Similarities and differences in the properties of alfalfa endonucleases

Recently, both acid and neutral nucleases acre separated and purified from germinated alfalfa seeds (Yupsanis et al., 1996). In this work, the neutral alfalfa nuclease was further purified to near homogeneity with a modified purification procedure and additional properties of both enzymes were studied. Nuclease extracts of SDS/PAGE and IEF gels revealed that the acid as well as the neutral nuclease hydrolyzed both ssDNA and RNA and showed nicking action against plasmid DNA (pTZ18R). Also, both nucleases were accompanied by 3' nucleotidase activity. In comparison with the acid nuclease the mode of the neutral nuclease action towards ssDNA and a synthetic thirty-sixth-deoxynucleotide (d-(32)pGpApTpCpCpApG-pApAp GpTpApGpApApGpCpApGpApTpCpTpCpGpApTpCpCpApGppTpCpTpG) was more endonucleolytic. The order of bond hydrolysis of dir above deoxynucleotide was Cp down arrow C >Cp down arrow A >Gp down arrow T >Gp down arrow C for the acid nuclease and A down arrow pT>C down arrow pA = T down arrow pC for the neutral nuclease. Both enzymes consist of one polypeptide chain of 37krDa with pI 4.9 (acid nuclease) and of 41 kDa with pI 5.3 (neutral nuclease). Based on the properties of alfalfa endonucleases we concluded chat their structural conformation should have common aspects bur different catalytic sites.