Figure 3.

Radio-HPLC analyses of the biochemical assay for IPMS2. Results for IPMS1 had a similar pattern. A, Incubation of IPMS2 with 500 μm [¹⁴C]acetyl-CoA and 3 mm 2-oxoisovalerate shows [¹⁴C]-2-isopropylmalate (IPM) as the only radioactive labeled product. B, In the absence of 2-oxo acid substrate, a small amount of [¹⁴C]acetate (Ac) is formed, whereas most of the [¹⁴C]acetyl-CoA (Ac-CoA) remains intact and is hardly retained in the HPLC column. C, UV trace (230 nm) of the HPLC showing the elution pattern of a standard solution containing 10 mm acetic acid (Ac) and 5 mm 2-isopropylmalate (IPM), and the injection peak at 15 min. D, Incubation of the IPMS2 gene product with 4-methylthio-2-oxobutyrate and [¹⁴C]acetyl-CoA yields a small amount of [¹⁴C]2-(2′-methylthio)ethylmalate (MTEM), whereas most of the [¹⁴C]acetyl-CoA (Ac-CoA) remains.