Main

CT (EC 2.3.1.9), T2, T1 (EC 2.3.1.16), mitochondrial TFP, and PT are present in human cells(1–3), and all but TFP(4) have thiolase activity toward AACoA. Such being the case, it is difficult to evaluate individual activities of T2 or CT. T2 is the only thiolase that is activated by the potassium ion(1), and the enzymatic diagnosis of T2 deficiency is carried out by comparing AACoAT activities in the presence/absence of potassium ion(5). We correctly diagnosed 21 patients with T2 deficiency using this method. Moreover, a substrate specific for T2, 2-methyl-AACoA, can also be used for T2 enzyme assay(6). Various groups of workers deduced CT activity from total AACoT activity; however, what percent of AACoAT activity is derived from CT in human fibroblasts is not clear. Bennet et al.(7) reported a case of CT deficiency based on about a 50% decrease of AACoAT activity in the presence of potassium ion and normal activity toward 2-methyl-AACoA.

CT has a role in AACoA formation from acetyl-CoA, at the first step in cholesterol biosynthesis in the cytosol(1). Because AACoA itself cannot pass through the mitochondrial membrane and is an essential precursor of cholesterol biosyntheisis, the synthesis of AACoA has to occur in the cytosol. Data on only two patients with CT deficiency have been reported(7, 8), and severe mental retardation, hypotonus, and persistent ketosis were present in these patients. We recently developed an anti-human CT antibody and cloned human CT cDNA(9).

We now report the contribution of each thiolase to the whole AACoAT activity in human fibroblasts, determined in an immunotitration analysis with antibodies against T2, CT, or T1. Only 26-38% of the total activity in the absence of potassium ion derived from CT, therefore, the immunotitration analysis provides a more accurate evaluation of CT activity in human fibroblasts and precise clinical diagnosis and treatment can be given.

METHODS

Fibroblasts. Six control fibroblast cell lines were cultured in Eagle's minimum essential medium containing 10% FCS and routinely maintained at 37°C in a 5% CO2 incubator. The fibroblasts were harvested with 0.05% trypsin/0.01% EDTA 2 d after reaching confluency. After three washes in 0.9% sodium chloride, cell pellets were stored at -80°C until use.

Enzyme assay. Pellets of the fibroblasts were freeze-thawed and suspended in 50 mM sodium phosphate (pH 8.0), 0.1% Triton X-100. After sonication and centrifugation at 10 000 × g for 10 min, the supernatant was used for enzyme assay. AACoAT activity was assayed with 15μM of AACoA and 50 μM of CoA in 0.1 M Tris-Cl (pH 8.0), 25 mM MgCl2, 0.5 mM DTT, with/without 50 mM potassium chloride(10). Enzyme activity was monitored by a decrease of AACoA at 303 nM. Protein concentration was determined by the method of Lowryet al.(11) using BSA as a standard.

Immunotitration analysis. Anti-[rat T2]antibody (2), anti-[human CT]antibody(9), and anti-[rat T1] antibody(2), were used. The 40 μL of extract of fibroblasts (6-10 mg/mL of protein concentration) were incubated with various amounts of antibody for 1 h at 4°C. After adjusting the volume to 80 μL with 50 mM sodium phosphate(pH 8.0), 0.1% Triton X-100, we added 20 μL of protein A-Sepharose(containing about 20 μg of protein A, Sigma Chemical Co., St. Louis, MO), and the preparation was incubated at 4°C for 1 h. The preparation was centrifugated at 10 000 × g for 10 min, and the supernatant was used for enzyme assay. After immunotitration, the pellet and the supernatant corresponding to 30 μg of fibroblast protein were applied to SDS-PAGE, using a 12% gel(12). Immunoblot analysis was performed according to Towbin et al.(13), using the ProtoBlot Western Blot AP Systems (Promega, Madison, WI). A mixture of the anti-CT and anti-T2 antibodies was used as the first antibody.

RESULTS

Standard enzyme assay. We assayed AACoAT activity under conditions of 15 μM AACoA and 50 μM CoA. As shown inTable 1, the ratio of AACoAT activity in the presence of potassium ion to that in the absence of the ion was about 1.9 in six control fibroblasts. Total activities of AACoAT in both the absence and presence of potassium ion had wide ranges.

Table 1 Acetoacetyl-CoA thiolase activity in human fibroblasts
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Immunotitration assay with anti-CT antibody. Polyclonal anti-[humanCT] antibody immunotitrated about 30% total AACoAT activity in the absence of potassium ion and almost the same activity was titrated in the presence of the ion (Fig. 1A). Immunoblot analysis of supernatants and pellets after the immunotitration confirmed that almost all the CT protein was immunoprecipitated in the presence of 1 μL of the antibody (Fig. 1B). T2 was not captured with the anti-CT antibody in immunotitration analysis, although this antibody did have a slight cross-reactivity to T2 in immunoblot analysis(9), hence it can react with the denatured T2 subunit but not with a native tetramer of T2. We used the same amount of CT antibody in the following experiments. As shown in Table 1, in six control fibroblasts, AACoAT activity immunotitrated with the anti-CT antibody was 1.3-2.4 nmol/min/mg of protein, a value that corresponds to 26-38% of total activity in the absence of potassium ion.

Figure 1
figure 1

Immunotitration assay with anti-[human CT] antibody.(A) AACoAT activities in immunotitration analysis with respect to the amount of anti-CT antibody. Control 1 fibroblasts were used. (B) immunoblot analysis of the supernatant (S) and pellets (P) in immunotitration assay. After immunotitration, aliquots of the supernatant and the pellets corresponding to 30 μg of fibroblast protein, were applied to 12% SDS-PAGE. The first antibody used was a mixture of anti-T2 and anti-CT antibodies.

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Immunotitration assay with antibodies. Antibodies against rat T2 and rat T1 immunotitrated human T2 and T1, respectively, with 1 μL of antibody being sufficient for the analysis (data not shown). Figure 2 shows a typical result, when the antibodies were used in combination. The 91 and 95% total AACoAT activities in the absence and presence, respectively, of potassium ion were immunotitrated when the three antibodies were used in combination. Thus, the contribution of PT to total AACoAT activity is not significant. After immunotitration with the anti-T2 antibody, residual activities were much the same in the presence and absence of potassium ion; therefore, T2 is the only thiolase activated by the potassium ion, and T2 activity in the presence of potassium ion was about 6-fold of that in the absence of the ion. Data on immunotitration assays done using the three antibodies and three control cell lines are summarized schematically in Figure 3. The contribution of T2, CT, and T1 to AACoA activity, in the absence of potassium ion, was 11-20%, 26-38%, and 40-47%, respectively. Less than 9% was not immunotitrated with the three antibodies.

Figure 2
figure 2

Typical immunotitration with various combinations of antibodies.

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Figure 3
figure 3

Contribution of thiolases to a total AACoAT activity determined by immunotitration with antibodies directed to individual thiolases.

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DISCUSSION

This is apparently the first report showing the contribution of three thiolases (T2, CT, and T1) to AACoAT activity in human fibroblasts. When evaluating AACoAT activity, as shown in Table 1 and in our previous studies(14, 15), one should keep in mind that total AACoAT activities in both the presence and absence of potassium ion have a wide range. The activities can differ when the same cells are cultured for different times: for example, in the absence of potassium ion, control 4 showed activities of 7.8 and 5.5 nmol/min/mg of protein in this study and in the previous assay, respectively. Thus, it is difficult to evaluate CT defects only on a decrease of the whole AACoAT activity, even in the absence of potassium ion. To diagnose CT deficiency, it is necessary to know the extent to which CT contributes to the total AACoAT activity. Middleton(1) reported the contribution of T2, CT, and T1 to the total AACoAT activity in rat organs, based on separation of the enzymes on column chromatography. However, this method is not so appropriate for an enzymatic diagnosis of T2 or CT deficiency using human fibroblasts because a large amount of these connective tissue cells is needed. Development of an anti-CT antibody(9) has made it feasible to evaluate the contribution of CT to the whole AACoAT activity.

Human fibroblasts have five known thiolases(1–3). Among them, TFP has three known enzyme activities, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and thiolase, toward long acyl-CoA substrates(3). Kamijoet al.(4) found that the human enzyme has no AACoAT activity, hence total AACoAT activity is a sum of the other four thiolases, T1, T2, CT, and PT. Anti-rat PT antibody did not immunotitrate human PT (T. Hashimoto, unpublished data); however, in immunoblot analysis, human PT was recognized by an anti-[rat PT] antibody(16). Hence in this report, PT activity was estimated as the residual activity not immunotitrated with a combination of three other antibodies. These four thiolases may have different kinetics for AACoA and CoA, as is the case of rat enzymes(1–3). Our standard conditions of 15 μM AACoA and 50 μM CoA were used throughout the experiments. T2, CT, T1, and PT activities in the absence of potassium ion were 0.7-1.2, 1.6-2.5, 2.4-3.0, and less than 0.5 nmol/min/mg of protein, respectively. Not only activity of CT but also that of T1 could be evaluated in this immunotitration assay, although 3-keto-hexanoyl-CoA or 3-keto-octanoyl-CoA thiolase activity is more satisfactory than AACoAT activity for evaluating T1 activity.

CT catalyzes AACoA formation from acetyl-CoA and cleavage of AACoA to acetyl-CoA in cytosol(1). This enzyme has an important role in AACoA synthesis, the first step of cholesterol biosynthesis in the cytosol(1). Cytosolic AACoA synthetase also catalyzes AACoA synthesis from acetoacetate, and the enzyme activity in most rat tissues is scanty(17). If CT is deficient, cholesterol biosynthesis may be affected and brain development could be impaired. In two CT-deficient patients reported previously, severe mental retardation, developmental delay or deterioration, and hypotonus were described, and in one of them a decreased cholesterol synthesis in the fibroblasts was noted(7, 8). In another patient, disorder in cholesterol biosynthetic pathway, mevalonic aciduria, psychomotor delay, and hypotonia were also noted(18). Interestingly, in both CT-deficient patients, colitis cystica superficialis, a rare intestinal condition developed. Pathognomonic metabolites in blood and urine were absent; however, both CT-deficient patients developed a persistent elevation in blood ketones, a condition being a well known characteristic in succinyl-CoA:3-ketoacid CoA transferase deficiency, another disorder of ketone body metabolism(19). This finding may also provide a clue for the clinician to suspect CT deficiency in a patient with severe neurologic findings. Unfortunately, the two cell lines(7, 8) have become extinct.

Our data show that less than 40 and 20% total AACoAT activity in the absence and presence, respectively, of potassium ion accounts for CT activity. We propose that immunotitration analysis with anti-CT antibody be used for accurate diagnosis of CT deficiency.