484

Biochimica et Biophysica Acta, 587 (1979) 484- -494 © Elsevier /Nor th-Hol land Biomedical Press

BBA 29048

CHARACTERIZATION OF RAT TESTICULAR GUANYLATE CYCLASE DURING DEVELOPMENT *

W. AUSTIN SPRUILL, ALTON L. STEINER and H. SHELTON EARP, III

University of North Caroline School of Medicine, Chapel Hill, NC 27514 (U.S.A.)

(Received March 8th, 1979)

Key words: Guanylate cyclase; Testicular development; Cyclic GMP level; (Rat)

Summary

The biochemical characteristics of rat testicular guanylate cyclase were inves- tigated and the activity and subcellular distribution of the enzyme was deter- mined during testicular development. Examination of the effects of metal ions, nucleotides, detergents and other in vitro activators on the activity of guanylate cyclase revealed that the testicular enzyme is similar in most respects to guany- late cyclase isolated from other mammalian tissues. Changes in the total activ- ity of guanylate cyclase during testicular development paralleled changes in the tissue concentration of cyclic GMP; i.e. guanylate cyclase activity and tissue cyclic GMP were highest during the early stages of development. Subcellular fractionation revealed that the activity of the soluble form of guanylate cyclase was best correlated with tissue cyclic GMP. Biochemical analysis of the soluble enzyme prepared from testes of neonatal and adult rats did not reveal any sig- nificant differences in the characteristics of the enzyme during ontogeny with the exception of a 2.5 fold increase in V noted in the neonatal testis. The results of this study are consistent with a molecular mechanism that allows independent regulation of the different forms of guanylate cyclase.

Introduction

Numerous studies have documented alterations in tissue cyclic GMP levels following hormonal stimulation and during processes such as growth and development [1,2]. However, no definitive relationship between hormone action and alterations in tissue cyclic GMP has been established, nor it there a

* This work w as presen te d in part at the F e d e r a t i o n o f A m e r i c a n S o c i e t i e s for E x p e r i m e n t a l B io logy m e e t i n g in April 1977, Chicago, IL.

485

consistent alteration in the direction and magnitude of tissue cyclic GMP levels during growth and development. In light of the difficulty in understanding the role of cyclic GMP some investigators have sought insight by studying the regu- lation of the activity and subcellular localization of guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) [3--6]. The control of guanylate cyclase activity during processes in which cyclic GMP might have functional significance has been the topic of several reports [7--20]. Data from this laboratory have demonstrated that in the rat testis cyclic GMP levels are ele- vated during early development and decline with maturation [21]. In an at tempt to understand the regulation of these growth-related alterations in cyclic GMP concentration, a study of the activity of testicular guanylate cyclase during development was undertaken.

In order to understand the control of guanylate cyclase activity a detailed analysis of the biochemical characteristics of testicular guanylate cyclase was performed. Knowledge concerning the effect of metal ions, nucleotides, deter- gents and other activators on the in vitro activity of the different forms of guanylate cyclase has allowed us to study the activity and subcellular localiza- tion of the enzyme during testicular development. The data reveal that the activity of guanylate cyclase was altered during testicular development in a fashion that paralleled the change in tissue cyclic GMP levels, i.e., guanylate cyclase activity measured in vitro was elevated early during development and decreased with maturation. Secondly, when subcellular fractionation was per- formed the activity of the soluble guanylate cyclase was most closely correlated with the tissue concentration of cyclic GMP. Analysis of the biochemical char- acteristics of the guanylate cyclase isolated from testes of 5~lay-old rats did not demonstrate any significant alterations between the enzymes with the exception of an increased V observed in the soluble guanylate cyclase prepared from 5<lay~)ld rats.

Methods

Male Sprague-Dawley rats (Charles River CD ®) were housed in a tempera- ture-controlled suite with 12 h of light each day. Rats were fed and watered ad libitum. Rats less than 20 days of age were obtained and maintained as litters. In the experiments measuring guanylate cyclase activity during development, one sample from each age group (5, 10, 20, 30, 45, and 60 days) was used to determine homogenate, soluble and particulate guanylate cyclase activity in a single experiment. At least five such experiments (including a sample from each time point) were performed. In the experiments comparing the characteristics of the enzyme from neonatal and adult testes, samples from 5<lay-old and 60- day,ald rats were used for tandem analysis.

Radioimmunoassay of cyclic GMP. Rats of specific ages were killed by cer- vical dislocation, and samples of tissue (50--200 mg) were removed from decap- sulated testis and immediately frozen in liquid nitrogen. Frozen tissues were weighed and homogenized in 1 ml of cold trichloroacetic acid by a glass homo- genizer and motor<lriven glass pestle. After centrifugation at 2400 rev./min for 20 min, a 750 gl portion of the supernate was aspirated and washed three times with 10 vols. of diethyl ether saturated with water. Samples were evaporated

486

with nitrogen at 60°C. Dried samples were reconstituted in 1 ml of 0.05 M sodium acetate buffer, pH 6.2, and aliquots used directly in the radioimmuno- assay for cyclic GMP [22]. Specificity and accuracy of the radioimmunoassay for testicular cyclic GMP has been described [21].

Guanylate cyclase assay. Fresh tissue samples (100--300 mg) obtained from decapsulated testes were homogenized in 20 vols. of 0.25 M sucrose, containing 10 mM Tris-HCl buffer (pH 7.6) at 4°C, using a Brinkmann Polytron (Brink- mann Instruments, Inc., Westbury, NY) equipped with a PT-10 Probe. Homo- genization at a setting of 4 for 20 s produced complete cellular disruption as determined by light microscopic examination of homogenized samples. Soluble and particulate fractions were separated by centrifugation at 105 000 × g for 60 min. Supernates were decanted, and the pellets were washed and resus- pended in the original volume of buffer. Resuspended pellets were then dis- persed with the Polytron before assay (setting of 5 for 15 s).

Guanylate cyclase activity was determined in the assay developed by Kimura and Murad [3] as described previously [16]. The reaction mixture (150 pl) contained 50 mM Tris-HC1 buffer (pH 7.8), 3 mM MnC12, 15 mM phospho- creatine, 20 gg creatine phosphokinase, 0.2 mM 3-isobutyl-l-methylxanthine and 30--100 pg protein from the enzyme preparation. Guanylate cyclase assays were initiated by addition of 1 mM GTP and incubated for 10 rain at 37°C. Assays were terminated by the addition of 0.9 ml of a 50 mM sodium ace- tate buffer (pH 4.0) and heating for 2 min at 90°C. The cyclic GMP generated was determined by radioimmunoassay. Addition of 0.2 mM 3-isobutyl-1- methylxanthine to the assay mixture was sufficient to allow recovery of greater than 90% of exogenously added cyclic GMP. In the experiments testing the effects of Ca 2+ on guanylate cyclase activity, 3 mM CaC12 was added to the incubation mixture in the presence of 0.6--3 mM MnC12. Similarly, varying con- centrations (0.1--1.0 mM) of ATP were added to the incubation mixture to test the effects of ATP on testicular guanylate cyclase activity. When designated, 1 mM NaN3 was added to the reaction mixture (in the presence of 5 pg bovine catalase) and preincubated with the enzyme preparation for 10 min at 37°C before the addition of GTP. The effect of Triton X-100 on guanylate cyclase activity was determined after the preincubation of sample fractions with 1% Triton X-100 for 1 h at 4°C. The activity of guanylate cyclase in all fractions was linear with respect to protein concentration (up to 150 ~g) and time (up to 20 min). Protein was determined by the method of Lowry et al. [23]. DNA determinations were made according to the method of Burton [24]. Statis- tical analysis was performed by the Student's t-test for unpaired observations.

Results

Characterization of testicular guanylate cyclase Unlike the well-studied adenylate cyclase which is found in the particulate

fraction of most tissues, the subcellular distribution of guanylate cyclase varies from tissue to tissue [6]. The partitioning of guanylate cyclase in untreated homogenates into soluble and particulate fractions has been characterized from a number of tissues and varies from 90% particulate in the rat intestine [25] to 80% soluble in the rat lung [4]. In the adult rat (60 days old) centrifugation of

4 8 7

testicular homogenates at 105 000 ×g reveals that the guanylate cyclase activity is approximately equally distributed between the soluble and par- ticulate fraction (13.23 pmol cyclic GMP/mg protein per min and 16.9 pmol cyclic GMP/mg protein per min, respectively). Experimental analysis of the bio- chemical characteristics of soluble and particulate guanylate cyclase reveals the following:

Metal ion dependence. As with guanylate cyclase preparations from other tissues, the activity in the presence of Mn 2÷ is greater than that observed with other divalent cations [3,4]. The activity with either Ca 2÷ or Mg 2÷ alone is less than 10% of that observed with Mn 2÷, with the optimal activity of both the soluble and particulate guanylate cyclase observed at 3 mM Mn 2÷ (Fig. 1). In other tissues Ca 2÷ stimulates the activity of soluble guanylate cyclases when suboptimal concentrations of Mn 2÷ are used [3,4]. In experiments with the tes- ticular soluble guanylate cyclase, this phenomenon has also been observed. In the presence of 0.6 mM Mn 2÷ the activity of soluble guanylate cyclase is less than 20% of that seen with 3 mM Mn 2*. The addition of 3 mM Ca 2. produces a four-fold rise in the soluble guanylate cyclase activity. When the assay is per- formed with 1 mM Mn 2÷ the addition of 3 mM Ca 2÷ produces a two-fold rise.

Effect of nucleotide trophosphates. Fig. 2 demonstrates the kinetic analysis of soluble and particulate guanylate cyclase with respect to GTP in the pres- ence of optimal Mn 2÷. The soluble guanylate cyclase exhibits hyperbolic satura- tion curves. The Lineweaver-Burk plot (insert, Fig. 2) is linear with an apparent Km for GTP of 60 gM. Particulate guanylate cyclase, assayed after solubiliza- tion in Triton X-100, exhibits a sigmoidal saturation curve. Positive coopera- tive behavior with a non-linear Lineweaver-Burk plot was obtained and the apparent Km for GTP was 200 pM (insert, Fig. 2). These kinetic characteristics are typical of those observed for guanylate cyclase from other sources [3,4].

Concentrations of 0.1--1 mM ATP inhibit guanylate cyclase activity in other

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Fig . 1. E f f e c t s o f Mn 2+ o n so luble and part icu late gu an y la te c y c l a s e ac t iv i ty f r o m rat tes t i s . E n z y m e act iv- i ty w a s m e a s u r e d as descr ibed in M e t h o d s in the p r e s e n c e o f 1 m M GTP and varying c o n c e n t r a t i o n s o f M n C I 2 . T he e n z y m e preparat ions ( s o l u b l e = 3 0 ~ g ; p a r t i c u l a t e = 6 0 / ~ i p r o t e i n ) w e r e i n c u b a t e d for 1 0 m i n

at 3 7 ° C . T h e data is e x p r e s s e d as c G M P f o r m e d and each p o i n t represent s t h e m e a n o f tr ip l icate de ter - m i n a t i o n s .

488

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15.0

14.0

13.0

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90

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4.0

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SOLUBLE

/ 2~, ,,~, ~ ~ ,~o

I

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160

150

140

130

120

I10

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90

80

70

60

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P A R T I C U L A T E

,12

I -ff

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Fig. 2. Kinet ic analysis o f tes t icu lar guany la te c y c l a s e . So luble and part icu late fract ions o f test i s h o m o g e - nates w e r e separated as descr ibed in the M e t h o d s s e c t i o n . The part icu la te e n z y m e wa s assayed af ter so lubf l i za t ion in 1% Tr i ton X - 1 0 0 for 1 h a t 4 ° C . Th e act iv i t ies o f the so luble and part iculate guanyla te c y c l a s e s w e r e d e t e r m i n e d in the presence o f 3 m M MnC12 and varying c o n c e n t r a t i o n s o f GTP as indi- ca ted . Each p o i n t represent s the m e a n o f tr ipl icate d e t e r m i n a t i o n s . Inserts are double -rec iproca l p lo t s o f cyc l i c GMP f o r m a t i o n as a f u n c t i o n o f the GTP c o n c e n t r a t i o n .

tissues [3 ,26] . Inhibition by ATP is a characteristic o f the testicular soluble enzyme also. At a concentration of 0.3 mM ATP inhibition is half-maximal. Additional experiments with 5'-AMP (0.1--1.0 mM) have demonstrated inhibi- tion of soluble guanylate cyclase activity by an adenine monophosphate . How- ever, cyclic AMP does not inhibit the basal activity of the soluble enzyme (unpublished observations).

The effect o f non-ionic detergents. The incubation of soluble and particulate guanylate cyclase with non-ionic detergents such as Triton X-100 results in dif- ferential activation of guanylate cyclase activity [3 ,4] . In the rat testis incuba- tion of the particulate guanylate cyclase results in the solubilization of the enzyme and a marked increase in activity. In the adult rat, the activity is increased by six-fold (17.75 _+ 1.31 versus 107.12 + 16.16 pmol cyclic GMP/mg protein per min, n = 5). Incubation of the soluble enzyme in 1% Triton X-100 for 1 h results in a less than 50% stimulation.

In vitro activation. Several investigators have uncovered experimental con- ditions or agents which result in a significant activation of gnanylate cyclase in vitro. The discovery that compounds containing or capable of conversion to nitroso groups can activate guanylate cyclase is an example of this type of acti- vation [27 ,28] . Sodium azide (NAN3), a potent nucleophile, was observed by Murad and coworkers [29 ,30] to activate (10--30-fold) gnanylate cyclase from

489

some, but not all, tissues. Their findings indicate that NaN3 is converted to an active compound in the presence of a protein factor. In several tissues the fac- tor has been demonstrated to be the enzyme catalase [31,32]. In the testis, preincubation of the enzyme with 1 mM NaN3 for 10 min prior to the initia- tion of the guanylate cyclase assay results in a small stimulation of both soluble and particulate guanylate cyclase (50% activation or less). In contrast, preincu- bation of testicular soluble guanylate cyclase with both NaN3 and 5 pg bovine catalase results in a 16-fold increase in activity, a stimulation comparable to that seen with liver tissue preincubated with NaN3 alone. The testicular par- ticulate enzyme activity is increased by 250% in the presence of catalase and NAN3. Thus, the enzyme is capable of responding in vitro to active nitroso- containing compounds.

Guanylate cyclase from certain tissues undergo spontaneous activation when incubated at 37°C for 30 min prior to assay [33]. This phenomenon of auto- activation was not observed in preparations of testicular guanylate cyclase. Again, this may be a product of differences in tissue metabolism rather than a difference in the guanylate cyclase molecule.

Guanylate cyclase activity during testicular development The tissue concentration of cyclic GMP, measured at intervals during rat tes-

ticular development, is depicted in Fig. 3. The initial elevation persists from 5 to 20 days of age. By 30 days of age the tissue concentration of cyclic GMP characteristic of the adult rat is reached. To determine whether the capacity to synthesize cyclic GMP correlated with the tissue cyclic GMP concentration guanylate cyclase activity was assessed in rats of varying ages (5--60 days old). Fig. 3 demonstrates that the homogenate guanylate cyclase activity is highest on day 5; a subsequent decline is seen on day 10, and by day 20 the adult level is approximated.

As discussed previously, guanylate cyclase is partitioned into at least two subcellular fractions, the soluble and particulate, the activity of which has been demonstrated to change independently during growth of certain tissues [7,8, 16]. Therefore, the distribution and activity of the guanylate cyclases was determined in homogenates centrifuged for 1 h at 105 000 × g. The activity of the particulate guanylate cyclase paralleled that observed in the homogenate, i.e. the highest activity found on day 5 followed by a decrease apparent by day 10. In contrast, the soluble guanylate cyclase activity remained elevated on days 5, 10 and 20. Only after day 20 did the guanylate cyclase level decline towards the adult level. From examination of Fig. 3 it can be seen that the levels of tissue cyclic GMP were best correlated with the activity or cyclic nucleotide level/mg tissue protein. When the DNA content of testis from 5 and 60<lay~ld rats was determined, the results showed that the ratio of DNA con- tent to tissue protein was three times higher in the 5~lay-old rat. Thus, when the data are expressed per mg DNA the activity of soluble guanylate cyclase from 5 and 60<lay-old rats is approximately equivalent. The discrepancy in the activities expressed per mg protein or per mg DNA in all likelihood reflects the presence of ceils of varying DNA complement seen early in development [34]. The fact remains, however, that the guanylate cyclase activity/unit of soluble protein is higher in the infantile stage.

490

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Fig. 3. Guanylate c y e l a ~ activity and t issue cycl ic GMP levels during test icular deve lopment . Guanylate cyclase activit ies and tissue concentra t ions o f cycl ic GMP were determined as described in Methods . Points represents the m e a n of the activity or level , and vertical l ines the S.E. The number of determina- t ions is s h o w n above vertical l ines. Asterisks d e n o t e significant di f ferences ( P < 0 . 0 1 ) b e t w e e n activities or levels in neonata l test is (5 day) and subsequent t ime points .

The alteration in the specific activity/mg protein may represent an alteration in the number of enzyme molecules present or a change in the catalytic activ- i ty/molecule (or both). This question has been approached indirectly by com- paring the biochemical properties o f the soluble and particulate guanylate cyclase prepared from testes of 5 and 60~iay-old rats.

Table I compares the effect o f Ca 2÷ and varying Mn 2÷ concentrations on soluble guanylate cyclsse activity from testes of 5 and 6 0 ~ l a y ~ l d rats. The results demonstrate that the sample have a proportional decrease in activity whe incubated with suboptimal Mn 2÷. The subsequent stimulation by Ca 2÷ addition are equivalent in the enzyme preparation from 5 and 60~lay-old rats. This, there is little change in the metal ion response of the soluble form of the testicular enzyme during development.

The kinetic behavior with respect to GTP was analyzed to test for a change in the activity state o f the enzyme. Fig. 4 demonstrates that the Km for GTP of soluble guanylate cyclases from both 5 and 60~lay-old rats was 60 ~M. The dif-

491

T A B L E I

E F F E C T S OF CaCI 2 ON S O L U B L E G U A N Y L A T E C Y C L A S E A C T I V I ' I ~ IN T E S T I S F R O M N E O N A - T A L A N D A D U L T R A T S

Neona t a l a n d adu l t rats w e r e 5 a nd 6 0 days o f age, respec t ive ly . Soluble preparat ions w e r e assayed in the p r e s e n c e or abs ence o f 3 m M CaCI 2 a nd the c o n c e n t r a t i o n o f MnCI2 was varied as ind ica ted . Th e va lues represent the m e a n ± S.E. o f f ive e x p e r i m e n t s .

R e a c t i o n m i x t u r e Cyclic GMP f o r m e d ( p m o l l m g pro te in per ra in)

Neona t a l test is Ad u l t test is

0.6 m M Mn 2+ 4 .12 ± 0 . 1 8 0 .86 ± 0 .12 0.6 m M Mn 2+ + 3.0 m M Ca 2+ 19 .66 + 0 .21 3.97 ± 0 .81 1.0 m M Mn 2+ 9 .42 ± 1,96 3.81 ± 0 .92 1.0 m M Mn 2+ + 3 .0 m M Ca 2+ 24 .17 ± 3 .25 7.77 ± 1.23 3.0 m M Mn 2+ 25 .98 ± 2.49 7.11 ± 0 .82 3.0 m M Mn 2+ + 3 .0 m M Ca 2+ 38 .61 + 4 .80 8.77 ± 0 .78

ference in kinetic behavior noted was the 2.5-fold increase in the V exhibited by the enzyme from the 5~lay-old rat. This different kinetic behavior could be explained by an increase in the enzyme concentration or by the presence of an activator or an inhibitor that would alter the V but not the K m of the enzyme. To determine whether a transferable activator or inhibitor was present, mixing experiments were performed. In these experiments the enzymes prepared from testes of 5 and 60<lay~ld rats were incubated separately and together. In three experiments, no significant stimulation or loss of activity was observed in the mixed preparations, i.e. greater than 85% of the activity was present in the mixed enzyme incubations. This indicates that neither a transferable activator or inhibitor was present.

However, activation of the enzyme could occur in vivo and the effect persist in vitro [35]. In order to approach this possibility, experiments were per- formed to assess the ability of guanylate cyclase from testes of 5 and 60~lay-

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Fig. 4. Kine t ic a_n_aJyfis o f so luble guany la t e cyclase f r o m n e o n a t a l an d adu l t testis. Soluble f rac t ions w e r e p r e p a r e d as descr ibed in M e t h o d s . Th e doub le - rec iproca l p lo t s ex pres s cycl ic GMP f o r m a t i o n in the pres- e n c e o f 3 m M MnCI 2 a n d varying c o n c e n t r a t i o n s o f GTP as ind ica ted . T h e prote in c o n c e n t r a t i o n for the n e o n a t a l (5 days ) a n d adu l t (60 days ) preparat ions w e r e 59.5 #g an d 51.5 pg, r espec t ive ly . Each p o i n t represents the m e a n o f tr ipl icate d e t e r m i n a t i o n s f r o m t w o e x p e r i m e n t s ,

492

old rats to respond to a known in vitro activator, NAN3. The conditions that produce a maximally activated enzyme in adult testis were used. When the soluble enzyme from 5~lay~)ld rats was preincubated with catalase and NAN3, a 15.9-fold activation was observed {52.9 -+ 10.3 vs. 841.3 + 78.6 pmol cyclic GMP/mg protein per min, n = 5). Similarly treated soluble enzyme from 60- d a y ~ l d rats was stimulated by 15.5-fold {21.9 -+ 1.4 vs. 341.1 +_ 16.7, n = 5). This proport ionali ty would argue against an explanation that the enzyme from the 5<lay~ld rat had been preactivated in the tissue or during enzyme isola- tion.

In order to assess the total content of the particulate enzyme, it is necessary to solubilize it with a non-ionic detergent. Experiments performed in which the particulate guanylate cyclase from 5 and 60<lay-old rats were preincubated with Triton X-100 at 4°C for 1 h prior to enzyme assay. Preincubation of par- ticulate enzyme from 5<lay-old rats within Triton resulted in a 6.5-fold enhancement in activity {26.9 _+ 2.1 vs. 174.8 + 20.4 pmol cyclic GMP/mg pro- tein per min, n = 4). A similar increase (7.2-fold) in enzyme activity was noted when particulate enzyme from 60~lay-old rats was pretreated with Triton (15.1 -+ 1.5 vs. 109.8 _+ 15.5, n = 4). This would suggest that particulate guany- late cyclase from the 5<lay-old rat had not been depleted during the prepara- tion of the enzyme; which if had occurred, might explain the higher soluble ac- tivity at 5 days. In addition, the kinetic properties of the soluble enzyme from 5<lay-old rats (Fig. 4) are characteristic of soluble guanylate cyclases and not those characteristic of solubilized particulate guanylate cyclase (Fig. 2).

Discussion

The biochemical characterization of the soluble and particulate testicular guanylate cyclase demonstrates that the testicular enzymes are very similar to those studied in other mammalian tissues. The lack of stimulation by NaN3 alone and the absence of autoactivation are probably a product of the dif- ference in the enzyme profile of testicular tissue rather than any difference in the guanylate cyclase.

During development the concentrat ion of tissue cyclic GMP remains elevated through day 20 but decreases with maturation. Examination of guanylate cyclase activity in subcellular fractions revealed a correlation between tissue cyclic GMP levels and the soluble guanylate cyclase activity measured in vitro. However, the tissue level of cyclic GMP reflects a dynamic balance between synthesis and destruction. Others have demonstrated that an increase in cyclic nucleotide phosphodiesterase activity occurs during testicular development [36] and thus it is difficult to determine to what extent the observed elevation in the soluble guanylate cyclase contr ibutes to the elevated cyclic GMP levels.

We have examined the relationship between tissue cyclic GMP and guanylate cyclase activity in another testicular model, unilateral cryptorchidism [17]. In contrast to the reduction in soluble guanylate cyclase activity observed during maturation, an increase in soluble enzyme activity is observed in the testis rendered surgically cryptorchid. In these experiments, within 10 days post- operatively, the surgically placed abdominal testis exhibits a five-fold higher content of tissue cyclic GMP and a three-fold higher soluble guanylate cyclase

4 9 3

activity when compared to the scrotal testis from the same rat. In cryptorchi- dism the elevations in tussue cyclic GMP and the soluble guanylate cyclase activity are equivalent when expressed either as per mg protein or per mg DNA. Therefore, both during testicular development and atrophy a similar relation- ship is observed, a correlation between changes in tissue cyclic GMP and soluble guanylate cyclase activity:

In the course of studies characterizing a soluble form of adenylate cyclase present in adult testis, Braun et al. [37] also measured guanylate cyclase activ- ity during testicular development. Three time periods were examined (13, 27 and 90 days). The data presented from the one published experiment show a lower activity for cytosol guanylate cyclase (20 000 × g supernatant) in the 13- day-old rat than the 27<lay-old rat. Bu 90 days of age the activity had declined. Other than differences in the preparation of the soluble fraction, we do not have any explanation for the discrepancy observed during the 10--20 day time period.

The characteristics of the soluble guanylate cyclase isolated from testes of 5 and 60<lay-old rats have been examined with respect to kinetic properties, response to metal ions, and response to in vitro activation. These experiments did not reveal any significant difference between the two enzyme preparations. The stimulation by Ca 2÷ is proportional, as is the decrease in enzyme activity in the presence of suboptimal Mn 2÷ concentrations. The Km for GTP of the two enzyme preparations is identical. The presence of a transferrable factor that would explain the difference in V observed has been ruled out by the mixing experiments. However, a more difficult problem to assess was whether activa- tion of the enzyme occurs in the neonatal testis or whether metabolic dif- ferences between the 5 and 60<lay-old testes lead to differential activation during homogenization and centrifugation. Our approach to this question was to study the maximal activity produced by incubation with catalase and NAN3. The fact that each preparation is activated proportionally (15-fold) suggests that preactivation of the 5<lay<)ld enzyme had not occurred in situ or during isolation.

In summary, the data comparing guanylate cyclase activity in testes from 5 and 60<lay~ld rats are most consistent with an increase in the concentration of enzyme molecules rather than activation of preexisting soluble guanylate cyclase. However, to definitively prove this point immunoprecipitation of radioactively labelled guanylate cyclase will be necessary. I f indeed there is an induction of the soluble form of this isoenzyme during early testicular develop- ment and in response to unilateral cryptorchidism, then these two systems would seem to be appropriate models for studying the molecular mechanism underlying the apparent independent regulation of the different forms of guanylate cyclase.

Acknowledgements

We would like to acknowledge the technical assistance of Jane Aghajanian and the assistance of Celeste Layton and Vonnie Coombs in preparing this manuscript. This research was supported in part by grants AM19796, AM17438 and AM0551 from the National Institutes of Arthritis, Metabolism and Diges- tive Diseases and a University of North Carolina Research Council Award.

494

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