| Research Abstract |
Our present purposes are to clarify the enzyme (s) responsible for the biosynthesis of the P-like antigen, and consequently to understand the human P blood group system well. Previously, we found in human serum and urine an N-acetylgalactosaminyltransferase (GalNAc-transferase), catalyzing the formation of the P-like antigen [Takeya et al. (1992), (1995)]. However, kinetic studies suggested the possibility that the enzyme activity is due to the N-acetylglucosaminyltransferase (GlcNAc-transferase) which catalyzes the formation of the blood group i antigen [Takaya et al.(1993), (1995)]. Thus, these studies have suggested a novel relationship between these two distinct human blood group systems. As future investigations will be focused on the donor substrate specificity of the enzyme, the use of highly purified donor substrates is necessary for accurate and reliable assays. Indeed, different values have been obtained for the activity of the GalNAc-transferase in human serum [Takaya et al.
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(1995)].The discrepancy might arise from different purity of labeled UDP-GalNAc preparations used for assays as a donor substrate.
To obtain the accurate value for the enzyme activity, we developed affinity chromatographic systems using columns of immobilized lectins for the separation and quantification of nucleotide sugars. We first found that GlcNAc-specific Psathyrella velutina lectin has a much highe affinity for UDP-GlcNAc than other GlcNAc-specific lections tested [Takaya et al. (1996) Carbohydrate Letters 2,109-113]. Further, we found that GalNAc-specific Vicia villosa B_4 lectin has a much higher affinity for UDP-GalNAc than other GalNAc-specific lectins tested [Takaya et al. (1997) Carbohydrate Letters 2,237-240]. Moreover, we determined the binding constant between these molecules, and studied about its powerful inhibitory effect on hydrolysis of the nucleotide sugar (Takaya et al., submitted). Using highly purified donor substrates, the activity of the GalNAc-transferase in human serum was determined as about a tenth of the activity of the GlcNAc-transferase. Less
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