A beta-(1----6)-D-galactosyltransferase has been purified over 2000-fold by affinity chromatography on UDP-p-aminophenyl-Sepharose. The enzyme, from a pellet fraction (8000 x g) of Helix pomatia albumen gland, catalyzes transfer of D-galactose from UDP-galactose to a (1----6) linkage on acceptor H. pomatia galactogen. Three other polymers served as acceptors: beef lung galactan, Lymnaea stagnalis galactogen and arabinogalactan from larch wood. To determine the linkage specificity of the enzyme, it was incubated with UDP-D-galactose and acceptor galactogen that had been tritiated previously by treatment with galactose oxidase and [3H]KBH4. The [3H]galactogen reaction product was recovered, methylated, hydrolyzed and acetylated; tritiated derivatives were identified by mass spectroscopy of effluent fractions separated by gas chromatography. This analysis revealed that (1----6)-linked galactosyl groups had been added to the enzyme-treated acceptor galactogen. Also identified was a hydrolytic enzyme that removed terminal alpha 1,2-linked L-galactosyl residues from H. pomatia galactogen.

Animals↗

PubMed1987

Calcium-induced opacification is dependent upon lens pH.

The intracellular pH of a normal lens is 6.8 in the cortex and remains unchanged during culture in media buffered at pH 7.2. Incubation of rabbit lenses in calcium enriched media, either at 24 degrees C or 37 degrees C, results in lens opacification provided that the lens pH remains slightly acidic. Opacities are prevented in cultured lenses with an alkaline interior (pH 7.1-7.3) despite the accumulation of calcium (1.3 mM). The mechanism by which an intracellular pH shift from 6.8 to 7.1 prevents opacification in the presence of excess calcium is not known, but does not appear to depend upon the total level of bound calcium. This study provides the first data that opacification caused by calcium is associated with lens pH.

Animals↗

PubMed1984

Substrate specificity of D-galactose oxidase. Evidence for the oxidation of internally linked galactosyl residues of Helix pomatia galactogen.

Linkage analysis of the carbohydrate portion of glycoproteins and glycolipids is widespread. Sequential treatment with D-galactose oxidase and tritiated borohydride is a standard method for incorporation of radioactive marker into what has been assumed to be exclusively terminal residues of D-galactose or N-acetyl-D-galactosamine. The data presented here establishes the ability of D-galactose oxidase to act upon a specific subterminal D-galactosyl residue, [----2)-D-Gal(1----], as well as upon terminal nonreducing galactosyl residues. Helix pomatia galactogen, a high molecular weight galactose homopolymer, was sequentially treated with D-galactose oxidase and tritiated borohydride. The 3H-galactogen was recovered and analyzed to determine which galactosyl units carried radioactive label. After complete methylation and then acid hydrolysis of 3H-galactogen, its partially methylated galactosyl components were reduced and acetylated for identification by gas chromatography and mass spectroscopy. Radioactivity was located by collection of effluent fractions during gas chromatography. The only subterminal residue to be labeled was the 2-linked D-galactose, although another with a free oxidizable 6-carbon was present, 3-linked D-galactose, [----3)-D-Gal(1----]. Linkage analysis of internal radiolabeled galactosyl residues could be used to detect changes in saccharide structure during cellular events.

Animals↗

PubMed1978