262

Biochimica et Biophysica Acta, 544 ( 1 9 7 8 ) 2 6 2 - - 2 7 2 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 28717

MODULATION OF ADENYLATE CYCLASE ACTIVITY BY SULFATED GLYCOSAMINOGLYCANS I. INHIBITION BY HEPARIN OF GONADOTROPINSTIMULATED O V A R I A N A D E N Y L A T E CYCLASE

YORAM SALOMON, YEHUDITH AMIR, RIKI AZULAI and ABRAHAM AMSTERDAM

Department o f Hormone Research, The Weizmann Institute o f Science, Rehovot (Israel) ( R e c e i v e d April 21st, 1 9 7 8 )

Summary Heparin inhibits (/so = 2 pg/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5'-(~,~/-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by heparin (Is0 = 6 gg/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was n o t eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Heparin (3 ~g/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did n o t change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged heparin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.

Introduction

Heparin and various sulfated mucopolysaccharides have been shown to be widely distributed in b o d y fluids, cells and tissues [1,2]. These highly charged macromolecules have been proposed to be lubricants of cell surfaces [1] and Abbreviations: p(NH)ppG, guanosine 5t-(~,7-imido)triphosphate; hCG, human chorionic gonadotropin; LH, luteinizing hormone; PMSG, pregnant mare serum gonadotropin; TSH, thyroid stimulating hormone.

263 to play a role in the initiation and control of cell division [3,4]. Heparin is best known as an anticoagulant, inhibiting the process of blood clotting (for review see ref. 1). Furthermore, heparin has been shown to stimulate the activity of lipoprotein lipase and as such has an antilipemic activity [1,5]. Various enzymes [6], such as acid phosphatase [7], ribonuclease [8], a-amylase [9], fumarase [10] and hormone-independent adenylate cyclase from lung alveolar tissue [11] have been shown to be inhibited by heparin, but the biological significance of these findings has n o t been established. We have shown that heparin-like substances are synthesized and accumulate in the rat ovary [12--14]. In search of a functional role for ovarian glycosaminoglycans we recently observed an inhibitory action of these substances on the activity of luteinizing hormone- and human chorionic gonadotropin-stimulated adenylate cyclase [15]. The mechanism by which heparin affects hormonal stimulation of ovarian adenylate cyclase was therefore studied. It will be shown that this action of heparin is associated in part with an interference with the binding of the hormone to its specific receptor. It is postulated that heparin may restrict the mobility of cell membrane components or affect their conformational state and thus interfere with the process of receptor enzyme coupling. Materials and Methods

Materials. [a-32p]ATP, 3',5'-cyclic [8-3H]AMP and Na'2SI (carrier free)were purchased from the Radiochemical Centre, Amersham, U.K. Guanosine-5'-(~,7imido)triphosphate (p[NH]ppG) from International Chemical Corp. ATP, GTP, 3',5'-cyclic AMP, phosphocreatine, creatine phosphokinase, sodium fluoride, bovine serum albumin and sodium heparin grade, I, 162 units/mg were products of Sigma Chemical Co. Ovine luteinizing hormone (oLH, NIH-LHS18; specific potency 1.03 × NIH-LH-S1 by ovarian ascorbic acid depletion test) was kindly supplied by the U.S. National Institutes of Health. Pregnant mare serum gonadotropin (PMSG; Gestyl, N.V.) and human chorionic gonadotropin (hCG: Pregnyl N.V.) were purchased from Organon, Oss, The Netherlands. Highly purified hCG (12 700 I.U./mg) for radioiodination was a product of Serono, Rome, generously made available through Dr. Aliza Eshkol. All other chemicals and solvents were of analytical grade. Animals. Wistar-derived female rats from the departmental colony were housed in air-conditioned quarters (23°C) with 14 h light per day and allowed free access to pelleted food (Purina Chow; Ralston Purina Co., St. Louis) and water. Tissue preparation and purification of plasma membranes. Hyperstimulated ovaries were obtained 48 h after administration of PMSG (15 I.U.) as described previously [ 16]. Ovarian plasma membranes were purified according to Neville [17] as modified by Mintz et al. [18]. Adenylate cyclase. ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1) was assayed by measuring the formation of cyclic [32p]AMP from [a32p]ATP. Unless otherwise indicated, incubation mixture contained the following in a final volume of 50 pl: 25 mM Tris acetate, pH 7.6; 5 mM magnesium acetate; 0.5 mM [a-32p]ATP (2--6 • 106 cpm); 0.05 mM cyclic AMP; 0.01 mM GTP; 1 mM dithiothreitol; 5 mM creatine phosphate; 50 units/ml creatine phosphokinase;

264 bovine serum albumin, 0.1 mg/ml. Where indicated, the following stimulants were added; 0.4 nM--0.1 gM LH, 10 pM p(NH)ppG and 10 mM NaF. The reaction was initiated by the addition of purified ovarian plasma membranes (2--5 ~g protein} and terminated after 15 min incubation at 30°C by adding 100 pl of a stopping solution as described earlier [18]. Cyclic [8-3H]AMP {approx. 15 000 cpm) in 50/11 buffer was added as recovery standard and the tubes were placed in a boiling water bath for 3 min. Assay blanks containing no enzyme were processed similarly. Blank values were 1--5 cpm per 106 cpm of substrate added to the incubation medium. Isolation of the labelled cyclic [32p] AMP was done according to Salomon et al. [19]. Recovery of cyclic [32P]AMP was 75-85%. Counting was performed in a 30% Triton/toluene based scintillation fluid mixture using a Packard Tricarb spectrometer. Enzyme assays were done in duplicates; results of these differed by less than 5% from their mean. Basal activity was measured in the absence of any stimulants. The activity determined in the presence of hormone is termed "hormone stimulated activity". The net increment in enzyme activity due to the hormone is therefore obtained by subtracting the value obtained in its presence from that obtained in its absence. Inhibition of hormone stimulation is expressed as a percentage of the net increment only. Iodination of hCG and binding of ~2SI-labeled hCG to ovarian plasma membranes were as described previously [16]. Results

The selective effect of heparin on hormone-dependent adenylate cyclase activity Adenylate cyclase activity was determined in purified ovarian plasma membranes. The addition of 0.1 pM LH to the assay medium increased enzyme activity (pmol cyclic AMP/15 min per mg protein) from 400 (basal) to 2250. NaF increased enzyme activity to 1100 only (Fig. 1). Enzyme activity was

I

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LH

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NaF

I

I

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X X

0

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~-0

?

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I

?

I0

20

30

40

Heparin [~q/ml ]

Fig. 1. I n h i b i t i o n

of LH sensitive adenylate cyclase by heparin. Adenylate cyclase activity was determined as d e s c r i b e d u n d e r M e t h o d s i n t h e p r e s e n c e o f 1 0 m M N a F o r 0 . 1 / ~ M o L H a n d i n t h e p r e s e n c e o f h e p a r i n at the indicated concentrations. Each assay system contained 3.9 ~g of purified ovarian plasma membranes.

265 TABLE I THE EFFECT CONDITIONS

OF HEPARIN

ON OVARIAN

ADENYLATE

CYCLASE UNDER

VARIOUS

ASSAY

A d e n y l a t e c y c l a s e a c t i v i t y w a s d e t e r m i n e d in t h e p r e s e n c e o f s t i m u l a n t s as d e s c r i b e d in M e t h o d s ; h o w ever, G R P w a s o m i t t e d f r o m t h e basic assay m i x t u r e s . T h e c o n c e n t r a t i o n o f LH and h C G w a s 1 0 n M , G T P or p ( N H ) p p G w a s 1 0 p M and o f N a F , 1 0 r a M . E a c h a s s a y s y s t e m c o n t a i n e d 3 ~g o f p l a s m a m e m b r a n e protein.

Additions

A d e n y l a t e c y c l a s e a c t i v i t y ( c y c l i c A M P , p m o l / 1 5 m i n per m g p r o t e i n )

No heparin

Heparin (10 #g/ml)

I n h i b i t i o n (%)

None GTP p(NH)ppG NaF

179 431 966 1473

84 152 254 1070

43 73 78 24

LH LH + GTP LH + p(NH)ppG

526 2095 2158

91 263 411

98 93 87

hCG hCG + GTP hCG + p(NH)ppG

639 2070 2326

104 378 599

96 86 74

inhibited when 1--40 gg/ml heparin was included in the assay medium. The effect of heparin was more pronounced when the enzyme was stimulated by the hormone, showing a maximal inhibition (92%) at a heparin concentration of 10 ~g/ml. In contrast, the inhibitory effect of heparin was small (15--24%) when enzyme activity was stimulated by NaF (Fig. 1, see also Table I). In the absence of stimulants (basal activity) maximal inhibition by heparin was approx. 50%. The dose-dependent inhibition under all conditions was in the range of 1--10 ~g heparin/ml. No further change was observed when heparin concentration was further increased to 40 ~g/ml. In experiments reported in this study (Figs. 1--6), adenylate cyclase activity was determined in the presence of GTP. It was of interest to determine the effect of heparin on enzyme activity in the absence of GTP or in the presence of its analog p(NH)ppG (Table I). Enzyme activity stimulated by either LH or hCG in the absence of added guanosine nucleotide were nearly abolished in the presence of heparin (10 pg/ml). In the presence of guanosine nucleotide, hormone stimulated activity was somewhat more resistant to the inhibitory action of heparin. Adenylate cyclase stimulated by NaF in the absence of GTP was much more resistant to the action of heparin. As can be learned from Table I, enzyme activity stimulated by GTP or p(NH)ppG alone was more susceptible to inhibition by heparin where compared to activity stimulated by NaF or in the absence of any stimulant.

The inhibition o f hormone binding to ovarian plasma membranes by heparin Due to the rather selective effect of heparin on hormone-dependent adenylate cyclase activity it was of interest to examine whether heparin interferes with the interaction of the hormone with its specific receptor. This interaction

266

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Heparin /g/ml of h C G sensitive a d e n y l a t e

Fig, 2. I n h i b i t i o n c y c l a s e a n d 125 l-labeled h C G b i n d i n g b y h e p a r i n . A d e n y l a t e eyclase ( c A M P ) a c t i v i t y was d e t e r m i n e d u s i n g 4.7 # g m e m b r a n e p r o t e i n a n d h C G (3.2 nM). h C G b i n d i n g was d e t e r m i n e d using 1 0 ~ g m e m b r a n e p r o t e i n a n d 1 2 5 I - l a b e l e d h C G ( 3 1 . 6 f m o l ; 49 0 0 0 c p m ) , as described u n d e r Methods.

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Fig. 3. T h e e f f e c t of h e p a r i n o n a c t i v a t i o n o f r a t o v a r i a n a d e n y l a t e c y c l a s e at v a r y i n g L H c o n c e n t r a t i o n s , A d e n y l a t e c y c l a s e ( c A M P ) a c t i v i t y w a s d e t e r m i n e d in t h e p r e s e n c e of L H a t t h e i n d i c a t e d c o n c e n t r a t i o n w i t h ( o ) or w i t h o u t ( e ) h e p a r i n a t a final c o n c e n t r a t i o n o f 2 btg/ml. O v a r i a n p l a s m a m e m b r a n e p r o t e i n was 3.6 ~ g / a s s a y . All o t h e r c o n d i t i o n s w e r e as d e s c r i b e d u n d e r M e t h o d s . T h e a c t i v a t i o n c o n s t a n t ( K a ) refers to t h e a c t i v a t i o n o f a d e n y l a t e c y c l a s e b y L H in t h e a b s e n c e of h e p a r i n .

267

is thought to lead to enzyme activation. '2SIllabeled hCG binding was therefore determined in the presence and absence of increasing concentrations of heparin Fig. 2). It is shown that inhibition of hormone binding increases with increasing heparin concentrations, in a manner similar to that describing the inhibition of hCG stimulated adenylate cyclase activity. In the presence of 10 pg/ml heparin, binding of 12SI-labeled hCG was inhibited by 65% while hCG-stimulated adenylate cyclase activity was inhibited by 87%. We investigated further the relationship between hormone concentration and heparin concentration by determining the kinetics of the inhibitory effect of heparin in hormone binding and hormone stimulation of adenylate cyclase. In Fig. 3, adenylate cyclase activity was determined at varying LH concentrations, in the absence or presence of 2 pg/ml heparin. Heparin was found to exert an inhibitory effect of a mixed type when data were plotted according to Lineweaver and Burk [20]. It can be seen that inhibition by heparin could not be overcome by excessive hormone concentrations. Furthermore, we also show that a similar relationship exists between the concentration of hormone and the degree of binding with respect to the inhibitory action of heparin (Fig. 4). Inhibition of '2SI-labeled hCG binding was also of a mixed type when plotted in a double reciprocal fashion [20]. Moreover, the inhibitory action of heparin cannot be overcome even at saturating hormone concentrations.

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Fig. 4. T h e e f f e c t o f h e p a r i n o n the b i n d i n g o f 1 2 S I - l a b e l e d hCG to ovarian p l a s m a m e m b r a n e s . Binding o f 12 S I - l a b e l e d h C G w a s d e t e r m i n e d in the • b s e n c e o f h e p a r i n ( e ) or in the P r e s e n c e o f 1 2 / ~ g / m l h e p a r i n (o) at v a r y i n g c o n c e n t r a t i o n s o f h C G as i n d i c a t e d . Each assay s y s t e m c o n t a i n e d 1 0 # g o f m e m b r a n e protein. All o t h e r details w e z e as d e s c r i b e d in t h e M e t h o d s s e c t i o n . I n d i c • t e d in t h e figure are the values calc u l a t e d for m a x i m a l 12 S I - l a b e l e d h C G b i n d i n g ( B m a x ) and the d i s s o c i a t i o n c o n s t a n t ( K D ) o b t a i n e d in the a b s e n c e o f heparin.

268 2000

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Time (rain.) Fig. 5. T h e e f f e c t o f s e q u e n t i a l a d d i t i o n of LH a n d h e p a r i n o n t h e i n h i b i t o r y e f f e c t o f h e p a r i n o n a d e n y late c y c l a s e a c t i v i t y . Basal e n z y m e a c t i v i t y ( o ) w a s d e t e r m i n e d i n t h e p r e s e n c e o f h e p a r i n ( H e p ) f r o m z e r o t i m e ( a ) o r in t h e p r e s e n c e o f h e p a r i n a d d e d at 7 rain a f t e r i n i t i a t i o n o f t h e r e a c t i o n ( 6 ) . A d e r t v l a t e c y c l a s e a c t i v i t y in t h e p r e s e n c e of LH (o) w a s d e t e r m i n e d in t h e p r e s e n c e o f h e p a r i n f r o m z e r o t i m e (*), a n d in t h e p r e s e n c e o f h e p a r i n a d d e d at 7 rain (0). LH w a s a d d e d a t 7 rain t o a n assay s y s t e m in w h i c h h e p a r i n w a s i n c l u d e d at t i m e z e r o (A) a n d t o a n assay s y s t e m c o n t a i n i n g n o s t i m u l a n t s (basal) (m). LH c o n c e n t r a t i o n w h e n a d d e d w a s 0.1 pM, h e p a r i n w a s 3 p g / m l a n d m e m b r a n e c o n c e n t r a t i o n w a s 3 ~g/ml. A d e n y l a t e c y c l a s e a c t i v i t y w a s m e a s u r e d as d e s c r i b e d u n d e r " M e t h o d s " b y s a m p l i n g 50 pl aliquots at t h e i n d i c a t e d t i m e s o u t o f a n assay s y s t e m c o n t a i n i n g initially 8 t i m e s 50-pl volume.

Is heparin's action an "ordered" one? In all the previously described experiments the enzymic reaction was initiated by adding the enzyme into a preformed mixture which already contained heparin in addition to the appropriate ingredients and hormone. We therefore carried out the experiment described in Fig. 5, in which some possible sequences of addition were tested. The rate of cyclic AMP synthesis was determined in assay systems in which heparin was present from zero time with or without LH. In addition LH was added after initiation of the reaction (7 min) to an assay system in which heparin was present from zero time. Heparin was also added at the same time (7 min) to an assay system which was previously challenged with LH at zero time. In all the combinations tested, the degree of inhibition obtained with heparin 3 pg/ml was 60%. It can also be seen from Fig. 5 that the effect of heparin is fast and occurs within 2 min, the shortest time interval tested. Control experiments in which LH or heparin were added to the assay systems on their own after initiation of the reaction (7 min) show that the responsiveness of adenylate cyclase to LH and heparin was not changed during this time interval.

269

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T

T

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A

I- Heparin +-~--" A • o

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Basal

--T

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5.5

6.0

6.5

?.0 pH

7.5

8.0

8.5

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, 2

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[Mg ÷÷] mM

Fig. 6. T h e e f f e c t o f heparin on the a c t i v i t y o f rat ovarian a d e n y l a t e c y c l a s e in the p r e s e n c e o f LH or h C G at v a r y i n g p H and m a g n e s i u m i o n c o n c e n t r a t i o n . A d e n y l a t e c y c l a s e activity w a s d e t e r r a i n e d in the a b s e n c e ( c l o s e d s y m b o l s ) or in t h e p r e s e n c e o f 2 # g / m ] hepaxin ( o p e n s y m b o l s ) . T h e c o n c e n t r a t i o n s o f LH and h C G w e r e 10 nM and e a c h assay t u b e c o n t a i n e d 4 # g o f m e m b r a n e p r o t e i n .

The effect of pH and magnesium ion concentration on adenylate cyclase activity in the presence of heparin Due to the fact that heparin is a highly charged polyanion at the pH of the reaction (pH =:7.5) it was logical to test whether its inhibitory action is a result of a possible shift in the pH optimum for adenylate cyclase activity. As seen in Fig. 6A, the activity of adenylate cyclase in presence of hormone is optimal at pH 7.0 while basal activity is rather insensitive to the change in pH in the range 5.9--8.3. The a~dition of heparin to the reaction mixture inhibited the adenylate cyclase activity in the presence or absence of LH, but had no effect on the general pattern' pf the activity vs. pH relationship. The optimum pH for LH stimulation was unaffected by the presence of heparin. However, the degree of inhibition of the LH stimulated activity seems to be somewhat lower at pH 5.9 than at the higher pH values. Increasing the concentration of magnesium ions in the reaction mixture from 1 to 100 mM has a biphasic effect on LH stimulated activity, showing an optim u m at a magnesium concentration of 5 mM. As seen in Fig. 6B, the addition of heparin did riot affect this optimal value for magnesium concentrations, thus suggesting that the mechanism which underlies the inhibitory action of heparin does not involve a change in the relationship of enzyme activity with magnesium ions.

270

Discussion In this study, and in other reports [15,21], we demonstrate that adenylate cyclase activity is inhibited by heparin. Heparin exerted a similar and pronounced effect on adenylate cyclase activity stimulated by LH or hCG, and had a weaker effect on hormone-independent activity measured in the absence of stimulants (basal) or in the presence of NaF. Interestingly, activity stimulated by NaF was more resistant to inhibition by heparin than was activity stimulated by either GTP or p(NH)ppG. Stimulation by all of these agents is believed to occur by a hormone receptor-independent mechanism [22,23]. In line with these findings are the observations by Wolff and Cook [24] who demonstrated inhibitory effects of dextran sulfate on thyroid-stimulating hormone-sensitive adenylate cyclase from bovine thyroid. Furthermore, the stimulatory effect exerted by polycations such as ribonuclease was also shown to be small when tested on basal or NaF stimulated activity, but was found to be larger when adenylate cyclase was stimulated by the hormone (TSH). These investigators suggested that polyions exert their effects by a general electrostatic perturbation of the conformation of the membrane. Heparin may conceivably exert its effect on ovarian adenylate cyclase by interfering at three levels; (a) the hormone receptor; (b) the catalytic subunit of the enzyme; or (c) the coupling between these two functions. As shown in this report, heparin interferes mainly with hormonal regulation of enzyme activity, as demonstrated by its inhibitory effect on the binding of hormone to specific receptor sites in this membrane preparation. However, hormone binding activity was less sensitive to inhibition (Is0 = 6 pg/ml) than was the adenylate cyclase activity stimulated by hCG (/s0 = 2 pg/ml). It is therefore concluded that inhibition of hormone stimulated activity can n o t be attributed solely to the interference of heparin with hormone binding. In addition, an effect exerted on the catalytic c o m p o n e n t and possibly interference with coupling between receptor to enzyme should be considered. According to the fluid mosaic model for biological membranes proposed by Singer and Nicolson [25], one could postulate that enzyme receptor coupling involves lateral mobility of these components within the membrane. This has recently been suggested by the studies of Orly and Schramm for the catecholamine receptor [26]. It is likely, therefore, that by adsorbing to opposite charges on the plasma membrane, heparin might restrict the mobility of receptor and enzyme, and thus interfere with their normal function. Alternatively, perturbation of receptor enzyme coupling might result from conformational changes induced by their interaction with heparin. Interestingly, heparin was effective to the same extent regardless of whether it was added to the reaction mixture simultaneously with the hormone, or after the enzyme was already stimulated by the hormone. Furthermore, preincubation of the membranes with heparin prior to the addition of LH did not affect the degree of inhibition achieved (Fig. 5). The inhibition by heparin is fully expressed within 2 min, the shortest time interval tested. Enzyme activity in the presence of heparin was linear and extrapolated to the time of addition, suggesting that the effect of heparin is instantaneous (Fig. 5}. The inhibition by heparin of adenylate cyclase activity was of a " m i x e d "

271 type as shown by determining its effect in the presence of increasing concentrations of hormone up to 6 nM (Fig. 3). Substantial inhibition by heparin was also evident at higher LH concentrations (i.e., 0.1 gM). Heparin 2 ~g/ml inhibited approx. 50% of LH stimulated adenylate cyclase activity using either 6 nM LH (Fig. 3) or as high as 100 nM (Figs. 1 and 6). It seems unlikely, therefore, that heparin exerts its inhibitory effect by complexing with free hormone and thus neutralizing its activity. Nevertheless such an interaction cannot be ruled out and may thus contribute to some extent to the observed effects of heparin. As shown by us elsewhere [15,21], the effect of heparin is a general phenomenon observed with several adenylate cyclase systems in which regulation is exploited by hormones of a diverse chemical nature, i.e., catecholamines, prostaglandins, peptide hormones and gonadotropins. Despite the suggested electrostatic interaction of heparin with the adenylate cyclase system, it was interesting to find that these do not affect the pH vs. activity relationship or the dependence of enzyme activity on Mg2÷ concentration. The possibility exists that access of the hormone and substrate to the relevant domains in the adenylate cyclase system might be limited due to shielding by heparin or due to heparin-induced formation of membrane aggregates. This possibility could not account for the inhibitory effects of heparin on adenylate cyclase activity for the following reasons: (1) Heparin inhibits stimulation by hormone and NaF to a different degree; (2) the degree of inhibition was independent of the order at which heparin and hormone were introduced into the assay system; (3)as shown elsewhere [21], heparin stimulated dopamine-sensitive brain adenylate cyclase at a concentration range similar to that used in this study. Heparin-like substances have recently been identified by us in the rat ovary [ 12--14]. Therefore, their possible role in the modulation of adenylate cyclase activity in ovulation related events should be considered. Acknowledgments We thank Drs. H.R. Lindner, C. Londos and J. Wolff for helpful discussions. This work was supported in part by a grant to H.R.L. by the Ford Foundation and the Population Council, Inc., New York, and by grants to Y.S. and A.A. by the United States-Israel Binational Science Foundation, Jerusalem. Y.S. is the incumbent of the Charles W. and Tillie Lubin Career Development Chair. A.A. is the incumbent of the Gestetner Career Development Chair. We thank Mrs. Lea Zilberstein and Mrs. Millicent Kopelowitz for excellent secretarial assistance. References 1 Sharon, N. (1975) in Complex Carbohydrates, their Chemistry, Biosynthesis and Function, pp. 258-281, Addison-Wesley, L o n d o n 2 Jeanloz, R.W. (1970) in The Carbohydrates, Chemistry and Biochemistry (Pigman, W.E. and Horton, D,, eds.), IIB, pp. 590--619, Academic Press, New Y o r k 3 Chiarugi, V.P. and V a n n u c c h i , S. (1976) J. Theor. Biol. 6 1 , 4 5 9 - - 4 7 5 4 Lippman, M. (1965) Trans. N.Y. Acad. Sci. 2 7 , 3 4 2 - - 3 6 0

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