A solid phase assay for galactosyl transferases. Evidence for an active site of less than 14 kDa |
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Authors: | Yves Plancke Béatrice Delpouve Jean Montreuil |
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Institution: | (1) Laboratoire de Chimie Biologique et Laboratoire Associé au CNRS No. 217, Université des Sciences et Techniques de Lille Flandres-Artois, 59655 Villeneuve d'Ascq Cédex, France |
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Abstract: | Galactosyltransferase (GalTase) prepared from human milk was found to exist as a complex with e-lactalbumin as demonstrated by crossed immunoelectrophoresis against specific antibodies raised against the complex. GalTase activity was stable to proteolysis and, when subjected to gel filtration on Ultrogel AcA54, the enzyme activity eluted as a single peak. A second peak of activity was found to be adsorbed to the column matrix and was eluted with buffer containing 1 M NaC1. The hydrophobic fraction represented 5% of the total GalTase activity in human milk. After polyacrylamide gel electrophoresis the main enzyme activity peak was represented by polypeptides of 67kDa molecular weight and of 14kDa molecular weight. Electroblotting of these peptides onto a nitrocellulose membrane followed by determination of GalTase activity showed activity for 45–55 kDa and for 14 kDa peptides. The hydrophobic fraction from the AcA54 column was resolved into polypeptides of 110 kDa-45 kDa molecular weight, all of which contained GalTase activity after blotting. It is supposed that the GalTase from non-proteolyzed milk is composed of a 14 kDa polypeptide containing the active site together with another part of the polypeptide backbone which is involved in the regulation of GalTase activity by -lactalbumin, a third part of the polypeptide is responsible for the membrane insertion.Abbreviations UDP-Gal
uridine diphosphatidyl galactose
- GlcNAc
N-acetylglucosamine
- Glc
glucose
- PAGE
polyacrylamide gel electrophoresis
- GalTase
galactosyl transferase (EC 2.4.1.22)
- -ovo
pronosac digest fraction of hen ovomucoid
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Keywords: | active site galactosyl transferase solid phase assay |
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