19] Preparation of yeast mitochondria (Saccharomyces cerevisiae) with good P/O and respiratory control ratios

Abstract

This chapter describes two methods: mechanical cell rupture and cell wall digestion by snail gut juice. It provides an account of new modifications of the two methods, the advantages—greater simplicity and shorter manipulation time. Both, the mechanical and the enzymatic methods have specific advantages and drawbacks. The main advantage of the mechanical method is speed; this is of critical importance in certain cases, for example, when a long physiological study of freshly prepared mitochondria is planned. The preparation of mitochondria by the mechanical methods takes only two hours as compared to about six hours for methods involving enzymatic cell wall digestion. The main advantage of the latter over the mechanical method is that mitochondria of higher integrity are obtained. The respiratory control ratios are higher for mitochondria obtained by the enzymatic method, although the mechanical method yields similar phosphate/oxygen (P/O) ratios and properly oriented mitochondrial membranes. Representative electron micrographs for the enzymatic and mechanical methods are illustrated in the chapter.