106] Pantetheine kinase

Abstract

Publisher Summary The enzymatic phosphorylation of pantetheine to form 4'-phosphopantetheine is measured by converting the product to CoA by a second incubation with a protamine-treated extract of acetone-dried pigeon liver. Such an extract is no longer able to carry out the phosphorylation of pantetheine, but it is still effective in synthesizing CoA from phosphopantetheine. This extract also contains the enzymes for the acetylation of sulfanilamide and thus the conversion of phosphopantetheine to CoA and the quantitative measure of the latter may be carried out simultaneously. One unit of enzyme is defined as that amount which results in the synthesis of one unit of phosphopantetheine, expressed in CoA units, in 30 minutes. Specific activity is expressed as units per milligram of protein. Protein is determined by the turbidimetric method of Bucher. The steps in the purification procedure are: (1) preparation of crude extract, (2) protamine treatment, (3) elution from protamine sulfate, and (4) adsorption and elution from calcium phosphate gel. Attempts to purify the enzyme by further employing ammonium sulfate or cold ethanol fraction invariably result in large losses of activity. The purified enzyme seems to be specific for pantetheine.