Permeabilisation of hepatocytes with α-toxin

Abstract

In large part, current concepts of eukaryotic cell metabolism are based on results obtained with disrupted cells and subcellular fractions. These preparations have been preferred to isolated cell suspensions and cell cultures for analysing enzymatic steps in different pathways, since the plasma membrane makes the interior of intact cells inaccessible to various substrates and modulators. However, apart from disrupting the normal spatial relationship between cellular organelles, homogenisation may also disintegrate functional enzyme units in the cell, to the detriment of complex reaction sequences and control systems. As a consequence, the coupling of individual steps in metabolic pathways, e.g. “channelling” of intermediates between enzymes, is obscured [1]. Evidence for such “topodynamic regulation” of intermediary metabolism has accumulated over the past years and has led to a dramatic increase in our knowledge of metabolic regulation [2–4]. Another important, and sometimes overlooked, drawback of homogenates and subcellular fractions is the risk for proteolytic or other alteration of the proteins, enzymes or other macromolecules, that are the subject of investigation.