Selective Release of Proteins from Spirillum itersonii by Tris(hydroxymethyl)aminomethane and Ethylenediaminetetraacetate

Abstract

Treatment of Spirillum itersonii with tris(hydroxymethyl)aminomethane (Tris)-ethylenediaminetetraacetate (EDTA) results in the quantitative release of alkaline phosphatase and ribonuclease into the surrounding medium. At the same time, about 90% of the total cellular soluble cytochrome c is liberated. This process occurs within 1 min of treatment at both 24 and 4 C. Release of these proteins by Tris-EDTA treatment is highly selective, since only 9% of the total cell protein is liberated, concomitantly with less than 5% ribonucleic acid, deoxyribonucleic acid, and malate dehydrogenase. Different sigmoidal curves are obtained for release of proteins as a function of EDTA concentration. The order of liberation with increasing EDTA is as follows: alkaline phosphatase, protein, soluble cytochrome c , and ribonuclease. Treatment of cells with Tris-EDTA under conditions which cause extensive loss of alkaline phosphatase, soluble cytochrome c , and ribonuclease results in cell death, with cessation of protein and ribonucleic acid synthesis. Cells treated with EDTA in phosphate buffer (in the absence of Tris) liberate a large portion of their soluble cytochrome c , but negligible amounts of alkaline phosphatase and ribonuclease. Addition of Tris to cells pretreated with phosphate-buffered EDTA releases high levels of alkaline phosphatase, but not ribonuclease. These results suggest that a common surface alteration is not solely responsible for release of periplasmic proteins. More likely, each protein of the periplasm is bound in an independent and specific manner.