CO~IXLTICATIOSS TO THE EDITOR
36U4
Previous work7 demonstrated that following hepatectomy, no DNA is synthesized or labeled by orotic acid for the first 18 hours following the operation. An experiment was therefore designed in which homogenates were prepared from lobes removed at partial hepatectomy, as well as from liver allowed to regenerate 15 hours, and 24 hours and incubated in the presence of radioactive thymidine. The results presented in Table I show the incorporation observed in these preparations,. TABLE 1 INCORPORATION OF THYMIDISE IXTO D S A HOMOCENATES
OF
1
2 I
2 :i
C.p.m./ Homogenate from
Lobes removed a t partial hepatectomy Lobes removed a t partial hepatectomy Liver 15 hours after operatiori Liver 2 1 hours after operatioii Liver 24 hours after operatioii
plate (actual")
D S A a S p . act.mg./ c.p.m./mg. plate DNA
350
0.35
1,000
270
.32
810
360
.30
1,200
12,300
4,iMI
.28 44,000 .%j
cident with an ultraviolet quenching spot, with Rr and spectrum of t h ~ m i n e . ~ (9) Cf.G. R . W y a t t i n E. Chargaff and J. X. Davidson, "The h-ucleic Acids," Vol. I , Academic Press, New York, N . Y., 1955, PP. 243-205. (IO) Postdoctoral Fellow ni t h e Kational Cancer Institute. 'This investigation was also supported b y a grant ( S o . C-646) from t h e National Cancer Institute, National Institutes of Health, Public Health Serrice.
MCARDLEhfEMORIAL I2AB0R.4TOR'2 UNIVERSITYOF \TISCOSSIN MADISOX 6, WISCONSIN RECEIVEDMAY21, 1957
F. J. BULLLJM~" R . POTTER
J-AN
R A T LIVER
Each 50-ml. flask contained: 2 ml. of 2554 r a t liver homogenate; approximately 3 X 106 c.p.m. ( 3 micrograms) of thymidine*: 20 p M . each of K fumarate, K glutamate and I( pyruvate; 5 p M . disodium A T P ; 100 p M . nicotinamide; 12 pM.MgSO,; 12 p M . I; phosphate, pH 7.6; 10 pk1. KCI; 20 p M . Tris:HCI buffer, p H 8; and 500 pkl. of sucrose in a final volume of 2.55 m l . ; incubation, for one hour with shaking, 38', O2 i i i the gas phase. Reaction was stopped b y placing the flasks in ail ice-bath. Sociiurri nucleates extracted and DXA purified as described previously.' A trace of radioactivity associated with RNA4 is attributed t o slight degradation of radioactive D S A during the mild alkaline hydrolysis required t o remove R X A . Rat no.
Vol. 79
FORMATION OF L-XYLULOSE FROM I.-GULONOLACTONE IN RAT KIDNEY
L-Xylulose, a sugar excreted by patients with essential pentosuria, is present normally in urine of man,' guinea pig' and rat.2 Conversion of Dand glucuronolactone to L-xylulose occurs in guinea pig.' L-Gulonic acid, which is formed from D-glucuronolactone in the rat and guinea pig,4 has been postulated to be an intermediate in this reaction.' This communication reports the presence of an enzyme system in rat kidney which catalyzes the formation of L-xylulose from L-gulonolactone. I t has been shown previously that L-gulonolactone is converted to L-ascorbic acid in r a t ~ . j - ~ Carboxyl labeled and uniformly labeled Lgulonolactones were incubated with the soluble fraction of rat kidney (Table I).9 Carboxyl labeled L-gulonolactone yielded 30 to BOY', of the added C14 in COz.'o Uniformly labeled L-gulonolactone yielded about one-sixth the amount of CI4 in CO?.
1(\,90(1'
Tritiurn \vas asstyeti i i i winrtowlcss flow counters. Since approsirnatcly the satnc amount ( i f 1 ) S A was placed on each plate iio correction has been made for self-absorption. Crude self absorption curves for tritium, using DN.4 a s absorber, indicate a correction o f about -'-fold could be apColorimetric analj plied to the D S A saniples. the Dische dipheriylamine reagent. This homogenate aged for 2 hours a t 0" before incubation. All others incubated immediately after preparation.
It is apparent that a low level of incorporation is present in the zero hour and 15 hour regenerated preparations, and that the 24 hour regenerating liver shows about a 50-fold increase (Rat no. 2) in ability to incorporate thymidine into DNA. Thus the results with the homogenates are in agreement with previous studies with orotic acid in vivo' and in rat liver slices8 It is our opionion that the marked incorporation seen in the 24-hour livers and the low incorporation in other samples argues against simple exchange reactions, since in all cases the same amount of tissue was incubated with the labeled precursor under identical conditions. T h e distribution of tritium in the isolated DNA was examined by hydrolysis with 989; formic acid for 2 hours a t 165' and chromatography of the bases in 2-propanol : HCl on S. and S. no. 389 filter paper. The chroinatograrn showed only one radioactive spot, coiw 17) I.. I . lIeclil and V I
