628 PRELIMINARY NOTES

metabolism in the lens has thus been demonstrated. These results, i f confirmed, would indicate a stimulatory effect of parathyroid hormone upon glucose utilization, as proposed previously z.

This investigation was supported in part by grant B-3oI6 from the National Institute of Neurological Diseases and Bhndness, U.S. Public Health Service. The author would like to express his appreciation to Dr. G. W. IRwin and the Eli Lilly Company for a generous gift of parathyroid extract which was used in these studies.

Department of Ophthalmology, H . E . FIRSCHEI~ New York University School of Medicine, New York, N.Y. (U.S.A.)

x H. E. FIRSCHEIN, W. F. NEUMAN, G. R. MARTIN AND B. J. MULRYAN, Recent Progr. in Hormone Research, 15 (1959) 427 . H. E. FIRSCHEIN, Trans. N .Y . Acad. Sci., in t he press.

a H. E. FIRSCHEIN, Conf. on Connective Tissue, W a y n e Sta te Univ. , Detroi t , in the press. 4 D. B. KIRBY, Trans. Am. Acad. ophthalmol., (193 o) 154. 5 C. S. O'BRIEN, Arch. ophthalmol., 7 (1932) 71. 6 L. JACQUES, Am. J. med. Sci., 175 (1928) 185. "l L. R. DRAGSTEDT, A. C. SUDAN, AND K. PHILLII~S, Am. J. Physiol., 69 (1924) 477. 8 E. I. EVANS, AND R. KERN, Am. J. ophthalmol., 14 (1931) lO29. 9 S. E. BROLIN, Acta Ophthalmol., 31 (1953) 485 .

10 j . NORDMANN, P. MANDEL, AND M. ACHARD, Brit. J. Ophthalmol., 38 (1954) 673. 11 W. W. UMBRI~IT, 1{. H. BURRIS, AND J. F. STAUFFER, in Manometric Techniques, 3rd ed., Burgess

Pub. Co., Minneapolis, 1957, p. 149. 12 G. GUIDOTTI, J. P. COLOMBO, AND P. P. FOA, Anal. Chem., 33 (1961) 15I. 13 S. B. BARKER, AND W. H. SUMMERSON, J. Biol. Chem., 138 (I94 I) 535.

Received February 9th, 1962 Biochim. Biophys. Acta, 58 (1962) 626--628

Reversal by pyruvate of fluoride inhibition in Aspergillus terreus

The Embden-Meyerhof reactions appear to represent the major pathway for the dissimilation of glucose in Aspergillus terreus 1. In strains producing itaconic acid, fluoride has been shown to inhibit the utilisation of glucose as well as the formation of itaconic acid 1, ~. In this communication we describe some unusual phenomena as- sociated with fluoride inhibition in one such strain, Asp. terreus NRRL 265.

The mold was grown in surface culture at 320 on 5o ml of a medium (pH 3) containing cane sugar (approx. 15 %), NH4NO3 (0.2 %), MgSO 4. 7H~O (o.I %), and KHzPO4 (o.o2 %), in 25o-ml conical flasks. After 6-8 days, the sugar-depleted medium was replaced by 50 ml of fresh medium to which NaF and sodium pyruvate had beet~ added ir~ the required concentration. After 8 days, the mats from 2 identically treated flasks were analysed for their content of itaconic acid and of reducing sugar b y ti~e methods of FR~EDKIN s and NATELSON et a~. 4, respectively.

At a coneemtrati~a of 0.4 mM, fluoride had very little inhibitory effect on the utilisation of sugar b y the mold, although the formation of itaconic acid was inhibited b y over 9 ° %. As the concentration of fluoride was increased, sugar utihsation was ~ e s s i v e l y inhibited; 4 mM fluoride inhibited almost completely the utilisation of sugar as well as the formation of itaconic acid, as reported for Asp. terreus NRRL 196o ~y BENTLEY AND THIESSEN 1.

Biochim. Bio~hys. Acta, 58 (1962) 628-630

PRELIMINARY NOTES 629

Pyruva t e reversed the inhibi t ion of sugar ut i l isat ion as well as of i taconic acid formation by fluoride. The results of two typical experiments, in which the effect of the addi t ion of various concentra t ions of pyruva te to cultures of the mold inhibi ted by fluoride at several concentrat ions was studied, are given in Table I. The a m o u n t of pyruva te required to reverse the inhibi t ion by fluoride of sugar ut i l isat ion and of i taconic acid formation was found to depend on the concentra t ion of fluoride in the culture. The reversal of the inhibi t ion of sugar ut i l isat ion did not, however, go hand

TABLE I

EFFECT OF PYRUVATE ON F L U O R I D E - I N H I B I T E D CULTURES OF AN

ITACONIC ACID-PRODUCING STRAIN OF .ASp. te~'~'e~A$

For composition of the basal medium, see text. The initial sugar concentration in the 2 experiments was 14. 9 % and 15. 7 %, respectively. All the values in the last 2 columns for each experiment,

are for 2 flasks, each containing 5 ° ml of the medium, pooled together.

Expt. x Expt. 2

Additions to basal medium

Fluoride Pyruvate (raM) (raM)

Sugar Itaconic acid Additions to basal medium Sugar Itaco~ic acid consumed formed Fluoride Pyruvat~ consumed formed

(g) (g) (raM) (raM) (g) (g)

o o 12.1 4.5

2 O 0 . 2 0 . 2

2 45 9.7 i . i 2 91 12.2 3.1 2 136 13-6 4.4 2 182 14.8 5.4 2 273 14. 9 6.2

o o 13. 5 5-5

I O 2. 9 0 , 2

I 91 15.7 5,o I 182 15. 7 5.8

1.33 o 2.1 0.2 1.33 91 14.5 4.2 1.33 182 15. 7 5.5

2 o 1.4 o.2 2 91 14.5 3.7 2 182 15. 5 5.4

4 o 1.6 0.2 4 91 12.o I. 5 4 182 13. 3 2.9

IO O 0 . 9 0 . 2

IO 91 8.o o.6 IO 182 12. 3 I.I

in hand with the reversal of the inhibi t ion of i taconic acid format ion; more pyruva te was required for complete reversal in the la t ter case than in the former, at all the fluoride concentra t ions tried (I-IO raM). For example, in the presence of 2 m M fluoride, in a typical experiment (Table I, Expt . I), 45 m M pyruva te reversed the inhibi t ion of sugar ut i l isat ion and of i taconic acid formation by 78 and 25 %, re- spectively; with 91 m M pyruvate , the reversals were Ioo and 69 %, respectively. 136 m M pyruva te completely reversed the inhibi t ion of i taconic acid formation, and effected the ut i l isat ion of 12 % more sugar t han in the control lacking fluoride and pyruvate . In the presence of o.273 M pyruvate , the amoun t of sugar util ised and the total yield of i taconic acid were, respectively, 23 and 38 % in excess of thos, obta ined in the control.

Biochim. Biophys. Acta, 58 (1962) 628-63 °

6~0 PRELIMINARY NOTES

The above observations on the inhibition of sugar utilisation and of itaconic acid formation by fluoride, and on the reversal of these inhibitions by pyruvate, suggest that fluoride has two sites of action in itaconic acid-producing strains of Asp. terreus, One of these sites must be a step in the normal glycolytic scheme, presumably that catalysed by enolase [D-2-phosphoglycerate hydro-lyase (phosphopyruvate hydra- tase), EC 4.2.1.11] which has been reported to be the enzyme most susceptible to fluoride out of all the enzymes so far tested. The other site of action would appear to be some step in the conversion of pyruvate to itaconic acid. Since this step is inhibited by concentrations of fluoride which do not appreciably inhibit glycolysis, and since the block in the biosynthesis of itaconic acid remains even after the inhi- bition of sugar utilisation is completely reversed, it would appear that enohse is not the enzyme which is most susceptible to fluoride in itaconic acid-producing strains of Asp. terreus. The nature of the "second" site of fluoride action in this mold is not yet clear, and is under investigation. Since alI the enzymes involved in the con- version of pyruvate to c/s-aconitate via acetyl-coenzyme A and citrate are known to be less susceptible to fluoride than is enolase, and since cis-~comtate decarboxylase (EC 4.1.1.6) 1 is unlikely to be mote suscept~le to fluoride tharr is enolase, itaconic acid would appear to be formed in Asp. tz~eus from pyruvate ~. without the inter- mediate formation of ace~yl-coenzyme A and/or citrate. As the work of BENTLEY AND THIESSEN 1 leaves no doubt that c/s-ac~mitate is the immediate precursor of itaconic acid, our observati~ts impl3~that in A ~ . temeus, cis-aconitate can b~ formed. from pyruvate by a pathwa~ other t~mn the umial one involving acetyl-coermyme & and citrate.

The complete reversal b y ]~rruvatm of fluolfiile inlu'bition does not appeaar to have, been reported earlier, and ii~ mechamism is not yet understoocL Pyruvate ma~ mediate in the conversion of fffw~ide t ~ a n inactive derivative. For ¢~tample, pyruvai~e could be first metabolised thremgh the~mormal oxidative patltway (~particul~rly if tlm itaconic acid pathway is blocimd), ano~ the ATI~ formed during tl~e oxidation couP! react with fluoride in the presence of t ~ pyruvate kinase (EE 2.7=x.4o) of the moki s, to give FH~POa. The fluoroplmsphate thus formed may hOlt be am inhibitmr of glyco- lysis. This possibility is being currently investigated. Although pyruvate~ is known to inhibit the fluorokinase activity o~ pyruva~e kinase, tiffs inMbition occurs oaly at high concentrations of pyruvate {for a 4~z% inhibition, o~o34 M pyruvate is requited in a purified systemS).

We are very grateful to Dr. S. HUSAIN 7~W~_R for his encouragemeat.

R~mmd R~utrd~ Laboratory, Hydembad (I~Zi4) M o ~ LAL P. M. BHXSGAVX

R. ~ m m ~ t - ~ - ~ e . T m m m m , J . Biot. C/u~., 226 (x957) 673, 689, 7o3. t p_ ms. B~kw~UtVA. K. RAMAC~ANDRXN. S. RAo, M. Lax. xm~ S. H. ZAHEmt, in O. Hc~x~m~t-

O s a m ~ m ~ Pm¢. IV. I ~ . Coa~. Biochem., V i~ t ta , I958 , Pergamon Press, Inc., London,

s ] K . ~ I a ~ L E a t g . Chem., x7 (x945) 637. * S ~ ~ J. ]3. PINcu$ A ~ J. K. LUGOVOY0 J . B/o/. Chem., I75 (x948) 745. • A. Tnrrf f i -m~ S~ ~ . Am~h. Biochem. B io~ys . , 78 (x958) 477.

R e c e i ~ ' ~ ~ . x#2

Biochim. Biophys. Acta, 58 (x962) 628-630