15. 12. 1974

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High Incorporation of Labeled Acetate into Yeast Ergosterol Durin~ Sporulation ~ T h e a v a i l a b i l i t y of ~aC-NMR-spectrometers w i t h fast F o u r i e r t r a n s f o r m accessories h a s g r e a t l y increased t h e p o s s i b i l i t y of o b t a i n i n g d e t a i l e d i n f o r m a t i o n on biosynthetic pathways without extensive and time-consuming c h e m i c a l d e g r a d a t i o n of t h e m e t a b o l i t e of interest. R e c e n t l y BURLINGAME et al. 2 i n c o r p o r a t e d E2-1~Qa c e t a t e into y e a s t p a l m i t o l e i c acid (30% laC e n r i c h m e n t ) d u r i n g aerobic g r o w t h a n d s h o w e d t h e a l t e r n a t e c a r b o n s to be labeled. I n c o n n e c t i o n w i t h our s t u d i e s on t h e b i o s y n t h e s i s of t h e y e a s t sterol, ergosterol, f r o m glycine a n d serine, we h a v e b e e n i n t e r e s t e d in u s i n g s u b s t r a t e s labeled w i t h carbon-13 as well as carbon-14. Carbon-14 has b e e n e m p l o y e d as a tool for e l u c i d a t i n g ergosterol b i o s y n t h e s i s a . H o w e v e r t h e fate of t h e a c e t a t e p r e c u r s o r in t h e sterol requires c h e m i c a l d e g r a d a t i o n such as d o n e b y HANAHAN a n d WAKIL 4 on t h e C2~ t o C~2 ergosterol f r a g m e n t i n d i c a t i n g t h e labeling p a t t e r n in one p a r t of t h e molecule. If ergosterol can be b i o s y n t h e s i z e d f r o m [1 or 2-~sC]-acetate, or [1,2-1aC~-acetate, t h e f a t e of e v e r y a c e t a t e c a r b o n could be l e a r n e d f r o m t h e ~aC-NMRs p e c t r u m since t h e c h e m i c a l shifts for ergosterol c a r b o n s are k n o w n S - L Parallel e x p e r i m e n t s w i t h ~aC-labeled glycine, serine a n d o t h e r s u b s t r a t e s w o u l d t h e n be simplified. C u r r e n t l y 2 t e c h n i q u e s are available for o b t a i n i n g h i g h l y labeled ergosterol f r o m yeast. KLEIN et al. s f o u n d t h a t y e a s t g r o w n u n d e r a n a e r o b i c c o n d i t i o n s will accelerate b i o s y n t h e s i s w h e n a e r a t e d a n d p r o d u c e d a 10-fold increase in sterols r e l a t i v e to a n a e r o b i c a l l y g r o w n yeast. This p r o c e d u r e has r e c e n t l y b e e n used b y FRYBERG et al. 3 to s t u d y t h e i n t e r m e d i a t e s in ergosterol b i o s y n t h e s i s b y yeast. If r a d i o a c t i v e glucose is p r o v i d e d d u r i n g aeration, over 75% of t h e specific a c t i v i t y was f o u n d in t h e nonsaponifiable f r a c t i o n 9. W h i l e no d a t a was g i v e n for a c e t a t e i n c o r p o r a t i o n , KLEIN i n d i c a t e d t h a t a c e t a t e could be used a l t h o u g h longer a e r a t i o n t i m e w o u l d be necessary. W e h a v e e x a m i n e d t h e labeling of ergosterol f r o m [2-~4C]-acetate in S. cerevisiae SK1 using KLEIN'S a n a e r o b i c - a e r o b i c p r o c e d u r e (Table I). W h i l e t h e h i g h e s t yield of r e c o v e r e d ergosterol was for t h e 20 g (wet cells)/ 100 m l (aeration m e d i u m ) , t h e h i g h e s t specific a c t i v i t y was for t h e lower sterol y i e l d i n g cell densities. T h e second e n r i c h m e n t t e c h n i q u e d e p e n d s on t h e o b s e r v a t i o n t h a t t o s u p p l y c o e n z y m e A to p a n t o t h e n i c

acid deficient y e a s t c u l t u r e s p u r s t h e de n o v o b i o s y n t h e s i s of lipids i n c l u d i n g sterols. HANAHAN a n d WAKIL ~~ f o u n d t h a t ~4C-labeled a c e t a t e s u p p l i e d a t t h e t i m e of t h i s a c c e l e r a t e d b i o s y n t h e s i s results in t h e labeling of 1/, of t h e ergosterol carbons. KLEIN e t a l . n , x2 s h o w e d t h i s p r o c e d u r e to yield a several 100% increase in t o t a l lipids i n c l u d i n g sterols r e l a t i v e to t h e u n e n r i c h e d m e d i u m . Our results w h i c h e m p l o y e d a m o d i f i e d HANAHAN a n d WAKIL p r o c e d u r e i n c o r p o r a t i n g t h e KLEIN a n a e r o b i c aerobic t e c h n i q u e are p r e s e n t e d in T a b l e II. T h e s e results i n d i c a t e t h e i n a d e q u a c y of t h i s a p p r o a c h for our purposes. R e c e n t w o r k has s h o w n t h a t d u r i n g y e a s t s p o r u l a t i o n i n d u c e d b y acetate, t h e r e is a d r a m a t i c rise in lipid b i o s y n t h e s i s ~3, ~. Therefore, it a p p e a r e d likely t h a t y e a s t s p o r u l a t i o n in a labeled a c e t a t e m e d i u m w o u l d yield ergosterol w i t h a h i g h specific a c t i v i t y . This result was realized as s h o w n in Table I I I . E x p e r i m e n t B w h i c h e m p l o y s a n a e r o b i c p r e s p o r u l a t i o n g r o w t h a n d aerobic s p o r u l a t i o n p r o d u c e s s i g n i f i c a n t l y m o r e ergosterol w i t h a g r e a t e r i n c o r p o r a t i o n of label.

1 Studies on Biosynthesis. Part VII. For Part VI, see A. K. Bose and B. L. HTJNCUND, Experientia 29, 939 (1973). 2 A. L. BURLINGAME,B. BALOGH,J. WELCH, S. LEWISand D. WAL~S, J. chem. Soc. (D) 7972, 318. 3 M. FRYBERG,A. C. OEHLSCHLAGERand A. M. UNRAU,J. Am. chem. Soe. 95, 5757 (1973) ; this work is the most recent. D. J. HANAHANand S. J. WAKIL,J. Am. chem. Soc. 75, 273 (1953). H. J. REICH, lV[.JAUTE~AT,M. T. MESSE,F. J. WEmERT and J. D. Roberts, J. Am. ehem. Soe. 91, 7445 (1969). 6 A. G. MeINNES, D. G. SMITH, C. WA% L. C, VINIXG and J. L. C, WRIGHT, Chem. Commun. 7974, 281. A. C. MCINNES, D. G. SMITH, C. WAT, L. C. VINING and J. L. C. WRIGHT, Chem. Commun. 1974, 282. 8 H. KLEIN, J. Bact. 69, 620 (1955). 2 H. KLEIN, N. R. EATON and J. C. ~'IURPHY, Biochim. biophys.

Acts 73, 591 (1954). 10 D. J. HANAHANand S. J. WAHIL,Arch. Biochem. Biophys. 37, 167 (1952). n H. KLEIN, Fedn. Proe. 70, 209 (1951). 12 H. KLEIN and F. LU~MANN,J. biol. Chem. 203, 95 (1953). 11t

R. F. ILLINGWORTH, A. H. NOSE and A. BECKETT, J. Baet. J 13, 373

(1973). 14 S. A. HENRY a n d H. O. HALVORSON, J. Baet. 71d, 1158 (1973).

Table I. Biosynthesis of labeled ergosterol from [2-14C]-acetate in S. cerevisiae SK1 using anaerobic-aerobic procedure. Anaerobic Growth in YPD a,b. Aeration suspension~ (0.1% glucose, 0.1 M [2-14C]-acetate, 0.1 M phosphate, pH 7.0) Ergosterol recovered a (rag)

Incorporation efficiency ~

Overall incorporation f

(g wet cells/100 ml aeration medium) 5 10 15 20 25 30

4.8 3.9 4.1 6.5 3.3 3.0

10 13 5 5 3 4

0.08 0.20 0.08 0.11 0.04 0.04

Cell concentration

YPD is 1% yeast extract, 2% peptone, 10% dextrose (Difco). b 0.5 i 2-day-old innoeulation of 15 1 culture grown for 5 days at 30~ with mechanical stirring and N2 purging followed by centrifugafion and washing twice with sterile distilled water, c Aerated for 24 h by shaking at 200 rpm at 30 ~ a Ceils saponified, extracted with Petroleum ether, TLC oH AgNOa-impregnated silica gel, Muted with CHC1a. " dpm/mole ergosterol recovered/dpm/mole acetate substrate • 1 mole ergosterol/15 mole acetate • 100 = % incorporation efficiency. ~ Total dpm of ergosterol recovered/total dpm of acetate substrate • 100 = % overall incorporation.

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Table II. Biosynthesis of labeled ergosterol from [2-14C]-acetate in S. cerevisiaeSKI using CoA-enrichment and anaerobic-aerobic procedure. Anaerobic Growth in Coenzyme-A deficient medium~, b. Aeration suspension c (0.1% glucose, 0.1 M [2-14C]acetate, 0.1 M phosphate, pH 7.0, 1.0% YNB containing 25 mg CoAa Cell concentration (g wet cells/100 ml aeration medium)

Ergosterol recovered e (mg)

Incorporation efficiency ~

Overall incorporation

20

4.8

4.5

0.08

1% Wickerham's Yeast Nitrogen Base (YNB) without pantothenic acid, 10% dextrose, and 17 mg/ml Tween 80 and 20 [xg/ml tetracycline b Same as (b) in Table I. o Same as (c) in TabIe I. a Grade II-A from Sigma. e Same as (d) in Table I. ~ Same as (e) in Table I. g Same as (f) in Table I.

Table III. Biosynthesis of labeled ergosterol from [2-14C]-acetate in S. cerevisiae SKI during sporulation. Sporulation Conditions for A and B: aerobic, 1% labeled acetate, 27 ~ vigorous mechanical stirring, 4 • 107 cells/rot (1 1 total volume, corttaining 5.5 g wet cells in 6 1 flask) Sporulation yield Ergosterol recoveredb (mg) Incorporation efficiency o 0 verall incorporation a

(A) 99% asci in 3 days 4.4 71 0.11

(B) 90% asei in 5 days 7.9 76 0.21

Presporulation growth conditions: (A) aerobic, YPD ~, 30 ~ 2 days 1 1.from loop, shaking at 200 rpm. (B) anaerobic, YPD, 30 ~ 2 days 1 1 from loop, shaking at 200 rpm. ~ Same as (a) in Table I. b Same as (d) in Table I. ~ Same as (e) in Table I. a Same as (f) in Table I.

As y e a s t s p o r u l a t i o n in a liquid m e d i u m is carried out a t a p p r o x i m a t e l y 4 • 107 cells/ml, a liter of 1% a c e t a t e is r e q u i r e d to o b t a i n t h e ergosterol yields we r e p o r t . H o w e v e r , since t h e s p o r u l a t i o n m e d i u m c o n t a i n s only a c e t a t e , t h e r e c o v e r y of u n i n c o r p o r a t e d label can be a c c o m p l i s h e d b y a simple e x t r a c t i o n . D a t a of labeled s u b s t r a t e i n c o r p o r a t i o n into a m e t a b olite can be s t a t e d in v a r i o u s ways. W e h a v e r e p o r t e d firstly t h e overall i n c o r p o r a t i o n as a n i n d i c a t i o n of t h e m e t a b o l i c m a g n i t u d e of ergosterol b i o s y n t h e s i s f r o m a c e t a t e . Secondly, t h e i n c o r p o r a t i o n efficiency expresses t h e p e r c e n t a g e of ergosterol c a r b o n s derived f r o m acetate. The f a c t o r of 1/15 (cf. T a b l e I, f o o t n o t e e) in t h e calculat i o n of i n c o r p o r a t i o n efficiency allows for t h e b i o s y n t h e s i s of 1 mole of ergosterol f r o m 15 moles of a c e t a t e . W e h a v e a s s u m e d t h a t a c e t a t e c o n t r i b u t e s t o all ergosterol c a r b o n s

~s H. DANIELSSONand K. BLOCH, J. Am. chem. Soc. 79, 500 (1957). le G. J. ALEXANDER,A. M. GOLD and E. SCHWENK,J. Am. chem. Soc. 79, 2967 (1957).

e x c e p t C2s w h i c h arises f r o m single c a r b o n donors (e.g. f o r m a t e ) b y t r a n s m e t h y l a t i o n v i a m e t h i o n i n e 15,1% The d a t a in T a b l e I I I clearly i n d i c a t e s t h a t t h e sporulat i o n t e c h n i q u e gives a m o r e efficient i n c o r p o r a t i o n t h a n t h e 2 earlier m e t h o d s described. F u r t h e r w o r k o n bios y n t h e s i s in y e a s t using 13C-labeled s u b s t r a t e s is in p r o g r e s s in our l a b o r a t o r y .

Zusammen[assung. E s w e r d e n drei M e t h o d e n fiir die D u r c h f i i h r u n g y o n V e r s u c h e n m i t l~C-markierten Subs t r a t e n ( E i n b a u m a r k i e r t e r A c e t a t e in Hefeergosterol) verglichen : 1. A n a e r o b i s c h e s W a c h s t u m m i t n a c h f o l g e n d e r Beltiffung u n d Markierung. 2. A n a e r o b i s c h e s W a c h s t u m in N/ihrl6sung m i t u n g e n i i g e n d e m C o A - G e h a l t m i t n a c h folgender Beltiftung u n d M a r k i e r u n g in einer m i t CoA a n g e r e i c h e r t e n L6sung. 3. S p o r e n b i l d u n g m i t m a r k i e r t e n Acetaten. A. K. B o s e a n d J. J. SAVARESE Department o/ Chemistry and Chemical Engineering, Stevens Institute o/ Technology, Hoboken (New Jersey 07030, USA), 23 July 7974.

A New Method for Recording Migratory Restlessness in Caged Birds M a n y b i r d s t h a t are n o r m a l l y d a y - a c t i v e m i g r a t e a t night. If k e p t in a cage, t h e y d e v e l o p n o c t u r n a l a c t i v i t y d u r i n g t h e m i g r a t o r y seasons w h i c h m a n i f e s t s itself in an i n t e n s e h o p p i n g off a n d o n t o perches, a f l u t t e r i n g a b o u t in t h e cage or a vigorous b e a t i n g of t h e wings ('whirring'). This m i g r a t o r y restlessness or ' Z u g u n r u h e ' has o f t e n b e e n t a k e n for an e x p r e s s i o n of t h e m i g r a t o r y d r i v e in birds. Q u a n t i t a t i v e l y , h o w e v e r , it was e i t h e r o n l y t h e h o p p i n g or general l o c o m o t o r a c t i v i t y t t l a t was m e a s u r e d so far 1-4,

b u t n e v e r t h e w h i r r i n g itself w h i c h m i g h t be a m o r e specific e x p r e s s i o n of t h e m i g r a t o r y drive. Moreover, in rec o r d i n g p e r c h h o p p i n g , b o t h d a y - and n i g h t - a c t i v i t y of a

1 2 3 4

U. ATTILA, Ornis fenn. 14, 38 (1937). j . S. SZYMANSKI, Pflfiger's Arch. ges. Physiol. 758, 343 (1914). H. O. WAGNER, Z. vergl. Physiol. 12, 703 (1930). L. SIIVONEN, Ornis fenn. 73, 67 (1950).