Gorgosterol biosynthesis: Localization of squalene formation in the zooxanthellar component of various gorgonians

Comp. Biochem. Physiol. Vol. 81B, No. 2, pp. 423428, 1985

0305-0491/85 $3.00 +0.00 ~ 1985 Pergamon Press Ltd

Printed in Great Britain

GORGOSTEROL BIOSYNTHESIS: LOCALIZATION OF SQUALENE FORMATION IN THE ZOOXANTHELLAR COMPONENT OF VARIOUS GORGONIANS DAVID G. ANDERSON Department of Biochemistry, University of Miami School of Medicine, Miami, FL 33101, USA Tel: (305) 547-6265 (Received 12 October 1984) Abstract--1. Four species of gorgonians: three related pseudoplexaurids Pseudoplexaura porosa, P. flagellosa and P. wagenaari; and the unrelated Pseudopterogorgia americana, are sources of zooxanthellae

capable, in purified broken cell preparations, of converting [J4C]labeled farnesyl pyrophosphate into squalene. 2. More extensive studies with P. porosa and P. americana zooxanthellae preparations characterize the conversion as enzymatic and demonstrate farnesyl pyrophosphate and reduced pyridine nucleotide as substrates. NADPH and NADH are essentially equivalent. Anaerobic conditions are not required. 3. Despite numerous attempts zooxanthellae formation of sterols, including gorgosterol, from radioactive substrates was unsuccessful. By enzyme studies, we can show only the conversion of mevalonate into squalene as a zooxanthellae capability.

INTRODUCTION

Gorgosterol, isolated first from gorgonians by Bergmann et al. (1943), is found in those tropical marine invertebrates, predominantly coelenterates, that contain symbiotic zooxanthellae or in the predators of such creatures (Ciereszko and Karnes, 1973). The structure of gorgosterol was reported to be a cholesterol derivative with an exhaustively methylated side chain (assuming the biosynthetic equivalency of methyl and cyclopropane) by Hale et al. (1970), with the absolute configuration established by Ling et al. (1970). It is generally assumed that the zooxanthellar (dinoflagellate) partner in the symbiosis is '_he site of all or at least most of the gorgosterol pathway. The biosynthetic pathway to this totally unprecedented sterol side chain has engaged the interest of a number of researchers. Despite this continuing interest, no gorgonian or zooxanthellar cell-free system capable of forming gorgosterol or any digitoninprecipitable sterol has been reported. Instead, evidence for the pathway of gorgosterol has depended on information derived from whole organism studies. Sterol distribution patterns in whole gorgonians (Kokke et al., 1979 and Withers et al., 1979a) have served to implicate dinosterol, or alternatively, sidechain methylated cholesterol derivatives as proximate precursors of gorgosterol. The two side-chain methyl groups of dinosterol in an axenically grown freeliving dinoflagellate were shown to arise from doubly iabeled methyl groups of methionine (Withers et al., 1979b). Changes in sterol distribution upon aposymbiotic growth of zooxanthellae isolated from a number of species, supported zooxanthellar localization of sterol biosynthesis but led to the suggestion that the gorgonian partner was required for complete biosynthesis of gorgosterol (Kokke et al., 1981). Withers et al. (1982) have reported on a unique zooxanthellar isolate from a Pacific anemone which on aposymbiotic growth, is capable of forming gor-

gosterol. The concurrent presence of 23-desmethylgorgosterol in this system suggested that this compound and not dinosterol is the more direct precursor. They report, however, that aposymbiotically grown zooxanthellae from all other sources lacked both gorgosterol and desmethylgorgosterol. Our interest in isoprenoid metabolism and its role in the gorgonian-zooxanthellae symbiosis has led us to include examination of gorgosterol biosynthesis as a complement to our studies of crassin acetate (Papastephanou and Anderson, 1982) and gorgonian sesquiterpene (Anderson, unpublished) biosyntheses. In none of our cell-free systems; gorgonian, zooxanthellar or mixed, some capable of supporting complex isoprenoid biosynthetic pathways or parts thereof; did we find any evidence for the formation of digitonin-precipitable sterols, let alone the formation of the more complex gorgosterol. However, we have been able to show that zooxanthellae isolated from a number of gorgonian species are capable of forming squalene, the linear precursor of sterols in all known systems. Broken cell preparations of zooxanthellae purified from these gorgonians convert labeled farnesyl pyrophosphate into squalene in the presence of appropriate co-substrates. This finding in conjunction with earlier findings that the pathway from mevalonate to farnesyl pyrophosphate is found in the zooxanthellae of P. porosa (Papastephanou and Anderson, 1982), confirms the proposal that at least a major portion of the pathway of sterol, and presumably gorgosterol, biosynthesis is sited in the zooxanthellar partner. The question of the participation, direct or indirect, the gorgonian (animal) partner in the formation of the final product(s) remains open. MATERIALS AND METHODS

Chemicals and biochemicals

In addition to those previously described (Papastephanou and Anderson, 1982), NAD, NADH and NADPH were

423

424

DAVID G. ANDERSON

Fig. 1. The final purification state of zooxanthellae derived from Pseudopterogorgia americana, zooxanthellae average 15/~m in diameter. products of Sigma, St Louis, as were HEPES and MES, Good buffers. Eastman supplied mercaptoethanol. Baker was the supplier of N-acetyl cysteine, maleic acid and all other chemicals, which were of reagent grade where applicable. Solvents used in extractions and chromatographic separations were from Baker with the exception of petroleum ether (30-60) which was from Mallinkrodt and further purified as previously described. Radioactive mevalonic acid and radioactive biosynthetic terpenyl pyrophosphates as a mixture, approximately 45~o farnesyl pyrophosphate, (equivalent to 90~o conversion of the proper isomer of mevalonate), were supplied and prepared as before (Papastephanou and Anderson, 1982). Isolation o f zooxanthellae

Pseudoplexaurid sources of zooxanthellae were collected as before (Rice et al., 1970). Pseudopterogorgia americana, abundant in the tidal swash seaward of cuts between the upper Florida Keys, were collected from such locations. Zooxanthellae of P. porosa were isolated as previously described (Rice et al., 1970) by homogenization, followed by repeated centrifugations and differential resuspension in 50~o sterile sea water. The first homogenization employed a mix of 50 g of gorgonian sections in 200ml of liquid. Zooxanthellae from other pseudoplexaurid sources, P. flagellosa and P. wagenaari, were isolated by the same technique. Isolation of zooxanthellae from P. americana presented particular problems, in the field, this gorgonian is distinguished by its extreme sliminess, and on homogenization yields a suspension of such viscosity as to preclude such simple operations as pouring a sample into another vessel. The cause of the viscosity is a sulfated mucopolysaccharide as characterized by Ciereszko et al. (1972). Numerous trials of liquefying agents led to the use of N-acetyl cysteine (Sheffner, 1963). Zooxanthellae were isolated by adding 20 g of freshly minced P. americana to 400 ml of sterile 50~, sea-water containing 2~ o w/v N-acety} cysteine, pH adjusted to 8.0 with 2.0 M NaOH, and homogenized at full speed in a Waring blender for 30sec. Following passage through cheese cloth and centrifugation at 3000 x g, the supernatant was discarded and the zooxanthellar pellet was resuspended 0.5~o N-acetyl cysteine, pH 8.0, and recentrifuged in the same medium following the procedure of Rice et al. (1970).

The final zooxanthellar fraction was predominately vegetative dinoflagellate with occasional lamellar "white bodies' which continued to exude slime. This contaminant was removed by running the zooxanthellar suspension slowly down an inclined glass plate. The bodies adhered and the zooxanthellae ran flee. The resultant preparation is virtually pure zooxanthellae, see Fig. 1. Zooxanthellar broken cell preparations

Isolated zooxanthellae were resuspended in an appropriate breaking medium, three volumes of breaking medium to one volume of packed cells, and frozen in a chilled Hughes press at - 8 0 ' C . The frozen pellet was forced through the slit orifice by striking the plunger with a heavy hand mallet. One of the following breaking media was used. (I) Maleate medium--0.1 M NaC1, 0.05 M KCI, 0.05 M maleate and 0.01 M mercaptoethanol, adjusted to pH 6.3. (If) HEPES medium as above except replace maleate with 0.05 M HEPES and adjust to pH 8.0. (Ill) MES medium--as in (1) above except replace maleate with 0.05 M MES and adjust to pH 6.3. (IV) Phosphate medium--as in (I) above except replace maleate with KPO 4 buffer, 0.05 M, pH 6.3. (V) Tris medium--as in (I) above except replace maleate with 0.1 M Tris buffer at pH 8.0. (VI) Sucrose-Tris medium, 0.2 M sucrose buffered with 0.1 M Tris at pH 8.0, mercaptoethanol 0.01 M. Following breakage, the preparation was centrifuged at 27,000g for 30min. The particulate portion derived from 1.0 ml of packed zooxanthellae was resuspended in 4.0 ml of appropriate medium, typically the breaking medium. Certain of the above media were sometimes further modified by the addition of PVP-40 (soluble) or Polyclar AT (particulate) when a particulate (PVP-40) or soluble (Polyclar AT) zooxanthellar fraction was sought. These polyvinylpyrrolidones protect against autoxidation and tanning (Loomis and Battaile, 1966). Reaction mixture f o r squalene production

Modifications of the standard reaction mixture for the production of squalene are indicated where appropriate. The standard mixture contained 0.04mM labeled biosynthetic [~4C]farnesyl pyrophosphate, 1.1 x 105 cpm (produced from [2-~4C]mevalonate in a preincubation catalyzed

Squalene formation by zooxanthellae

425

Table 1. Sources and characteristics of zooxanthellar systems for squalene biosynthesis Medium, Counts/min in Item Source conditions squalene 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

A A A A B C C C C C C C C C C C C C

I (no KCI)-~I (no KCI) I~I V---~V VI---~VI I--,I I--* I II ~ II III---~III I V ~ IV V--~V V--*I VI--* I I ~ Frozen Frozen~ I I (heavy)---~I I (light)--~ I I (sup) I ~ I (same day)

130 256 0 0 185 334 50 78 110 0 106, 132 0 110 257 303 48 0 3710

The sources are A--Pseudoplexaura wagenaari, B--Pseudoplexaura flagellosa, and C Pseudoplexaura porosa. Breaking (incubation) media are listed in Materials and Methods. 1 4 1 indicates Hughes Press breakage in Medium I followed by resuspension of 27,000 × g particulates in Medium I for incubation. See Materials and Methods for conditions of incubation and squalene characterization. In items 13 and 14, I ~ F r o z e n indicates breakage in Medium I followed by freezing, and F r o z e n ~ I indicates freezing of purified zooxanthellae in 50% seawater followed by transfer into and breakage in Medium I on thawing. In Items 15, 16 and 17, heavy refers to particulates gathered at 1000 x g for 2 min; light to particulates between 1000 x g and 27,000 x g for 30 min; and sup to the 27,000 x g supernatant.

by a rat liver preparation, heat-inactivated following incubation); an NADPH generating system consisting of 0.10 mM NADP, 0.01 M glucose 6-phosphate and 2.0 IU of glucose 6-phosphate dehydrogenase; 0.1 ml of an antibiotic mixture containing 2% K penicillin, 0.4% streptomycin SO4, 0.2% chloramphenicol and 0.02% neomycin SO4; and 1.0 ml of a zooxanthellar preparation, equivalent to 0.25 ml of packed zooxanthellae. The final volume was 2.5 ml. Incubations originally lasted 18 hr but when attention centered on P. porosa and P. americana, time was reduced to 4 hr. The reaction was stopped by adding 2.5 ml of methanol. Isolation and characterization of squalene. The inactivated reaction mixture was extracted with 2.5 ml of petroleum ether, which extract was combined with the two subsequent petroleum ether phases resulting from extraction with two 5ml portions of 50:50, petroleum ether:methanol. The combined petroleum ether extracts were reduced to 3.0 ml under N 2 and dried over Na:SO 4. Squalene was tentatively identified by its chromatographic behavior on alumina. Columns of Merck's alumina for chromatography measured 10 x 1.4 cm and were developed with increasing concentrations of ethyl ether, i.e. 2, 5, 10 and 20%, in petroleum ether. With this system squalene elutes behind and well separated from cyclic sesquiterpene hydrocarbons, abundant in gorgonians. P. americana contains fl-gorgonene, a sesquiterpene with two exomethylene groups, which might spill into the squalene fractions. That it did not was proven by silicic acid chromatography which separates squalene and fl-gorgonene readily. Certain identification of squalene was attained by repeated crystallizations to constant specific radioactivity of the hexahydrochloride derivative and the constant specific radioactivity of the squalene on repeated regeneration from the thiourea adduct. The first of these was prepared according to Langdon and Bloch (1953), after the addition of 100 mg of carrier squalene (Eastman) purified by saponification and passage over alumina. Clathrate formation followed the procedure of Rabourn et al. (1954) with 100 mg of squalene as above. Squalene was regenerated by dispersing the cla-

thrate crystals in water and extracting squalene into petroleum ether. Squalene was brought to constant weight by evaporating under nitrogen followed by storage in vacuum over paraffin wax.

RESULTS

In a survey o f the ability o f various cell-free a n d b r o k e n cell p r e p a r a t i o n s o f pseudoplexaurid-derived zooxanthellae to form squalene, first indications of success came on trial o f a b r e a k i n g m e d i u m with maleate as a buffer a n d the use o f a Hughes press for cell breakage, see Table 1. The source of zooxanthellae in this case was Pseudoplexaura wagenaari. The other pseudoplexaurids, P. flagellosa a n d P. porosa also provided active p r e p a r a t i o n s w h e n their zooxanthellae were similarly treated. Since P. porosa typically yields more zooxanthellae a n d is c o m m o n a n d easily identified in the field, its zooxanthellae were routinely employed in subsequent studies. Regardless of the source of zooxanthellae, maximally active p r e p a r a t i o n s required b o t h NaC1 a n d KC1 in the b r e a k i n g medium. Breaking media employing buffers o t h e r t h a n maleate gave less active or inactive preparations. This is not solely a p H effect since b o t h m e d i u m III ( M E S buffer) a n d m e d i u m IV (phosphate) are at the p H o f the maleate medium. Reduced activity is in part the consequence o f inadequate breakage or of a n inhibition of the squalene synthetic activity since transfer from b r e a k i n g m e d i u m V (tris) to a maleate i n c u b a t i o n m e d i u m results in m o d e r a t e activity. The activity survived freezing whether frozen as intact zooxanthellae or as the b r o k e n maleate p r e p a r a t i o n , but with substantial loss over the period of a week. C o n s e q u e n t l y only fresh p r e p a r a t i o n s were employed.

426

DAVID G. ANDERSON Table 2. Reproducibility of assay for incorporation of [t4C]farnesyl pyrophosphate into squalcne Counts/min in Replicate Squalene Experiment 1 3430 I 2 3680 3 3280 4 3160 1 689 2 630 3 728 4 688 Replicates were run on two different zooxanthellar preparations, derived from separate collections of P. porosa. See Materials and Methods for details of incubation and product characterization.

A t t e m p t s to e s t a b l i s h t h e i n t r a c e l l u l a r l o c a l i z a t i o n o f s q u a l e n e f o r m a t i o n , typically m i c r o s o m a l in h o m o g e n a t e s , give n o i n d i c a t i o n o f t h e o r g a n e l l e inv o l v e d . T h a t p a r t o f t h e p r e p a r a t i o n w h i c h is m o s t active s e d i m e n t s at 1000 x g w i t h i n 2 m i n . N o disc e r n i b l e activity r e m a i n s in t h e s u p e r n a t a n t f o l l o w i n g c e n t r i f u g a t i o n at 27,000 x g for 30 m i n . T h e interm e d i a t e f r a c t i o n s e d i m e n t i n g b e t w e e n 1000 a n d 27,000 × g s h o w s r e d u c e d activity. In early e x p e r i m e n t s we d i d n o t a t t e m p t c o n t r o l o f collection c o n d i t i o n s or t h e t i m e i n v o l v e d in z o o x a n t h e l l a e i s o l a t i o n . In later e x p e r i m e n t s , s a m e d a y collection, z o o x a n t h e l l a e i s o l a t i o n a n d i n c u b a t i o n r e s u l t e d in a s u b s t a n t i a l i n c r e a s e in i n c o r p o r a t i o n , see T a b l e 1, i t e m 18. H o w e v e r , in spite o f a t t e m p t s to standardize conditions, the extent of incorporation i n t o s q u a l e n e w a s still h i g h l y v a r i a b l e f r o m collection to collection, t h o u g h h i g h l y c o n s i s t e n t w i t h i n a single p r e p a r a t i o n as i n d i c a t e d in T a b l e 2. T h a t t h e c o n v e r s i o n is e n z y m a t i c is i n d i c a t e d b y t h e h e a t lability o f t h e i n c o r p o r a t i o n ( T a b l e 3). F u r t h e r m o r e , t h e t i m e c o u r s e o f i n c o r p o r a t i o n is linear for at least t h e first h o u r a n d is also directly d e p e n d a n t o n the amount of broken zooxanthellar suspension used. S u b s t r a t e r e q u i r e m e n t s for t h e i n c o r p o r a t i o n a r e listed in T a b l e 3. It is e v i d e n t t h a t labeled f a r n e s y l p y r o p h o s p h a t e , e i t h e r in t h e m i x t u r e u s e d in its

Table 4. Representative derivatizations of squalene with demonstration of constant specific radioactivity Recrystallizations of hexahydrochloride Exp A m p Counts/min/mg 1 109-I 10 21.2 2 108-1 l0 14.0 3 109-1 il 18.0 4 109-111 17.0 Exp B 1 107 110 2107 111 3111 113 4109 iII 5 111-112

27.0 25.0 22.6 24.8 23.8

Regenerations of squalene from thiourea adduct Exp C Counts/min/mg 1 37.5 2 33.8 3 35.2 Exp D 1 2 3

40.5 42.4 41.3

Exps. A and C present derivatization of squalene derived from incubations of P. porosa zooxanthellae in the standard system employing medium I. Exps. B and D represent derivatization of squalene derived from incubation of P. americana zooxanthellae broken in Medium IV supplemented with 2% PVP-40 and resuspended following breakage in Medium IV. See "Materials and Methods' for details of the derivatizations.

f o r m a t i o n o r c h r o m a t o g r a p h i c a l l y purified, is req u i r e d . A l s o r e q u i r e d is a n N A D P H - g e n e r a t i n g syst e m . N A D P H or N A D H s u b s t i t u t e , b u t a n N A D H generating system with alcohol dehydrogenase and e t h a n o l is ineffective for u n k n o w n r e a s o n s . T h e e x p e c t a t i o n o f t h e r e q u i r e m e n t for a n a n a e r o b i c s y s t e m is n o t b o r n e out. T y p i c a l l y , t h e c r u d e s y s t e m s s u c h as this, s q u a l e n e f o r m a t i o n is r a p i d l y f o l l o w e d by its c y c l i z a t i o n to f o r m t h e sterol n u c l e u s , c a t a l y z e d by m i x e d f u n c t i o n o x i d a s e s r e q u i r i n g b o t h N A D P H a n d 0 2 . W e find t h e effect o f o x y g e n (air) to be m i n i m a l in t h e s e s y s t e m s , p r o b a b l y c o n s e q u e n t to t h e effective a b s e n c e o f s u c h activities. H o w e v e r , r e s u l t s d i d a p p e a r less v a r i a b l e e m p l o y i n g a n a e r o b i c c o n d i t i o n s a n d this c o n d i t i o n w a s a d a p t e d as r o u t i n e . J u s t i f i c a t i o n for t h e u s e o f t h e s y s t e m a d o p t e d m a y be f o u n d in t h e e x c e l l e n t r e p r o d u c i b i l i t y d e m o n s t r a t e d in T a b l e 2. Proof that the hydrocarbon produced was indeed s q u a l e n e is p r e s e n t e d in T a b l e 4. C o n s t a n t specific radioactivity of the hexahydrochloride derivative f r o m s q u a l e n e f o r m e d in selected e x p e r i m e n t s is a p p a r e n t . H o w e v e r , since p o s s i b l e r a d i o a c t i v e c o n -

Table 3. Characterization of the system for squalene formation by zooxanthellar preparations. Counts/min in Incubation system squalene Complete system 3320 Purified farnesyl pyrophosphate (FPP) replaces pre-incubation mixture 2940 Components of FPP-forming system without enzyme replaces FPP 12 Zooxanthellae preparation boiled 0 Minus NADP, NADPH-generating system present 51 Minus NADPH generating system, NADP present 140 NADPH replaces NADP and NADPH-generating system 3410 NADH replaces NADP and NADPH-generating system 1430 Air replaces N 2 in gas phase 2360 Volume of zooxanthellae preparation reduced as indicated 0.05 ml 622 0.10ml 1070 0.15ml 1340 Term of incubation reduced as indicated 20 min 340 40 min 1110 60 min 1570 The standard system consists of [~4C] farnesyl pyrophosphate (a preincubation mix), NADP, an NADPH-generation system, broken zooxanthellae from P. porosa equivalent to 0.25 ml of packed ceils, suspended in 1~0 ml of breaking medium I and an antibiotic mix. See 'Materials and Methods" for details. N 2 replaces air in the gas phase. Where indicated, in the absence of a reduced pyridine nucleotide generating system the reduced nucleotide was added at 0.5 mM.

Squalene formation by zooxanthellae taminants include hydrochlorides of cyclic unsaturated sesquiterpenes which might coprecipitate with squalene hexahydrochloride, a second derivative, the thiourea clathrate (adduct) was also employed. Clathrate formation depends on the linear nature of all-trans-squalene (Langdon and Bloch, 1953), not found in cyclic sesquiterpenes. Since clathrate formation is basically a purification technique and required the addition of carrier squalene, we report specific radioactivity of the regenerated squalene as a criterion of identification and purity. In subsequent experiments in which major changes were made in the zooxanthellar preparation or in the conditions of incubation, the product identification was validated by clathrate formation. The system most active in converting [14C]farnesyl pyrophosphate into squalene has been derived from the zooxanthellae of P. americana. They must be isolated as soon after collection of the colony as possible. Specimens collected near the peak of the incoming tide appear to provide more readily isolated zooxanthellae (see Methods), which in turn provide more active preparations. These tentative conclusions have not been critically examined. Isolation of P. americana zooxanthellae requires a liquefying (mucolytic) agent in order to combat the overwhelming viscosity of the homogenate. Among the variety of agents tested, acetamide, urea, streptokinase, Maraspers and N-acetyl cysteine, only the latter provides successful dispersion of the homogenate. As a breaking medium for these zooxanthellae, phosphate medium (IV) supplemented with 2% PVP40, a soluble polyvinylpyrrolidone, proved most effective in terms of squalene formation. PVP-40 was absent from the resuspending medium. Controlled comparison of broken cell preparations of zooxanthellae from P. porosa and P. americana collected and prepared on the same day showed the latter at least 60-fold more efficient in incorporation of label into squalene, 19,400 compared with 390 cpm. Subsequent experiments routinely employed P. americana zooxathellae. The dependence of squalene formation on the presence of a source of reduced pyridine nucleotide, indicated in Table 3, is further elucidated in Fig. 2 which presents results of the examination of the concentration dependence on both NADPH and NADH for the incorporation of [14C]farnesyl pyrophosphate into squalene. Incorporation into squalene is strictly dependant on and linear in the range shown with NADPH concentration. That the points generate a straight line is a further validation of the assay. NADH is almost as effective a source of reducing equivalents required for squalene formation. At concentrations higher than shown, 0.5mM for both NADPH and NADH, NADPH causes a decrease in incorporation (8800 counts/min) while stimulation by NADH is greater (13,400 counts/min). As indicated above, validation of the product as squalene in each of the above incubations was attained by thiourea 'clathrate formation. In each case, at least 90% of the chromatographically isolated radioactivity was present in squalene by this criterion. In this as well as earlier experiments, that part of radioactivity in the petroleum ether extract which is present in squalene represents at most some 10-20%

427

10.0

i; 5.0

g l.O

0.05 0.10 0.15 0 . 2 0 0.25 Pyridine Nucleotide Concentration, mM

Fig. 2. Dependence of squalene formation on concentrations of NADPH and NADH. ×, NADPH; O, NADH The system consisted of purified P. americana zooxanthellae broken in Medium IV supplemented with 2% PVP-40 and resuspended and incubated in Medium IV following centrifugation at 27,000 × g for 30 min. Indicated concentrations of NADPH or NADH substituted for the NADPHgenerating system of the standard incubation which is detailed in 'Materials and Methods'.

of the total extracted into petroleum ether. In the standard chromatography on alumina, at least three separate peaks in addition to squalene appear later in the elution pattern. While none of these peaks has been definitively identified, it is also the case that none of them show any dependence on pyridine nucleotide presence or concentration. For this reason they are probably unrelated to squalene production or to its cyclization into the sterol nucleus since both processes require the participation of a reduced pyridine nucleotide.

DISCUSSION The discovery of squalene production by broken cell preparations of zooxanthellae was a byproduct of the search for a cell-free system capable of forming the sesquiterpene hydrocarbons abundant in many gorgonians (Ciereszko and Karnes, 1973). The incidental finding of the formation of hydrocarbon(s) with chromatographic characteristics different to those of the sesquiterpenes, an interest in sterol, especially gorgosterol, biosynthesis and preliminary gas-chromatographic evidence that the unsaponifiable lipids of zooxanthellae include squalene, led us to a more directed search for squalene formation. This was accomplished by including a NADPH-generating system in the survey, since squalene formation typically requires a reduced pyridine nucleotide. In the presence of 02, such a system would also allow for sterol synthesis, with consequent depletion of squalene, hence the survey routinely employed an N 2 gas phase. This precaution proved unnecessary since none of our zooxanthellae preparations were capable of cyclizing squalene to digitonin precipitable sterols. Before successful use of the Hughes Press, a variety of cell breaking systems provided zooxanthellae prep-

428

DAVID G. ANDERSON

arations with microscopic evidence of cell damage, but none that were active in converting the labeled farnesyl pyrophosphate supplied as substrate into a hydrocarbon, although some breaking systems brought about the apparent release of a phosphatase since free farnesyl, chromatographically identified, was produced in the survey incubation. Only cell breakage in a variety of media with the Hughes Press supplied a preparation capable of squalene formation from farnesyl pyrophosphate. We prefer to term this a broken cell rather than a cell-free preparation since the active low-speed precipitate consists largely of zooxanthellae which show but slight indications of damage on microscopic examination. Other breaking procedures such as the French Press are more successful in providing a cell-free preparation, but these are inactive in both squalene and sterol formation. One impetus for this publication is our continued inability to develop a gorgonian-derived system capable of converting labeled acetate, mevalonate, farnesyl pyrophosphate or squalene into any digitoninprecipitable sterol. We have tested more than a dozen cell-breaking techniques, tried more than twenty breaking media, a variety of mixes of gorgonian and zooxanthellae preparations, and supplementation with a number of additional possible substrates and co-factors, all with no success. We have now abandoned any systematic attempt to find a preparation capable of forming gorgosterol, despite the lure of this sterol's biosynthetic uniqueness. The results reported here demonstrate that squalene can be formed from labeled farnesyl pyrophosphate by zooxanthellar systems derived from P. porosa, other pseudoplexaurids and particularly from zooxanthellae derived from P. americana, which can form squalene in 20~o yield from the radioactive substrate supplied. Taken in conjunction with previous results showing the zooxanthellar (P. porosa) location of the conversion of mevalonate into farnesyl pyrophosphate (Papastephanou and Anderson, 1982), we may conclude that zooxanthellae from P. porosa are capable of at least that part of the pathway from mevalonate to gorgosterol which encompasses the formation of squalene. The same conclusion probably holds for the zooxanthellae isolated from the other gorgonians listed, although the route from mevalonate to farnesyl pyrophosphate has not been demonstrated in such cases. The question of the symbiotic site of squalene cyclization and that product's eventual conversion into gorgosterol is still unanswered at least in terms of defined biochemical systems.

Acknowledgements--The technical acumen, diving skill and general enthusiasm of Carol Sadlek, the pilotage and patch reef knowledge of Clyde Kopp and the gorgonian identification capabilities of colleagues at the Rosenstiel School of Marine and Atmospheric Sciences of the University of Miami are all gratefully acknowledged as is support by grant GM-12229 from the General Medical Institute of the National Institutes of Health.

REFERENCES

Bergmann W., McMean M. J. and Lester D. (1943) Contributions to the studies of marine products. XII1. Sterols from various marine invertebrates. J. org. Chem. 8, 271 282. Ciereszko L. S. and Karnes T. B. (1973) Comparative biochemistry of coral reef coelenterates. In Biology and Geology of Coral Reefs. Vol. 2, Biology I (Edited by Jones O. A. and Endean R.), pp. 183-203. Academic Press, New York. Ciereszko L. S., Mizelle J. W. and Schmidt R. W. (1972) Occurrence of taurobetaine in coelenterates and of polysaccharide sulfate in the gorgonian Pseudopterogorgia americana. In Food-Drugs from the Sea, Proe. 1972 (Edited by Worthen L. R.), pp. 177-179. Marine Technology Society, Washington D.C. Hale R. L., Leclercq J., Tursch B., Djerassi C., Gross R. A., Jr, Weinheimer A. J., Gupta K. C. and Scheuer P. J. (1970) Demonstration of a biogenetically unprecedented side chain in the marine sterol, gorgosterol. J. Am. Chem. Soc. 92, 2179-2180. Kokke W. C. M. C., Fenical W., Bohlin L. and Djerassi C. (1981) Sterol synthesis by cultured zooxanthellae: implications concerning sterol metabolism in the hostsymbiont association in Caribbean gorgonians. Comp. Biochem. Physiol. 68B, 281-287. Kokke W. C. M. C., Fenical W., Bohlin L. and Djerassi C. (1982) Novel dinoflagellate 4ct-methylated sterols from four Caribbean gorgonians. Phytochemistry 21,881-887. Kokke W. C. M. C., Withers N. W., Massey I. J., Fenical W. and Djerassi C. (1979) Isolation and synthesis of 23-methyl-22-dehydrocholesterol--a marine sterol of biosynthetic significance. Tet. Letts. 3601-3604. Langdon R. G. and Bloch K. (1953) The biosynthesis of squalene. J. biol. Chem. 200, 129-134. Ling N. C., Hale R. C. and Djerassi C. (1970) The structure and absolute configuration of the marine sterol gorgosterol. J. Am. Chem. Soc. 92, 5281 5282. Loomis W. D. and Battaile J. (1966) Plant phenolic compounds and isolation of plant enzymes. Phytochem. 5, 423~,38. Papastephanou C. and Anderson D. G. (1982) Crassin acetate biosynthesis in a cell-free homogenate of zooxanthellae from Pseudoplexaura porosa (Houttyun); implications to the symbiotic process. Comp. Biochem. Physiol. 73B, 617-624. Rabourn W. J., Quackenbush F. W. and Porter J. W. (1954) Isolation and properties of phytoene. Arch. Biochem. Biophys. 48, 267-274. Rice J. R., Papastephanou C. and Anderson D. G. (1970) Isolation, localization and biosynthesis of crassin acetate in Pseudoplexura porosa (Houttyn). Biol. Bull. 138, 334-343. Sheffner A. L. (1963) The reduction in vitro in viscosity of mucoprotein solutions by a new mucolytic agent, Nacetyl-L-cysteine. Ann. N.Y. Acad. Sci. 106, 298-310. Withers N. W., Kokke W. C. M. C., Kohmer M., Fenical W. and Djerassi C. (1979a) Isolation of sterols with cyclopropyl-containing side chains from the cultured marine algae Peridinium foliaceum. Tet. Letts. 3605-3608. Withers N. W., Tuttle R. C., Goad J. L. and Goodwin T. W. (1979b) Dinosterol side chain biosynthesis in the marine dinoflagellate, Cryptothecodinium cohnii. Phytochemistry 18, 71-73. Withers N. W., Kokke W. C. M. C., Fenical W. and Djerassi C. (1982) Sterol patterns of cultured zooxanthellae isolated from marine invertebrates: synthesis of gorgosterol and 23-desmethylgorgosterol by aposymbiotic algae. Proc. natn. Acad. Sei. USA 79, 3764-3768.