Acetyl-coenzyme A Synthetase (Acs) Assay

Materials and Reagents

1. Extraction
   1. Potassium phosphate buffer 65 mM (pH 7.5) (Sigma-Aldrich, catalog number: P5379 )
   2. Cultured cells (approx. 10¹⁰ cells) (e.g., for Escherichia coli cells grown in M9 glucose minimal medium, 1 ml of OD₆₀₀ 1 corresponds to approx. 6 x 10⁸ cells)
2. Enzyme activity
   1. 100 mM Tris-HCl buffer (pH 7.8) (Sigma-Aldrich, catalog number: T1502 )
   2. 20 mM ATP  (Sigma-Aldrich, catalog number: A3377 )
   3. 2 mM Coenzyme A trilithium salt (CoASH) (Sigma-Aldrich, catalog number: C3019 )
      
   4. 60 mM β-nicotinamide adenine dinucleotide hydrate (NAD⁺)  (Sigma-Aldrich, catalog number: N7004 )
      
   5. 50 mM MgCl₂  (Panreac Applichem, catalog number:  131396 )
      
   6. 50 mM L-malate (Sigma-Aldrich, catalog number: 02288 )
      
   7. 1 M sodium acetate trihydrate (Sigma-Aldrich, catalog number: S8625 )
      
   8. 50 U/ml malate dehydrogenase (Mdh) from bovine heart (Sigma-Aldrich, catalog number: M9004 )
      
   9. 25 U/ml citrate synthase from porcine heart (Cs) (Sigma-Aldrich, catalog number: C3260 )
      Reagents 2-4 were prepared in milliQ water and components 5-7 in Tris-HCl buffer (100 mM, pH 7.8). All concentrated stocks (1-7) were prepared in advance and stored at -20 °C. Enzymes (8-9) were stored at 4-6 °C (following the instructions of the manufacturer) and diluted in Tris-HCl buffer (100 mM, pH 7.8) immediately before being used.
   10. 96 well flat bottom, non-treated, non-sterile transpartent plates
   11. Ice-cold phosphate buffer
       

Equipment

1. Refrigerated benchtop centrifuge (Eppendorf, catalog number: 5801 R ) 
   
2. Ultrasonic homogenizer (Sonics & Materials Inc., Vibra Cell VC 375) equipped with a 3 mm diameter probe
3. Spectrophotometric plate reader (Bio-Tek)
4. Novaspec Plus spectrophotometer (Amersham Bioscience GE Healthcare Europe GmbH)
   
5. Non-sterile transpartent plates
   
6. Water-ice bath
   

Procedure