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Biochimica et Biophysica Acta, 428 (1976) 525--531 © E l s e v i e r S c i e n t i f i c Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27838

SUBCELLULAR LOCALIZATIONS OF GUANYLATE CYCLASE AND 3',5'-CYCLIC NUCLEOTIDE PHOSPHODIESTERASE IN SEA URCHIN SPERM

MAMORU SANO

Section of Cy tochemistry, Department of Morphology, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03 (Japan) (Received October 30th, 1975)

Summary The subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella {tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 80% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucleotide phosphodiesterase in sperm flagella were also briefly described.

Introduction

It has recently been suggested that cyclic GMP * may be involved in biological functions such as cell division [1,2] and cholinergic neurotransmission [3, 4], although its physiological significance has remained unrevealed. Guanylate cyclase, the enzyme which catalyzes the formation of cyclic GMP from GTP, has chiefly been studied on the soluble fractions of several mammalian tissues [5--12]. Gray et al. [13] reported that this enzyme activity in sea urchin * The abbreviations used axe: cyclic GMP, guanosine 3',5'omonophosphate; cyclic AMP, adenosine 3',5'-monophosphate.

526 sperm was localized in the particulate fraction and more than 1000 times higher than in any mammalian tissues so far examined. On the other hand it was demonstrated that acetylcholine enhanced the motility of sea urchin sperm in the presence of dimethylsulfoxide [ 1 4 ] , and that the respiration and motility of bovine epididymal spermatozoa were markedly stimulated by cyclic nucleotide and also by inhibitors of cyclic nucleotide phosphodiesterase such as caffeine and papaverine [15]. These observations suggest the possibility that cyclic GMP may participate in some physiological function of sea urchin sperm. In the present study it was attempted to elucidate the subcellular localization of guanylate cyclase as well as cyclic GMP phosphodiesterase in sea urchin sperm and briefly to examine the properties of these enzymes. Methods Materials [8-3H] GTP, ammonium salt, cyclic [8-3H] GMP, ammonium salt and cyclic [8-3H] AMP, a m m o n i u m salt, were purchased from the Radiochemical Centre. GTP (sodium salt), cyclic GMP, cyclic AMP and snake venom (King Cobra), were obtained from Sigma. Neutral aluminium oxide was purchased from M. Woelm. Other chemicals were obtained from commercial sources. Subcellular fractionation o f sea urchin sperm Sea urchin sperm was fractionated by a slight modification of the m e t h o d described by Hayashi and Higashi-Fugime [ 1 6 ] . The sperm was ejaculated from sea urchin, Hemicentrotus Purcherrimus by the injection of 0.5 M KC1 into the periviseral cavity. All subsequent manipulations were carried o u t at 0--4 ° C. After the sperm was homogenized with 10 vol. of sea water in a Potter-Elvehjem Teflon-glass homogenizer at low speed to detach the tails from the heads, the suspension was centrifuged for 5 min at 1000 × g to separate the heads as the precipitate. From the resultant supernatant the tails (flagella) were collected by centrifugation for 20 min at 10 000 × g. For the purification of the flagella resuspension and centrifugation were repeated until the heads were n o t detected in the suspension under a phase-contrast light microscope. Unless otherwise specified, the isolated flagella were suspended in TMK buffer (20 mM Tris • HC1 buffer, pH 8.0, containing 3 mM MgC12 and 30 mM KC1) together with 0.25% Triton X-100 for 30 min at 0°C, followed by centrifugation at 10 000 X g for 20 min. The resultant precipitate was resuspended in TMK buffer and designated as the axonemal fraction, while the supernatant was denoted as the plasma membrane fraction which consisted of plasma membrane and matrix protein [17,18]. E n z y m e assays Guanylate cyclase activity was assayed as described elsewhere [12]. Unless otherwise specified, the standard reaction mixture contained 60 nmol of [8-3H] GTP (0.2/zCi), 0.3/amol of cyclic GMP, 30 #tool of Tris • C1 buffer (pH 7.7) and 10 to 20 #g of enzyme protein in a total volume of 0.15 ml. The mixture was incubated at 30°C for 10 min. The reaction was terminated by boiling

527 in a water bath for 2 min. The enzymatically produced [3H]cyclic GMP was isolated by the serial use of a neutral aluminium oxide-Dowex l-X2 column, as described in the preceding paper [12] except that cyclic GMP was eluted from D o w e x 1-X2 (chloride form) column with 3 ml of 0.5 M HC1, after washing the D o w e x column with 10 ml of 0.05 M HCI. Cyclic nucleotide phosphodiesterase activity was assayed by measuring radioactive nucleoside derived from [3H]cyclic GMP or [3H]cyclic AMP in the presence of snake venom 5'-nucleotidase as described elsewhere [12]. The standard reaction mixture contained 40 nmol of cyclic [8-3H]GMP or cyclic [8-3H]AMP, 2 pmol of MgCl~, 1.5 pmol of 2-mercaptoethanol, 16 #mol of Tris • HC1 buffer (pH 8.0), and 1 to 2 pg of enzyme protein in a final volume of 0.4 ml. De terminations The radioactivity of 3H samples was determined as described elsewhere [ 1 2 ] . Protein was determined by the m e t h o d of L o w r y et al. [19] with bovine serum albumin as a standard. Results and Discussion

The enzymic formation and degradation of cyclic GMP in sea urchin sperm were examined. As shown in Table I, the specific activities of both guanylate cyclase and cyclic GMP phosphodiesterase were much higher in the flagella (tails) than in the heads, and more than 70% each of these t w o enzyme activities in the sperm were found in the flagella. In agreement with the observation by Gray et al. [15], the addition of 1% Triton X-100 to the assay mixture enhanced more than 10 times the cyclase activity in the flagella as well as in the heads, whereas such addition rather inhibited the phosphodiesterase activity. When aliquots of the flagellar suspension were incubated with Triton X-100 in various concentrations at 0°C for 30 min, followed by centrifugation at 10 000 × g for 20 min, as demonstrated in Fig. 1, almost all of the cyclase activity in the flagella was recovered as the supernatant fraction and the cyclase TABLE I E N Z Y M I C F O R M A T I O N A N D D E G R A D A T I O N O F C Y C L I C GMP I N S E A U R C H I N S P E R M T h e i s o l a t e d h e a d s a n d flagella f r o m 1 m l of d r y s p e r m w e r e f u r t h e r h o m o g e n i z e d w i t h T M K b u f f e r b y a P o l y t r o n P T 1 0 h o m o g e n i z e r f o r 2 rain a t m a x i m u m s p e e d t o use as e n z y m e s o u r c e . T h e s t a n d a r d a s s a y c o n d i t i o n s w e r e e m p l o y e d e x c e p t that T r i t o n X - 1 0 0 was a d d e d in a final c o n c e n t r a t i o n of 1%. O n e u n i t is defined as t h a t a m o u n t o f e n z y m e w h i c h p r o d u c e s o r h y d r o l y z e s 1 n m o l e o f cyclic G M P p e r rain under the s t a n d a r d a s s a y c o n d i t i o n s .

Protein * (mg)

Heads Flagella

13.0 4.38

Guanylate cyclase (units/rag protein)

Cyclic GMP p h o s p h o d i e s t a r a s e (units/mg protein)

No a d d i t i o n

1% T r i t o n X - 1 0 0

No a d d i t i o n

1% T r i t o n X - 1 0 0

8.0 ( 1 0 4 . 0 ) 65.2 (286.0)

6.0 ( 7 8 . 0 ) 48.0 (210.2)

3.4 ( 4 4 . 2 ) 42.4 (185.7)

0.79 ( 9 . 7 5 ) 4.06 (17.8)

**

* T h e v a l u e s i n d i c a t e t h e a m o u n t s of p r o t e i n r e c o v e r e d in e a c h f r a c t i o n f r o m 1 m l o f d r y s p e r m . ** The values in p a r e n t h e s e s indicate the t o t a l activities (units) in each f r a c t i o n .

528 T A B L E II DISTRIBUTIONS OF GUANYLATE SPERM FLAGELLA

CYCLASE AND CYCLIC GMP PHOSPHODIESTERASE

IN THE

T h e i s o l a t e d flagella f r o m 1 m l o f d r y s p e r m w e r e f r a c t i o n a t e d i n t o t h e a x o n e m a l a n d p l a s m a m e m b r a n e f r a c t i o n s , as d e s c r i b e d in " M e t h o d s " . T h e s t a n d a r d a s s a y c o n d i t i o n s w e r e e m p l o y e d e x c e p t t h a t 1% Trit o n X - 1 0 0 w a s a d d e d f o r t h e c y c l a s e a c t i v i t y . T h e d e f i n i t i o n o f u n i t is d e s c r i b e d in T a b l e I.

Subfraction

Protein * (mg)

Axoneme Plasma membrane

2.40 1.02

Guanylate cyclase (units/rag protein)

Cyclic G M P phosphodiesterase (units/mg protein)

Spec. act.

Total activity

Spec. act.

Total activity

17.7 216.0

42.5 220.3

36.7 72.3

88.0 73.7

* T h e v a l u e s i n d i c a t e t h e a m o u n t s of p r o t e i n r e c o v e r e d in e a c h s u b f r a c t i o n f r o m 1 m l o f d r y s p e r m .

activity itself was n o t so inhibited by the detergent in higher concentrations. On the other hand the phosphodiesterase was also partially solubilized by Triton X-100. However, this enzyme appeared to be inactivated by the treatm e n t with the detergent in higher concentration. The sperm flagella were fractionated into the axonemal and plasma membrane fractions, as described in Methods. It can be seen from Table II that the

A

E E

7C

2

I

f

,

+

v E 3C

Urn

°T 0

C) 1C i

05 110 % Concentration of TritonX-lOO(v/v)

0

0,1

i

i

i

i

i

i

i

0.5 Mg 2+ o r CQ2+ ( mM )

i

i

1.0

Fig. 1. E f f e c t s o f T r i t o n X - 1 0 0 o n g u a n y l a t e c y c l a s e a n d c y c l i c G M P p h o s p h o d i e s t e r a s e i n t h e s p e r m flagella. A l i q u o t s ( 2 . 2 m g p r o t e i n ) o f t h e f l a g e l i a r s u s p e n s i o n in T M K b u f f e r w e r e i n c u b a t e d f o r 30 r a i n a t 0 ° C w i t h T r i t o n X - 1 0 0 in 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 s , f o l l o w e d b y c e n t r i f u g a t i o n a t 1 0 0 0 0 X g f o r 20 m i n . T h e r e s u l t i n g s u p e r n a t a n t ( o ) a n d p e l l e t ( e ) w e r e u s e d as e n z y m e s o u r c e s f o r g u a n y l a t e c y c l a s e ( A ) a n d c y c l i c G M P p h o s p h o d i e s t e r a s e (B). T h e s t a n d a r d a s s a y c o n d i t i o n s w e r e e m p l o y e d e x c e p t t h a t t h e ass a y m i x t u r e f o r t h e c y c l a s e w a s a d d e d T r i t o n X - 1 0 0 in a final c o n c e n t r a t i o n o f 1%. T h e d e f i n i t i o n o f u n i t is d e s c r i b e d in T a b l e I. F i g . 2. S t i m u l a t o r y e f f e c t s o f C a 2÷ a n d M g 2÷ o n f l a g e l l a r g u a n y l a t e c y c l a s e i n t h e p r e s e n c e o f M n 2+. T h e standard assay conditions were employed except that different concentrations o f e i t h e r C a 2. ( e ) or M g 2. ( o ) w e r e a d d e d w i t h 0 . 1 m M M n 2+.

529 specific activity of the cyclase was much higher in the plasma membrane fraction than in the axonemal fraction, and that approx. 80% of the cyclase activity was observed in the former fraction. Although the specific activity of the phosphodiesterase in the plasma membrane fraction was higher than in the axonemal fraction, the total activity of this enzyme was rather higher in the latter fraction. To examine whether the cyclase in the plasma membrane fraction originates in the plasma membrane itself or in the matrix protein, the frozen flagella were subjected to such fractionation as described in Table III. Even after the frozen flagella were thawed and further homogenized with the Teflon-glass homogenizer, the cyclase was recovered as the precipitate, and more than 90% of the activity was solubilized by incubation with 0.25% Triton X-100. These observations indicated that the cyclase activity appeared to be associated with the plasma membrane, whereas the phosphodiesterase activity was observed in both the axonemal and plasma membrane fractions. Using the plasma membrane fraction from the sperm flagella, the enzymic properties of the flagellar guanylate cyclase were examined. The apparent Km value for GTP, determined at various concentrations (10 #M to 1 mM), was calculated from the Lineweaver-Burk plot to be 2.8 • 10 -4 M. The enzyme activity showed a pH o p t i m u m at pH 7.7, using Tris • C1 buffers with different pH values under the standard assay conditions. Mn 2÷ was absolutely required for the enzymic formation of cyclic GMP and the maximum activity was obtained at 1 m M M n 2+. Neither Ca 2+ nor M g 2÷ could substitute M n ~+. However, when either Ca 2+ or M g 2+ was added to the assay mixture with less than 0.5 m M M n 2+, these two divalent cations stimulated the enzyme activity (Fig. 2). In the presence of 0.1 m M Mn2+the addition of Ca2+(0.5 m M ) or M g 2+ (0.8 m M ) increased the for-

T A B L E III THE SOLUBILIZATION OF GUANYLATE CYCLASE FROM FROZEN SPERM FLAGELLA T h e f r o z e n flagenar s u s p e n s i o n in T M K b u f f e r was t h a w e d as r a p i d l y as possible, f o l l o w e d b y c e n t r i f u g a t i o n a t 1 0 0 0 0 X g f o r 20 rain. T h e s u p e r n a t a n t (1st s u p e r n a t a n t ) w a s d i s c a r d e d . T h e resulting p r e c i p i t a t e (1st p r e c i p i t a t e ) was h o m o g e n i z e d w i t h T M K b u f f e r in a P o t t e r - E l v e h j e m Teflon-glass h o m o g e n i z e r a n d c e n t r i f u g e d at 26 0 0 0 × g f o r 30 rain. T h e s u p e r n a t a n t ( 2 n d s u p e r n a t a n t ) was d i s c a r d e d . T h e r e s u l t i n g p r e c i p i t a t e ( 2 n d p r e c i p i t a t e ) w a s r e s u s p e n d e d in T M K b u f f e r a n d a l i q u o t s o f this s u s p e n s i o n w e r e a d d e d T r i t o n X - 1 0 0 in v a r i o u s c o n c e n t r a t i o n s as s h o w n in T a b l e . A f t e r i n c u b a t i o n at 0 ° C for 30 m i n , t h e supern a t a n t s ( 0 . 0 1 - - 0 . 2 5 % T r i t o n s u p e r n a t a n t ) w e r e o b t a i n e d b y c e n t r i f u g a t i o n a t 26 0 0 0 × g f o r 30 rain. E n z y m e a c t i v i t y was a s s a y e d u n d e r t h e s t a n d a r d assay c o n d i t i o n s , e x c e p t that the assay m i x t u r e cont a i n e d 1% T r i t o n X - 1 0 0 . T h e d e f i n i t i o n of u n i t is d e s c r i b e d in T a b l e I. Fraction

Protein (mg)

Guanylate cyclase activity (units)

1 st s u p e r n a t a n t 1 st P r e c i p i t a t e 2nd supernatant 2nd preciPitate

2.5 16.8 0.6 16.0

12.2 184.8 8.6 220.8

0.9 1.6 2.2

15.7 77.0 206.1

0.01% Triton supernatant 0.05% Triton supernatant 0.25% Triton supernatant

530 1Iv x 10

B 6"

4-

A 60

>,40

I / v x 10

(~

> 20

I

I

I Cyclic

I

I I 50 nucleotide

I (pM)

I

I

I 100

-2

-1

0

1 2 3 [ 1 / S ] x 10-5M

4

Fig. 3. K i n e t i c a n a l y s i s of c y c l i c G M P a n d c y c l i c A M P h y d r o l y s i s b y t h e a x o n e m a l a n d p l a s m a m e m b r a n e f r a c t i o n s o f s p e r m flagella. A , E f f e c t s of substrate c o n c e n t r a t i o n s o n c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e a c t i v i t y o f the a x o n e m a l ( o ~ o , cyclic GMP; z --, c y c l i c A M P ) a n d p l a s m a m e m b r a n e f r a c t i o u s (o o, c y c l i c G M P ; • • , c y c l i c A M P ) . One unit o f v e l o c i t y is d e f i n e d as t h a t a m o u n t o f e n z y m e w h i c h h y d r o l y z e s 1 n m o l e o f c y c l i c n u c l e o t i d e s per m i n p e r m g p r o t e i n . B,C, D o u b l e r e c i p r o c a l p l o t s o f c y c l i c G M P (o) a n d c y c l i c A M P ( e ) h y d r o l y s i s b y t h e a x o n e m a l (B) a n d p l a s m a m e m b r a n e f r a c t i o n s (C).

mation of cyclic GMP a b o u t 3-fold. These stimulatory effects of Ca2+and Mg :÷ in the presence of Mn 2+ were consistent with the observations reported by Garbers et al. [20]. Recently it has been demonstrated that the accumulation of cyclic GMP in smooth muscle by cholinergic agents [21] as well as in the slices of mouse cerebellum by depolarizing agents [22] is dependent on Ca 2+. These observations suggest the possibility that, in addition to Mn 2+, the other divalent cations such as Ca 2+ and Mg 2+ may be responsible for in situ formation of cyclic GMP in sea urchin sperm. The axonemal fraction as well as the plasma membrane fraction from the sperm flagella hydrolyzed both cyclic GMP and cyclic AMP. However, when cyclic nucleotides in various concentrations were subjected to hydrolysis under the standard assay conditions, the hydrolyzed rates of cyclic GMP by these t w o fractions were always evidently higher than those of cyclic AMP, as exhibited by Fig. 3A. Using the axonemal and plasma membrane fractions, the apparent Km values for cyclic nucleotides, determined at various concentrations (1 pM to 1 mM), were calculated from the Lineweaver-Burk plots (Fig. 3B and 3C). As the phosphodiesterase both the axonemal and plasma membrane fractions had apparent K m value of approx. 18 #M for cyclic GMP, while they had t w o apparent Km values for cyclic AMP of a b o u t 3 and 100 #M. Namely, the apparent Km values for each cyclic nucleotide by the plasma membrane fraction were quite equivalent to those by the axonemal fraction.

Acknowledgements I am grateful to Dr. K. Nakazawa for his valuable discussions and critical reading of this manuscript, and also to Dr. M Hayashi, Nagoya University Faculty of Science for his valuable discussions. I am also indebted to Sugashima Marine Biological L a b o r a t o r y for generous supplies of sea urchin.

5

531

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