Effect of high of pressure of EcoRI reactions

Abstract

The effect of pressure on reactions of restriction endonucleases was investigated. No obvious irreversible (after) effect was observed for EcoRI, while a considerable irreversible inactivation was found for BamHI. Thus the EcoRI reactions against lambda DNA, pBR322 and pBluescript were studied under high pressure and little effect was observed on the overall reactions. The DNA concentration dependence of the kinetic data apparently fits the Michaelis-Menten type equation and the evaluated rate parameters were: Vmax = 6.2 +/- 0.24 and 7.0 +/- 0.22 (x 10(-2) nM/min) at 0.1 and 200 MPa, respectively; Km = 19 +/- 1.8 and 28 +/- 1.7 nM at 0.1 and 200 MPa, respectively. The apparent activation volume corresponding to kcat/Km was ca +1 mL/mol. A characteristic effect of pressure on the sequence specificity of these enzymes was seen in their star activity. Relaxed specificity was tightened by increasing pressure (200 MPa) with respect to that induced by low salt concentration or by the presence of organic solvent.