49] Measurement of matrix enzyme activity in Situ in isolated mitochondria made permeable with toluene

Abstract

This chapter discusses that studies on isolated enzymes cannot accurately reflect their precise in vivo behavior. As the knowledge concerning the concentrations of metabolic intermediates has increased, it has become possible to show that regulatory data obtained from studies on enzymes in vitro do not agree with the known metabolic behavior of enzymes in tissues. In an attempt to explain the apparent discrepancies, the existence of microenvironments within cells has been postulated. The major difficulty with testing such hypotheses in animal cells stems from the impermeability of cells to most cofactors and substrates so that behavior of enzymes in vivo cannot be tested. Toluene has been employed to make isolated microbial cells permeable to normally non-penetrating substrates and cofactors. Because these cells have rigid cell walls, no problem was encountered in relation to cell breakage. In a similar manner, yeast cells, which also possess a rigid wall, have been made permeable to metabolic intermediates by toluene treatment, and the control properties of several enzymes were studied. It presents a method of making rat mitochondria permeable to substrates and stabilizing them so that the individual properties of Krebs cycle enzymes can be studied.