Acta Biochimica et Biophysica 12. (1977)
1977 / 3. szám - Sándor, A.-Kerner, J.-Alkonyi, I.: Effect of Biguanide (Buformin) on Carnitine Polmitoyltransferase Activity
218 Sándor et al. : Effect of Bigiianide on Carnitine Palmitoyltransferase One-way reaction from left to right (see the equation in Results) to measure the L-(3H)-carnitine liberated from (-)palmitoyl-(3H)carnitine (Bremer, Norum, 1967/a). Samples were evaporated to dryness under an infrared lamp before dissolving them in Bray’s solution. Radioactivity was measured in a Beckman LS-230 counter. One-way reaction from right to left. In this newly developed system the SH group of released CoASH was detected by 4,4’-dithiobis-pyridine, a highly sensitive thiol reagent (e = 19.8). In the presence of dithiobis-pyridine we could follow the reaction continuously using a Specord UV VIZ spectrophotometer. Dithiobispyridine inhibits neither CPT (our observation) nor carnitine acetyltransferase (Ramsay, Tubbs, 1975). In other cases ditiobispyridine was added after stopping the reaction (this time the reaction was going at pH 6.8). (-)Palmitoyl-(3H) carnitine was prepared by the above mentioned exchange reaction and palmitoyl-CoASH was prepared also enzymatically as described by Kornberg and Pricer (1953). DL-(Me3H) carnitine was purchased from Radiochemicals (Amersham), Coenzyme A from Boehringer (Mannheim) 4,4’-dithiobispyridine from Fluka (Buchs). Buformin and metformin were gifts of Chinoin (Budapest) and Chemiewerk (Hamburg). Results First, the carnitine palmitoyltransferase reaction (-)palmitoylcarnitine + CoASH palmitoyl — SCoA + L-carnitine was studied by the exchange assay (Fig. 1). Buformin was found to enhance the incorporation of L-(3H)-carnitine without shifting the equilibrium. One-way reactions offer more detailed information. Figure 2 illustrates the results obtained by analyzing the one-way reaction from right to left. Substrate Incubation time (min) Fig. 1. Effect of buformin on the exchange reaction of carnitine palmitoyltransferase. The incubation mixture contained 240 fiM ( —) palmitoylcarnitine and L-carnitine, 100/rM CoA, 2 mM dithioerythritol, 250 000 cpm DL-(3H)-carnitine, approximately 90 mU enzyme, 0.1 M Tris-HCl (pH 7.5) in a total volume of 1.0 ml at 30 °C. The figure is representative of four experiments, о—о no addition; • 1 mM buformin; x —x 2.5 mM buformin Acia Biochimica ci Biophysica Academiae Scientiarum Hungaricae 12, 1977