Properties of Acyl-coA: cholesterol O-acyltransferase in aortic microsomes from atherosclerotic rabbits

A critical role for the histidine residues in the catalytic function of acyl‐CoA:cholesterol acyltransferase catalysis: Evidence for catalytic difference between ACAT1 and ACAT2

“…There might be a relationship to a minor degree between enzymatic activity and oleoyl-CoA binding activity on the basis of results indicating that the upward order of enzymatic activity and oleoyl-CoA binding activity is consistent, but the degree is too small to impact full ACAT activity. These invariant substrate-binding characteristics of histidine mutants are seen more clearly in the [4][5][6][7][8][9][10][11][12][13][14] C]cholesterol binding assay, where the cholesterol-binding capability of all histidine mutants and the WT-ACAT was almost the same ( Fig. 4C and D).…”

“…Saturation binding experiments were performed in 500 ll of enzyme assay buffer, using 50-100 lg of protein per tube and [1-14 C]oleoyl-CoA or [4][5][6][7][8][9][10][11][12][13][14] C]cholesterol, ranging between 10 and 80 lM, incubated at 37°C for 2 h, by which time the radiolabeled substrate had reached equilibrium. Each experiment was performed in triplicate.…”

“…[19], and linoleic amide [20] was determined. The inhibitory assay was carried out using microsomal fractions of ACAT isoenzymes and their mutants with substrates [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]oleoyl-CoA and cholesterol as described [8].…”

“…C]oleoyl-CoA (56 lCi/ lmol; Amersham Biosciences, UK) was used as a tracer of newly formed cholesteryl ester (CE), as described [14]. In assay method 2, [4-14 C]cholesterol (50 lCi/lmol; Amersham Biosciences, UK) was added to the assay as an indicator of CE products as described [16].…”

Effects of propranolol on atherogenesis in the cholesterol-fed rabbit.

“…Acyl CoAxholesterol acyltransferase activity (ACAT) was determined in aortic microsomes by measuring the rate of incorporation of [l- (Brecher and Chan, 1980). In the standard assay, 15-20 /xg of protein were incubated for 25 minutes at 25°C in 0.2 ml of solution containing 0.1 M potassium phosphate buffer (pH 7.5), 20 MM dithiothreitol, 0.3% of bovine serum albumin, and 5 ^M [l-' 4 C]oleoyl CoA.…”