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Analysis of the acid phosphatase activity of soybean vegetative storage protein
Soybean (Glycine max L. Merr.) vegetative storage proteins (VSPs) are glycoproteins found in paraveinal mesophyll cells. VSPs have also been reported to possess low acid phosphatase activity. To determine acid phosphatase activity of VSPα, VSPA cDNA was cloned into a glutathione-S-transferase (GST) expression vector and expressed in E. coli. As a positive control, a soybean root nodule ACP cDNA was also engineered into GST. Both fusions exhibited highest activity for 5′-GMP; however, VSPα : GST still showed low levels of acid phosphatase activity compared with nodule ACP fusions. In this E. coli expression system, the Km values of the ACP fusion were 0.9 mM for 5′-GMP and 8.8 mM for pNPP. The cause of low acid phosphatase activity of VSPs could be due to the lack of nucleophilic aspartate residue in VSPs, which was previously noted by others to be replaced by serine and glycine in G. max VSPα and VSPβ, respectively. VSP cDNAs from the cultivated soybean relatives, G. falcata and G. tomentella, were isolated and sequenced to examine whether the deduced amino acid sequences reveal the existence of nucleophilic aspartate residue. The predicted VSP-related protein sequences from these species also had serine in this position. Substitution of the Ser at the position 106 with Asp in VSPα increased the acid phosphatase activity about 20 fold higher than the wild type VSPα. The VSPα Ser106Asp had substrate preference for 5′-GMP. This finding demonstrates that Asp 106 is essential for acid phosphatase activity in the soybean VSP and at least partially explains the basis for low acid phosphatase activity previously noted for these proteins. ^
Leelaporn, Oranuch, "Analysis of the acid phosphatase activity of soybean vegetative storage protein" (2003). ETD collection for University of Nebraska - Lincoln. AAI3092568.