Detailed protocol and critical view for the analysis of control in metabolic systems by shortening and enzyme titration.

In this paper we give a general description of the 'shortening and enzyme titration method'. This method allows us to determine the Flux Control Coefficients of the different steps of a metabolic pathway in an in vitro experimental system. The system submitted to study is shortened in vitro by means of auxiliary enzymes, and the shortened pathway is titrated with extraneous enzymes. In this way we can modulate the activity of every enzyme of the system and thus every Flux Control Coefficient can be obtained. We criticize its different features in order to comment on the possibilities of its application to different types of systems. Our conclusion is that the method has a general applicability provided: a) that a correct definition of the metabolic pathway is given; b) that the system occurs in only one subcellular fraction and, c) that the dilution of the system by a given factor drives to the same reduction in every enzyme activity of the system.