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This study describes the kinetic properties of N-acetyl-D-galactosaminylttiansferase in serum from subjects with blood groups A, and A2. When the Al and A2 enzymes were compared, with lacto-N-fucopentaose I and 2'-fueosyllactose as acceptors, the enzymes differed in their cation requirements, pH optima, and Km values.The two acceptors competed for the same transferase.Mixing experiments showed that the lower activity of the AP enzyme could not be attributed to a modifier or inhibitor in serum. It Was concluded that the Al and A2 enzymes differ qualitatively.

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Reduction of interchain disulfide bonds converted some IgG incomplete antibodies to direct hemagglutinins. This conversion occurred whether antibody was free in solution or bound to the red-cell surface. Reduced antibody permitted to reoxidize in air no longer behaved as a direct agglutinin; reversion to an incomplete antibody did not occur when reoxidation was prevented by S-alkylation. These antibodies in low titer. Four sera containing an incomplete antibody of high titer were selected for the isolation and chemical modification of IgG; three (Gue, Les, and Tur) contained anti-D and one (Dai) contained anti-K. Other sera containing incomplete antibodies (anti-D, anti-C, anti-c, anti-E, anti-K, anti-S, anti-Fya, and anti-Jka) were tested without isolation of the IgG. The sera had not been heat-inactivated. Isolation and Characterization of IgG. Two methods were used for the isolation of IgG; in both, partial delipidation by centrifugation (20,000 X g for 30 min) was performed before isolation. In the first, serum Gue and serum Dai were fractionated by ammonium sulfate precipitation followed by anion-exchange chromatography on Whatman DE-52 cellulose equilibrated in 10 mM Tris-HCI/20 mM NaCl, pH 7.0. Monomeric IgG was obtained from the unadsorbed fraction by gel chromatography on LKB Ultrogel AcA 34 equilibrated in Tris-buffered saline (10 mM Tris-HCI/0.15 M NaCI/0.02% NaN3, pH 7.8). The purity of the isolated IgG fractions was established by immunoelectrophoresis in 1.3% agarose (Kallestad) in barbital buffer (pH 8.6, , = 0.05, containing 0.85 mM calcium lactate) against a sheep antiserum to whole human serum and class-specific antisera, and by 0.1% sodium dodecyl sulfate/polyacrylamide gel electrophoresis in 7.0% gels (10).Since in serum Gue anti-D activity was restricted to the fast 'y/f3 fractions isolated from DE-52 cellulose in the gradient fractions, as observed with another example of anti-D (11), the other two sera (Les and Tur) were fractionated on QAE-Sephadex A-50 (12) followed by gel filtration on Ultrogel AcA 34 in Tris-buffered saline. The IgG fractions were assessed for purity as described above and were found to contain a trace contaminant of Al3 mobility, identified as transferrin by Ouchterlony analysis. Before testing, all samples were concentrated by dialysis under reduced pressure against phosphate-buffered saline (40 mM in phosphate, pH 7.2, A = 0.15) containing 0.02% NaN3. The concentration of IgG in whole.sera and in the IgG fractions was determined by single radial immunodiffusion and spectrophotometrically from the absorbance at 280 nm using Alm = 14.0, respectively.Reduction and S-Alkylation of IgG. Each of the IgG samples (about 10 mg/ml) was adjusted to pH 8.6 with 2 M Tris base and mildly reduced with 10 mM dithioerythritol (Sigma Chemical Co.) at room temperature. After 45 min, iodoacetamide (Sigma, twice recrystallized) was added to a final concentration of 25 mM; the samples were left for 1 hr in the dark and then dialyzed into phosphate-buffered saline for testing. R...