Isolation and identification of 4 UDP-trisaccharides from the mammary gland and colostrum of the ewe

Abstract

4 UDP‐trisaccharides present in the mammary gland and colostrum of the ewe were isolated and identified. The nucleotides, extracted by means of trichloracetic acid, were purified in the following way : 2 ion‐exchange chromatographies on Dowex 1 (formate), filtration on Sephadex G‐25, paper electrophoresis, pH 5, followed by paper chromatography in 3 different solvents. Using these fractionation techniques, 4 uridine derivatives (X1′, X2′, Y1′, Y2′,) with positive reaction to the Warren neuraminic acid test, were obtained.UDP‐5′, which constitutes the nucleotidic part of the 4 derivatives, was defined more accurately after various acid or enzymatic analyses. The isolation of UDP‐5′ was made by ionexchange or paper chromatography and the total and labile phosphate was estimated. The Warren positive substances released from X1′, X2′, Y1′, Y2′, by neuraminidase or mild acid hydrolysis were identified as N‐glycolyl‐neuraminic acid for the X1′, Y2′ nucleotides, and N‐acetyl‐neuraminic acid for the X2′, Y2′ nucleotides.The behaviour of asialylated nucleotides to ion‐exchange and paper chromatography is described as well as that of asialylated oligosaccharides. The 4 asialylated oligosaccharides contain galactose and an N‐acetyl‐D‐hexosamine, the latter being N‐acetyl‐D‐glucosamine in the case of X1′ and X2′, and N‐acetyl‐D‐galactosamine in the case of Y1′ and Y2′. Total analysis of UDP‐oligosaccharides shows a 1:1:1:1 ratio between uridine, N‐acetyl‐D‐hexosamine, galactose, and neuraminic acid.The 1‐P‐trisaccharides and the trisaccharides were identified after phosphodiesterase hydrolysis followed by phosphatase hydrolysis. The N‐acetyl‐D‐hexosamine carried the reducing group (sodium borohydride). Some structural characteristics of the 4 nucleotides were determined using β‐galactosidase, neuraminidase, periodate, and periodate borohydride. Two of the nucleotides contain N‐acetyl‐glucosamine and are identical to those previously isolated from the goat colostrum. As for the two others, containing N‐acetyl‐galactosamine and appearing as new trisaccharide nucleotides, we may, on the basis of the results obtained, suggest the following sequences:a) UDP‐N‐acetyl‐D‐galactosamine (3←1) D‐galactose (6←2) N‐acetyl‐neuraminic acid;b) UDP‐N‐acetyl‐D‐galactosamine (3←1) D‐galactose (6←2) N‐glycolyl‐neuraminic acid. The possible role of these new nucleotides is discussed.