THE JOURNALOF VITAMINOLOGY5, 261-268 (1959)
INFLUENCE OF SUGARS, VITAMIN B6 AND VITAMIN K ON THE PHOSPHORYLATION PROCESS OF RIBOFLAVIN
SHIN WATANABE
National Sanatorium, Toneyama Hospital, Toyonaka, Osaka
(Received June 23, 1959)
Studies on the effects of various vitamins, such as thiamine , nicotinic acid, folic acid, vitamin B6 and vitamin K, on the phosphorylation process of ri boflavin which has been reported in the previous report (1), were carried out . Of the vitamins tested, pyridoxal and vitamin K have been found to be most effective in the phosphorylation of riboflavin. Further, pyridoxal has been shown to activate the process of riboflavin→flavinyl-glucoside→flavin-adenine dinucleatide (FAD), in the course of the phasphorylation process of riboflavin , whereas vitamin K to activate the process of riboflavin→flavin mononucleo tide (FMN)→FAD.
EXPERIMENTAL
The riboflavin contained in the whole bodies of mice was determined just in the same manner as described in the previous report (1). The vitamin K used was K4, i.e., 2-methyl-1, 4-naphthohydroquinone.
RESULTS
1. Effect of Sugars on the Formation of Flavinyl-Glucoside
TABLE Ⅰ Effects of Disaccharides Amount of riboflavin loaded: 40μg/g
261 262 WATANABE 1959
The amount of riboflavin was measured immediately after riboflavin was loaded together with maltose, sucrose and lactose, respectively. In the case of maltose alone, the more flavinyl-glucoside was produced, the more FAD was formed. Maltose is therefore assumed to take some part in the forma tion of flavinyl-glucoside (Table Ⅰ). The amounts of flavinyl-glucoside and FAD produced were maximum, when the amount of maltose was five times as large as riboflavin (40μg/g) on a molar basis. For comparing other sugars with maltose, effects of equi molar amounts of glucose, glucose-1-phosphate, glucose-6-phosphate, fructose and galactose were tested. In all the cases, the amount of flavinyl-glucoside obtained was smaller than in the case of maltose (Table Ⅱ).
TABLE Ⅱ Effects of Monosaccharides
2. Effect of Pyridoxal on the Phosphorylation of Riboflavin
When the amount of riboflavin was measured immediately after 40μg/g of riboflavin and pyridoxal were simultaneously loaded, the amounts of flavin yl-glucoside and of FAD produced were increased, with the rise of the dose of pyridoxal (Table Ⅲ).
TABLE Ⅲ Effect of Pyridoxal on the Phosphorylation of Riboflavin
The figures indicate μg/g. Also the same in the following Tables.
Observations made immediately after, and one hour after, administering riboflavin alone or riboflavin plus pyridoxal revealed that percentage forma tion of flavinyl-glucoside to the total riboflavin loaded was higher, when pyridoxal was loaded (Table Ⅳ). Pyridoxal seems to increase the formation of flavinyl-glucoside. Vol. 5 PHOSPHORYLATION OF B2 263
TABLE Ⅳ Effect of Pyridoxal One Hour after Loading with Riboflavin
3. Effect of ATP After loading with riboflavin and pyridoxal together with adenosine tri phosphate (ATP), the amounts of flavinyl-glucoside and of FAD were increased in the presence of ATP immediately after the loading. In the absence of pyridoxal, less amount of flavinyl-glucoside was produced. After loading with adenosine-5-phosphate (AMP) in place of ATP, the amount of FAD form ed was decreased, without any distinct change in flavinyl-glucoside. From these findings, ATP is assumed to take some part in the formation of flavinyl glucoside (Table Ⅴ).
TABLE Ⅴ Effect of ATP
The amounts loaded: riboflavin, 40; pyridoxal, 180; ATP, 50; AMP, 35μg/g
4. Effect of Pyridoxal Loaded Simultaneously with Each Type of Riboflavin Each type of riboflavin was determined immediately after each type of riboflavin and pyridoxal were simultaneously loaded. For the control each type of riboflavin alone was loaded. When FAD was simultaneously loaded with pyridoxal, the amount of flavinyl-glucoside produced was larger and that of FAD smaller than the controls. After loading with FMN together with pyridoxal, a small amount of flavinyl-glucoside was detected (Table Ⅵ). From these findings, pyridoxal is considered to be involved in the formation of flavinyl-glucoside. 5. Effects of Glutamate and Alanine When both riboflavin and pyridoxal were loaded together with glutamic 264 WATANABE 1959
TABLE Ⅵ Effect of Pyridoxal Lauded with Each Type of Riboflavin
The amounts loaded: riboflavin, 40μg/g; FMN and FAD, equimoles of riboflavin; pyridoxal, 180μg/g (10 moles of riboflavin).
acid or alanine, the ratio of flavinyl-glucoside to total riboflavin was rather reduced, whereas the ratio of FMN plus FAD to total riboflavin was elevated
(Table Ⅶ). Therefore, these amino acids seem not to be participated with the production of flavinyl-glucoside by the action of pyridoxal.
TABLE Ⅶ Effects of Glutamic Acid and Alanine
The mounts loaded: riboflavin, 40; pyridoxal, 180μg/g.
6. Effect of Vitamin K on the Phosphorylation of Riboflavin When both riboflavin and vitamin K were loaded, the ratio of esterified
TABLE Ⅷ Effect of Vitamin K on the Phosphorylation of Riboflavin Vol. 5 PHOSPHORYLATION OF B2 265 riboflavin produced to the total riboflavin was elevated, with the increase of the amount of vitamin K administered, suggesting the participation of vita min K with phosphorylation of riboflavin (Table Ⅷ). 7. Effect of ATP When both riboflavin and vitamin K were loaded together with ATP, and the amount of riboflavin was measured immediately after loading, scarcely any effect was recognized, except for the decrease in FAD when 10 times as large amount of ATP as riboflavin on a molar basis, was given to the subjects (Table Ⅸ). Vitamin K is considered therefore to promote the phos phorylation of riboflavin, regardless of the presence of ATP.
TABLE Ⅸ Effect of ATP
The amounts loaded; riboflavin, 40; vitamin K, 310μg/g .
8. Effect of Vitamin K Loaded Together with Each Type of Riboflavin When riboflavin was determined immediately after loading with both each type of riboflavin and vitamin K, values for FMN and FAD were lower than those without vitamin K. When FMN was loaded together with vitamin K, the amount of FAD was much larger than those without vitamin K , no riboflavin being produced in this case. When, however, FAD was loaded together with vitamin K, most of it turned into FMN, and FAD itself was far smaller than those without vitamin K (Table Ⅹ). From these findings ,
TABLK Ⅹ Effect of Vitamin K Simultaneously Loaded with Each Type of Riboflavin
The amounts loaded: riboflavin, 40μg/g; FMN, FAD, equimoles of riboflavin; vitamin K, 310μg/g (10 moles of riboflavin). 266 WATANABE 1959 vitamin K seems to take some part in controling the equilibrium between FMN and FAD. 9. Effects of Succinate, Fumarate and Ascorbic Acid When small amount of succinate, fumarate, or ascorbic acid was loaded together with riboflavin and vitamin K, the amounts of flavinyl-glucoside, FMN and FAD were increased (Table Ⅹ Ⅰ, Ⅹ Ⅱ).
TABLE Ⅹ Ⅰ Effects of Succinate and Fumarate
The amounts loaded: riboflavin, 40; vitamin K, 310μg/g.
TABLE Ⅹ Ⅱ Effect of Ascorbic Acid
The amounts loaded: riboflavin, 40; vitamin K, 310μg/g.
TABLE ⅩⅢ Effect of Pyridoxal Simultaneously Loaded with Riboflavin and Vitamin K
The amounts loaded: riboflavin, 40; vitamin K, 310μg/g. Vol. 5 PIIOSPHORVLATION OF B2 267
10. Effect of Pyridoxal in Case of Loading with Both Riboflavin and Vitamin K When riboflavin, vitamin K and pyridoxal were simultaneously loaded, the amount of FAD was decreased, with the increase of pyridoxal. When ten times as great molar amount of pyridoxal as riboflavin was used, only traces of FMN and FAD were detected (Table Ⅹ Ⅲ).
DISCUSSION
In the previous experiment, the phosphorylation process of riboflavin was studied, and in the present experiment, investigations were carried out on the effects of sugars and vitamins on the phosphorylation of riboflavin. At first, the effects of various sugars were tested. The amounts of both flavinyl-glucoside and FAD were found to be increased, accompanied with the rise of that of maltose, and from this, flavinyl-glucoside seems to be produced from riboflavin and maltose. Since the production of flavinyl-glucoside and of FAD was increased with the rise of the amount of pyridoxal loaded together with riboflavin, it is concluded that pyridoxal promotes the formation of flavinyl-glucoside. How ever, the amount of FAD was small, when ten times as great molar amount of pyridoxal as riboflavin was used, possibly because of the lack of ATP indis pensable for the formation of FAD. The effect of ATP was therefore stud ied, and the participation of ATP in the formation of flavinyl-glucoside was again ascertained. When pyridoxal was loaded together with FAD, little FAD, but much flavinyl-glucoside were found. The glucoside was not found in the control. It was thus assumed that FAD was degraded into riboflavin and adenosine diphosphate (ADP) and at the same time pyridoxal is combined with ADP forming pyridoxal-adenine dinucleotide (PAD), which serves as a coenzyme for flavinyl glucoside-forming enzyme. The reason why the amount of FAD was smaller than the control, when pyridoxal was loaded together with ri boflavin seems to be as follows. For utilizing a great amount of riboflavin to produce flavinyl-glucoside, PAD is needed and for producing PAD, degra dation of FAD is indispensable. As can be seen in Tabie Ⅵ, the amount of flavinyl-glucoside is increased by the administration of ATP, contrary to AMP. It can easily be explained if PAD is taken into consideration, but PAD itself has not been confirmed. Further, the formation of flavinyl-glu coside is no increased after loading with glutamic acid or alanine together with riboflavin and pyridoxal. Therefore, the effect of pyridoxal phosphor ylation coupled with amino acid metabolism can possibly be excluded. When both riboflavin and vitamin K were loaded at the same time, the decrease of flavinyl-glucoside and the increase of FMN and FAD were ob served, with the rise of the amount of vitamin K administered. Assuming the participation of ATP in the reaction, the effects of various amounts of ATP was examined, and it was found that the formation of flavinyl-glucoside was unchanged, while the amounts of FMN and FAD were reduced. From these findings, it is suggested that ATP has nothing directly to do with the 268 WATANABE 1959 action of vitamin K, though it is necessary for phosphorylation. In case of loading with each type of riboflavin together with vitamin K, the amount of FAD became larger in comparison with the control value, and no riboflavin was detected when FMN was loaded, whereas most of the loaded vitamin was converted into FMN, when FAD was loaded. These findings suggest that vitamin K promotes the degradation and synthesis of FAD, and inhibit the degradation of FMN to regulate the quantitative equilibrium of FAD and FMN. Furthermore, when small amount of succinate, fumarate and ascorbic acid are loaded together with riboflavin and vitamin K, the rise of the ester ified riboflavin over the control was recognized, suggesting some relationship of these compounds with respiratory systems. In short, pyridoxal is considered to influence the phosphorylation of ribo flavin by coupling with carbohydrate metabolism, possibly in the active form of PAD. Further, vitamin K seems to influence the phosphorylation of riboflavin by coupling with respiratory systems. Pyridoxal and vitamin K have a contrasting action toward the phosphorylation of riboflavin, i.e., the former activates the system: riboflavin→flavinyl-glucoside→FAD, whereas the latter activates the system: riboflavin→FMN→FAD.
SUMMARY
1. When maltose was loaded together with riboflavin, flavinyl-glucoside was produced, accompanied with the rise of FAD production. From this, it is suggested that the flavinyl-glucoside of animal tissues is produced from ribo flavin and maltose. 2. Contrasting actions of pyridoxal and vitamin K toward the phosphoryla tion of riboflavin were ascertained, i.e., the former activates the system; riboflavin→flavinyl-glucoside→FAD, and the latter activates the system, riboflavin→FMN_??_FAD.
ACKNOWLEDGMENT
The author wishes to thank Dr. Saburo Watanabe, President of Toneyama Hospital, who had offered his kind guidances in support of this research.
REFERENCE
1. Watanabe, S., J. Vitaminol. 5, 254 (1959).