Sialyl Lactose Research Articles

Inhibition of the fertilizing capacity of rabbit spermatozoa by sialoproteins

Partially purified Cowper's gland mucin of the boar, fetuin and sialyl lactose inhibited fertilization of rabbit ova by capacitated spermatozoa in vitro. The mucin and fetuin also inhibited fertilization in vivo, but sialyl lactose failed to do so. A dose response was observed between the amount of glycoprotein and inhibition of fertilization. Removal of sialic acid from glycoproteins reduced their antifertility effect. Corona penetrating enzyme was completely inhibited by the mucin and fetuin. These studies provide the first experimental evidence for glycoprotein inhibitors of fertilization.

Properties of decapacitation factor and presence in various species.

A delay between the optimum time for insemination and the time of ovulation a uterine incubation period for spermatozoa (capacitation) observed in some species is related to the presence of a decapacition factor (DF) in seminal plasma which functionally reverses the capacitation process. Spermatozoa were removed by centrifugation from rabbit bull stallion boar monkey rooster dog and human semen. A DF pellet of partially purified DF was prepared from monkey and bull seminal plasma. In other species whole seminal plasma was assayed. Capacitated spermatozoa recovered from the uterus of a doe 11 hours after mating were incubated with bull DF pellet and rabbit plasma at a level of 1 mg biuret-reactive-material (BRM)/100000 spermatozoa with monkey DF pellet at 2 mg BRM/100000 and with seminal plasma of the remaining species at 5 mg BRM/100000 spermatozoa. DF treated capacitated spermatozoa were introduced into one oviduct of a doe 12 hours after administration of an ovulatory dose of LH (3.1 mg.) An equal number of untreated capacitated spermatozoa were inseminated into the second oviduct. Recapacitation was demonstrated in does inseminated with untreated capacitated spermatozoa in one horn and with washed ejaculated spermatozoa in the other. Does were killed 26 hours or less after ovulation. The uteri of does in seminated with bull rabbit boar stallion and monkey seminal plasma treated spermatozoa showed no fertilized ova while control uteri for each group showed mean percent fertilized ova of 54.8 87.4 35.8 70.0 and 85. 2% respectively. Rooster dog and human seminal plasma showed no DF activity. No fertilized ova were found after insemination with capacitated spermatozoa and seminal plasma incubated with crude-neuraminidase-lecithinase (Vacholera filtrate) pronase lysozyme alpha-amylase glucose oxidase or hyaluronidase compared to 37.6 to 63.7% of ova fertilized in the control oviducts. Beta-amylase destroyed DF activity with 53.4% ova fertilized compared to a mean 50.8% in the control oviducts. When a DF pellet replaced seminal plasma alpha-amulase and beta-amylase destroyed the activity of partially purified DF with 5.9% and 26.2% ova fertilized respectively and 59.5% and 50.8% ova fertilized in the control oviducts. It has been observed that amylase content of uterine fluid elevated at the time when capacitation normally occurs. Incubation of capacitated spermatozoa with sialic acid previous to incubation with a DF factor did not prevent decapacitation after insemination. Incubation of uterine spermatozoa with sialic acid sialyl lactose or maltose before insemination did not prevent normal capacitation. It is possible that capacitation is required in humans but the DF is to specific to decapacitate rabbit spermatozoa.

Release of carbohydrates from sphingoglycolipid by osmium-catalyzed periodate oxidation followed by treatment with mild alkali

The carbohydrate moiety of sphingoglycolipid, after preliminary acetylation, can be released by periodate oxidation catalyzed by a trace amount of osmium tetroxide, followed by alkaline treatment. Cerebroside, lactosyl ceramide, hematoside, globoside, and gangliosides were degraded to yield, respectively, galactose, lactose, sialyl lactose, a tetrasaccharide, and various oligosaccharides containing sialic acid. Oligosaccharides were separated by paper chromatography and paper electrophoresis. The procedure is useful for characterizing micromolar amounts of sphingoglycolipids.

AN IMMUNOLOGICAL STUDY OF AVIAN, VIRAL AND BACTERIAL NEURAMINIDASE BASED ON SPECIFIC INHIBITION OF ENZYME BY ANTIBODY.

SUMMARY: Antisera were prepared to several strains of influenza virus (grown in the chick embryo), to purified Vibrio cholerae neuraminidase and to partially purified neuraminidase from chick chorioallantois. The ability of the antisera to inhibit the action of each enzyme on substrates of different molecular weight was tested. The substrates used and their molecular weights were sialyl lactose (640), fetuin (48,000) and ovine submaxillary gland mucin, OSM (1 × 106). Antiserum to V. cholerae and avian neuraminidase inhibited strongly the action of the homologous enzyme on each substrate. Antiserum to viral neuraminidase inhibited almost completely homologous action on fetuin and OSM but only partially the action on sialyl lactose. LEE influenza virus was grown in embryonated eggs and in cultures of calf kidney cells and from each preparation a soluble neuraminidase was isolated. Antiserum to the egg-grown virus inhibited the enzyme of both viruses not only in the intact virus particle, but also after separation in the form of a soluble, low molecular weight product. There was little or no serological cross-reaction between any enzyme and heterologous antisera, with one exception. Antiserum to avian neuraminidase partially inhibited (fetuin, but not sialyl lactose, as substrate) the soluble enzyme derived from egg-grown LEE virus, but not from virus grown in cultures of calf kidney cells. It was concluded that the soluble enzyme prepared from LEE virus is a virus specific product but also carries some antigenic determinants characteristic of host specificity.

Purification and properties of avian neuraminidase.

1. 1. Neuraminidase ( N -acetylneuraminate glycohydrolase, EC 3.2.1.18) from the chorioallantois of the 19-day-old chick embryo has been isolated and studied. 2. 2. The isolation procedure consisted of mild extraction of the washed membranes, heat treatment, precipitation with ammonium sulphate and chromatography on a column of hydroxyl apatite. The final product is about 200-fold purer than the starting fluid. 3. 3. The purified preparation is stable at o° but unstable at 37°. It has a sedimentation coefficient of about 3.3 S and is inactivated by sulfhydryl reactants. The pH of optimum activity on three substrates, sialyl lactose, fetuin and ovine submaxillary gland mucin is between 5 and 4. Values for K m and maximum rates of action on these substrates have been determined.

Purification and properties of avian neuraminidase

1.1. Neuraminidase (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) from the chorioallantois of the 19-day-old chick embryo has been isolated and studied.2.2. The isolation procedure consisted of mild extraction of the washed membranes, heat treatment, precipitation with ammonium sulphate and chromatography on a column of hydroxyl apatite. The final product is about 200-fold purer than the starting fluid.3.3. The purified preparation is stable at o° but unstable at 37°. It has a sedimentation coefficient of about 3.3 S and is inactivated by sulfhydryl reactants. The pH of optimum activity on three substrates, sialyl lactose, fetuin and ovine submaxillary gland mucin is between 5 and 4. Values for Km and maximum rates of action on these substrates have been determined.

The enzymatic synthesis of sialyl-lactose

Sialyl-lactose (SL) was first isolated from lactating rat mammary gland by Trucco and Caputto (1954). In subsequent extensive studies on milk oligosaccharides, Kuhn and coworkers ( Kuhn and Brossmer, 1958; Kuhn, 1958) obtained two isomers of SL from human milk, one being identical to SL from bovine colostrum. Bovine SL was shown to be N-acetylneuraminyl-(2 → 3)-β-D-galactopyranosyl-(1 → 4)-D-glucose, i.e., SL-(2 → 3). The other SL from human milk is N-acetylneuraminyl-(2 → 6)-β-D-galactopyranosyl-(1 → 4)-D-glucose, i.e., SL-(2 → 6). We now wish to report the enzymatic synthesis of sialyl-lactose by a particulate preparation from rat mammary gland. Chromatographic and periodate oxidation data indicate that the product is SL-(2 → 3). In addition, preliminary evidence shows that this preparation catalyzes the synthesis of other trisaccharides, such as sialyl-galactosyl-acetylglucosamine. Materials and Methods .

Purification and Properties of Neuraminidase from Vibrio cholerae

SUMMARY: A method is described for the purification of neuraminidase from culture fluids of Vibrio cholerae. Five steps are involved: fractionation with methanol, adsorption to and elution from human red cells, fractionation with ammonium sulphate, chromatography on columns of hydroxyl apatite, crystallization. From 35 l. of culture filtrate, an average yield of 21% of the original enzyme activity was obtained as crystals. The degree of purification was about 5000 fold. Purified neuraminidase possessed 12·6 × 106 units of biological activity/mg. dry weight, and gave a value for E 1% 280 mμ of 8·96, measured at 0·025% (w/v). Enzyme activity was stimulated by calcium ions and inhibited by ethylenediaminetetra-acetate. In the presence of 0·001 M-CaCl2, neuraminidase showed maximum activity at pH5·6. With sialyl lactose as substrate, a value of 1·2 x 10-3M was found for the Michaelis constant. At an enzyme concentration of 0·16 μg./ml., V max. was 0·021 μM N-acetylneuraminic acid/min./ml. The enzyme was stable when dried from the frozen state and stored under vacuum at 0°. A suspension of crystals in water also retained activity when stored at 0°. Solutions of crystalline neuraminidase showed a small increase in activity when stored at 0° for several weeks. This effect was greatest at pH 6·7 and 8·5 but was barely detectable at pH 4·6. At pH 5·6 or 6·7, the enzyme lost about 20% of its activity over a period of 2 hr. at 37° (concentration = 15 μg./ml.) No proteolytic activity nor N-acetylneuraminic acid aldolase activity was detected in the crystalline preparation.

Stimulation of the production of neuraminidase in Vibrio cholerae cultures by N-acetylneuraminic acid and sialyl-lactose.

SUMMARY: A simple medium is described which allows adequate growth of Vibrio cholerae with the production of only trace amounts of neuraminidase. When added to this medium to give a final concentration of 600 μ m, sialyl lactose or sialyl-N- acetylgalactosamine, two oligosaccharides which contain ketosidically bound N-acetylneuraminic acid and which are substrates of neuraminidase, caused at least a 100-fold increase in the amount of neuraminidase produced during growth of the organisms. N-Acetylneuraminic acid was also found to stimulate neuraminidase production. Galactose and N-acetylgalactosamine had no effect. N-Acetylneura- minic acid was metabolized by V. cholerae as indicated by the disappearance from such enriched medium of substances giving the direct Ehrlich reaction. Evidence is presented which suggests that neuraminidase is an extracellular enzyme.