Acta Physiologica 26. (1965)
3. szám - BIOCHEMIA - Gánti T.-Fodor J.: Studies on the Kinetics of NAD-decomposition
Biochemia STUDIES ON THE KINETICS OF NAD-DECOMPOSITION By T. Gánti and J. Fodor YEAST FACTORY, BUDAPEST (Received May 18, 1963) An attempt has been made to establish the optimum conditions of nicotine amide adenine dinucleotide (NAD) extraction from yeast, taking into account the kinetics of extraction and decay of the compound. The results supported the findings of Lowry et al. (1961), as regards the rate of decomposition of NAD. The Arrhenius-diagram, plotted on the basis of computed А-values (constant of the decomposition rate of NAD) has a straight line. Gradient of temperature, energy of activation and frequency-factor calculated from the measured data, were within the expected range. The constant of the decomposition rate of NAD in pure aqueous solution showed a deviation from the rate in the extracted yeast. The characteristic constants were: Temperature gradient 2.5/10° C; activation energy 26.98 kcal/moles; frequency factor 0.59 X 1016; optimum temperature 80° C; duration of extraction 5 minutes. The processes recommended for the preparation of nicotine amideadenine dinucleotid (NAD) are based on the extraction of the compound from the cell hy heat treatment. The different processes show considerable divergencies in the degree of temperature, or in the length of time of heat treatment; at 90—92° C for 8 —15 minutes (Le Page 1949); at 90° C for 4 — 7 minutes, and subsequently at 94° C for 1 — 2 minutes (Korhberc and Sprice 1953); at 90° C for 5 minutes (Neilands and Aiceson 1951); at 85° C for 10 minutes (Shin et al. 1960); at 82° C for 10 minutes (Okunuki et al. 1955); at 80° C for 5 minutes (Tadokoro and Takasugi 1939) is the yeast treated. Our own observations, as well as data in the literature (Lowry et al. 1961; Rauen 1956) show that decomposition is not negligible at the temperature of extraction. In view of this, it has been attempted to establish the optimum conditions, taking into account the kinetics of the extraction and decay of NAD. Material and Methods In the experiments, fresh pressed yeast from the 2nd plant of the Yeast Factory, Budapest (Budapesti Élesztőgyár) was used. The NAD used was produced from pressed yeast according to Kornberg and Sprice (1953), with some modifications. The ADH used was a product of REANAL, Budapest. Measurement of TSAD-contenl : 4.4 ml buffer (4.5 g of Na2H2P207. 10 H20 -f- 3 ml alcohol, made up to 100 ml with distilled water; pH 9.3) is added to 0.5 ml of material. The photometer is set to zero at 340 mft in a 1 cm thick cuvette. 0.1 ml crystalline alcohol dehydrogenase-enzym 10 mg/ml is added, then 3 minutes later the extinction of the solution is read. Extinction of 0.01 = 10.0454 /tg NAD.