Life Sciences, Vol . 21, pp . gg7-1008 Printed in the U.S .A .

Pergamon Press

OUAFYLATE CYCLABE FROM DICTY08TELIUM DISCOIDEUM Anamarie Ward and Michael Brenner Department of Biology, Harvard University Cambridge, Mass .

02138

(Received in final form August 29, 1977) 8u~ary Cruanylate cyclase Prom crude homogenates of vegetative Dict stelium diecoideum has been characterized. It has a pH optimum of .0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity . It strongly prefers Mn~ to Mgt ae divalent cation, requires ~++ in ezceee of GTP for detect able activity, and ie inhibited by high Mn~ concentrations . It has an apparent I4a for GTP of approzimatelyr 51i uM at 1 mM excess Mn~ . The specific activity of guanylate cyclase is vegetative homogenates ie 50-80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found is the supernatant of a 100,000 z g spin (8100) . The enzyme is relatively unstable . It loses 40x of its activity after 3 hours storage oa ice. Enzyme activity vas measured Prom cells that had been shaken in phosphate buffer for various times . It tree found that the specific Activity changed little for at least 8 hours . Cyclic AMP at 10- M did not affect the guar~rlate cyclaee activity from crude hamogeaetea of vegetative or 6 hour phosphate-shaken cells . Cfuanoeiae 3' :5'-cyclic moaophosphate (cCMP) has been implicated ss a coatrolliag element for eueh diverse eukaryotic ftimctions as suppression of cardiac contractility, stimulation of uterus contraction, and control of the cell cycle (1,2) . Recent evidence suggests that eGMP may oleo play en improtant role in the life cycle of the cellular slime mold, Dictsostelium diecoideum . Hence, this organism may provide a convenient model system for studying the effects and metabolism of this nucleotide . During its growth phase, D . discoidr sum exists u a population of unicellular amoebae ; but vhea starved, cells aggregate chemotactively to form a multicellular organism composed of up to 105 cells . The aggregate may migrate for a period of time dd a slug-like peeudoplaemodium before completing development by lifting a sphere of spores on a cellular stalk. A possible role for cGMP in controlling the earliest stages of development ie suggested by the finding of McMahon and Goldberg thnt cGMP levels fall 20-fold during the first hour of starvation (D . McMahon and N . Goldberg, is preparation) . The only other event haorra to occur this early is the organism's development is a marked reduction is polyeomes (3) . cGMP may eleo act together ~rl.th adenosine 3' :5'-cyclic moaophosphate (CAMP) to control eomexàat later developmaatal events . It is already knoira that CAMP is the chemoattrsetant for D. discoideum (4) . A pacemaker cell initiates aggregation by emitting CAMP in pulses . Ad,)acent cells detect each pules and respond in tvo x~ye ; they move towards the source and emit their awe pulse of the nucleotide . In this manner, each signal travels outxard from the center, at497

998

Guanylate Cyclase from D . discoidewrt

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trecting cells inward . However, these CAMP pulses appear to control early differentiation events as well as chemotaxie . It has recently been shown that cells stirred in suspension autonomously produce cAMP pulses just as they do during aggregation oa a surface (4) . P~rthermore, when suspended cells are given an artificial pulse of CAMP, they synthesize and then secrete their own pulse of cAMP is response, just as expected from the chemotactic mechanism (6) . By srtificially pulsing suspended cells with CAMP before they would normally begin autonomous oscillations, certain early developmental events can be accelerated, like increases in CAMP phoaphodieeterase, in membrane-associated CAMP binding sites, and in membrane sites mediating cellular adhesions (7) . In addition, certain mutants usable to aggregate can be phenotypically cured by supplying CAMP pulses to the suspended cells (8) . Quite recently, Wurster, et al . (9) have discovered that iamtediete],y following the applied CAMP pulse, but preceding the cell-produced inereaee is cAMP, there is a transitory 10-fold increase in cGMP levels . Hence the first known response of the cells to an extrecellular CAMP signal is the rapid synthesis of eGMP . The ezteat to which this eGMP pulse mediates the effects elicited by the external CAMP aignale is not yet known . Ia order to understand the mechanism by which the 20-fold drop in cGMP levels occurs early is starvation, ae well as the mechanism controlling the eGMP oscillations during aggregation, a detailed understanding of the proper ties of the enzymes involved in its metabolism is required . Here we present as initial charneterization of the guanylate cyclase from DictYOetelium discoideum . Methods sad Materiale Growth of cells and re oration of crude homo seats . Amoebae of axenic strain Aa3 were grown at 2 C is HL5 10 og a rotating shaker at 200 rpm . Cello were harvested during log phase (2-6 x 10 /ml) and washed twice is lq mM potassium phosphate buffer, pH 6 .0, at 0°C by centrifl~gation at 2500 x g for 1 minute . Homogenization and subsequent steps were carried out nt ice bath temperatures . Vegetative enzyme was obtained by resuspending a pellet of washed cells at 4-q x 10 /ml in a small volume of homogenizing buffer consisting of 0 .1 mM EDTA, 1 mM dithiothreitol (DTT) (Calbiochem), 0 .2 mM MgC12, 1 .5 M sucrose, sad 10 mM TES-NaOH, pH 8 .0 (N-tris I$Ydroxymethyl] metYLyl-2-amiaoethane sulfonic acid) . Cells were Dounce homogenized to at least 90x breakage as determined by counting on a hemocyta~meter . Homogenates were used immediately for assay . Starved cells were prepared by reeuependiag washed vegetative cells in 1~ mM potassium phosphate buffer, pH 6 .0, at 2 .5 x 10~/ml and shaking them at 22° C on a rotary shaker (11) at 200 rpun in a flask whose volume ie approximately 5 times the volume of the cell suspension . At intervals, cells were harvested, washed, and homogenized as described for vegetative cells . Guauvlate cYClase aseaYS and column procedure for purification of eGMP . Unless otherwise stated, guan~ylate cyclase aseeys were carried out in 90 or 180 ul reaction volvmea containing 50 mM TES-NeOH, pH 8 .0, 1 mM D'PP, 2 mM cGMP (Sigma), 500 yM GTP (Sigma or PL) 1 .5 mM MnCl2, 16T ug/ml bovine serum albumin (Sigma, fraction V) and 2-5 x lOb epm a~P-GTP (New England Nuclesr, 19-29 Ci/mmole) . Reactions were started by addition of 15 or 30 u1 of a fresh preparation of crude homogenate which was previously diluted 1 :2k in homogenizing buffer . Aliquots were taken at various times, added to 100 ul of e diluting solution containing 1 mM cGMP, 1 .5 mM GTP, 10 mM Tris-HC1 (Tris hydroxymethyleminomethane), pH q .5, and approximately 3000 cpm 3H-cOMP (New England Ruclear, 8 .~4 Ci/mmole) (to allow calculation of cGMP recoveries) and stored oa ice until co~letion of the experiment . eGMP wen purified by column chromatography on neutral alumine (Sigma) followed by DEAF cellulose (Whatmaa DE-52) . 4 em neutral alumiaa columns and 1 cm DEAF columns, both equilibrated in 10 mM Tris-

vol . 21, No . 7, 1977

Guaeylate Cyclase from D . discoide~can

999

HC1, pH T .5 (column buffer), `rare prepared is advance is disposable Pasteur pipettes . Samples were loaded onto the neutral alumiaa columns and washed with 9 ml of column buffer . The neutral alumina columns were then placed over the DEAF cellulose columns sad washed with 8 ml column buffer to elute the eGMP onto the DEAF cellulose . The cGMP vas eluted from the DEAF cellulose is 1 ml 0 .1 x HCI, and radioactivity vas determined by dissolving the slants is 11 mls of a scintillation fluid containing 25Z Triton A-100 and fluors, as previously described (12) . eGMP recoveries, calculated from the x 3H-eGx~ recovered, ranged from 30-k5x . Guat~Ylste cyclsse setivitiee were calculated by subtrac tion of the column background fletermiaed i~om a zero time point . The zero time point samples never differed significantly from a blank lacking enzyme . With a32P-GTP type xEG 006 from xew England Nuclear, colu~ backgrounds ranged from a roximatelry 30 to 80 eprn for 1-3 z 10 cpm input a P-GTP (approximately 0 .0025 . Other souroes of a9~P-C~PP were tried (REA ftEG 006%, Amersham/Seeds PB146), but often gave higher column backgrounds and also partially inhibited enzymic activity . All experiments were done at leant twice with comparable results . Each assay point vas done in duplicate, the duplicates averaging lass than a 10Z difference between each other . xon-enzymatic formation of c01~ from GTP has been reported (13), but vas not observed under our reaction conditions . Protein determinations were by the method of Lowry, et al . (14), using bovine serum albumin ss standard . Pho hodieeterase di estioaa of ~P reaction roduct . Ia order to prove that the ~P-containing reaction product eluted from the columns vas indeed cGMP, eluatee from four reactions were cambiaed, evaporated to dryness, reeuspended is k00 ul 10 mM Trie-HC1, pH T .S, adjusted to pH 6-8 with xaOH and digested with beef heart phosphodiesterase (Sigma) in a 185 yl reaction volume contaiaiag 81 mM MgC12, 81 mM Trie-HC1, pH T .S, 270 U8/~ phoaphodiesternee, 5600 or 9900 cpm 3ü-eGMP sad 1g0 yl sample (approximately 2500 cpm ~P) . Reactions were carried out at 35 C . 25 U1 aliquots were taken at intervals sad added to 5 U1 50f trichloroacetic acid . 20 yl samples plus 5 u1 of a solution contaieiag 1 mM each GMP, cGMP, and guanosine were spotted oe Whatmaa 3Mid paper and run by ascending chromatography for 4 .5 hours in 1M ammonium acetate :95x ethanol, 3 :8 . Spots were visualised under ultraviolet light, cut out, sad eluted with 1 ml distilled voter for 20 minutes . After addition of 11 ml of the Trit a %-100 containing scintillation fluid, the samples were counted for ~P and ~ . GTP hsdrolseis determinations . Hydrolysis of ~P-GTP vas monitored by thin layer chromatography on cellulose 300-PEI plates (Brinkman) . 5-10 yl sables were spotted along with 5-10 amoles each of GTP, GDP, GMP, and eGMP standards onto PEI-cellulose sheets that had bees previousl.,q washed is water and air dried . Chromatograms were rue to a height of T .5 em in 1 .0 M LiCl, followed by further development to 15 cm is 1 .6 M LiCl (15) . Spots were visualized is ultraviolet light, cut out and coveted directly in POP-toluene scintillation fluid (12) . p="el;minarv fractionation . Crude homogenate was diluted 1 :3 is ho~mogemiziag buffer end centrifuged at 5000 z g for 10 minutes at 11 o C . S~ernatae~, including parti " _lly sadimeated particulate matter, van removed sad the ps3let (P5) vas resuspsnded in hamogsaisiag buffer to a final volume that was approximately one fourth the volume of the original homogenate . The supernatant (85) wu further centrifuged at 100,000 x g for 30 minutes at 4° C . Clear supernatant (5100) was removed sad the pellet (P100) vas resuspendsd is hamogeniziag buffer to a final volume of approximately one fourth that of the 85 . All samples were further diluted 1 :4 in hamogeniziag buffer and assayed for guanplate cyclase setivity as described above .

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Guanylate Cyclase from D . diaoaideum

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Results Proof reaction product is eGMP . To prove that the ~P-labeled product obtained from the guaaylate cyclase reaction is indeed cGMP, it was digested with beef heart phophodiesterase in the presence of authentic 3H-cGMP . It can be seen from the results in Figure 1 that both radioactive substances disappeared Pram the cGMP spot and accumulated in the 5'GMP spot with the same kinetics, indicating that the 3 2Pproduct, like the 3H-standard, is eGMP .

a Z c7 u ô a z c~ _c u ;~

15

mlnutp

30

45

FIG . 1 Co-digestion of 32 P reaction product and 3H eGMP standard with beef heart phoaphodiesterase . Phosphodiesterase reactions were erformed as described in Methods . Activity ie eapressed as ~ cpm ~H (/ /) or ~P in GMP (/ ~) or cGMP (/ 0) of total 3H or ~P cpm in GMP + cGMP . No significant accumulation of radioactive label was observed in the gueaosine spot . 82x of the input 3H cpm and 88x of the input ~P cpun were recovered as cGMP or GMP .

(~ 0)

Optimization of reaction conditions Protein concentration, specific activity, stability . The guanylate cyclase activity is approximately proportional to protein concentration between 13-60 ug protein/90 ul reaction volume for the crude homogenate enzyme (Figure 2) . At these protein concentrations, activity is approximately linear for 20 minutes (Figure 3), and the specific activity of the enzyme is generally 50-80 pmoles cGMP formed/min/mg protein . Thin is within the range found for guanylate cyclases from many eukaryotes (16) . The enzyme is relatively unstable . It loses approximately 40x of its activity after storage on ice for 3 hours . Temperature, pH, buffers . Maximum activity for the D . discoideum guanylate cycleae was obtained . at pH 8 .0 (Figure 4A), althoughthe activity changea little between pH 7 .0 to 8 .0 . A pH of 8 .0 was chosen for routine asagy conditions . At this pH the affinity of divalent cation for C~fP ie large enough to permit the assumption that the free divalent cation concentration is equal to the concentration in excess of the G'fP concentration (17) .

.c E â u w m

D. disooidewrt

Guanylata Cyclase from

Vol . 21, No . 7, 1977

4

a

E n 2

100

200

300

400

Ny protelnl90Nl

FIG.

Gueaürlate cyclase activity is crude hamogeaates of vegetative cells ne e function of protein concentration. Reactions ~rere performed at 22°C for 10 miauten under standard reaction conditions (eee Methods) .

too

eo

a

u

60

ô ~0

20

0

10

minufM

20

ä0

FIG. 3 Kinetics of guagylate cyclaee reaction . Reactions were done under standard conditions (see Methods) at 25°C using protein concentratioaa of 2k Ug/90 ul (O ), k8 ue/90 u1 (~) or no added eaayme (~> .

1001

Guaaylate Cyclaae from D . discoidewn

1002

Vol . 21, No . 7, 1977

Several buffers Whose pK'e fall is the range of ~-8 were tested for their effect oa guanylate cyclese activity . All were at a concentration of 50 mM . HEPSS-NnOH (N-2-hyflro~eti~yl-piperazine-N'-2-ethane eulfonic acid), TES-NaOH (N-trig [hydrozymethyl] methyl-2-aminoetheae sulfoaic acid), and triethanolemiae-HC1 all gave opt ime`1 activity ; TES-NaOH was chosen for our routine reaction conditions . At the same concentrations, Trie-HC1 gave a 20Z reduction is activity, and Tricine-NaOH (N-trio [hydro~gmethyl] methyl glyrciae) a 50~ reduction . A temperature optimum of 25°C was found (Figure 4B), which is close to the optimal temperature for growth and development of the organism (22° C) (18) .

60

60

O E

c_

~E

40

~ 40

É à

a C7 u m

20 É a

6

7

8

9

PH

0

70

20

30

t~mp~ratun fC)

FIG . 4 pH and temperature optima . Reactions were done under standard conditions (see Methods) nt 40 ug Protein/90 u1 except that (A) the buffer was MFG-NaOH (O ) or TES-NnOH (~) at the indicated pH, or (B) reactions were performed at the indicated temperatures . GTP rep[eneratin[~ sYStem, DTP, cGMP . Use of n GTP regenerating system was avoided because a pronounced inhibition of guaaylate cyclase activity was found in the presence of either 5-15 mM phosphoenolpyruvate (Calbiochem) (approximately q5K inhibition with 5 mM and 95x inhibition with 15 mM), or 15 mM crear tine phosphate (Calbiochem) (approximately 65x inhibition) . The inhibitory effect was somewhat reduced in the presence of the corresponding kinase . In the absence of a regenerating system it was necessary to monitor GTP levels in the reaction whoa conditions were varied to insure that altered GTPaee activity was sot the actual cause of observed changes is guagqlate cyclaee activities . This was dose ae described is Methods . MazinnTn guar~glate cyclase activity was found with 1-2 mM dithiothreitol . Activity wse 32~ lower in the presence of 0 .1 mM DTT, and 68f lower is its cadets absence .

Vol . 21, No . 7, 1977

Guanylate Cyclase from

D. disoaidewn

1003

su

resetioas to protect against Noa-radioactive eGMP (2 mM) was added to For the vegetative phoephodiesterase degradation of the 32P-eGMP generated . eaayme this level of eGMP reduced activity about 15x relative to that obtained However, the 2 mM eGMP vas retained, since assays of cells at is its absence. veaious developmental stages were contemplated, and it ie ]moan that cellaasociated phoephodiesterase levels increase markedlyr during the aggregation stage of development (19) . Phoephodiesteraee inhibitors routinely used against mammalian phosphodiesterases (theopbylliae, caffeine) are ineffective against the D. diecoideum ensyme (M . Brenner, unpublished observation) . Divalent cation . Mn~ and Mgt were tested for their effectiveness as divalent catione for the crude guanylate cyclase . It can be seen from the results is Figure 5 that this easyme exhibits specificity for Mn~ Addition of Mgt nt As hen been comparable concentrations resulted in ao detectable activity . found for moat other guanylate cyclaees, the Mn~ concentration must be at least equimolar with the GTP concentration for detectable activity, but high concentrations of Mn~ (above 2 mM for the D. discoidaum easyme) are inhi bitor Breakdown of GTP was checked, since divalent cation concentrations were varied during this ezperimeat, and GTPeee requires divalent canons for activity . The amount of degradation wan found to be lees than

lx .

~ so

a 6 d 2

r . ~ 1 2

+ 3

i 4

eonc~ntmtlan ImMI

i 5

FIG. 5 ~++

and Mgt concentration curves . Reactions were carried out at 25°C under standard conditions (see Methods) using 50 pg protein/ 90 U1, except that the indicated concentrations of MnC12 (O) or MgC12 (~) were used . Activities are calculated from 10 minute reactions. Substrate. The concentration of GTP was varied from 0.1 mM to 2 mM, and the ~eatratioa varied in parallel to maintain a constant excess Mn~ conInitial reaction rates were plotted by the method of centration of 1.0 mM . E~trapolatioa of the double reciprocal plot Lineweavar and Burie (Figure 6) . All revealed an apparent I~ for GTP of 517 uM for the crude homogenate easyme . other e~eriments reported is this paper were carried out at 500 utd GTP. Maintenance of the substrate at subsaturatiag concentrations alloue detection of changes is sasyme activity due to alterations is the Rm .

Guanylate Cyclase from D. discoic%um

1004

Vol . 21, No . 7, 1977

s

3

2

~ 11ßTP (mM )

9

1ß

FIG. 6 Km determination for GTP. Reactions were carried out at 25 °C is 180 u1 reaction vol~ee containing 50 mM TES-NnOH, pH 8 .0, 1 mM DTT, 2 mM cGMP, 16T yg/ml BSA, 100 ug protein, and e constant excess Maw concentration of 1 .0 mM . Velocity is expressed as pmoles eGMP/min/mg homogenate protein . GTP l~yrdrolysis was not greater than 8x for the time points used to determine initial reaction velocities for atoy of the GTP concentrations used . Distribution . Simple fractionation by centrifugation was performed oa the crude homogenate from vegetative cello to determine the distribution of enzyme activity . The data presented in Table I are typical . About T6x of the initial activity was found in the supernatant following a low speed centrifugation (5,000 x g) . Of the activity remaining following high speed centrifugation (100,000 x g), about 90~ is found in the aupernntant . However, npprozimately 45x of the activity in the low speed supernatant was lost during the 100,000 x g spin, and it cannot be ruled out that the lost activity was exclusively from the particulate fraction . Thus we cea only seky that at least 50x of the original activity is is the supernatant of the 100,000 x g spin . A reconstitution experiment was performed to determine if some component required by the enzyme in one fraction were segregated into the other by the high speed centrifugation . However, mixing of P100 and 5100 did not result in activity significantly different from the sum of the individual activities . GuaRylate cyclaae nativity in celle shaken is phosphate buffer . It ha~ been shown that marry of the early events in develop~meat also occur when cells are shaken in phosphate buffer is the absence of nutrients (19) . The specific activity of guarLylate cyclase was measured in crude ha~mogenates of vegetative cells sad cells that hnd been shaking in 17 mM potassium phosphate buffer, pH 6 .0, for various lengths of time . The reaulte are presented is

Vol . 21, No . 7, 1977

Guaaylate Cyclase from D. disooiderart

1005

Table Î Frsetioaation of Crude Homogenate by CeatrüLigntion fraction

total activity (pmoles/ min)

x total total activity protein (mg)

hamogeaate P5 $5

4230 520 3226

(100) 12 76

85 P100 sloo

302 147 1500

100 5 50

130 .8 17 .9 92 .6 21 .7 53 .4

x total protein (100) 14 71

~00) 25 62

specific activity (pmolee/ min/mg)

protein/ g0 ul reaction

32 .34 29 .05 34 .80

80 ug 40 ug

6.77 28 .10

7o ug 6o ug

7o ug

Reactions Fractions were prepared ns described in Methods . were carried out under ntandard reaction conditions (see Methods) at 25°C for 10 minutes. A11 f~setionn were diluted 1:12 before addition to the reaction mizture. Assays of all fractions were carried out at the same time, after the fractionation procedures were completed (1 .5 hours after cell harvest) . Activities obtained after high speed spin were normalized to that present in the S5 . Figure 7. The activity decreases to approzimately 87x of the vegetative activity at 2 hours, and then iacreeaee to a peak of appro:imately 1 .5 times the GTPase activity was found not to change eignivegetative activity at 6 hours . ficant]y during this time . 150

`ô s

~ô ô

i

2

i

L hours

v

6

ii

8

FIG . 7 Guanylate cyclaee activity is crude homogenates of cells shaken Reactions were in phosphate buffer for various lengths of time . carried out in 90 ul volumes under standard reaction conditions (see Methods) at 25° C for 6 miauten with 40-60 ug hamogeaate protein . Activities are ezpressed as x of vegetative activity (0 hour) .

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Guanylate Cyclase from D. disaoidewe

Vol . 21, No . 7, 1977

Effect of CAMP The alternating cGMP-CAMP oscillations observed by Wurster, et e1 . (9) auggßst that SAID may affect gueaylate cyclaee activity . To teat this, CAMP (10- and 10 - M) was added to reaction miaturee containing crude homogenate Pram either vegetative c~lls or Pram 6 hour phosphate-shaken cells . A lOx activity increase at 10- M CAMP was the largest effect observed . Discussion The guar~ylate cyclaee from crude homogenates of vegetative Dictyostelium diecoideum shares s cumber of characteristics with gue,~lnte cyclases of several over Mgt (1T), the other evkaryotee . These include the preference for bEn requirement that the Mn~ concentration be greater than that of GTP, inhibition by high levels of Mn~, inhibition by phosphoenolpyruvate and creatine phosphate, stimulation by a reducing agent, and a pH optimum in the range of q .0 8.0 . The D. discoideum enzyme is also similar to that of other eukaryotee in that a large fraction of the activity is found in the supernateat after a 100,000 x g spin . In contrast, the adenylate cyclaee from _D . discoideum (A . Ward, unpublished observation), and other eukaryotes (1~) appears to be mainly confined to the particulate fractions . The considerable loss of gunnylate cyclaee activity during high speed centrifugation ie eo far unexplained . The D. diacoideum guagylate cyclaee differs from those of moat other eukaryotee in its low temperature optimum (25°C) . However., this temperature is near that optimal for growth sad development of the organism (22 C), and is similar to the optimum for several other D. discoideum enzymes, including the adegylate cyclaee (20) . The Km for GTP of 51q uM is in the range of the highest velues so far reported for a guanylate cyclaee (400 tiiM for the soluble enzyme for rat kidney) (li) . This places the Km above the intracellular GTP concentration which in D. discoideum is approximately 200 yM (J . Geller and M . Bramer, in preparation) . The results obtained from the phosphate-shaken cells suggest that no dramatic changes in guar~ylnte cyclaee activity occur as the cells undergo starvation and become aggregation-competent . The twenty-fold decrease in intracellular eGMP concentration observed by McMahon sad Goldberg is not due to a simple decrease is specific activity of the enzyme . The activity we report here for phosphate-shaken cells may represent a minimal activity . Rooa et e1 . (21) and Klein (22) have found that the adenylate cyclaee activity of phosphate-shaken cells °scillates in phase with the CAMP concentratioa. The stimulated activity, obtained during the peak of an oscillation, is extremely unstable . _In vitro it decays is lees than a minute to a 7-fold lower basal activity (11) Since cGMP concentrations also oscillate in phosphate-shaken cells, it is possible that the gunRylate cyclaee behaves like the ßder~ylate cyclaee, and oscillates between an unstable acti vated state and a basal state of much lower activity . This is made particularly likely eiace the concentration of substrate, GTP, does not change over an oscillation (J . Geller and M. Brenner, in preparation) . If such oacillationa in enzyme activity occur, we would detect only the basal activity, as twenty minutes elapse during preparation of the crude extracts . The observation of Wurster et al . (9) that an increase in the intracellular cGMP concentration is induced by cAt~, suggests that guar{ylate cyclaee may be stimulated by CAMP . We have not been able to detect such a stimulation in vitro in extracts prepared from either vegetative or 6 hour phosphate-shaken cells .

Vol . 21, No . 7, 1977

Guanylate cyclase from D . tiiaooideun

1007

Ackaowledgmeate Thin work was supported by great A" PCM T6-19929 and BMS 72-01830 A02 from NSF . A .W . wan supported by NIH Training Grant 5-TO1-HD00415 . Refereacea l. 2.

3. 4. 5" 6. 7. 8. 9. 10 . ll . 12 . 13 . 14 . 15 . 16 . 17 .

18 . 19 . 20 . 21 . 22 .

N . D . OOLDBERG, M . K . HADDOX, S . E . NICOL, D . B . GLASS, C . H . SAAFORD, F . A . KUEHL, JR ., and R . ESTERSEN, Advan . Cyclic Nucleotide Res . 307-330 (1975) . R . D . GOLDBERG, M . K . HADDOX, E . DUNHAM, C . LOPEZ, and J . W . HADDEN, is The Cold ri Harbor sium oa the Re atioa of Proliferation in Animal Cells B . Clarkaoa end R . Baserga, eds . , pp . 09- 25, Cold Bpring Harbor Laboratory, Cold Spring Harbor, N . Y . (1973) . H . F . IADISH, T . ALTON, J . MARGOLSI~S, R . DOTTIIP, and A . WEINER, is Proceeds s of the 1 ICN-UCLA sium oa Devel tal Biolo (D . McMahoa and C . F . Fox, eds . , pp . 3 3 3, W . A . Benjamin, Inc ., 1975) . T . M . KONIJN, Advan . Cyclic Nucleotide Rea . 1 17-31 (1972) . G . GERISCH, and v . WICK, Biochem . Bioptüra . Rea . commun . ~ 364-370 (1975) . W . R008, V . NANJUNDIAH, D . MALCHOW, and G . GERISCH, FERS letters ~ 139-142 (1975) " G . GERISCH, D . MALCHOW, A HUF80EN, V . NANJUNDIAH, W . R008 and V . WICK, in Procea of the 1 ICN-UCLA aium on Develo tal Biolo (D . McMahon and C . F . Fox, eds . pp " 7 , W . A . Henjemin, Inc ., 1975) " M . DARNION, P . BRACHST, and L . H . P . DASILVA, Proc . Nat . Acad . 8ci . UBA Z 3163-3166 (1975) . B . WURSTER, K . SCHUBIGER, V . WICK, and G . GERISCH, FERS letters 76 141-144 (1977) . D . J . WATTS, and J . M . ABHWORTH, Biochem . J . 1~ 171-174 (1970) . H . BEUG, F . E . KATZ, ana G . GERISCH, J . Cell Biol . ~6 647-658 (1973) " 'M . BRELPRER, J . Biol . Chem . in preen . H . KIIrIIJRA, and F . MURAD, J . Biol . Chem . 2~ 32¢331 (1974) . 0 . H . LOWRY, A . J . ROSENBROUGH, A . L . FARR, and R . J . Rexnerr . , J . Biol . Chi . 1,265-275 (1951) . K . RANDERATH, and E . RANDERATH, is Methods is Ea l0 12A~(L . Groeamea and K . Moldave, eds .), pp " 323-347, Academic Press, R . Y . 1967) . P . V . SUL~AKHE, S . J . SULARHE, N . L . LEUNG, P . J . 6T . LOUIS, and R . A . HICKIE, Biochem . J . 1~ 705-712 (1976) . D . L . GABBERS, T . D . CHRISMAN, and J . G . HARDMAN, in L~karyotic Cell Function and Grovth . Re ation Intracellular lic Rucleotidea, J . E . Dumoat, B . L . Brown, and N . J . Marsha]1, eds . , pp " 155-193, Pleaium Preen, N . Y . (1976) . W . F . LOOMIS, Dirt atelium diecoideum A Devel tal tem, Academic Preen, R . Y . (1975 D . MALCHOW, H . RAGELE, H . SCHWARZ, and G . GERIBCH, Eur . J . Hiochem . 28 136-142 (1972) " C . KLEIR, FEBB Letters 68 125-128 (1976) . W . 8008, c . SCHNEIDEGGER, and G . OERISCH, Nature 266 259-261 (1977) " C . KLEIN, P . BRACHET, and M . DARMON, FERS ktters Z 145-147 (1977) "