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The authors made a detailed comparison of the core oligosaccharide units from …

Biology Articles » Biochemistry » Carbohydrate Biochemistry » Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated » Materials and methods

Materials and methods
- Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated

Materials. Ion exchange resins and gel filtration materials came from Bio-Rad and phenylmethanesulfonyl fluoride (toluenesulfonyl fluoride) was froim Sigma. Bacterial alkaline phosphatase was from Worthington, jack bean a-mannosidase was prepared according to Li (10), and endo-p-N-acetyl-D-glucosaminidase H was purified from Streptomyces plicatus (American Type Culture Collection 27800) according to Tarentino et al. (11). Carboxypeptidase Y was isolated from compressed bakers' yeast (Universal Foods, San Francisco) by the procedure of Kuhn et al. (12). Reference oligosaccharides were from an earlier study (13).

Methods. Alkaline phosphatase digestion was done for 24 hr in 1.0 M Tris-HCl (pH 8.0); a-mannosidase digestion was for 24 hr in 50mM NaOAc, pH 4.5/0.1 mM ZnCl2; and endoglucosaminidase H digestion was for 96 hr in 50 mM Na citrate (pH 5.5). All reactions were carried out at 37°C. Carboxypeptidase Y was treated with toluenesulfonyl fluoride according to Kuhn et al. (14). Mannose 6-phosphate was determined by a coupled enzyme assay (15) as modified by Hashimoto et al. (16). A correction was made for the amount of NADPH produced by an acid hydrolysate of bovine serum albumin. Partial acetolysis of oligosaccharides was carried out in acetic acid/acetic anhydride/ sulfuric acid, 10:10:1 (vol/vol) at 40°C for 7 hr (17), and the recovered acetylated products were deacetylated in methanol containing Na methoxide. Mild acid hydrolysis to cleave glycosylphosphate bonds was done in 0.01 M HC1 at 100°C for 30 min. Carboxypeptidase Y was methylated by a standard procedure (13).

Oligosaccharide samples (50-500 ,ug) were reduced in 500 *,l of 0.1 M NaHCO3 by adding 200 uCi (1 Ci = 3.7 X 10"° becquerels) of NaB3H4 as a dimethyl sulfoxide solution (4 mCi/ ml). After 12 hr at 23°C, 5 mg of NaBH4 was added and the reaction was allowed to proceed for another 12 hr. Dowex 50 (H+) was added until effervescence ceased, the tube contents were filtered through a Pasteur pipette plugged with glass wool, and the filtrate was evaporated to dryness under reduced pressure. One drop of glacial acetic acid and 1 ml of methanol were added to the residue, and the mixture was refluxed for 1 min and then evaporated to dryness under a stream ofN2. This treatment was repeated four times. For neutral samples, an acidic noncarbohydrate radioactive contaminant was removed by passing a water solution of the reduced product through a small column of Dowex 1 (acetate). Acidic reduced oligosaccharides were purified on a Bio-Gel P-4 (>400 mesh) column (1 X 25 cm) in 0.1 M acetic acid. Finally, the reduced compounds were desalted by passage through a Sephadex G-10 column (0.5 x 25 cm) in water.

Oligosaccharide sizes were estimated by gel filtration on a calibrated Bio-Gel P-4 column (>400 mesh) (0.6 x 115 cm) in 0.1 M NH4OAc containing 0.02% NaN3 to prevent microbial growth. Fractions of 3 drops were collected, and the elution positions were determined relative to the reference compounds ,/Man-*4GlcNAcH2, aMan--3(3Man-*4GlcNAcH2, aMan--> 2aMan--2aMan--3/3Man--*4GlcNAcH2, Man6GlcNAcH2 (from ovalbumin) (13), Man8GlcNAcH2 (from IgM) (13), and Man,,GlcNAcH2 (from yeast mannoprotein) (7). On this same column, bovine serum albumin was eluted at fraction 60 and mannose was eluted at fraction 176.

Polyacrylamide gel (10%)/0. 1% NaDodSO4 electrophoresis was done by the discontinuous system of Laemmli (18), with a running buffer ofTris HCl (pH 7.2). Samples were reduced with 1% mercaptoethanol/1% NaDodSO4 at 100°C for 3 min. Gels were stained for protein with Coomassie brilliant blue R-250. 'H NMR and 31P NMR were carried out as described (13, 16).

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