Abstract
UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) catalyzes the addition of N-acetylglucosamine in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-linked sugar chains. The enzyme has been purified over 153,000-fold in 1.5% yield from a Triton X-100 extract of rat kidney by fractionation procedures utilizing QAE-Sepharose, Cu(2+)-chelating Sepharose, and affinity chromatography on UDP-hexanolamine and substrate-conjugated Sepharose. The purified protein migrates as one major and one minor band with apparent molecular masses of 62 kDa and 52 kDa, respectively. The purified enzyme was digested with trypsin, and the amino acid sequences of four peptides were determined. Oligonucleotide primers were designed according to those amino acid sequences and used in the polymerase chain reaction. Screening for the cDNA for GnT-III was carried out by plaque hybridization using a rat kidney cDNA library (lambda gt10) and a polymerase chain reaction product as the probe. Rat kidney GnT-III has 536 amino acids and three putative N-glycosylation sites. There is no sequence homology to other previously cloned glycosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases. The GnT-III activity in transiently transfected COS-1 cells was found to be about 500-3600-fold as compared to that in non- or mock-transfected cells.
Highlights
Oligonucle- GnT-I11 activity was first observed in hen oviduct (1).High otide primers weredesigned according to those amino activity of the enzyme has been alsoreportedinhepatic acid sequences and used in thepolymerase chain reac- nodules of rat liver during hepatocarcinogenesis ( 7, 8 ),Novition
We reported that normal adult lriavter tissues exhibited very little activity while high levels were found in type I1 transmembrane protein like the othgelyr cosyl- AH-66 hepatoma ascitescells (13) and ratkidney (12)
The GnT-I11activity in transiently trans-results agreed with theprevious observation that thbeisecting fected COS-1 cells was found to be about 500-3600- GlcNAcwas presentin y-glutamyltranspeptidases purified fold as compared to that in non- or mock-transfected from AH-66 hepatoma cells (16) andfrom rat kidney (6)
Summary
Step I-Kidneys of male Donryu rats (2,222 g) were homogenized in 4 volumes of 10 mM Tris-HC1 buffer, pH 7.4, containing 0.25 M sucrose, 1mM benzamidine hydrochloride, and 20 p M APMSF with a Polytrohnomogenizer (Brinkmann Instruments). Step 3-The above extract was applied to a column (5 X 46 cm) of QAE-Sepharose which had been equilibrated with pH 7.4, 20 mM sodium phosphate buffer, containing 1mM benzamidine hydrochloride, 20 PM APMSF, and 0.1% Triton X100. Step 4-A hydroxylapatite column (14 x 13 cm) was equilibrated with pH 6.8,50 mM sodium phosphate buffer, containing 0.1% Triton X-100. Step 5-The GnT-I11 fraction was applied directly to a Cu2+-chelatingSepharose column (5 X 15 cm) which had been equilibrated with pH 8.0, 20 mM Tris-HC1 buffer, containing 0.5 M NaCl and 0.05% Triton X-100. After washing the column with the same buffer until the eluate was essentially free of protein, elution was carried out with a linear gradient established with 900 ml of the starting buffer and 900 ml of this buffer containing 0.1 M glycine (Fig. l b ). Elution was started at the point indicated by the arrow
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