Identification and characterization of beta-D-galactosyl-transferase in chick corneas.
OBJECTIVE
To purify beta-galactosyltransferase, which is involved in the biosynthesis of keratan sulfate, from 2-day-old chick corneas.


METHODS
The activity was assayed using pyridylaminated GlcNAcbeta1-3Galbeta1-4Glc as acceptor substrate.


RESULTS
The beta-galactosyltransferase did not bind to several ion exchange and affinity columns. In particular, it did not bind completely to an alpha-lactalbumin-agarose column. The partially purified enzyme showed an optimum pH at 7.0 [(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (HEPES) buffer]. Pyridylaminated GlcNAcbeta1-3Galbeta1-4GlcNAc, as well as the pyridylaminated GlcNAcbeta1-3Galbeta1-4Glc, served as the acceptor substrate of the enzyme, but not p-nitrophenyl-beta-GlcNAc. The crude extract from chick corneas contained a high activity of the beta-galactosyltransferase, which transfers Gal to pyridylaminated 6-sulfo-GlcNAcbeta1-3Gal, but the activity was almost all lost during the purification procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme fraction showed many bands. When each of the five main band proteins was partially analyzed for the amino acid sequence, none showed homology with the recently reported chick beta-galactosyltransferases 1 and 2.


CONCLUSIONS
beta-Galactosyltransferase, which transfers Gal to 6-sulfo-GlcNAc end, was identified in chick corneas. The chick corneal beta-galactosyltransferase(s) may be a novel one.
Sashima
Susumu
S
Radiochemistry Section, Faculty of Pharmacy, Meijo University, Nagoya, Japan.
Takahashi
Ikuko
I
Nakazawa
Kiyoshi
K
D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003315:Cornea / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005700:Galactosyltransferases / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008297:Male; D012685:Sepharose; D013379:Substrate Specificity
D012685:Sepharose; D005700:Galactosyltransferases