Inhibition of yeast growth by fluoride and its reversal by phosphoric esters: an effect of pH

482

SHORT COMMUNICATIONS

A crystalline sample of 3'-deoxyadenosine was kindly provided b y Dr. L. GOOI)MAN, Stanford Research Institute. Dr. D, C. DEJONGH would like to thank Parke, Davis and Co. for a research grant for partial support of this investigation.

Parke, Davis and Company, Research Laboratories, Ann Arbor, Mich. (U.S.A.) Wayne State University, Department of Chemistry, Detroit, Mich. (U.S.A.) Baylor University College of Medicine Department of Biochemistry, Houston, Texas (U.S.A.) I

2 3 4 5 6 7 8 9 Io ii 12 13 14

STEPHEN HANESSIAN

DON C. DEJON6H JAMES A. McCLOSKEY

K. G. CUNNINGHAM, S. A. HUTCHINSON, W. MANSON AND F. S. SPRING, J. Chem. 5"oe., (1951) 2299. H. R. BENTLEY, K. G. CUNNINGHAM AND F. S. SPRING, J. Chem. Soe., (1951) 23Ol. R. A. RAPHAEL AND C. M. ROXBURGH, J. Chem. Soc., (1955) 3405 • S. HANESSIAN, Advan. Carbohydrate Chem., 21 (1966) in the press. P. W. KENT, M. STACEY AND L. F. WIGGINS, .]. Chem. Soe., (1949) 1232. S. MUKHERJEE AND A. R. TODD, dr. Chem. Sot., (1947) 969 . E. A. KACZKA, E. L. DULANEY, C. O. GITTERMAN, H. n. WOODRUFF AND K. FOLKERS, Biochem. Biophys. Res. Commun., 14 (1964) 452. E. A. KACZKA, N. R. TRENNER, B. ARISON, R. \V. WALKER AND K. FOLKERS, Bioehem. Biophys. Res. Commun., 14 (1964) 456. W. W. LEE, A. BENITEZ, C. D. ANDERSON, L. GOODMAN AND B. R. BAKER, ]. Am. Chem. Soc., 83 (196I) 19o6. V. NHAFIZADEH,Advan. Carbohydrate Chem., i1 (1956)263. H. R. BENTLEY, Methods Carbohydrate Chem., I (1962) 266. R. K. HULYALKAR, J. K. N. JONES AND M. B. PERRY, Can. J. Chem., 43 (1965) 2085. K. BIEMANN AND J. A. MCCLOSKEY, J. Am. Chem. Soe., 84 (1962) 2005. J. A. MCCLOSKEY, P h . D . Thesis, M a s s a c h u s e t t s I n s t i t u t e of Technology, 1963.

Received December 27th, 1965 Biochim. Biophys. A eta, 117 (t966) 480-482

BBA 23 206

Inhibition of yeast growth by fluoride and its reversal by phosphoric esters: an effect of pH Several years ago NICKERSON AND CHUNG 1 reported that glucose I-phosphate and mannose I-phosphate, but not glucose 6-phosphate, were able to revert the inhibition of yeast growth by fluoride 1. In consequence, they advanced the hypothesis that the phosphoglucomutase reaction was the site of fluoride inhibition and that sugar 1-phosphates could enter the cell and give rise to the cell-wall polysaccharides, thus obviating the need for the blocked step. Since, apparently, both glucose and mannose 1-phosphate were equally efficient as precursors of glucan and mannan 2, an interconversion between the two e s t e r s - - o r derivatives t h e r e o f - - w a s postulated. The subject has been reviewed by TREVELYANa. The interesting possibility of such an interconversion led us to reinvestigate this problem. Unexpectedly, however, our findings point to a different interpretation of the results of !NTICKERSONAND CHUNG1. Biochim. Biophys. Aeta, i 17 (1966) 482-485

483

SHORT COMMUNICATIONS

The yeast strains used in this study were Saccharomyces cerevisiae 197 (C~tedra de Microbiologia, Facultad de Agronomla y Veterinaria, Universidad de Buenos Aires) and Saccharomyces carlsbergensis74 S (National Collection of Yeast Cultures, Nutfield, Surrey, England). Both strains gave essentially identical results. The basal growth medium was that described by NICKERSON AND CHUNG1 but with 0.33 M glucose. As shown in Table I, we were able to reproduce the reversion of fluoride inhibition by glucose 1-phosphate, but glucose 6-phosphate was equally effective. In addition, the following esters were tested and found to be active: mannose 1-phosphate, mannose 6-phosphate, galactose 1-phosphate, galactose 6-phosphate, fructose 1-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, 2-phosphogiycerate and a-glycerophosphate. It can also be seen in Table I that a-glycerophosphate was ineffective if added after incubation of the cells with fluoride. TABLE I REVERSION

Expt.

OF

FLUORIDE

INHIBITION

OF

YEAST

GROWTH

Additions to basal medium

No.

BY

--

O

IO mM K F IO mM K F + IO mM glucose I - p h o s p h a t e IO mM K F + IO mM glucose 6 - p h o s p h a t e

0.002 O.Ol 5 0.02

-IO IO IO io

0.24 0.24 0.24 0.24 o.24

KF K F + IO mM a - g l y c e r o p h o s p h a t e KF c¢-glycerophosphate

PHOSPHATES

Absorbance at 660 m #

t=o

mM mM mM mM

SUGAR

3h

24h 0.430 0.078 0.430 o.441

0.57 0.34 0.50 0.34* 0.46**

1.24 0.36 1.22 0.46 1.18

* A t t h i s t i m e i o mM a - g l y c e r o p h o s p h a t e was added. ** At t h i s t i m e IO mM K F was added.

Proof that glucose 1-phosphate did not act in the manner suggested by CHUNG AND NICKERSON2 was obtained by measuring the incorporation of radioactivity in the cell-wall polysaccharides, when either the glucose 1-phosphate or the glucose of the growth medium was labeled. The results shown in Table II indicate that in every case the precursor of both glucan and mannan was the glucose of the medium. The suspicion then arose that the phosphoric esters might act by changing the final pH, since their solutions had a pH of about 8.3 while the growth medium was at pH 5.2-5.4. In fact, the pH of the medium with added glucose 1-phosphate was found to be 6.2-6. 4. If the same change in pH was brought about by adding inorganic phosphate, rather than a phosphate ester, reversion of the fluoride inhibition took place, as shown in Table III. Conversely, addition of glucose I-phosphate, previously brought to pH 5.2, did not modify the effect of fluoride. This pH effect probably results from the impermeability of the yeast cells to fluoride at the more alkaline pH (ref. 5), as confirmed by the data of Table IV. A similar explanation for the effect of glucose 1-phosphate on fluoride penetration in Acacia roots was independently proposed by PETERS, MURRAY AND SHORTHOUSE7. In conclusion, the reversion of fluoride inhibition by phosphoric esters can be Biochim. Biophys. Acta, 117 (1966) 482-485

484 TABLE

SHORT COMMUNICATIONS II

INCORPORATION OF RADIOACTIVITY IN GLUCAN AND MANNAN A f t e r g r o w t h w i t h t h e a d d i t i o n s i n d i c a t e d below, t h e cells were c e n t r i f u g e d off a n d w a s h e d w i t h w a t e r . G l u c a n a n d m a n n a n were t h e n i s o l a t e d from 5 m g of y e a s t (wet weight) as i n d i c a t e d b y TREVELYAN A N D H A R R I S O N 4, r e s u s p e n d e d in w a t e r a n d t r a n s f e r r e d t o s t a i n l e s s - s t e e l p l a n c h e t s . R a d i o a c t i v i t y w a s c o u n t e d w i t h a w i n d o w - l e s s flow c o u n t e r .

Changes and additions to basal growth medium

Radioactivity incorporated in polysaccharides

0.33 M L14C~glucose (3000 c o u n t s / m i n p e r / , m o l e ) 0.33 M [14C]glucose + i o mM fluoride 0.33 1V[ [14Qglucose + i o mM fluoride + i o mM glucose I - p h o s p h a t e IO mM fluoride + IO mM [14C]glucose 1 - p h o s p h a t e (IOOOO c o u n t s / r a i n p e r t~inole)

TABLE EFFECT

p H ON FLUORIDE

INHIBITION

OF YEAST

918 o

1324 o

890

I962

o

o

GROVCTH

--

mM mM mM mM

Mannan (counts/miu)

III OF

Additions to basal growth medium

IO IO io io

Glucan (eounts/mi~)

p H oJ the complete medium

A bsorbance at 24 h

5-4

0.274 o.oi8 o.133 0.032 0.227

5,4

KF K F + IO mM glucose I - p h o s p h a t e (pH 8.2) K F + i o mM glucose 1 - p h o s p h a t e (pH 5.2) K F + i o mM p o t a s s i u m p h o s p h a t e (pH 8.6)

6.2 5-4 6.2

TABLE IV PENETRATION OF FLUORIDE IN YEAST CELLS UNDER DIFFERENT CONDITIONS Y e a s t cells (13. 7 mg, d r y weight) were s u s p e n d e d in i ml of a m e d i u m t h a t c o n t a i n e d 6 o~/ glucose, o. 3 % K H 2 P O ~, 0.o25 ~o MgSOa' 7 H 2 0 a n d Io mM KF. A f t e r 5o m i n a t 2o ° t h e cells were collected on Millipore m e m b r a n e s (pore o.6/z), a n d w a s h e d w i t h t h e p e n e t r a t i o n m e d i u m m i n u s glucose a n d fluoride. The cells were t h e n e x t r a c t e d for 3 ° m i n a t l o o ° in p o l y p r o p y l e n e t u b e s w i t h 8 ml of w a t e r c o n t a i n i n g one drop of o.5 N N a O H . F l u o r i d e w a s d e t e r m i n e d a ft e r p u r i f i c a t i o n of t h e e x t r a c t w i t h D o w e x - I columns, as d e s c r i b e d b y NIELSEN 6.

Addition to the medium

p H of medium

re#moles of fluoride in cells

--

5.2

--

6. 4

o

IO mM glucose I - p h o s p h a t e

6. 4

o

125

ascribed to a change in the p H of the g r o w t h m e d i u m with the a t t e n d a n t decrease in fluoride p e n e t r a t i o n . The site or sites of fluoride action in the cell still r e m a i n undetermined. T h e a u t h o r s wish to express their g r a t i t u d e to Dr. L u I s F. LELOIR for his cont i n u e d interest a n d s u p p o r t of this work. T h e y are also i n d e b t e d to their colleagues Bioehim. Biophys. Acta, 117 (1966) 482-485

SHORT COMMUNICATIONS

485

at the Instituto de Investigaciones Bioquimicas for useful discussions and criticism. This investigation was supported in part by a research grant (GM-o3 442) from the National Institutes of Health, U.S. Public Health Service; by the Rockefeller Foundation; and by the Consejo Nacional de Investigaciones Cientificas y T@cnicas, Argentina. L. B. R. was a fellow and E. C. a carreer investigator of the Consejo Nacional de Investigaciones Cientificas y T@cnicas (Argentina).

Instituto de Investigaciones, Bioquimicas "Fundaci6n Campomar" and Facultad de Ciencias Exactas y Naturales, Buenos Aires, (Argentina)

LUCIA B . ROTHMAN ENRICO CABIB

I "~V. J. lXTICKERSON AND C. W . CHUNG,Am. J. Botany, 39 (1952) 669. 2 C. W. CHONG AND W. J. NICKERSON, J. Biol. Chem., 2o8 (1954) 395. 3 X¥. E. TREVELYAN,in A. H. COOK, Chemistry and Biology of Yeasts, A c a d e m i c Press, N e w York, 1958, p. 396. 4 W. E. TREVELYAN AND J. S. HARRISON, Biochem. J., 5 ° (1952) 298. 5 M. MALM, Naturwissenschaften, 28 (194 o) 723 . 6 H. M. NIELSEN, Anal. Chem., 3 ° (1958) lOO9. 7 R. A. PETERS, L. R. MURRAY AND M. SHORTHOUSE, Bioehem. J., 95 (1965) 724 •

Received November 29th, 1965 Biochim. Biophys. Acta, 117 (1966) 482-485

BBA 23 213

Amino acid composition of telomycin-producing streptomyces 3-Hydroxyproline, a heretofore unknown naturally occurring amino acid was discovered in collagen z-a and shown to be incorporated in the polypeptide chain as judged by degradation studies with collagenase~. At about the same time the two diastereoisomers of 3-hydroxyproline were isolated from telomycin4, 5, an antibiotic s which contains one residue each of the following amino acids: aspartic acid, serine, threonine, allo-threonine, alanine, glycine, trans-3-hydroxyproline, cis-3-hydroxyproline, erythro-fl-hydroxyleucine, tryptophan and fl-methyltryptophan7. Thus, it was of interest to examine the amino acid composition of the organism that makes telomycin to ascertain if its proteins and cell wall contained 3-hydroxyproline. Accordingly, washed cells of telomycin-producing streptomyces C-5o9 (kindly furnished by Dr. I. R. HOOPER, Bristol Laboratories, Syracuse, New York) were lyophilized. The dried cells were finely ground and extracted with methanol in a Soxhlet apparatus for 24 h to remove any telomycin. The material was dried in a vacuum desiccator over P~05. Total N---- 8.75 %. The mycelium, 147 mg was hydrolyzed in a sealed tube with 20 ml of constant-boiling HC1 for 24 h at IiO °. After filtration and removal of HC1 in vacuo the residue was dissolved in IO.O ml of distilled water. This solution was used for analyses. The cyclic amino acids were determined following the 2,4,6-trinitrobenzene sulfonate method described in the isolation of cisand trans-3-hydroxyprolines 2. An automatic amino acid analyzer (Phoenix Precision Instrument Company) was used for the amino acid analysis. Identification of muramic acid was performed by actual isolation employing a combination of paper and column chromatography. Tert.-amyl alcohol-2,4-1utidine-H~O (178:178:iio, by vol.) was Biochim. Biophys. Acta, 117 (1966) 485-486