Requirement of pyridoxal in the algao
1029
for pyridoxol. H e n c e , the v i t a m i n B6 r e q u i r e m e n t a p p e a r s to be more b a s i c and p r o b a b l y
o c c u r r e d at an earlier p o i n t in the evolution of t h e s e catalytic proteins than that for A M P .
It is of interest, too, that the single p h o s p h o r y l a s e p r e s e n t in Cyanidiurn caldarium, a2,
a l t h o u g h not sensitive to A M P , nonetheless exhibited the same r e q u i r e m e n t for the v i t a m i n
as the i s o z y m e s p r e s e n t in Oscillatoria princeps and Chorella pyrenoidosa.
C h e l a t i o n of the essential m a n g a n e s e ion per se does not a p p e a r to play a m a j o r role as
witnessed by the inactivity of the a n a l o g o u s c o m p o u n d , NSC-1942 in the biosynthesis of
active p h o s p h o r y l a s e proteins.
E X P E R I M E N T A L
The algae, Oscillatoria princeps, Chlorellapyrenoidosa and Cyanidium caldarium were cultured in meaia
previously described. 24 Extracts were prepared and fractionated with ammonium sulfate. The phosphorylases
were separated on 8 % polyacrylamide gels in art E-C No. 470 Vertical Cell and protein staining and a histo-
chemical method for the visualization of phosphorylase applied.24,25 All techniques have been described.2.
NSC-68955 was dissolved in the culture media to give final concentrations of 10 -2, 10 -a and 10 -4 M.
When vitamin Be (pyridoxine hydrochloride, Lilly) was used, it was added to the media in the same concen-
trations. Those cultures to which NSC-1942 was added, did not contain the vitamin. The 5-hydroxy-2-
(hydroxymethyl)-4H-pyrone (kojic acid, Matheson) was re~rystallized twice before use and added in the same
concentrations as NSC-68955.
Pyridoxal phosphate (Sigma) was added directly to the algal extracts and incubated for 1 hr at 30° at a
concentration of 0"1 M.
Preparation of NSC-68955
60 g of kojic acid (Matheson) were heated in a pressure flask with 100 ml cone. NI-I,OI-I (d = 0-88) in a
boiling water bath for 4 hr. The brown mixture was steamed to drive offexcess ammonia and the residue was
extracted several times on a boiling water bath with a total of 1 I. of methanol. The extracts were com-
bined and treated with activated carbon (Darco). After 1 hr, the extract was filtered through iron-free paper
and the filtrate evaporated to one-third its original volume on a water bath. The crystals were redissolved in
methanol and r~rystallized three times. They were dried over silica gel. The pale brown crystals showed a
m.p. of 237-238°.
Acknowledgements--This work was supported by a research grant from the George B. Dodge Cancer Mem-
orial Fund of the Dodge Institute for Advanced Studios. We are indebted to the National Cancer Institute
of the National Institutes of Health for the screening of NSC-68955 and the determination of its activity
against lymphoid leukemia, L-1210.
2,, j. F. FREDRICK, Phytochem. in press (1971).
25 C. H. DAVIS, L. H. SCHLISELFELD, D. P. WOLF, C. A. LEAVIT'F and E. G. K ~ a s , J. Biol. Chem. 242, 4824
(1967).
Fredrick et al.
