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1 2 7 0 M. METZLER, K. H. DAHM, D. MEYER, AND H. RÖLLER

tides. Origin and nature of these substances, which are characterized by their lacking binding capacity for charcoal, are not yet clear. CHOU and SCHER-B A U M 3 1 working with Tetrahymena described an accumulation of acid-soluble phosphorylated deoxy-sugars by release from existing structures after a heat treatment. The question, if degradation pro-ducts from D N A (e. g. deoxynucleosides, cf. 1. c. 3 2 ' 3 3 ) are formed also in yeast after X-irradia-tion, needs further investigation.

A n interpretation of the nearly undisturbed in-crease of "deoxyribosides" after X-irradiation by mechanisms other than periodical deoxynucleotide synthesis, e. g. by partial D N A degradation and re-

pair, seems difficult, however, for it would imply similar periodicity and similar timing of the basic events.

Taken together our data indicate, that the rhyth-mic increase of "deoxyribosides" in synchronized growing yeast is regulated independently from D N A replication. On the other hand, the intracellular level of "deoxyribosides" apparently does not trigger the rate of D N A synthesis.

The investigations were kindly supported by the Deutsche Forschungsgemeinschaft. I am indepted to Prof. HILZ for stimulating discussions and generous aid, and to Miss K. LOGES for excellent technical assistance.

On the Biosynthesis of Juvenile Hormone in the Adult Cecropia Moth

MANFRED METZLER, KARL HEINZ DAHM, DIETRICH MEYER , and HERBERT ROLLER

Institute of Life Science, Department of Biology, Texas A&M University, College Station, Texas 77843, U.S.A.

(Z. Naturforsch. 26 b, 1270—1276 [1971]; received June 5, 1971)

Juvenile Hormone, Biosynthesis, Cecropia Moth

In adult males of the giant silk moth Hyalophora cecropia (L.) the amount of juvenile hormone (JH) extractable from the abdomens increases sharply between the first and fourth day of adult life; 4 — 8 day-old moths contain up to 6/ug. During the biosynthesis, L-methionine provides the ester methyl group of both JH and its lower homologue JH-II. It does not contribute to the for-mation of the carbon skeleton. Farnesol, farnesyl pyrophosphate, and propionate are not utilized. Mevalonate is extensively incorporated into £r<ms,£rares-farnesol, but not into the sesquiterpene-like hormone. This result indicates that JH is not synthesized via mevalonate in the adult moths. Label of 2-14C-acetate was recovered in both JH and farnesol; the incorporation rate, however, was very small. The label of JH was located in the carbon skeleton.

The juvenile h o r m o n e 1 ' 2 ( J H ; 1) is one of the major endocrine components which regulate post-embryonic growth and development in insects. This hormone and its lower homologue 3 (JH — I I ; 2 ) seem to be closely related biochemically to acyclic sesquiterpenes like farnesol ( 3 ) . Most remarkable, however, are the ethyl groups at positions C -7 and C - l l . This type of substitution is without precedence among natural isoprenoid compounds. Ethyl side chains have been found in some phytosterols 4 but in no case do they replace a methyl group biosyn-

Requests for reprints should be sent to Dr. H. ROLLER, In-stitute of Life Science, Department of Biology, Texas A&M University, College Station, Texas 77843, U.S.A.

1 H . R O L L E R , K . H . D A H M , C . C . S W E E L E Y , a n d B . M . T R O S T , Angew. Chem. 79, 190 [1967] ; Angew. Chem. Int. Ed. 6, 179 [1967].

thetically derived from the methyl group of mevalo-nate.

JH R=C2H5 1 FARNESOL 3 JH-D R=CH3 2

If JH is indeed a product of the mevalonate metabolism as its structure seems to indicate, two biochemical mechanisms could explain the introduc-tion of the additional C-atoms. The biosynthesis

2 H. ROLLER and K. H. DAHM, Recent Progr. Hormone Res. 24,651 [1968].

3 A . S . M E Y E R , H . A . S C H N E I D E R M A N . E . H A N Z M A N N , a n d J. H. Ko, Proc. nat. Acad. Sei. USA 60, 853 [1968].

4 E. LEDERER, Quart. Rev. (chem. Soc., London) 23, 453 [1969].

BIOSYNTHESIS OF JUVENILE HORMONE 1 2 7 1

starting from mevalonate m a y follow the usual path-way except that during an intermediary stage ethyl groups are formed by methylation at the appro-priate positions. A n alternate route would start with the incorporation of propionate into homomevalo-nate, two molecules of which would combine with one molecule of mevalonate to form JH. JH — II may be produced by einher pathway. Since no specific hormonal function different from that of JH has been demonstrated for JH — II, it may be considered a side product of the JH biosynthesis.

In order to investigate the two indicated biosyn-thetic routes we determined the incorporation into JH of labelled mevalonate, farnesol, farnesyl pyro-phosphate, propionate, and, as a source of C^-Units, methionine. For control purposes acetate was used as a less specific precursor which may also be in-corporated through an entirely different biosynthe-tic pathway.

JH has been isolated from abdomens of Hyalo-phora cecropia3'5, H. gloveri6, Samia cynthia7

and from organ cultures of corpora allata8 . The technique of culturing the endocrine glands in vitro would have been best suited for our studies, but practical difficulties and the small yield of JH pre-cluded this approach. Adult males of H. cecropia contain the highest amount of the hormone and a refined purification technique 6 makes it possible to isolate JH from a single abdomen. Therefore, the male cecropia moth was chosen as the experimental animal for our investigation.

R e s u l t s

I. J u v e n i l e H o r m o n e T i t e r i n A d u l t M a 1 e H. cecropia

In order to find the optimal time for the admin-istration of precursors, the amount of ether ex-traotable JH during the adult life of H. cecropia was determined (Fig. 1 ) . Both JH and JH — II were extracted from moths of different age groups, puri-fied 6 , and determined quantitatively by gas chro-matography ( G L C ) . In one series with 5 abdomens per experiment the recovery of JH was determined through addition of 2 - 1 4 C-JH to the crude extracts and was found to be between 53 and 6 3 % in all

5 H . R O L L E R , J . S . B J E R K E , a n d W . H . M C S H A N , J . I n s e c t Physiol. 11,1185 [1965].

8 K . H . D A H M a n d H . R O L L E R , L i f e S e i . P t . 2 , 9 , 1 3 9 7 [1970].

(ECDYSIS) DAYS OF ADULT L I F E —

Fig. 1. Amounts of JH in the abdomens of male H. cecropia. In one series ( A — A JH, • — • JH —II) the recoveries of JH have been determined experimentally through addition of 2-14C-JH. In the second series ( A ~ A JH, O - O J H - I I ) the amounts present have been calculated assuming a recovery of

55 per cent.

cases. The major loss during the purification can be attributed »to the deep temperature precipitation where 3 0 % of the JH was enclosed in the precipitate. This material can be recovered by a change of the solvent ratio and its amount or by a repetition of the precipitation. If this is done, however, the puri-fication faotor is decreased and the subsequent puri-fication steps become more tedious. On the other hand, a nearly quantitative isolation of JH was not necessary for the purpose of our investigation.

With the help of a bioassay, GILBERT et a l . 9

found the steepest increase of the JH titer around the time of adult ecdysis. According to our deter-mination, the JH titer increases most drastically between the first and the fourth day of adult life (Fig. 1 ) . JH was always accompanied by 1 0 — 3 0 % JH — I I ; we did not detect a significant age-depen-dent change in this ratio. The deposition of JH in the fat body of the adult moth may reflect an en-hanced biosynthetic rate as well as a decreased bio-logical degradation. Since nothing was known about the rate of JH metabolism in H. cecropia we de-cided to administer possible precursors during the first few days after emergence.

II. I n c o r p o r a t i o n o f P r e c u r s o r s i n t o J u v e n i l e H o r m o n e

Radioactively labelled L-methionine, farnesol, far-nesyl pyrophosphate, mevalonate, propionate and

7 D . M E Y E R , K . H . D A H M , a n d H . R O L L E R , in p r e p a r a t i o n . 8 H . R O L L E R a n d K . H . D A H M , N a t u r w i s s e n s c h a f t e n 5 7 , 4 5 4

[ 1 9 7 0 ] , • L . I . G I L B E R T a n d H . A . S C H N E I D E R M A N , G e n . c o m p .

Endoer. 1,453 [1961].

1 2 7 2 M. METZLER, K. H. DAHM, D. MEYER, AND H. RÖLLER

Precursor Moth JH Spec. act. Dose/moth num- Age Appli- Incu- isolated Spec. act.

ber cation bation per moth Spec. act.

[dpm/^mole] me- period

per [dpm///molc]

[dpm/^mole] [dpm] x 106 [days] thod a [hours] l>g] [dpm] XlO 3

L-methionine [3H-methyl] 7 .33x109 47.4 3 < 1 a 17 0.8 9900 3500 L-methionine [ 3 H -methyl ] 7.33 X10 9 47.4 3 2 a 17 1.3 28500 6400 L-methionine [ 3 H -methyl ] 7.33 X10 9 47.4 2 2 a 5 1.2 20500 5000 L-methionine [ 3 H -methyl ] 7.33 XlO 9 47.4 2 4 a 15 1.4 44600 9400 L-methionine [1 4C -methyl] 0.12 XlO 9 5.5 10 < 1 a 84 1.3 760 172 2-14C-farnesol 11.1 XlO 6 0.22 10 1 ,2 £ 24 0.6 3 2 2-14C-farnesol 11.1x106 0.75 4 1 ,2 e 24 1.0 5 2 2-14C-farnesol 11.1 XlO 6 0.49 4 2 Ö 24 0.7 5 2 2-14C-farnesol 11.1 XlO 6 0.68 5 1 ß 15 1.2 0 0 2-14C-famesol 11.1 x l O 6 1.32 5 1 ,2 s 16 1.2 6 2 2-14C-farnesyl pyrophos- 1.11 XlO 6 0.54 Y 0 0 phate + 4 2

Y 16 2.1

L-methionine [ 3 H -methyl ] 7.33 X10 9 23.7 a 27000 3800 5-3H-mevalonate 7.77 XlO 9 58.7 4 2 , 3 a 16 3.6 0 0 5-3H-mevalonate 7.77 XlO 9 58.7 3 3 a 14 3.5 47 4 2-14C-mevalonate 10.7 XlO 6 5.5 10 < 1 a 84 1.5 20 4 l-14C-propionate 57.2 X 1 0 6 5.5 10 < 1 a 84 1.4 20 4 2-14C-acetate 1.12 X 108b 57.8 4 1 ,2 a 15 2.1 1700 240 2-14C-acetate 1.12 x 1 0 s 63.0 3 2 a 13 1.6 1350 250 2-14C-acetate 1.12 x 1 0 s 98.0 3 2 a 16 1.1 1300 350 l-14C-acetate 1 .21x10 s 5.5 10 < 1 a 84 1.4 50 10

Table 1. Incorporation of possible precursors into JH. a See Experimental. b In this experiment the fatty acids were isolated (see Table 3).

acetate were administered as possible precursors in the biosynthesis of JH (Table 1) and JH - II . Water soluble compounds were dissolved in insect R i n g -e r - solution and injected. Farnesol in most experi-ments was applied as an oily solution behind the brain close to the corpora allata, where the pumping action of the heart was most likely to distribute the oil within a short time throughout the body. This proved to be the case, since in control experiments with 2-1 4C-farnesol and 2 - 1 4 C-JH the label could be extraoted from all parts of the body 2 — 3 hours after application. In all experiments with labelled precursors JH and JH — I I were isolated and their specific activities determined.

L -Methionine: The methyl group of L-methionine was extensively incorporated into both JH and JH — I I . U p to 0 . 1 % of the administered dose could be recovered in JH. The incorporation of the label into JH (Fig. 2 , 1 ) was confirmed by catalytic hy-drogenation and isolation of the resulting labelled methyl 7-ethyl-3.11-dimethyl-tridecanoate ( 4 ) . After cleavage of 4 with lithium aluminum hydride the label was found in the methanol, isolated as methyl 3.5-dinitrobenzoate ( 6 ) ; the 7-ethyl-3.11-dimethyl-tridecanol ( 5 ) was unlabelled.

COOCHj

0 % °H

I R = C0Hk: 100% ̂ H 2 R = CH3 = 100% 3H

R = C2H5= 98% H 4o R = CH, « 93 % 3H

C00CH-3

6 (from 4) = 79% 3H (from 4a): 93% 3 H

Fig. 2. Degradation of JH and JH —II after incorporation of the label from [methyl-3H] -L-methionine. The yields after each reaction were determined by GLC — in the case of 6 by weighing — and the recovery of the label (3H) was calcu-

lated for a 100% yield.

Degradation of JH — II (Fig. 2 , 2 ) demonstrated that in this case also the label was located exclusively in the ester methyl group.

Farnesol and farnesyl pyrophosphate: Neither farnesol nor farnesyl pyrophosphate were converted into juvenile hormone. Farnesol was extensively metabolized to compounds that according to their

BIOSYNTHESIS OF JUVENILE HORMONE 1 2 7 3

chromatographic properties were different from those produced after application of 2 - 1 4 C-JH.

Mevalonate: N o significant incorporation into JH and JH — II was detected after application of mevalonate. A considerable portion of the label, however, proved to be incorporated in trans,trans-farnesol which was isolated from the same animals. The ability of insects to synthesize farnesol was first demonstrated for Samia cynthia10, another Satur-niid moth. GOODFELLOW and GILBERT 1 1 found in extracts of cecropia 5 — 15 jug farnesol per gram lipid which corresponds to about 2 jug per moth. Ac-cording to our results abdomens of adult male cecro-pia contain less than 0 . 2 jug farnesol. In control experiments the recovery throughout the purification was checked by addition of 2-1 4C-farnesol to the crude extracts. In spite of the small amounts of far-nesol isolated from the moth, up to \ % of the ad-ministered label was incorporated (Table 2 ) . T o confirm the identity of the material we converted samples of the biosynthetically labelled farnesol by hydrogenation and by acetylation into the respective derivatives, both of which retained the label through GLC-purification. In one experiment natural 3H-far-nesol was mixed with synthetic 2-1 4C-farnesol and hydrogenated; the perhydro-farnesol after GLC-separation had the same 3 H / 1 4 C ratio as the starting material.

Propionate: N o incorporation into JH and JH — II was detected after application of labelled propionate. This result must be considered preliminary, since in this case no compound known to be formed from propionate was isolated as a control. The applied dose might have been too small to allow detection of incorporation.

Acetate: Acetate was definitely incorporated into JH and JH — II , but only to a small extent

( < | 0 . 0 0 3 % of the dose) . The labelled JH was de-graded in the same way as in the methionine experi-ment (Fig. 2 ) . Hydrogenation led to the saturated ester 4 which retained 2 / 3 of the radioactivity. The loss of radioactivity was due to highly labelled contaminants of JH which could be collected during the GLC purification of 4 . After cleavage with L i A l H 4 the aqueous phase containing the methanol derived from the ester methyl group was inactive; the methanol therefore was not further isolated. In contrast to the experiments with methionine, the radioactivity was recovered with the 7-ethyl-3 .11-dimethyl-tridecanol ( 5 ) . The small amount of label-led JH used for the degradation did not allow fur-ther localization of the label.

Acetate was also significantly incorporated into trans,ira/is-farnesol (Table 2 ) . Acetate is utilized for the synthesis of a broad variety of lipids and the moth contains only a small amount of farnesol. Therefore, it was greatly anticipated that part of the activity of the isolated material might be due to highly labelled contaminants. The specific activities of the farnesol determined in the first two experi-ments (Table 2 ) are certainly too high. In the third experiment we added a second thin layer chromato-graphy ( T L C ) to the purification scheme and used a carbowax column instead of an X E - 6 0 column for the final isolation by GLC. The small number of counts available after this isolation precluded any further identification through derivatives.

T o obtain more information about the utilization of acetate in adult H. cecropia the crude abdominal extract was transesterified with B F 3 / C H 3 O H . The methyl esters of palmitic, palmitoleic, stearic, oleic, linoleic, and linolenic acid were then isolated (Table 3 ) . The highest incorporation was found for the saturated fatty acids, whereas the polyunsatura-

Precursor Juvenile hormone trans, trans-Farnesol Spec. act. Dose/moth isolated per moth Spec. act. isolated per moth Spec. act. [dpm///mole] [dpm] [M g] [dpm] [dpm/^mole] M [dpm] [dpm//imole]

XlO 6 XlO 3 XlO 3

5-3H-mevalonate 7.77 X10 9 58.7 3.6 0 0 250000 5-3H-mevalonate 7 .77X10 9 58.7 3.5 47 4 < 0 . 1 3 313000 535000

2-14C-acetate 1.12x108 57.8 2.1 1700 240 0.15 2200 3300 2-14C-acetate 1.12x108 63.0 1.6 1350 250 0.10 660 1500 2-14C-acetate 1.12 x 1 0 s 98.0 1.1 1300 350 0.15 230 340

Table 2. Incorporation of 5-3H-mevalonate and 2-14C-acetate into JH and trans,trans-iaTneso\.

10 P. SCHMIALEK, Z. Naturforsch. 18 b, 462 [1963] . 11 R. D. GOODFELLOW and L . I. GILBERT, Amer. Zool. 3, 5 0 8 [1963].

1 2 7 4 M. METZLER, K. H. DAHM, D. MEYER, AND H. RÖLLER

Fatty acid methyl ester Isolated/abdomen Isolated/abdomen % of dose Spec. act. [mg] [dpm] [dpm/^mole] X10 3

Methyl palmitate 10.5 1070000 1.85 27 Methyl stearate 2.8 280000 0.49 30 Methyl palmitoleate 27.8 21000 0.04 0.2 Methyl oleate 116.9 180000 0.31 0.4 Methyl linoleate 4.9 0 0 0 Methyl linolenate 77.7 0 0 0

Table 3. Incorporation of 2-14C-acetate into fatty acids of H. cecropia (1 and 2 day-old male moths injected with 57.8 x 106

dpm/moth and sacrificed 15 hours later).

ted acids were devoid of label. STEPHEN and GIL-BERT 1 2 have reported a similar pattern of acetate incorporation into the fatty acids of H. cecropia pupae. The specific activity of the saturated fatty acids is approximately one order of magnitude be-low that of JH. One reason for this difference may be the dilution of newly synthesized labelled fatty acids by the presumably large amount of unlabelled fatty acids stored in the adult moth. In the case of JH the ratio of newly formed to already present hormone is expected to be smaller.

Discuss ion

JH is synthesized in the adult moth by a process including the transfer of methyl groups from methionine to form the ester methyl group of JH. AKAMATSU and L A W 1 3 have discovered an enzyme from Mycobacterium phlei which catalyzes the syn-thesis of fatty acid methyl esters from fatty acids and S-adenosyl-methionine. W e do not yet know whether or not a free acid is a precursor of JH * . During the time that JH is synthesized in the insect, the methionine pool is not used, however, as a source of C^-Units f o r the homologization of iso-prenoid methyl groups. The male cecropia moth produces farnesol from mevalonate at the same time that JH is synthesized without incorporation of this precursor (Table 2 ) . The dose used would have al-lowed us to detect an incorporation of mevalonate into JH even if, at the time when the precursor was available, the rate of the JH biosynthesis had been only 1 / 1 0 0 0 that of the farnesol biosynthesis. W e therefore conclude that in the adult moth JH is not formed from mevalonate. The second hypothesis

12 W . F . STEPHEN and L. I. GILBERT, J. Insect Physiol. 15 , 1 8 3 3 [ 1 9 6 9 ] .

13 Y . AKAMATSU and J. H. LAW , J. biol. Chemistry 245 , 709 [1970].

mentioned in the introduction which postulates the biosynthesis of homomevalonate from propionate cannot be ruled out on the basis of our experiment with propionate because the administered dose of propionate might have been too small. This path-way, however, is extremely unlikely also since it re-quires the incorporation into JH of one molecule of mevalonate in addition to the two molecules of homomevalonate.

The hypothesis that JH is formed biochemically like an isoprenoid compound is so far based only on the similarity of its structure with that of acyclic sesquiterpenes. If this hypothesis is to be upheld, it must be concluded that the carbon skeleton of JH is already synthesized during the larval or pupal stage of H. cecropia and that in the adult only minor conversions are necessary to complete the synthesis, one of these conversions being esterification using methionine as the methyl donor. The alternative, namely that JH is not synthesized through the iso-prenoid pathway, finds some support by our finding that the incorporation of acetate is a hundred times higher than that of mevalonate (Table 1 ) . The per-centage of acetate incorporation, however, is so small that more extensive investigations are neces-sary before this theory can seriously be advocated.

E x p e r i m e n t a l

Animals: H. cecropia were bought as diapausing pupae from Richter Butterfly Farm, N.Y. , and were kept at 4 ° for 3 to 12 months. Adult development was initiated by exposure to 2 5 ° .

Radiochemicals: [Methyl-3H]-L-methionine (3.3 Ci/ mmole), 5-3H-mevalonate (3.5 Ci/mmole) and 2-14C-acetate (50.6 mCi/mmole) were purchased from

* Note added in proof: Up to 10% of 10-epoxy-7-ethyl-3,ll-dimethyl-2,6-tridecadienoic acid injected into adult male Hyalophora cecropia is converted to JH. — M. METZLER, D . M E Y E R , K . H . D A H M , H . R O L L E R , a n d J. B . S I D D A L L , submitted for publication.

BIOSYNTHESIS OF JUVENILE HORMONE 1275

Schwarz Bioresearch Inc., N.Y. — [Methyl-14C] -L-me-thionine (53.7 mCi/mmole) and l-14C-acetate (54.7 mCi/mmole) were purchased from Amersham Searle Corporation, Des Piaines, 111. — 2-14C-DL-mevalono-lactone (4.82 mCi/mmole) and l-14C-propionate (25.8 mCi/mmole) were purchased from Nuclear Chicago Corp., Des Piaines, 111. — 2-14C-farnesol (40% trans, trans, 22% cis,trans, 31% trans,cis, 7% cis,cis), 5 mCi/ mmole) was synthesized by Dr. CHRISTIAN SCHLATTER, Universität Bern, Switzerland (The fraction consisting of pure trans,trans-farnesol was lost through autoxi-dation). — 2-14C-farnesyl pyrophosphate (0.5 mCi/ mmole) was prepared according to POPJÄK et al . 1 4 ' 1 5 , from 2-14C-farnesol after dilution with unlabelled irons,Zrans-farnesol. — 2-14C-juvenile hormone (spec, act. 1.3 mCi/mmole) was a gift from Zoecon Corp., Palo Alto, California.

Radioassay: Radioactivity was measured by liquid scintillation counting with a TriCarb 3375 (Packard Instrument Co., Downers Grove, 111.). Scintillators were dioxane/10% naphthalene/0.7% PPO or toluene/0.5% PPO/0.01% dimethyl-POPOP. The dpm were calcu-lated using the automatic external and internal stan-dards.

Administration of precursors: a: in R i n g e r - solu-tion according to Finlayson (7.5 g NaCl, 0.35 g KCl, and 0.21 g CaCl2 per 1 distilled water): 20, 30, or 40 pi of the solution were injected through the inter-segmental membrane between the 6th and 7th abdomi-nal segment.

ß: in olive oil. 2 /u\ injected like a. y: in Tris/HCl buffer pH 8.5, 60 injected like a. 3 : in olive oil. A triangular tab was cut out of the

forehead of the animal, some crystals of streptomycin and reduced glutathion were applied, and 1 or 2 pi of the olive oil solution was administered behind the brain with a round tip syringe. The tab was closed and sealed with a mixture of paraffin and olive oil.

e: in cecropia oil from 6 — 8 day-old males after purification by deep temperature precipitation 6, other-wise like <5.

C: in acetone. 2 pi of the solution was applied topi-cally on the intersegmental membrane between the 3rd and 4th abdominal segment.

Isolation of JH and JH — II: Purification was achieved as previously reported6 with the exception that acetone/benzene (1 : 1) was used instead of me-thanol/benzene ( 1 : 1 ) for chromatography on LH-20. JH and JH — II were isolated by preparative GLC. [Thermal conductivity detector, 180 cm glass column, inside diameter 4 mm, 3% XE-60 on Gas Chrom Q (Applied Science Lab., Inc., State College, Pa.).] In some cases after the yield of JH and JH — II had been determined unlabelled JH was added as a carrier prior to the final GLC-separation.

Isolation of trans,trans-farnesol: 1. Column chro-matography of crude oil on Sephadex LH-20 in ben-

1 4 G . P O P J Ä K , J . W . C O R N F O R T H , R . H . C O R N F O R T H , R . RYHAGE, and DEW. S. GOODMAN, J. biol. Chemistry 2 3 7 , 5 6 [ 1 9 6 2 ] .

zene/acetone (1 : 1 v/v) ; 2. TLC on silica gel in chloroform/ethylacetate ( 2 : 1 v/v) ; 3. TLC on silica gel in benzene/ethyl acetate (9 : 1 v/v) ; 4. GLC on XE-60 or Carbowax 20M. The identity of the isolated material was confirmed: 3H-farnesol biosynthetically labelled with 3H-mevalonate was mixed with synthetic 14C-farnesol and hydrogenated with Pd/BaS04 as the catalyst. The reaction product — 3.7.11-trimethyl-do-decanol — after purification by GLC on XE-60 had the same dpm 3H/dpm 14C-ratio (1.93 : 1) as the star-ting material.

Isolation of fatty acid methyl esters: Crude oil was transesterified with BF 3 /CH 3OH 1 2 and the methyl esters were resolved by subsequent TLC on silica gel, TLC on silver nitrate impregnated silica gel, and GLC on EGSS-X.

Degradation of 3H-JH from experiments with [methyl-3H]-^-methionine

Methyl 3.11-dimethyl-7-ethyl-tridecanoate (4 ) : 3.8 pg JH (64,000dpm, spec. act. 4.95 x 106 dpm/^mole) in 400 pi CH3OH were hydrogenated at room temperature for 20 min with Pd-black as the catalyst. The solution was decanted, the catalyst washed three times with CH3OH, and the combined solutions were evaporated to dryness under vacuum. Purification by GLC on XE-60 afforded 2.8 pg 4 (76%, 48,000 dpm, spec. act. 4.89 x 106 dpm/amole).

3.11-dimethyl-7-ethyl-tridecanol (5) : 2.6 pg 4 (44,600 dpm) and 18 pg of unlabelled 4 in 100 pi ether were cleaved by an excess of LiAlH4 in 50 pi ether. After addition of 100 pi 10% H 2 S0 4 the ether layer was separated, washed with 50 /ul HoO and re-solved by GLC on XE-60 to yield 11.5 pig 5 (62%, 0 dpm).

Methyl 3.5-dinitrobenzoate (6) : 95% of the com-bined aqueous phases of the foregoing reaction after addition of 100 pi CH3OH were dissolved in 1.5 ml pyridine. 3 g 3.5-dinitrobenzoyl chloride were added in portions, the reaction mixture heated to give a clear solution, after cooling decomposed with 30 ml 20% HCl and ice, and extracted with ether. The extract was washed three times with 30 ml saturated NaHC03-solution, once with H 2 0 , and dried on CaCl2. Eva-poration of the ether left 421 mg 6 (75%, 25,100 dpm, spec. act. 13,500 dpm/mmole). 6 was crystallized twice from CH 3 0H/H 2 0 . First crystallization: fraction I had a spec. act. of 15,050 dpm/mmole, fraction II 14,950 dpm/mmole; recrystallization of fraction I: 14,700 dpm/mmole.

Degradation of 3H — JH — II from experiments with [methyl-3H]-methionine: The 3H-labelled JH — II from several experiments was combined (2.5 pg, 43,000 dpm) and hydrogenated after addition of 22.4 pg of ulabelled JH — II (spec. act. 4.8 x 105 dpm/umole).

1 5 P . W . H O L L O W A Y a n d G . P O P J Ä K , B i o c h e m . J . 1 0 4 , 5 7 [1967].

1 2 7 6 E. STEUDLE UND U.ZIMMERMANN

Purification by GLC afforded 17 pg (71%, 28 ,400 dpm, spec. act. 4.5 x 105 dprn/^mole) of methol 3.7.11-trimethyltridecanoate (4 a) . 16.8 pg (28 ,100 dpm) 4 a were cleaved with L i A l H 4 . The 3.7.11-trimethyltri-decanol (5 a) after purification by GLC (12.5 pg, 83%) did not contain any of the label. The methanol was isolated as described above from 9 / 1 0 of the aqueous phase as the methyl 3.5-dinitrobenzoate (6) (376 mg, 66%, 15,500 dpm, spec. act. 9,300 dpm/ mmole). Crystallization from C H 3 0 H / H 2 0 afforded fraction I (spec. act. 8 ,700 dpm/mmole) and fraction II (8,900 dpm/mmole). Fraction I recrystallized: 8 ,400 dpm/mmole.

Degradation of UC-JH from experiments ivith 2-14C-acetate: 2 pg JH diluted with unlabelled JH to 45 pg (2,300 dpm, spec. act. 15 ,000 dpm/^mole) were hydro-

genated. During GLC purification besides 35 pg 4 (80%, 1,100 dpm, spec. act. 8 ,900 dprn/^mole) some labelled byproducts (520 dpm) were collected. 32 pg (1,000 dpm) 4 were cleaved with L i A l H 4 . The aqueous phase after acidification and extraction with ether was inactive. The ether phase contained only 9 pg (32%) 5 which was purified by GLC (8 pg, 260 dpm, spec. act. 8 ,300 dpm/^mole).

The 2- 1 4C -JH was kindly donated by Dr. JOHN B. SIDDALL, Zoecon Corp., Palo Alto, Calif. W e thank Dr. CHRISTIAN SCHLATTER, Universität Bern, Switzerland, for synthesizing the 2-14C-farnesol. Our work was generously supported through grants from the National Science Foundation (GB-7941) and the Cotton Pro-ducers Institute (CPI 69-139) .

Zellturgor und selektiver Ionentransport bei Chaetomorpha linum Cell Turgor Pressure and Selective Ion Transport of Chaetomorpha linum

E . STEUDLE und U . ZIMMERMANN

Institut für Physikalische Chemie, Kernforschungsanlage Jülich GmbH, Jülich

(Z. Naturforsch. 26 b, 1276—1282 [1971]; eingegangen am 3. April 1971)

The littoral alga Chaetomorpha linum is especially able to maintain a constant turgor pres-sure in the cell by regulating the internal osmotic pressure, if the salt content of the sea water changes. Experiments in artificial isotonic sea water with a constant sodium concentration, but variable potassium concentrations (from 1 to 50 mMol/1) prove, that the decrease or increase of the potassium concentration in the medium (CK) is an essential cause for this regulation of the turgor pressure besides the change of the osmotic pressure of the medium, which was thought to be the predominant cause till now. In the examined concentration range the ratio CK to Ck (po-tassium concentration in the cell) depends linear on CK in the steady state. At low values of Ck ( < 10 mMol/1) the decrease in Ck is compensated by a reversible sodium uptake only in part, and this leads to partly high changes in the cell turgor pressure, although the osmotic pressure of the medium remains constant. The results are discussed on the basis of carrier models.

Meeresalgen, die in Gebieten mit wechselndem Salzgehalt des Seewassers (Flußmündungen, Gezei-tenzonen usw.) wachsen, verfügen über mehr oder minder stark ausgeprägte Regulationsmechanismen, die den Turgor trotz erheblicher Änderungen des osmotischen Druckes in der Umgebung in engen Grenzen konstant halten Als Turgor wird der hy-drostatische Druck im Innern der Algenzelle bezeich-net, der im stationären Zustand der osmotischen Druckdifferenz zwischen innen und außen entspricht. Auch viele Meerestiere können durch Regulierungs-vorgänge Änderungen im Salzgehalt des Außen-mediums auffangen, im Unterschied zu Algen — vor

Sonderdruckanforderungen an Dr. U . ZIMMERMANN und Dipl.-Chem. E. STEUDLE, Institut für Physikalische Chemie der Kernforschungsanlage Jülich, D-5170 Jülich, Postfach 365.

1 H. KESSELER, Kieler Meeresforsch. 15, 51 [1959]. 2 A. KROCH, Z. vergleich. Physiol. 25, 335 [1938].

allem bei höherer Organisationsstufe — halten sie aber den osmotischen Druck im Innern ihrer Zellen bzw. in den Körperflüssigkeiten annähernd konstant.

Ausführliche Darstellungen hierüber finden sich bei KROGH 2 , POTTS und PAMY 3 sowie bei FLOREY 4 .

Für die Turgorregulation bei Algen werden zur Zeit drei Formen diskutiert: 1. Regulation durch Synthese oder Abbau osmotisch aktiver Stoffe in den Zellen (sog. Ana- bzw. Katatonose). 2 . Regulation durch Ausscheidung von Wasser durch pulsierende Vakuolen. 3 . Regulation durch Ionentransport. Alle drei Regulationsarten konnten bei Algen gefunden werden. KAUSS 5>6 schließt z. B. aus seinen Ver-

3 W. T. W. POTTS U. G. PAMY, Osmotic and ionic regulation in animals, Pergamon Press, Oxford 1964.

4 E. FLOREY, Lehrbuch der Tierphysiologie, Georg Thieme-Verlag, Stuttgart 1970.

5 H. KAUSS, Z. Pflanzenphysiol. 56, 453 [1967]. 6 H. KAUSS, Nature [London] 214,1129 [1967].