ARCHIVES
OF
BIOCHEMISTBY
Guanylate
AND
Cyclase
of Sea Urchin
J. PATRICK Department
of .Pharmacology,
172, 31-38 (1976)
BIOPHYSICS
Faculty
GRAY2
AND
Sperm: GEORGE
Subcellular
Localization’
I. DRUMMOND3
of Medicine,
The Universty of British V6T 1 w5 Received May 14, 1975
Columbia,
Vancouver,
Canada
The intracellular location of guanylate cyclase was examined in sperm from two species of sea urchin, Strongylocentrotuspurpuratus and Lytechinuspictus, and from the tube worm Chaetopterus uariopedatus. Cells suspended in a medium isotonic with sea water were passed repeatedly through a 23-gauge hypodermic needle to break flagella from heads. This preparation was then fractionated by two methods, one based on centrifugation over a 25% sucrose medium and the other involving repeated differential centrifugation, to resolve flagella from heads. Guanylate cyclase specific activity was increased 3.5-4.5fold in the flagellar fraction relative to the starting sperm homogenate. Relatively little activity was present in the head fraction where specific activity was V~O-~/~OOthat of the flagella. Plasma membranes were separated from axonemal microtubules by dialyzing flagella against low ionic strength buffer, followed by centrifugation over a 40% sucrose medium. Although the overall recovery of guanylate cyclase was low, the specific activity in the plasma membrane fraction was increased two- to threefold over the dialyzed flagella, and over 90% of the recovered activity resided in this fraction. Thus the flagellar plasma membrane is a site rich in guanylate cyclase. It could not be determined, however, whether this is the only intracellular locale of the enzyme.
Guanylate cyclase exists in both particulate and soluble forms in many mammalian tissues (l-6). Evidence has been presented by Chrisman et al. (7) that the mammalian particulate enzyme, after solubilization with Triton X-100, is chromatographically separable from the enzyme in tissue supernatant fractions; the two activities were also distinguished kinetically. The finding of highly active, totally particular guanylate cyclase in invertebrate sperm (8, 9) is suggestive that some metabolic process in which cyclic GMP is involved may reside in these cells. Elucidation of the subcellular location of’the enzyme could be of value in determining a role for this cyclic nucleotide in these cells. This report presents data suggesting that ’ This work was supported by a grant from the Medical Research Council of Canada. 2 An MRC Postdoctoral Fellow. 3 Present address: Biochemistry Group, Department of Chemistry, The University of Calgary, Calgary, Alberta T2N lN4, Canada. Inquiries should be sent to G.I.D. at this address.
the flagellar plasma membrane is a primary locale of the enzyme. Some of these results have been presented in preliminary form (10, 11). MATERIALS
METHODS
The sources and specifications for all reagents and the methods for collecting and washing sperm from sea urchins, Strongylocentrotus purpuratus and Lytechinus pictus, and tube worms, Chaetopterus uariopedatus, were given in a previous paper (9). The packed pellet of washed sperm was usually stored overnight at 4°C prior to use. Zsolation of sperm fractions. Method I. All procedures were carried out at 4°C. Sperm pellets were suspended in nine volumes of “19:l + Ca*+” medium’ (475 mM NaCl, 25 mM KCl, 1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 1 mM CaCl,). Heads were separated from flagella by repeated passage of the suspension through a 23-gauge hypodermic needle according to the procedure of Shelanski and Taylor (12). As determined by phase contrast microscopy, lo-12 passes were required for 4 Abbreviation used: 19:l + Ca2+ medium, 475 mM NaCl, 25 m&f KCl, 1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1 mni dithiothreitol and 1 mM CaCl,. 31
Copyright 0 19’76by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
32
GRAY
AND
greater than 95% breakage of flagella from heads. The suspension of “cut” sperm was then diluted twofold with 19:l + Ca2+ medium. Twenty-milliliter aliquots were layered over lo-ml volumes of 25% sucrose (w/v), 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM dithiothreitol and 1 mM CaCl, in swinging-bucket tubes and centrifugation was carried out at 636g for 15 min in a Beckman SW 25.1 rotor (12). Three fractions were collected based on examination of tube contents by phase contrast microscopy. A fraction containing mainly flagella was collected by removing the top 16 ml of fluid from each tube. A mixed interface fraction consisting of heads heavily contaminated with flagella or flagellar plasma membranes was recovered from the next 10 ml of fluid. A third fraction comprising the bottom 4 ml plus the tightly packed pellet consisted of heads with some contaminating membranes. These are referred to as flagella, mixed-interface and head fractions, respectively. In some experiments, the mixed-interface fraction was subjected to several cycles of passage through the hypodermic needle followed by centrifugation on a sucrose layer for further resolution of flagella from heads. The resulting flagella and head layers were then added to the respective original fractions. Each fraction was diluted with 19:l + Ca2+ medium to reduce sucrose concentration and the particles were collected by centrifugation at 37,OOOg for 1 hr. The resulting pellets were then homogenized using a Ten Broeck homogenizer in nine volumes (based on wet weight of original sperm) of 2 mM glycylglycine, pH 7.5, containing 10 mM NaCl, 10 mM KCl, 5 FM EDTA and 0.1 mM dithiothreitol. Aliquots were usually stored at -80 or +4”C for several days before being assayed for enzyme activity. Method ZZ. This procedure involved repeated differential centrifugation. Exactly 40 ml of diluted “cut”sperm suspension was placed in 105 x 29-mm round-bottom tubes and centrifuged at 636g for 15 min in a fixed-angle rotor. The top 26 ml of fluid was carefully removed using a 50-ml syringe with a 20gauge needle and transferred to clean tubes. An additional 14 ml of 19:l + Ca2+ medium was added to each of these to restore the starting volume and centrifugation was repeated. Again, the top 26 ml from each tube was removed and set aside (flagella 1). The bottom 14 ml of fluid from these tubes was also removed and set aside (flagella 2). The bottom 14 ml from each tube of the first centrifugation (containing mainly heads) were combined, diluted to starting volume, subjected to the hypodermic needle treatment and centrifuged as above. Fractions were obtained exactly as above. This cycle was repeated five more times. After seven cycles, the combined flagella 1 fraction was virtually free of heads (observed by phase contrast microscopy). The combined flagella 2 preparation was contaminated with heads.
DRUMMOND This fraction received two successive centrifugations at 636g (15 min). The supernatant fluid was virtually free of heads and was combined with the flagella 1 fraction. The head-enriched lower portions of the first centrifugation from cycle number 7 was diluted to three times the starting volume, layered over 25% sucrose, 10 rnM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM dithiothreitol and 1 mM CaCl, and centrifuged at 63% for 15 min. Flagella, mixedinterface and head fractions were obtained as in Method I. Particles from these were sedimented, suspended in 2 mM glycylglycine, pH 7.5, containing 10 mM KCl, 5 PM EDTA and 0.1 mM dithiothreitol in the usual manner, and aliquots of the mixed interface and head fractions were stored at -80°C. The flagella fraction was added to the combined flagella fraction above. Flagellar particles were then collected in 300-ml centrifuge tubes at 13,000g for 1.5 h and were suspended in 1.0 mM Tris-HCl, pH 8.0, containing 0.1 mM EDTA, 0.1 mM dithiothreitol and 0.2 mM CaCl,. Aliquots were removed for assay and the remainder used for preparation of flagellar subfractions. Preparations of flagellar subfractions. Flagellar suspensions (from above), usually in 50-ml aliquots, were dialyzed for up to 36 h against 6 liters of 1.0 mM Tris-HCl, pH 8, 0.1 mM EDTA, 0.1 mM dithiothreito1 and 0.2 mM CaCl,, the dialysis solution being changed after 14-20 h. The dialysis tube contents were collected, sedimented material was resuspended by using a Ten Broeck homogenizer, and the suspension was diluted with one volume of the above buffer. Aliquots were removed for enzyme assay. Dialysis against this low ionic strength buffer effectively freed flagellar plasma membranes from around the axonemal microtubules, this process having been initiated earlier by the hypodermic needle treatment. Dialysis solubilized (as determined by centrifugation at 100,OOOgfor 1 hr) about 20% of the particulate flagellar protein. Twenty-milliliter-volumes of the dialyzed suspension were layered over lo-ml volumes of 40% sucrose (w/v) containing 10 m&f Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM dithiothreitol and 1 mM CaCl*, and the tubes were centrifuged in a Beckman SW 25.1 rotor at 16,OOOgfor 1 h. This procedure effectively separated axonemal microtubules, which sedimented, from flagellar plasma membranes, which were largely trapped at the sucose-buffer interface) as described for Chlamyo!omonas flagella by Witman et aE. (131. The first subfraction which contained solubiiized protein and small membrane vesicles was collected by removing the top 16 ml from each tube with a Pasteur pipet. A second fraction comprising the next 10 ml, similarly withdrawn, contained numerous membrane vesicles and a thick white fluffy layer of free flagellar plasma membranes trapped at the sucrose-buffer interface. A third fraction was recovered consisting
SEA URCHIN
SPERM
GUANYLATE
CYCLASE
33
tion it was 0.75; the total recovery of enzyme from this source was 80%. Table I also contains data from experiments in which L. pi&us sperm were fractionated by Method II. This method, which involved repeated centrifugation and washing, was an attempt to improve the resolution of heads from flagella, especially by avoiding the relatively large amount of unresolved material in the mixed-interface fraction. The method also had limitations in that only 65% of the total activity was recovered. Protein recovery was also low. Nevertheless 60% of the original activity and more than 90% of the recovered activity were in the flagellar fraction; the increase in specific activity relative to the starting materisl was 3.5-fold. Only 4 and 1.2% of the recovered activity were present in the mixed interface and head fraction, respectively. Specific activity in the head fraction was reduced 30-fold relative to the original sperm. Both of the above fractionation attempts resulted in more than half of the guanylate cyclase activity being distributed in the flagella fraction; specific activities in these were increased up to fourfold relative to the original sperm. Although there was much variation in activity in the head fraction, specific activity was reduced to as RESULTS much as l/lo that of the starting homogeTable I presents data on the distribution nate, and was ‘/lo-l/loo that of the flagella of guanylate cyclase in sperm fractions pre- fraction. Phase contrast microscopy in evpared from S. purpuratus and L. pi&us by ery case revealed that flagella fractions Method I. The recovery of activity from S. were virtually free of heads. The amount purpuratus sperm was 87%, but with L. of flagella and flagellar-derived mempictus it was lower, 55%. In both cases the branes that contaminated the head prepaonly fraction having increased specific ac- ration could not be easily determined. A tivity was the flagella where it was in- complicating factor was the loss of activity creased 4.5- and 3.7-fold in preparations during fractionation. This loss was not due from S. purpuratus and L. pictus, respec- to release of soluble enzyme during sperm tively, relative to the starting homoge- breakage. In spite of these limitations, the nate. Less than 15% of the recovered activ- data suggest that much of the guanylate ity was in the head fraction. In a similar cyclase activity of invertebrate sperm is experiment (data not shown) with sperm located in the flagellum. from the tube worm C. uariopedatus, more Guanylate Cyclase in Flagellar than half of the recovered activity was in Subfractions the flagellar fraction, representing a 3.4Flagella, prepared as above, were then fold increase in specific activity relative to fractionated to separate plasma memthe original sperm. Specific activity in the branes from axonemal microbutules (Matehead fraction relative to the homogenate rials and Methods). Distribution of activwas 0.45 and for the mixed-interface frac- ity in the recovered fractions in the three of the bottom 4 ml of tube contents plus the brownish pellet of microtubules. These three subfractions (upper, middle and lower) were diluted several-fold with 1.0 mM Tris-HCl, pH 8.0, containing 0.1 mM EDTA, 0.1 mM dithiothreitol and 0.2 mM CaCl, and centrifuged l.!j h at 100,OOOg.Pellets from the upper and middle subfractions were combined to constitute the plasma membrane fraction. Pellets were suspended in fresh buffer and stored as described for the sperm fractions. The 100,OOOgsupernatant fluid recovered from the upper subfraction was also stored and assaved. ‘The three subfractions recovered are designated supernatant, plasma membranes and microtubules. Guanylate cyclase assays. Guanylate cyclase was assayed using WJGTP as substrate (9). Unless otherwise indicated the reaction mixture contained 3 mM MnCl,, 1.0% Triton X-100, a GTP-regenerating system containing 22 units/ml of creatine kinase and 20 mM creatine phosphate. The incubation temperature was 25”C, and l-30 pg of protein was used depending on the activity of the fraction. Guanylate cyclase activity was linear with respect to time and protein concentration in all fractions assayed. Protein. Protein was determined by the method of Lowry et al. (14). Electron microscopy. Aliquots of appropriate fractions were sedimented at 100,OOOg for 1 h and the pellets were fixed with glutaraldehyde (15, 16) and osmium tetroxide and embedded in Epon-Araldite mixture. Sections, 500-600 A, were stained with uranyl acetate and lead citrate (17) and examined in a Philips 300 electron microscope.
34
GRAY
AND
DRUMMOND
preparations is shown in Table II. Seventy to eighty-five percent of the activity survived the dialysis. Values are expressed on the basis of recovery from the original sperm preparation from which the flagella were prepared (Table I). In this way it is possible to see the yield of protein and the activity throughout the entire fraetionation procedure and the enrichment of enzyme activity in each fraction. Recovered activity was primarily in the plasma mem-
brane fraction. The soluble supernatant fraction, derived by centrifuging the upper subfraction at 100,OOOgand consisting of proteins solubilized during dialysis, contained almost no activity. Also little activity was present in the microtubule fraction. Overall recovery of activity throughout the fractionation was relatively low (24-50%). Nevertheless the plasma membrane fraction is clearly enriched with activity, the specific activities being in-
TABLE GUANYLATE
CYCLASE
Sperm fraction
IN FRACTIONS
S. purpuratus
I
OF S. purpuratus
SPERM”
L pictus
~-
Method I
Original homogenate Flagella Mixed-interface Head Total
AND L. pi&us
Method II
Method I
Protein (o/o)
Guanylate cyclase (%)
Activity relative to homogenate
Protein (o/o)
Guanylate cyclase (o/o)
Activity relative to homogenate
Protein (%b)
Guanylate cyclase (%)
Activity relative
100 11.6 17.4 64.3 93.3
100 51.9 16.4 20.1 88.4
1.00 4.49 0.94 0.31 -
100 9.7 20.5 34.9 65.1
100 35.6 11.7 8.1 55.4
1.0 3.67 0.57 0.23 -
100 16.9 3.6 38.0 58.5
100 59.7 3.7 1.2 64.6
1.0 3.53 1.01 0.03 -
hot:ogenate
a S. purpuratus and L. pictus sperm suspensions were subjected to hypodermic needle treatment and fractionated by Method I. For S. purpuratus sperm, the mixed-interface layer was subjected to three cycles of needle treatment and centrifugation over sucrose medium (Materials and Methods); for L. pictus sperm, five cycles were used. Activity in the starting S. purpuratus sperm homogenate was 26.7 nmol/mg of proteinlmin; in the starting L. pictus sperm homogenate fractionated by Methods I and II it was 46.3 and 41.2 nmollmg of proteimmin, respectively. TABLE DISTRIBUTION
Fraction
Flagella Dialyzed
flagella
Supernatant Plasma membranes Microtubules Total
OF GUANYLATE
II
CYCLASE
IN FLAGELLAR
S. purpuratus Protein (%)
Guanylate cyclase (%)
11.6 10.9
51.9 41.1
2.7 2.5 4.8 10.0
1.4 28.6 7.5 37.5
SUBFRACTIONS”
L. pictus (1) Protein (%lo)
Guanylate cyclase (%)
4.49 3.78
9.7 9.6
0.52 11.62 1.56 -
2.1 2.4 4.7 9.1
Activity relative to homogenate
L. pictus (2)
Activity relative to homogenate
Protein (o/o)
Guanylate cyclase (%)
35.6 31.6
3.67 3.27
16.9 17.0
59.7 56.8
3.53 3.35
0.5 17.1 6.4 24.0
0.24 7.13 1.37 -
2.3 3.0 8.1 14.3
0.2 34.6 16.0 50.8
0.06 11.5 1.96 -
Activity relative to homogenate
n Flagellar subfractions were prepared as described in Materials and Methods. Starting material for the preparation from S. purpurata and L. pictus (1) were the flagellar preparations in Table I prepared by Method I. Starting material for L. pictus (2) was the flagella preparation in Table I prepared by Method II. Values are expressed relative to those of the starting sperm homogenate (see text).
SEA URCHIN
SPERM
creased two- to threefold over the dialyzed flagella. The three preparations of plasma membrane represented a 11.6-, 7.1- and 11.5fold increase in specific activity relative to the corresponding starting homogenate with a yield of activity of 28.6, 17.1 and 34.6% in the three preparations. In these experiments fractionation was constantly monitored by phase contrast microscopy. The upper fraction was seen to consist of tiny membrane vesicles, the middle fraction, membrane vesicles of various sizes including some transparent menibrane sheets, and the lower layer contained some membranes but primarily consisted of fibrils (the axonemal microtubules) devoid of plasma membranes. Figure 1 shows electron photomicrographs of the subfractions from one preparation (L. pi&us (2) of Table II). The upper layer (Fig. 1A) consisted of small membrane vesicles that had failed to sediment to the sucrose interface. (These particles when sedimented were added to the plasma membrane fraction, Table II.) The middle fraction consisted primarily of large membrane vesicles which had become trapped at the sucrose-buffer interface during fractionation. The lower fraction is seen to consist primarily of microtubules but also contained some trapped membrane vesicles. Thus guanylate cyclase was distributed in a manner similar to the observed distribution of flagellar plasma membranes. As reported previously guanylate cyclase of sea urchin sperm is solubilized and activated by Triton X-100 (9). We considered it possible that if the enzyme was located in the flagellar plasma membrane, the effectiveness of a given level of Triton in solubilizing enzyme activity might correlate with its effectiveness in solubilizing these membranes. Gibbons and Gibbons (18) found that plasma membranes were completely removed from flagela by suspension of sea urchin sperm in Triton X100 in concentrations as low as 0.04%. In two experiments (with identical results) sea urchin sperm were suspended in various concentrations of Triton X-100 (18). Guanylate cyclase was determined in 100,OOOg(1 h) supernatant fractions. Fig-
GUANYLATE
CYCLASE
35
ure 2 reveals that the enzyme was progressively solubilized as Triton levels were increased from 0.02 to 0.07%. Approximately half the activity was recovered in the supernatant fluid when the Triton concentration was 0.04%, and maximal solubilization occurred at 0.08%. Electron microscopy of selected pellets showed that plasma membranes were loosened and released from the axonemes by the detergent at a concentration of 0.03%; some free unsolubilized membranes were still present when the concentration was 0.1%. No intact membranes remained when the Triton concentration was 1%. Thus the Triton concentration for solubilization of enzyme activity closely paralleled the degree of solubilization of plasma membranes. DISCUSSION
A discrete physiological role for cyclic GMP in any cell type has not yet been elucidated. The extraordinarily high level of guanylate cyclase in invertebrate sperm compared with the most active mammalian tissues (9) makes it very tempting to speculate that some unique role for the cyclic nucleotide exists in these cells. Sperm are highly specialized cells, having lost much metabolic machinery especially the endoplasmic reticulum, during maturation (19). Thus a higher percentage of total cell membrane material resides in the plasma membrane than in other cell types. A large percentage of sperm structure and function is related to its flagellum. From these results and those reported previously (9), one might be encouraged to consider a role for cyclic GMP in flagellar motility. There is no evidence for this however. Whatever the role, it is unique to invertebrae sperm because guanylate cyclase is totally lacking from sperm cells of higher vertebrate forms. The present efforts to elucidate the intracellular location of guanylate cyclase in sea urchin sperm were complicated by loss of enzyme activity during fractionation and by incomplete resolution of cellular organelles by the techniques used. However much of the activity clearly resided in the flagella, which were separated from head-midpiece fractions by two separate
GRAY
36
FIG. 1. used was Methods. represents
AND
DRUMMOND
Electron photomicrographs of flagellar subfractions from L. pictus. The preparation L. &us (2) (Table II). Subfractions were prepared as described in Materials and (A, B, C), Upper, middle and lower subfractions, respectively. The scale in (A) 0.5 6 m and also serves for. P (B) and (C).
SEA URCHIN
SPERM
FIG. 2. Solubilization of guanylate cyclase by Triton X-100. Sperm from S. purpurutus were suspended in 200 volumes of 150 mM KCl, 4 mM MgSO,, 0.5 mM EDTA, 0.5 mM dithiothreitol and 2 mM TriHCl, pH 8.0. Aliquots of the suspension were added to tubes containing an equal volume of medium containing Triton to give the final concentrations indicated on the abscissa. A control tube contained no Triton. Tubes were kept at 20°C for 30 s (181, then at 4°C for 30 min, then centrifuged at 100,OOOgfor 1 h. The upper portion of each supernatant fraction was assayed for guanylate cyclase in the presence of Triton X-100. Activities on the ordinate are expressed relative to the activity of the control which was subjected to Triton (1%) only during the assay. Activity in the control was 15.3 nmol/mg of protein/min. Selected pellets were examined by electron microscopy as described in the text,
techniques, one involving centrifugation of sperm suspensions over sucrose solutions, the second involving repeated differential centrifugation. When flagella were dialyzed against low ionic strength buffer and fractionated to separate plasma membranes from axonemal microtubules, most of the recovered enyzme resided in the plasma membrane fraction. In addition, the ability of the detergent Triton X-100 to solubilize guanylate cyclase from sperm cells paralleled its ability to solubilize plasma membranes as observed by electron microscopy. Clearly the flagellar plasma membrane is a rich source of the enzyme. Because of losses sustained during fractionation, it was not possible to ascertain whether this is the only locale of the enzyme, but this is considered a possibility. All evidence obtained so far suggests
GUANYLATE
CYCLASE
37
that only one molecular species of guanylate cyclase is present in invertebrate sperm, and this is totally particulate (9). This enzyme exists in both soluble and particulate forms in many mammalian tissues (l-6) and these may represent two distinct molecular species (7). The exact subcellular location of the mammalian particulate enzyme has not been established, but Chrisman (20) has found activity in fractions from rat liver enriched with smooth endoplasmic reticulum, rough endoplasmic reticulum, plasma membranes, nuclei or golgi apparatus; no activity was found in mitochondrial fractions. Kimura and Murad (6) have found activity in rat heart homogenates sedimenting at 1000, 10,000 and 100,OOOg.Thus the invertebrate sperm cell enzyme seems to possess several unique characteristics: It possesses extremely high activity, it exists as a single, totally particulate species, and may be located mainly in a single intracellular site, the flagellar plasma membrane. What this means in the functional economy of this cell type is not known at present. ACKNOWLEDGMENTS The authors are indebted to Dr. Donald M. McLean and Ms. Therese Walters, Department of Medical Microbiology, University of British Columbia, for assistance with the electron microscopy. REFERENCES 1. HARDMAN, J. G., AND SUTHERLAND, E. W. (1969) J. Biol. Chem. 244,6363-6370. 2. WHITE, A. A., AND AURBACH, G. D. (1969) Biochim. Biophys. Actu 191, 686-697. 3. GOLDBERG, N. D., O’DEA, R. F., AND HADDOX, M. K. (1973) Aduan. Cyclic Nucleotide Res. 3, 155-223. 4. HARDMAN, J. G., BEAVO, J. A., GRAY, J. P., CHRISMAN, T. D., PATTERSON, W. D., AND SUTHERLAND, E. W. (1971) Ann. N.Y. Acad. Sci. 185, 27-35. 5. HARDMAN, J. G., CHRISMAN, T. D., GRAY, J. P., SUDDATH, J. L., AND SUTHERLAND, E. W. (1973) in Pharmacology and the Future of Man. Proceedings of the 5th International Congress of Pharmacology, San Francisco, 1972, Vol. 5, pp. 134-145, Karger, Basel. 6. KIMURA, H., AND MURAD, F. (1974) J. Biol. Chem. 249,6910-6916. 7. CHRISMAN, T. D., GARBERS, D. L., PARKS, M. A., AND HARDMAN, J. G. (1975) J. Biol. Chem.
38
GRAY
AND
250, 374-381. 8. GRAY, J. P., HARDMAN, J. G., BIBRING, T., AND SUTHERLAND, E. W. (1970) Fed. Proc. 29, 608. 9. GRAY, J. P., DRUMMOND, G. I., LLJK, D. W. T., HARDMAN, J. G., AND SUTHERLAND, E. W. (1975) Arch. Biochem. Biophys. 172, 20-30. 10. GRAY, J. P., AND DRIJMMOND, G. I. (1974) Fed. Proc. 33, 1250. 11. GRAY, J. P., LUK, D., AND DRUMMOND, G. I. (1975) Advan. Cyclic Nucleotide Res. 5, 823Cabstract). 12. SHELANSKI, M. L., AND TAYLOR, E. W. (1967) J. Cell Biol. 34, 549-558. 13. WITMAN, G. B., CARLSON, K., BERLINER, J., AND ROSENBAUM, J. L. (1972) J. Cell Biol. 54,507-
DRUMMOND 539. 14. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193,
265-275. 15. KARNOVSKY, M. J. (1965) J. Cell Biol. 2’7, 137A. M. J., AND SPIRA, A. W. (1973) 16. HOLLENBERG, Amer. J. Anat. 137, 357-363. 17. REYNOLDS, L. W. (1963) J. Cell Biol. 17, 208. 18. GIBBONS, B. H., AND GIBBONS, I. R. (1972) J. Cell Biol. 54, 75-97. 19. MANN, (1964) The Biochemistry of Semen and of the Male Reproductive Tract, Methuen, London. 20. CHRISMAN, T. D. (1974) Ph.D. dissertation, Vanderbilt University, Nashville, Tenn.
OF
BIOCHEMISTBY
Guanylate
AND
Cyclase
of Sea Urchin
J. PATRICK Department
of .Pharmacology,
172, 31-38 (1976)
BIOPHYSICS
Faculty
GRAY2
AND
Sperm: GEORGE
Subcellular
Localization’
I. DRUMMOND3
of Medicine,
The Universty of British V6T 1 w5 Received May 14, 1975
Columbia,
Vancouver,
Canada
The intracellular location of guanylate cyclase was examined in sperm from two species of sea urchin, Strongylocentrotuspurpuratus and Lytechinuspictus, and from the tube worm Chaetopterus uariopedatus. Cells suspended in a medium isotonic with sea water were passed repeatedly through a 23-gauge hypodermic needle to break flagella from heads. This preparation was then fractionated by two methods, one based on centrifugation over a 25% sucrose medium and the other involving repeated differential centrifugation, to resolve flagella from heads. Guanylate cyclase specific activity was increased 3.5-4.5fold in the flagellar fraction relative to the starting sperm homogenate. Relatively little activity was present in the head fraction where specific activity was V~O-~/~OOthat of the flagella. Plasma membranes were separated from axonemal microtubules by dialyzing flagella against low ionic strength buffer, followed by centrifugation over a 40% sucrose medium. Although the overall recovery of guanylate cyclase was low, the specific activity in the plasma membrane fraction was increased two- to threefold over the dialyzed flagella, and over 90% of the recovered activity resided in this fraction. Thus the flagellar plasma membrane is a site rich in guanylate cyclase. It could not be determined, however, whether this is the only intracellular locale of the enzyme.
Guanylate cyclase exists in both particulate and soluble forms in many mammalian tissues (l-6). Evidence has been presented by Chrisman et al. (7) that the mammalian particulate enzyme, after solubilization with Triton X-100, is chromatographically separable from the enzyme in tissue supernatant fractions; the two activities were also distinguished kinetically. The finding of highly active, totally particular guanylate cyclase in invertebrate sperm (8, 9) is suggestive that some metabolic process in which cyclic GMP is involved may reside in these cells. Elucidation of the subcellular location of’the enzyme could be of value in determining a role for this cyclic nucleotide in these cells. This report presents data suggesting that ’ This work was supported by a grant from the Medical Research Council of Canada. 2 An MRC Postdoctoral Fellow. 3 Present address: Biochemistry Group, Department of Chemistry, The University of Calgary, Calgary, Alberta T2N lN4, Canada. Inquiries should be sent to G.I.D. at this address.
the flagellar plasma membrane is a primary locale of the enzyme. Some of these results have been presented in preliminary form (10, 11). MATERIALS
METHODS
The sources and specifications for all reagents and the methods for collecting and washing sperm from sea urchins, Strongylocentrotus purpuratus and Lytechinus pictus, and tube worms, Chaetopterus uariopedatus, were given in a previous paper (9). The packed pellet of washed sperm was usually stored overnight at 4°C prior to use. Zsolation of sperm fractions. Method I. All procedures were carried out at 4°C. Sperm pellets were suspended in nine volumes of “19:l + Ca*+” medium’ (475 mM NaCl, 25 mM KCl, 1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 1 mM CaCl,). Heads were separated from flagella by repeated passage of the suspension through a 23-gauge hypodermic needle according to the procedure of Shelanski and Taylor (12). As determined by phase contrast microscopy, lo-12 passes were required for 4 Abbreviation used: 19:l + Ca2+ medium, 475 mM NaCl, 25 m&f KCl, 1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1 mni dithiothreitol and 1 mM CaCl,. 31
Copyright 0 19’76by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
32
GRAY
AND
greater than 95% breakage of flagella from heads. The suspension of “cut” sperm was then diluted twofold with 19:l + Ca2+ medium. Twenty-milliliter aliquots were layered over lo-ml volumes of 25% sucrose (w/v), 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM dithiothreitol and 1 mM CaCl, in swinging-bucket tubes and centrifugation was carried out at 636g for 15 min in a Beckman SW 25.1 rotor (12). Three fractions were collected based on examination of tube contents by phase contrast microscopy. A fraction containing mainly flagella was collected by removing the top 16 ml of fluid from each tube. A mixed interface fraction consisting of heads heavily contaminated with flagella or flagellar plasma membranes was recovered from the next 10 ml of fluid. A third fraction comprising the bottom 4 ml plus the tightly packed pellet consisted of heads with some contaminating membranes. These are referred to as flagella, mixed-interface and head fractions, respectively. In some experiments, the mixed-interface fraction was subjected to several cycles of passage through the hypodermic needle followed by centrifugation on a sucrose layer for further resolution of flagella from heads. The resulting flagella and head layers were then added to the respective original fractions. Each fraction was diluted with 19:l + Ca2+ medium to reduce sucrose concentration and the particles were collected by centrifugation at 37,OOOg for 1 hr. The resulting pellets were then homogenized using a Ten Broeck homogenizer in nine volumes (based on wet weight of original sperm) of 2 mM glycylglycine, pH 7.5, containing 10 mM NaCl, 10 mM KCl, 5 FM EDTA and 0.1 mM dithiothreitol. Aliquots were usually stored at -80 or +4”C for several days before being assayed for enzyme activity. Method ZZ. This procedure involved repeated differential centrifugation. Exactly 40 ml of diluted “cut”sperm suspension was placed in 105 x 29-mm round-bottom tubes and centrifuged at 636g for 15 min in a fixed-angle rotor. The top 26 ml of fluid was carefully removed using a 50-ml syringe with a 20gauge needle and transferred to clean tubes. An additional 14 ml of 19:l + Ca2+ medium was added to each of these to restore the starting volume and centrifugation was repeated. Again, the top 26 ml from each tube was removed and set aside (flagella 1). The bottom 14 ml of fluid from these tubes was also removed and set aside (flagella 2). The bottom 14 ml from each tube of the first centrifugation (containing mainly heads) were combined, diluted to starting volume, subjected to the hypodermic needle treatment and centrifuged as above. Fractions were obtained exactly as above. This cycle was repeated five more times. After seven cycles, the combined flagella 1 fraction was virtually free of heads (observed by phase contrast microscopy). The combined flagella 2 preparation was contaminated with heads.
DRUMMOND This fraction received two successive centrifugations at 636g (15 min). The supernatant fluid was virtually free of heads and was combined with the flagella 1 fraction. The head-enriched lower portions of the first centrifugation from cycle number 7 was diluted to three times the starting volume, layered over 25% sucrose, 10 rnM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM dithiothreitol and 1 mM CaCl, and centrifuged at 63% for 15 min. Flagella, mixedinterface and head fractions were obtained as in Method I. Particles from these were sedimented, suspended in 2 mM glycylglycine, pH 7.5, containing 10 mM KCl, 5 PM EDTA and 0.1 mM dithiothreitol in the usual manner, and aliquots of the mixed interface and head fractions were stored at -80°C. The flagella fraction was added to the combined flagella fraction above. Flagellar particles were then collected in 300-ml centrifuge tubes at 13,000g for 1.5 h and were suspended in 1.0 mM Tris-HCl, pH 8.0, containing 0.1 mM EDTA, 0.1 mM dithiothreitol and 0.2 mM CaCl,. Aliquots were removed for assay and the remainder used for preparation of flagellar subfractions. Preparations of flagellar subfractions. Flagellar suspensions (from above), usually in 50-ml aliquots, were dialyzed for up to 36 h against 6 liters of 1.0 mM Tris-HCl, pH 8, 0.1 mM EDTA, 0.1 mM dithiothreito1 and 0.2 mM CaCl,, the dialysis solution being changed after 14-20 h. The dialysis tube contents were collected, sedimented material was resuspended by using a Ten Broeck homogenizer, and the suspension was diluted with one volume of the above buffer. Aliquots were removed for enzyme assay. Dialysis against this low ionic strength buffer effectively freed flagellar plasma membranes from around the axonemal microtubules, this process having been initiated earlier by the hypodermic needle treatment. Dialysis solubilized (as determined by centrifugation at 100,OOOgfor 1 hr) about 20% of the particulate flagellar protein. Twenty-milliliter-volumes of the dialyzed suspension were layered over lo-ml volumes of 40% sucrose (w/v) containing 10 m&f Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM dithiothreitol and 1 mM CaCl*, and the tubes were centrifuged in a Beckman SW 25.1 rotor at 16,OOOgfor 1 h. This procedure effectively separated axonemal microtubules, which sedimented, from flagellar plasma membranes, which were largely trapped at the sucose-buffer interface) as described for Chlamyo!omonas flagella by Witman et aE. (131. The first subfraction which contained solubiiized protein and small membrane vesicles was collected by removing the top 16 ml from each tube with a Pasteur pipet. A second fraction comprising the next 10 ml, similarly withdrawn, contained numerous membrane vesicles and a thick white fluffy layer of free flagellar plasma membranes trapped at the sucrose-buffer interface. A third fraction was recovered consisting
SEA URCHIN
SPERM
GUANYLATE
CYCLASE
33
tion it was 0.75; the total recovery of enzyme from this source was 80%. Table I also contains data from experiments in which L. pi&us sperm were fractionated by Method II. This method, which involved repeated centrifugation and washing, was an attempt to improve the resolution of heads from flagella, especially by avoiding the relatively large amount of unresolved material in the mixed-interface fraction. The method also had limitations in that only 65% of the total activity was recovered. Protein recovery was also low. Nevertheless 60% of the original activity and more than 90% of the recovered activity were in the flagellar fraction; the increase in specific activity relative to the starting materisl was 3.5-fold. Only 4 and 1.2% of the recovered activity were present in the mixed interface and head fraction, respectively. Specific activity in the head fraction was reduced 30-fold relative to the original sperm. Both of the above fractionation attempts resulted in more than half of the guanylate cyclase activity being distributed in the flagella fraction; specific activities in these were increased up to fourfold relative to the original sperm. Although there was much variation in activity in the head fraction, specific activity was reduced to as RESULTS much as l/lo that of the starting homogeTable I presents data on the distribution nate, and was ‘/lo-l/loo that of the flagella of guanylate cyclase in sperm fractions pre- fraction. Phase contrast microscopy in evpared from S. purpuratus and L. pi&us by ery case revealed that flagella fractions Method I. The recovery of activity from S. were virtually free of heads. The amount purpuratus sperm was 87%, but with L. of flagella and flagellar-derived mempictus it was lower, 55%. In both cases the branes that contaminated the head prepaonly fraction having increased specific ac- ration could not be easily determined. A tivity was the flagella where it was in- complicating factor was the loss of activity creased 4.5- and 3.7-fold in preparations during fractionation. This loss was not due from S. purpuratus and L. pictus, respec- to release of soluble enzyme during sperm tively, relative to the starting homoge- breakage. In spite of these limitations, the nate. Less than 15% of the recovered activ- data suggest that much of the guanylate ity was in the head fraction. In a similar cyclase activity of invertebrate sperm is experiment (data not shown) with sperm located in the flagellum. from the tube worm C. uariopedatus, more Guanylate Cyclase in Flagellar than half of the recovered activity was in Subfractions the flagellar fraction, representing a 3.4Flagella, prepared as above, were then fold increase in specific activity relative to fractionated to separate plasma memthe original sperm. Specific activity in the branes from axonemal microbutules (Matehead fraction relative to the homogenate rials and Methods). Distribution of activwas 0.45 and for the mixed-interface frac- ity in the recovered fractions in the three of the bottom 4 ml of tube contents plus the brownish pellet of microtubules. These three subfractions (upper, middle and lower) were diluted several-fold with 1.0 mM Tris-HCl, pH 8.0, containing 0.1 mM EDTA, 0.1 mM dithiothreitol and 0.2 mM CaCl, and centrifuged l.!j h at 100,OOOg.Pellets from the upper and middle subfractions were combined to constitute the plasma membrane fraction. Pellets were suspended in fresh buffer and stored as described for the sperm fractions. The 100,OOOgsupernatant fluid recovered from the upper subfraction was also stored and assaved. ‘The three subfractions recovered are designated supernatant, plasma membranes and microtubules. Guanylate cyclase assays. Guanylate cyclase was assayed using WJGTP as substrate (9). Unless otherwise indicated the reaction mixture contained 3 mM MnCl,, 1.0% Triton X-100, a GTP-regenerating system containing 22 units/ml of creatine kinase and 20 mM creatine phosphate. The incubation temperature was 25”C, and l-30 pg of protein was used depending on the activity of the fraction. Guanylate cyclase activity was linear with respect to time and protein concentration in all fractions assayed. Protein. Protein was determined by the method of Lowry et al. (14). Electron microscopy. Aliquots of appropriate fractions were sedimented at 100,OOOg for 1 h and the pellets were fixed with glutaraldehyde (15, 16) and osmium tetroxide and embedded in Epon-Araldite mixture. Sections, 500-600 A, were stained with uranyl acetate and lead citrate (17) and examined in a Philips 300 electron microscope.
34
GRAY
AND
DRUMMOND
preparations is shown in Table II. Seventy to eighty-five percent of the activity survived the dialysis. Values are expressed on the basis of recovery from the original sperm preparation from which the flagella were prepared (Table I). In this way it is possible to see the yield of protein and the activity throughout the entire fraetionation procedure and the enrichment of enzyme activity in each fraction. Recovered activity was primarily in the plasma mem-
brane fraction. The soluble supernatant fraction, derived by centrifuging the upper subfraction at 100,OOOgand consisting of proteins solubilized during dialysis, contained almost no activity. Also little activity was present in the microtubule fraction. Overall recovery of activity throughout the fractionation was relatively low (24-50%). Nevertheless the plasma membrane fraction is clearly enriched with activity, the specific activities being in-
TABLE GUANYLATE
CYCLASE
Sperm fraction
IN FRACTIONS
S. purpuratus
I
OF S. purpuratus
SPERM”
L pictus
~-
Method I
Original homogenate Flagella Mixed-interface Head Total
AND L. pi&us
Method II
Method I
Protein (o/o)
Guanylate cyclase (%)
Activity relative to homogenate
Protein (o/o)
Guanylate cyclase (o/o)
Activity relative to homogenate
Protein (%b)
Guanylate cyclase (%)
Activity relative
100 11.6 17.4 64.3 93.3
100 51.9 16.4 20.1 88.4
1.00 4.49 0.94 0.31 -
100 9.7 20.5 34.9 65.1
100 35.6 11.7 8.1 55.4
1.0 3.67 0.57 0.23 -
100 16.9 3.6 38.0 58.5
100 59.7 3.7 1.2 64.6
1.0 3.53 1.01 0.03 -
hot:ogenate
a S. purpuratus and L. pictus sperm suspensions were subjected to hypodermic needle treatment and fractionated by Method I. For S. purpuratus sperm, the mixed-interface layer was subjected to three cycles of needle treatment and centrifugation over sucrose medium (Materials and Methods); for L. pictus sperm, five cycles were used. Activity in the starting S. purpuratus sperm homogenate was 26.7 nmol/mg of proteinlmin; in the starting L. pictus sperm homogenate fractionated by Methods I and II it was 46.3 and 41.2 nmollmg of proteimmin, respectively. TABLE DISTRIBUTION
Fraction
Flagella Dialyzed
flagella
Supernatant Plasma membranes Microtubules Total
OF GUANYLATE
II
CYCLASE
IN FLAGELLAR
S. purpuratus Protein (%)
Guanylate cyclase (%)
11.6 10.9
51.9 41.1
2.7 2.5 4.8 10.0
1.4 28.6 7.5 37.5
SUBFRACTIONS”
L. pictus (1) Protein (%lo)
Guanylate cyclase (%)
4.49 3.78
9.7 9.6
0.52 11.62 1.56 -
2.1 2.4 4.7 9.1
Activity relative to homogenate
L. pictus (2)
Activity relative to homogenate
Protein (o/o)
Guanylate cyclase (%)
35.6 31.6
3.67 3.27
16.9 17.0
59.7 56.8
3.53 3.35
0.5 17.1 6.4 24.0
0.24 7.13 1.37 -
2.3 3.0 8.1 14.3
0.2 34.6 16.0 50.8
0.06 11.5 1.96 -
Activity relative to homogenate
n Flagellar subfractions were prepared as described in Materials and Methods. Starting material for the preparation from S. purpurata and L. pictus (1) were the flagellar preparations in Table I prepared by Method I. Starting material for L. pictus (2) was the flagella preparation in Table I prepared by Method II. Values are expressed relative to those of the starting sperm homogenate (see text).
SEA URCHIN
SPERM
creased two- to threefold over the dialyzed flagella. The three preparations of plasma membrane represented a 11.6-, 7.1- and 11.5fold increase in specific activity relative to the corresponding starting homogenate with a yield of activity of 28.6, 17.1 and 34.6% in the three preparations. In these experiments fractionation was constantly monitored by phase contrast microscopy. The upper fraction was seen to consist of tiny membrane vesicles, the middle fraction, membrane vesicles of various sizes including some transparent menibrane sheets, and the lower layer contained some membranes but primarily consisted of fibrils (the axonemal microtubules) devoid of plasma membranes. Figure 1 shows electron photomicrographs of the subfractions from one preparation (L. pi&us (2) of Table II). The upper layer (Fig. 1A) consisted of small membrane vesicles that had failed to sediment to the sucrose interface. (These particles when sedimented were added to the plasma membrane fraction, Table II.) The middle fraction consisted primarily of large membrane vesicles which had become trapped at the sucrose-buffer interface during fractionation. The lower fraction is seen to consist primarily of microtubules but also contained some trapped membrane vesicles. Thus guanylate cyclase was distributed in a manner similar to the observed distribution of flagellar plasma membranes. As reported previously guanylate cyclase of sea urchin sperm is solubilized and activated by Triton X-100 (9). We considered it possible that if the enzyme was located in the flagellar plasma membrane, the effectiveness of a given level of Triton in solubilizing enzyme activity might correlate with its effectiveness in solubilizing these membranes. Gibbons and Gibbons (18) found that plasma membranes were completely removed from flagela by suspension of sea urchin sperm in Triton X100 in concentrations as low as 0.04%. In two experiments (with identical results) sea urchin sperm were suspended in various concentrations of Triton X-100 (18). Guanylate cyclase was determined in 100,OOOg(1 h) supernatant fractions. Fig-
GUANYLATE
CYCLASE
35
ure 2 reveals that the enzyme was progressively solubilized as Triton levels were increased from 0.02 to 0.07%. Approximately half the activity was recovered in the supernatant fluid when the Triton concentration was 0.04%, and maximal solubilization occurred at 0.08%. Electron microscopy of selected pellets showed that plasma membranes were loosened and released from the axonemes by the detergent at a concentration of 0.03%; some free unsolubilized membranes were still present when the concentration was 0.1%. No intact membranes remained when the Triton concentration was 1%. Thus the Triton concentration for solubilization of enzyme activity closely paralleled the degree of solubilization of plasma membranes. DISCUSSION
A discrete physiological role for cyclic GMP in any cell type has not yet been elucidated. The extraordinarily high level of guanylate cyclase in invertebrate sperm compared with the most active mammalian tissues (9) makes it very tempting to speculate that some unique role for the cyclic nucleotide exists in these cells. Sperm are highly specialized cells, having lost much metabolic machinery especially the endoplasmic reticulum, during maturation (19). Thus a higher percentage of total cell membrane material resides in the plasma membrane than in other cell types. A large percentage of sperm structure and function is related to its flagellum. From these results and those reported previously (9), one might be encouraged to consider a role for cyclic GMP in flagellar motility. There is no evidence for this however. Whatever the role, it is unique to invertebrae sperm because guanylate cyclase is totally lacking from sperm cells of higher vertebrate forms. The present efforts to elucidate the intracellular location of guanylate cyclase in sea urchin sperm were complicated by loss of enzyme activity during fractionation and by incomplete resolution of cellular organelles by the techniques used. However much of the activity clearly resided in the flagella, which were separated from head-midpiece fractions by two separate
GRAY
36
FIG. 1. used was Methods. represents
AND
DRUMMOND
Electron photomicrographs of flagellar subfractions from L. pictus. The preparation L. &us (2) (Table II). Subfractions were prepared as described in Materials and (A, B, C), Upper, middle and lower subfractions, respectively. The scale in (A) 0.5 6 m and also serves for. P (B) and (C).
SEA URCHIN
SPERM
FIG. 2. Solubilization of guanylate cyclase by Triton X-100. Sperm from S. purpurutus were suspended in 200 volumes of 150 mM KCl, 4 mM MgSO,, 0.5 mM EDTA, 0.5 mM dithiothreitol and 2 mM TriHCl, pH 8.0. Aliquots of the suspension were added to tubes containing an equal volume of medium containing Triton to give the final concentrations indicated on the abscissa. A control tube contained no Triton. Tubes were kept at 20°C for 30 s (181, then at 4°C for 30 min, then centrifuged at 100,OOOgfor 1 h. The upper portion of each supernatant fraction was assayed for guanylate cyclase in the presence of Triton X-100. Activities on the ordinate are expressed relative to the activity of the control which was subjected to Triton (1%) only during the assay. Activity in the control was 15.3 nmol/mg of protein/min. Selected pellets were examined by electron microscopy as described in the text,
techniques, one involving centrifugation of sperm suspensions over sucrose solutions, the second involving repeated differential centrifugation. When flagella were dialyzed against low ionic strength buffer and fractionated to separate plasma membranes from axonemal microtubules, most of the recovered enyzme resided in the plasma membrane fraction. In addition, the ability of the detergent Triton X-100 to solubilize guanylate cyclase from sperm cells paralleled its ability to solubilize plasma membranes as observed by electron microscopy. Clearly the flagellar plasma membrane is a rich source of the enzyme. Because of losses sustained during fractionation, it was not possible to ascertain whether this is the only locale of the enzyme, but this is considered a possibility. All evidence obtained so far suggests
GUANYLATE
CYCLASE
37
that only one molecular species of guanylate cyclase is present in invertebrate sperm, and this is totally particulate (9). This enzyme exists in both soluble and particulate forms in many mammalian tissues (l-6) and these may represent two distinct molecular species (7). The exact subcellular location of the mammalian particulate enzyme has not been established, but Chrisman (20) has found activity in fractions from rat liver enriched with smooth endoplasmic reticulum, rough endoplasmic reticulum, plasma membranes, nuclei or golgi apparatus; no activity was found in mitochondrial fractions. Kimura and Murad (6) have found activity in rat heart homogenates sedimenting at 1000, 10,000 and 100,OOOg.Thus the invertebrate sperm cell enzyme seems to possess several unique characteristics: It possesses extremely high activity, it exists as a single, totally particulate species, and may be located mainly in a single intracellular site, the flagellar plasma membrane. What this means in the functional economy of this cell type is not known at present. ACKNOWLEDGMENTS The authors are indebted to Dr. Donald M. McLean and Ms. Therese Walters, Department of Medical Microbiology, University of British Columbia, for assistance with the electron microscopy. REFERENCES 1. HARDMAN, J. G., AND SUTHERLAND, E. W. (1969) J. Biol. Chem. 244,6363-6370. 2. WHITE, A. A., AND AURBACH, G. D. (1969) Biochim. Biophys. Actu 191, 686-697. 3. GOLDBERG, N. D., O’DEA, R. F., AND HADDOX, M. K. (1973) Aduan. Cyclic Nucleotide Res. 3, 155-223. 4. HARDMAN, J. G., BEAVO, J. A., GRAY, J. P., CHRISMAN, T. D., PATTERSON, W. D., AND SUTHERLAND, E. W. (1971) Ann. N.Y. Acad. Sci. 185, 27-35. 5. HARDMAN, J. G., CHRISMAN, T. D., GRAY, J. P., SUDDATH, J. L., AND SUTHERLAND, E. W. (1973) in Pharmacology and the Future of Man. Proceedings of the 5th International Congress of Pharmacology, San Francisco, 1972, Vol. 5, pp. 134-145, Karger, Basel. 6. KIMURA, H., AND MURAD, F. (1974) J. Biol. Chem. 249,6910-6916. 7. CHRISMAN, T. D., GARBERS, D. L., PARKS, M. A., AND HARDMAN, J. G. (1975) J. Biol. Chem.
38
GRAY
AND
250, 374-381. 8. GRAY, J. P., HARDMAN, J. G., BIBRING, T., AND SUTHERLAND, E. W. (1970) Fed. Proc. 29, 608. 9. GRAY, J. P., DRUMMOND, G. I., LLJK, D. W. T., HARDMAN, J. G., AND SUTHERLAND, E. W. (1975) Arch. Biochem. Biophys. 172, 20-30. 10. GRAY, J. P., AND DRIJMMOND, G. I. (1974) Fed. Proc. 33, 1250. 11. GRAY, J. P., LUK, D., AND DRUMMOND, G. I. (1975) Advan. Cyclic Nucleotide Res. 5, 823Cabstract). 12. SHELANSKI, M. L., AND TAYLOR, E. W. (1967) J. Cell Biol. 34, 549-558. 13. WITMAN, G. B., CARLSON, K., BERLINER, J., AND ROSENBAUM, J. L. (1972) J. Cell Biol. 54,507-
DRUMMOND 539. 14. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193,
265-275. 15. KARNOVSKY, M. J. (1965) J. Cell Biol. 2’7, 137A. M. J., AND SPIRA, A. W. (1973) 16. HOLLENBERG, Amer. J. Anat. 137, 357-363. 17. REYNOLDS, L. W. (1963) J. Cell Biol. 17, 208. 18. GIBBONS, B. H., AND GIBBONS, I. R. (1972) J. Cell Biol. 54, 75-97. 19. MANN, (1964) The Biochemistry of Semen and of the Male Reproductive Tract, Methuen, London. 20. CHRISMAN, T. D. (1974) Ph.D. dissertation, Vanderbilt University, Nashville, Tenn.
