Spectrophotometric assay for the condensing enzyme activity of the microsomal fatty acid chain elongation system.
A rapid and simple spectrophotometric method was developed to measure the activity of the condensing enzyme component of the microsomal fatty acid chain elongation system. The intermediate product of the condensation reaction is the beta-ketoacyl CoA which exists in two tautomeric forms, i.e., keto and enol. The addition of bovine serum albumin (BSA) to a cuvette cell containing a beta-ketoacyl CoA derivative resulted in the formation of a 303-nm absorbance peak, characteristic of enolate formation. The beta-ketoacyl CoAs with carbon chain length of 6 to 18 interacted with BSA to produce the 303-nm peak; acetoacetyl CoA was the only beta-keto compound tested which did not interact with BSA to produce the peak. Other compounds which were unaffected by BSA included CoA, free beta-keto acid, beta-hydroxyacyl CoA, acyl CoA, trans-2-enoyl CoA, and malonyl CoA. BSA could not be replaced by ovalbumin; furthermore, denatured (boiling) BSA could not induce the 303-nm peak. The specific activity of the condensing enzyme measured by the spectrophotometric method compares favorably with the activity obtained by the radioactive method. The apparent extinction coefficient (epsilon) for the absorbance peak generated by the beta-keto thioester varied from 5 to 30 mM-1 cm-1 depending on the beta-keto derivative. The spectrophotometric procedure can be used in the determination of the condensing enzyme activity in not only hepatic microsomes but also in kidney and brain microsomes both of which have significantly lower activity. The advantages of the novel method over the radioactive method are that (i) it does not involve the use of radioactive compounds, (ii) it is much less cumbersome and significantly less costly, and (iii) it is rapid and easy to perform.
Department of Pharmacology, University of Connecticut Health Center, Farmington 06032.
M N
MN
Nagi
L
L
Cook
S K
SK
Suneja
P
P
Osei
D L
DL
Cinti
D000818:Animals; D001923:Brain Chemistry; D005227:Fatty Acids / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013056:Spectrophotometry, Ultraviolet
D005227:Fatty Acids