22 

 dried on preweighed filters made from Miracloth in an oven 

 at 50°C and were weighed daily until constant dry weight 

 values were obtained. From these two methods, growth curves 

 were determined. 

 Preparation of C. sorokiniana extracts 



Frozen cell pellets of C. sorokiniana were suspended 

 into 50 mM PIPES buffer, pH 7.3, containing 1.0 mAf DTT, 1.0 

 mAf PMSF, 35 mM KCl , and were broken by 2 passes through a 

 French Pressure Cell at 20,000 psi. The slurry was 

 centrifuged at 18,000 x g, at 4°C for 15 min, and the 

 supernatant was next centrifuged at 150,000 x g for 1 h at 

 4°C. Extracts were either desalted and used for column 

 chromatography, or dialyzed with 50 mM EPPS-KOH buffer with 

 all protective components as listed above for use in enzyme 

 assays. (Pipes buffer plus KCl was used in place of EPPS 

 buffer for column chromatography in order to separate 

 isoenzymes of chorismate mutase for another study.) 

 Glycerol and PLP were added to column fractions to protect 

 several of the enzymes to be assayed (PAT and ADH) . 

 Preparation of bacterial extracts 



The mutant E. coli strains {aroD and aroE) carrying the 

 cDNA clones encoding functional proteins (DQT or SDH, 

 respectively) were grown in 200 ml of LB/Amp media, washed 

 and resuspended in KPO4 buffer containing 2 0% glycerol and 1 

 mM DTT at pH 7.6, and centrifuged at 150,000 x g, at 4°C for 



