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

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