Triton X-114, with nearly the same efficiency. These two transferases showed different pH optima, different cation and anion effects, and differential heat-inactivation patterns at 55°. Permethylation studies of the radioactive products isolated from both of the enzyme-catalyzed reactions using respective 3H-substrates and nonradioactive UDP-GlcNAc showed the presence of 2,4,6-tri- O-methylgalactose in the hydrolyzed products. This indicated the presence of a (1→3)-linked β- d-Glc pNAc group at the nonreducing end in both cases. The linkage of the β- d-Glc pNAc group to the subterminal d-Gal residue in the two products was confirmed by an almost 90% cleavage of the terminal [ 3H]GlcNAc group by purified clam and papaya β- d-hexosaminidases.

Biosynthesis of GM1b and similar neolactoseries gangliosides by a partially purified chicken skeletal muscle sialyltransferase. Effect of sphingomyelin and acetylcholine

An α2 → 3 glycolipid galactosyl sialyltransferase (SAT3/4) has been partially purified from embryonic chicken skeletal muscle. It is preserved in 50 mM Hepes buffer (pH 6.8) containing 1% Triton CF-54 and 20% glycerol at −70°C for a period of 6 months without loss of activity. The SAT3/4 preparation transfers sialic acid to nLcOse 4Cer, nLcOse 6Cer and GgOse 4Cer with respective K m values of 1.4, 0.83 and 0.45 mM. The activity is stimulated 2–3-fold at high substrate concentration and 6–8-fold at low substrate concentration; 0.01 and 0.005 μmol for asialo GM1 and 0.025 and 0.01 μmol for other glycolipids in the presence of phosphatidylcholine (PC) and sphingomyelin (SM) at an optimum concentration 0.75%. A higher concentration is inhibitory. SM from chicken muscle is more effective than that from bovine brain and the stimulation is qualitatively proportional to that of the saturated fatty acyl content of SM. Free fatty acids (palmitic and stearic), their sodium salts, other choline compounds including choline chloride, phosphorylcholine and acetylcholine either do not have any effect or are inhibitory. Acetylcholine, even in the presence of SM and PC, is strongly inhibitory (70%).

Isolation, partial purification and characterization of a potato peel ∞-solanine cleaving glycosidase

An enzyme which hydrolyzes the rhamnose from ∞-solanine was isolated and partially purified by ammonium sulfate precipitation from the peels of Kennebec and Wauseon potatoes. Enzyme activity was found to be present in the 60 to 80% ammonium sulfate fraction. Enzyme activity was increased by the use of non-ionic detergents with a mixture of Triton X-100:Triton CF-54 (2:1, w/w) providing the greatest increase in activity. The pH optimum for the rhamnosidase for both varieties was found to be 6.0 while of the temperatures evaluated 42 °C was the optimum temperature. The effect of substrate concentration on enzyme activity was determined for both varieties and the Vmax and Km were calculated from Lineweaver-Burke double reciprocal plots. The Vmax and Km for the Kennebec and Wauseon rhamnosidases were found to be 0.27 mg/ml and 5.4 x 10-4 M and 0.48 mg/ml and 8.2 x 10-4 M, respectively.

A Simple and Specific Assay of Glycosyltransferase and Glycosidase Activities by an Enzyme-Linked Immunosorbent Assay Method, and Its Application to Assay of GalactosyItransferase Activity in Sera from Patients with Cancer&lt;xref ref-type="fn" rid="fn1"&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;

A simple, sensitive, and specific assay method for glycosyltransferase and glycosidase activities has been established by means of an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody, H-11 directed to lactoneotetraosylceramide (nLc4Cer). Enzyme activity was determined by assaying the amount of reaction product, nLc4Cer with the ELISA method. For the assay of galactosyltransferase activity, lactotriaosylceramide (Lc3Cer) immobilized on a 96-well microtiter plate was incubated with bovine milk galactosyltransferase in cacodylate buffer (pH 6.8) containing Triton CF-54, Mn2+, and UDP-galactose. Optimum incubation conditions for the enzyme were determined. Glycosidase activity was also assayed by the ELISA method by using Clostridium perfringens sialidase and neolacto-series gangliosides as substrates, and the substrate specificities towards the gangliosides were examined. By this method, 3-100 pmol of reaction product could be determined. The assay method has several advantages as follows: 1, the method is simple; 2, separation of the reaction product is not required; 3, quantification and identification of the reaction product were done simultaneously; 4, naturally occurring substrates are available (especially for glycosidase); 5, many samples can be assayed in one microplate; 6, sensitivity is very high. The present method was applied for the detection of galactosyltransferase in human sera. Significant elevations of the galactosyltransferase levels were observed in the sera from cancer patients. The formation of nLc4Cer was confirmed by employing the TLC-immunostaining method for bands of Lc3Cer after incubation of the bands with serum and cofactors on an HPTLC plate.