with 10 nil. of 0.2 AI NaHC03 and the insoluble material was renioved by centrifuging a t 2,000 X g for 30 inin. Co X was removed from the supernatant by treatment with Dowex-l.x The crude extracts respond to the alumina procedure3 for the removal of significant quantities of bond thioctic acid from the enzymes. Although assayg shows that only about 507c of the thioctic acid is reiiioved by the alumina procedure, treated enzymes possess significantly less acetate activating activity, ;is measured by the hydroxamic acid method,10 than do untreated enzymes (Table I). This indicates t h a t although the cofactor has not been coiripletely removed, the concentration has been decreased to below enzyrnc saturation level. Activity of the acetate-activating system may be restored to alumina treated preparations by the addition of 7.5 M l g . of synthetic thioctic acid." Two fractions of the pigeon liver extracts have been separated by ammonium sulfate precipitation ; the fractions were collected by centrifugation a t 1S,O00 X g for 13 min. Fraction I (0-33y0 saturated) contains only very slight acetate activating activity. Fraction I1 (35-70y0 saturation) contains almost the entire activity contained in the crude extract; but it does not respond to alumina treatment. Removal of thioctic acid by the aluTABLE I Incubation mixture contained, per 1.3 nil. : 100 p M . Xa acetate; 20 units Co A ; 200 p M . T R I S buffer, PH 8.2; 10 p M . glutathione: 2110 phf. hydroxylamine; 10 p M . iYaATP; 10 p M . hfgCl:; 60 p M . S a F ; enzyme solution. Incubated 00 min. at 32". Glutathione, Sa-ATP and hydroxylamine were adjusted to PH 8 with 1 JI T R I S before use. Thioctic acid content of crude untreated material was 20 l f p g . per mg. protein; after alumina treatment the content was 11 h l p g . per mg. Fractions I and I1 contained 17 hlpg. thioctic acid per mg. protein before alumina treatment and 9 hlpg. per mg. after treatment. Alumina treatment of Fraction I1 alone resulted in no change in thioctic acid content. Additions and treatment
e R I . hpdroxamic acid FracFracCrude tion I tion I 1 (0.3 (0 2 (0.3 ml.) ml.) mi.)
Soiie 0.96 0.02 0 . 5 2 .95 .03 .50 7.5 Mpg. thioctic acid Alumina treated .42 .. .53 Alumina treated 7.5 M p g . thioctic acid .80 .. .. 2 p M . arsenite .88 .. .1x Alumina treated 7.5 hlpg. thioctic acid 2 pM. ar.. .. senite .51 Alumina treated 7.5 Mpg. 2 p M . arthioctic acid senite 20 pM. cysteine .49 , , .. Alumina treated 7.5 Mpg. thioctic acid 2 phf. arsenite 20 phf. addnl. glutathione .50 .. .. Alumina treated 7.5 Mpg. 2 p M . arthioctic acid senite 10 p M . BAL .84 .. ..
+
+
+ + + + + + + + + +
mina procedure is achieved upon combination of the two fractions. Heating Fraction I for 10 min. a t 6.5" completely destroys the activity. It thus appears t h a t Fraction I functions by splitting protein-bound thioctic acid from the enzyme ; liberated thioctic acid is then adsorbed and removed by the alumina. As anticipated12with a reaction requiring thioctic acid, the acetate-activating system is inhibited by arsenite, but not by arsenate. The arsenite sensitivity of systems containing alumina-treated enzyme plus added thioctic acid is much greater than is the sensitivity in mixtures with untreated enzymes containing an excess of the cofactor. The inhibition is reversed by BAL, but not by such monothiols as cysteine or glutathione. (12) I C Gunsalus J (10531
+
0.54 .53 .34
.47 .19
41 (buppl I )
113
CARTER PHYSIOLOGY LABORA~ORY USIVERSITYOF TEXAS MEDICALBRANCH GALVESTON, TEXAS GERALDK SEAMAN J A N U A R Y 20, 1954 RECEIVED THE 3-ACETYLPYRIDINE ANALOG OF BPN'
Sir : 3-Acetylpyridine has been reported to produce symptoms of nicotinamide deficiency in animals but is without effect on the growth of microtirganisms.? I t has been found that the DPNase from pig brain will catalyze an exchange between isonicotinic acid hydrazide and the nicotinamide moiety of D P N (I) to form the isonicotinic acid hydrazide analor of DPN.3 We have in a similar manner been able to demonstrate the synthesis of the 3-acetylpyridine analog of D P N (11) by the pig brain DPNase according to the equation 0
I1
formed Fractions I I1 (0.1 mi.) (O2ml.)
Cell C o r n p P h y s i u l
+
fJ-c-cH3
fyL* 0
'
