Separation and purification of both acid and neutral nucleases from germinated alfalfa seeds

Abstract

Two enzymes possessing endonuclease activity were isolated from alfalfa seedlings. These enzymes were further purified by ammonium sulphate precipitation, DEAE cellulose, Heparin Sepharose and ssDNA-cellulose chromatography. One of them was an acid and the other a neutral nuclease with optimal activity at pH 5.5 and 7.0, respectively. Both enzymes showed a similar molecular weight of about 40kDa, were thermostable, loss their activity with increasing concentrations of NaCl and were inhibited by sulphydryl compounds and polyamines. No significant inhibition was observed with sulphydryl reagent. Zn 2+ stimulated the acid and strongly inhibited the neutral nuclease activity. Alfalfa nucleases attacked natural nucleic acids and synthetic homopolyribonucleotides, and the relative rate of degradation was: polyU > ssDNA > polyA > RNA > polyC > dsDNA = polyG for acid and polyA 5= polyU > ssDNA > RNA > polyC > dsDNA = polyG for neutral nuclease. On the basis of hydrolysis of 5′ labelled deoxydecanucleotide d- 32 pCpCpTpGpGpCpApGpTpT the two enzymes differed in their preference for the susceptible phosphodiester bond. Thus, acid nuclease preferentially hydrolysed the Cp↓C, Tp↓C, Gp↓T and Tp↓T bonds, neutral nuclease the T↓pG, G↓pG and T↓pC bonds. The substrates (polyA or synthetic deoxydecanucleotide) products of acid nuclease were mononucleotides and oligonucleotides, while those of neutral nuclease were only oligonucleotides, bearing the monoesterified phosphate at the 3′ and 5′ positions respectively. Although the individual properties and specificity of investigated nucleases vary considerably, they must belong to plant nuclease type I enzymes (E.C. 3.1.30.X). However, our results indicate that a more detailed classification could be achieved by subdivision of the general plant nuclease category into groups containing enzymes with similar behaviours.