A SIMPLE MICROMETHOD FOR MEASUREMENT OF COASH AND ITS USE IN MEASURING INTRACELLULAR LEVELS OF COASH AND SHORT CHAIN ACYL-COAS IN ESCHERICHIA-COLI K12 CELLS

A simple micromethod for measurment of CoASH was established. CoASH was quantitatively converted to acetyl-CoA with phosphate acetyltransferase (EC 2.3.1.8), then acetyl-CoA originated from CoASH was measuring by the acyl-CoA cycling method using malonate CoA-transferase (EC 2.8.3.3). This method is highly sensitive and can detect 10(-12) mol of CoASH without any loss of CoA-thioesters, and it is specific for CoASH and 3'-dephospho-CoA. The established method was useful to define the rapid changes in the size and composition of in vivo CoA pool (CoASH, acetyl-CoA, and malonyl-CoA) in Escherichia coli K12. It was found that the CoA pool changed rapidly and dramatically in a minute order in ranges of CoASH; 40-300-mu-M (0.11-0.80 nmol/mg dry wt.), acetyl-CoA; 40-350-mu-m (0.12-0.93 nmol/mg dry wt.), and malonyl-CoA; 0-290-mu-M (0-0.79 nmol/mg dry wt.). When glucose in the medium was depleted, CoASH increased to be the major component (82%) of the CoA pool, while when sufficient amount of glucose (28 mM) was provided, CoASH was reduced to a minor component (15%). In contrast with this, acetyl-CoA increased to the maximal level of 350-mu-M (0.93 nmol/mg dry wt.) and to be the major component (66%) 10 min after 28 mM glucose was added. A remarkable demonstration was achieved in confirmation of the reverse relationship between CoASH and short chain acyl-CoAs, particularly acetyl-CoA, of which the in vivo behavior has been mere conjecture until now.