First in-gel detection and purification of human xylosyltransferase II.
Human xylosyltransferases I and II (XylT-I, XylT-II) are key enzymes in glycosaminoglycan biosynthesis. Knowledge about the in vivo molecular weight, oligomeric state or turnover number are essential characteristics which have been addressed in this study. XylT-II was purified from Pichia pastoris by fractionated ammonium sulfate precipitation, heparin affinity and ion exchange chromatography. XylT-II was purified over 7000-fold with a final yield of 2.6%. By utilizing mass spectra analysis we can prove its first in-gel detection showing a migration pattern behavior that confirms its in silico molecular weight of 95.8 kDa. We could determine a turnover number of 2.18 min(-1) or one transferred xylose molecule per one XylT-II molecule each 27.5s. The k(cat)/K(M) ratio was 0.357 min(-1)microM(-1) for XylT-II using the bikunin-homologous acceptor Bio-QEEEGSGGGQKK-F. The comparison to XylT-I derived from the same organism revealed a 2.4-fold higher catalytic efficiency (0.870 min(-1)microM(-1)) for XylT-I.
Casanova
Javier Carrera
JC
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstrasse 11, 32545 Bad Oeynhausen, Germany.
Roch
Christina
C
Kuhn
Joachim
J
Kleesiek
Knut
K
Götting
Christian
C
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D000097608:UDP Xylose-Protein Xylosyltransferase
D005782:Gels; D010430:Pentosyltransferases; D011994:Recombinant Proteins; D000097608:UDP Xylose-Protein Xylosyltransferase