A colorimetric assay method to measure acetyl-CoA synthetase activity: application to woodchuck model of hepatitis virus-induced hepatocellular carcinoma

Abstract

A new spectrophotometric method for quantitation of acetyl-CoA synthetase (ACAS) activity is developed. It has been applied for ACAS assay in the liver tissues of a woodchuck model of hepatitis virus-induced hepatocellular carcinoma (HCC). The assay is based on the established pyrophosphate (PPi) detection system. ACAS activity is indexed by the amount of PPi, the product of ACAS reaction system of activated form of acetate (acetyl-CoA) with ACAS catalysis. PPi is determined quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and 2-mercaptoethanol. PPi reacts with molybdate reagent to produce phosphomolybdate and PPi-molybdate complexes. 2-mercaptoethanol is responsible for color formation which has the peak absorbance at 580 nm. This method was sensitive from 1 to 20 nmol of PPi in a 380-mul sample (1-cm cuvette). A ten-fold excess of Pi did not interfere with the determination of PPi. To study the major metabolic pathways of imaging tracer [1-(11)C]-acetate in tumors for detection of HCC by Positron Emission Tomography (PET), the activity of one of the key enzymes involved in acetate or [1-(11)C]-acetate metabolism, ACAS was assayed by this newly developed assay in the tissue samples of woodchuck HCCs. A significant increase of ACAS activity was observed in the liver tissues of woodchuck HCCs as compared with neighboring regions surrounding the tumors (P<0.05). The respective ACAS activities in the subcellular locations were also significantly higher in HCCs than in the surrounding tissues (P<0.05) (total soluble fraction: 876.61+/-34.64 vs. 361.62+/-49.97 mU/g tissue; cytoplasmic fraction: 1122.02+/-112.39 vs. 732.32+/-84.44 mU/g tissue; organelle content: 815.79+/-100.77 vs. 547.91+/-97.05 mU/ g tissue; sedimentable fragment: 251.92+/-51.56 vs. 90.94+/-18.98 mU/ g tissue). The finding suggests an increase in ACAS activity in the liver cancer of woodchuck models of HCC as compared to that in the normal woodchuck liver. The developed assay is rapid, simple and accurate and is suitable for the investigation of ACAS activity under physiologic and pathophysiologic conditions.

Fig. 1

Standard calibration curve of PPi and Pi. Absorbance at 580 nm versus nmoles of PPi and Pi with 2-mercaptoethanol (2-ME) present or absent. The standard reaction mixture containing different amounts of 0.1 mM Na₄P₂O₇, 50μl of 2.5% molybdate reagent and 20 μl of Eikonogen in a final volume of 0.5 ml with 25 μmoles of 2-mercaptoethanol or without 2-mercaptoethnol was used. Following incubation at 37°C, the absorbance at 580 nm was spectrophotometrically measured within 10∼60 min. The data represent the average of the same standard reaction mixture performed in triplicate.

Figure 2

Mixed PPi and Pi assay. Fig. 1. Standard calibration curve for mixed PPi + 7 nmoles Pi systems. Absorbance at 580 nm versus nmoles of PPi and Pi with 2-mercaptoethanol (2-ME) present or absent. The standard reaction mixture containing different amount of 0.1 mM Na₄P₂O₇ plus 7 nmoles Pi, 50 μl of 2.5% molybdate reagent and 20 μl of Eikonogen in a final volume of 0.5 ml with 25 μmoles of 2-mercaptoethanol or without 2-mercaptoethnol was used. Following incubation at 37°C, the absorbance at 580 nm was spectrophotometrically measured within 10∼60 min. The data represent the average of the same standard reaction mixture performed in triplicate.

Figure 3

Dependence of ACAS activity on time. The experiment was carried out by withdrawing 380 μl reaction mixture from reaction mixture of ACAS assay at each 5 min interval. The increase in absorbance at 580 nm was recorded over time.

Figure 4

Effect of pH on acetyl-CoA synthetase activity in Tris buffer.

Figure 5

Acetyl-CoA Synthetase activities in the woodchuck livers

Figure 6

The Distribution of Acetyl-CoA Synthetase activities in the subcellular location of woodchuck livers