STUDIES ON FLAVIN ADENINE DINUCLEOTIDE BIOSYNTHESIS USING P32-LABELED FLAVIN MONONUCLEOTIDE

KAZUO HOTTA, ROKURO KATSUNUMA, KAZUO WATARAI, TOSIE UCHIDA, EIJI ISHIKAWA, JUNZI ITAYA AND NOBUHIKO KATSUNUMA

Department of Biochemistry, School of Medicine, Nagoya University, Showa-ku, Nagoya, and National Sanatorium Obuso, Obu

(Received December 16, 1955)

The formation of flavin adenine dinucleotide (FAD) by the enzymes of the brewer's yeast and liver has been reported using flavin mononucleotide

(FMN) and adenosine triphosphate (ATP) as substrates. Detailed reports of the enzyme itself have been presented by Trufanov (1), Kornberg (2) and Kearney (3). This paper presents the results, in confirmation with previous workers, ascertaining the reaction mechanism of the synthesis by the use of radioactive phosphoric acid. It has been presumed by previous investigators a following formula, FMN+ATP_??_FAD+Pyrophosphoric acid. If so, the reaction, P32-FMN being used as a substrate, is expected to be: Riboflavin-P32+Adenosine-P•`P•`P_??_Riboflavin-P32-P-adenosine+P•`P. The amount of P32 per mole of P32-FMN should be epual to P32 per mole of P32-FAD. The experiment has proved to be the case.

EXPERIMENTAL

Materials 1. Preparation of P32-FMN . 0.1mc of P32 as KH2PO4 was added to 1ml of FMN (10mg), adjusted to pH 7.2, and injected via the ear vein of the rabbit. Twenty hours later the animal was bled to death by severing the carotid artery. The riboflavin portion from the liver was concentrated by Crammer's method (4). It was hydrolyzed with 0.1N HCl for 15 minutes at 100•‹. By this method, the majority was found to have been converted into FMN. This mixed solution containing free riboflavin (FR), FMN and FAD was adjusted to pH 8.0-9.0, and immediately passed through a 100-200 mesh column of ion exchange resin, IRA-400 (Cl form). By washing with distilled water, pH 7.0, ER was eluted. Afer fluorescent substances disappeared from the elutate, 0.1 N HCl was passed through, whereby, as indicated in Fig. 1, FMN was

57 58 HOTTA, KATSUNUMA ET AL 1956 obtained. After FMN was entirely removed, a small quantity of undecom posed FAD was eluted by the addition of 1/5N HCl. By concentration of the FMN portion, P32-labeled FMN was obtained. In most cases the count of P32 in this P32-FMN was too strong in proportion to the amount of FMN, so that non-labeled FMN was added to bring the P32 count to a suitable strength, and this was used as a substrate. The fractionation of the ribofla vin compounds with ion exchange resin was carried out by the method of Hotta et al. (5). 2. Sodium Salt of ATP. A product of Zellstoff-Fabrik Waldhorf was used and dissolved in an isotonic phosphate buffer. 3. Enzyme Solution. A homogenate of rat's liver was centrifuged, and the supernatant was used. In order to bring the end concentration of the enzyme to 8mg N per 100ml, it was diluted with the isotonic phosphate buffer.

Methods 1. Condition of Reaction. P32-FMN, 16.2•~10-6M (590cpm/ml) or 9.2•~10-6M (427cpm/ml) 1ml; ATP-Na, 10•~10-4M (Control: without ATP) 1ml; MgCl2, 2.5M 1ml, en zyme solution 1ml, total volume 4ml, pH 7.4. It was incubated at 35•‹ for 30 minutes. 2. Stop of Reaction. The pH was at first brought to about 6.0, and the supernatant was sepa rated by centrifugation after heating for 5 minutes at 90•‹. 3. Determination of the P32-FAD Produced. According to the method previously described (5) for the preparation of P32-FMN, both FR and FMN were sufficiently removed by ion exchange resin; and P32-FAD was quantitatively separated, concentrated, and FAD was determined by measuring the optical density at 450mƒÊ. At the same time P32 was determined by counting. Corrections were always made for natural counts.

Results

The control without the addition of ATP produced practically no FAD, the amount corresponding to that contained in the enzyme. An important

point worthy of note was the fact that the FAD contained no P32 at all.

FIG. 1

Determination of FMN and FAD by Ion Exchange Resin.

The concentrate obtained by Crammer's method was hydro lyzed with 0.1N HCl at 100•‹ for 15 minutes, and subjected to ion exchange fractionation. Vol. 2 FLAVIN ADENINE DINUCLEOTIDE 59

TABLE ‡T Relation of FAD Produced to Amount of P32 Contained.

TABLE ‡U Relation of P32 per Mole of FMN Used to P32 per Mole of FAD Produced.

SUMMARY

The amount of P32 in FMN used as a substrate and that in FAD obtained enzymatically were proved to be equal. In other words, the phosphoric acid of FMN was incorporated in toto in FAD, according to the formula previously indicated. The determination of pyrophosphoric acid was fairly difficult; Schrecker and Kornberg (6), in the biosynthesis of diphosphopyridine nucleo tide, have demonstrated the formation of the nucleotide and pyrophosphoric acid by directly determining both substances from nicotinamide mononucleo tide and ATP. As for FAD, it has not been possible so far, to prove directly the reaction mechanism by determining pyrophosphoric acid. With the use of P32, it was possible to contribute to an analysis of this reaction mechanism.

REFERENCES

1. Trufanov, A. V., Biokhimiya 7, 188 (1942). 2. Kornberg, A., J. Biol. Chem. 182, 779 (1950). 3. Kearney, E. B., ibid. 194, 747 (1952). 4. Crammer, J. L., Nature 161, 349 (1948). 5. Hotta, K., Ishiguro, I., and Tomoda, M., Vitamins 8, 269 (1955). 6. Schrecker. A. W., and Kornberg, A., J. Biol. Chem. 182, 795 (1950).