JOURNAL OF BACTERIOLOGY, Mar. 1973, p. 1249-1255Copyright 0 1973 American Society for Microbiology

Vol. 113, No. 3Printed in U.S.A.

Metabolism of Adenosine 3', 5'-CyclicMonophosphate and Induction of Fruiting

Bodies in Coprinus macrorhizusISAO UNO AND TATSUO ISHIKAWA

Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo, Japan

Received for publication 13 September 1972

The adenyl cyclase and phosphodiesterase metabolizing adenosine 3', 5'-cyclic monophosphate (cyclic AMP) were detected in mycelia of strains ofCoprinus macrorhizus which form fruiting bodies, but not in those of strainswhich do not form fruiting bodies. The adenyl cyclase synthesized cyclic AMPfrom adenosine triphosphate. The phosphodiesterase degr- led cyclic AMP toadenosine-5'-monophosphate and was inhibited by adenosine-3'-monophos-phate, theophylline, and caffeine. The strains which form fruiting bodies incor-porated and metabolized cyclic AMP, but strains which do not form fruitingbodies did not. The possible participation of cyclic AMP in the induction offruiting bodies is discussed.

The metabolic pathway of adenosine 3', 5'-cyclic monophosphate (cyclic AMP) may in-volve two enzymes: adenyl cyclase and phos-phodiesterase. The adenyl cyclase catalyzesthe formation of cyclic AMP from adenosinetriphosphate (ATP), and the phosphodiester-ase degrades cyclic AMP to adenosine mono-phosphate (AMP). These enzymes may play animportant role in regulating the concentrationof cyclic AMP in vivo (5). Earlier studies (20,21) indicated that mycelial cells of particularstrains of Coprinus macrorhizus contain cyclicAMP which may play a role in the induction ofmonokaryotic fruiting bodies. The present re-port describes characteristics of adenyl cyclaseand phosphodiesterase obtained from myceliaof the strains of C. macrorhizus which formmonokaryotic fruiting bodies.

MATERIALS AND METHODSOrganisms. The following strains of C.

macrorhizus Rea f. microsporus Hongo were used:708-7 A7B8, 708-15 A8B7, fis-, fis+, and fisc. Theorigin and characteristics of these strains have beendescribed elsewhere (20).

Media. Fruiting assays, production of mycelia andfruiting bodies, and culture maintenance were car-ried out on a malt-yeast medium (MY medium)which contained 10 g of malt extract, 4 g of yeastextract, and 4 g of glucose per liter of deionizedwater. To prepare solid agar medium, 10 g of agarpowder was added to 1 liter of MY medium.Assay for FIS activity. Bioassays for fruiting-

inducing activity (FIS activity) were performed asdescribed elsewhere (20, 21).

Preparation of cell-free extract. Mycelial sus-pensions were inoculated and incubated in 100-mlErlenmeyer flasks containing 20 ml of MY mediumat 30 C for 5 to 14 days under continuous illumination(1,000 to 6,000 ergs per cm2 per s). Mycelia, fruitingbody primordia, and fruiting bodies were harvestedon a filter paper after 5 to 14 days, washed well withdistilled water, and macerated in 0.05 Mtris(hydroxymethyl)aminomethane (Tris)-hydro-chloride buffer, pH 7.4, with a Waring blender for 5min. The suspension obtained was further homoge-nized with a Braun homogenizer for 3 min. Theresulting homogenate was centrifuged at 1,000 x g for20 min. The supernatant fluid was used as the crudeenzyme preparation.

Gel filtration and ion-exchange chromatography.Gel filtration and ion-exchange chromatography wereperformed as previously described (21).Enzyme assays. The adenyl cyclase activity was

determined by the method described by Tao andLipmann (19). The phosphodiesterase activity de-grading cyclic AMP was measured by the two-stepassay method described by Butcher and Sutherland(2). The inorganic phosphate liberated was measuredcolorimetrically' according to the Fiske-Subbarowmethod (12).

Bray's solution. Radioactivity was determined byusing the following Bray's solution (1): naphthalene,60 g; 2,5-diphenyloxazole, 4 g; 1,4-di-2-(5-phenyl-oxazolyl)-benzene, 0.2 g; methanol, 100 ml; ethyleneglycol, 20 ml; and dioxane to 1 liter.

Protein measurement. Protein concentration wasdetermined by the method of Lowry et al. (13).

1249

on August 8, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

UNO AND ISHIKAWA

Chemicals. Malt extract was a product of Difco,and yeast extract was purchased from Kyokuto.Other special chemicals used were as follows: Dowex1 (Dow Chemicals), AG50W (Calbiochem), Se-phadex G-100 and Blue Dextran (Pharmacia), cel-lulose thin-layer sheets (Eastman), 3H-ATP and3H-cyclic AMP (Radiochemical Centre and NewEngland Nuclear Corp.), snake venom (SigmaChemical Co.), and ATP, cyclic AMP, and othernucleotides (Boehringer Mannheim).

RESULTSDetection and partial characterization of

adenyl cyclase. Crude mycelial extracts of fiscA8B7, fis+ A8B7, fis- A8B7, and the dikaryon(A 7B8 + A8B7) were prepared at various stagesof culture and assayed for adenyl cyclase activ-ity. As shown in Fig. 1, adenyl cyclase activitywas detected in the strains which are able toform fruiting bodies (fisc, fis+, and the dikar-yon), but not in the fis strain. The level ofadenyl cyclase activity varied in the course offruiting body formation. Prior to cell aggrega-tion or formation of fruiting body primordia,higher levels of adenyl cyclase activity weredetected. This activity decreased at the stageof fruiting body formation in all strains whichform fruiting bodies.The adenyl cyclase was partially purified by

ammonium sulfate precipitation and gel filtra-tion. A 35.2-g amount of ammonium sulfatewas added to 50 ml of crude extract prepared

-

0Q.4000IEU'

E 300-1

_ 200z

.2_LI.

4, 100)cL

0

5 10Culture time (days )

from 8-day-old mycelia of the fisc strain. Thesolution was left for 2 h at 4 C in an ice-waterbath, and the supernatant fluid was removedby centrifugation. The precipitate was dis-solved in 8 ml of 0.05 M Tris buffer, pH 7.4, andthe remaining precipitate was removed bycentrifugation. The supernatant fluid was fil-tered through a Sephadex G-100 column. Asshown in Fig. 2, the adenyl cyclase activity wasdetected in the void volume of the elutionbuffer. This means that the adenyl cyclase mayhave a molecular weight of more than 150,000.Fractions showing adenyl cyclase activity werepooled and used as the enzyme sample infurther experiments.To analyze the reaction products of adenyl

cyclase, 0.4 ml of the reaction mixture, con-taining 120 nmol of 3H-ATP (40 ,Ci), 20 ,umolof Tris buffer, pH 7.4, 8 ,Amol of MgSO4, andthe enzyme sample (0.63 mg of protein), wasincubated at 30 C for 30 min. The reaction wasterminated by heating in a boiling-water bathfor 3 min, and then 25 Aliters of 5% ZnSO, and25 Ailiters of 0.3 N Ba(OH)2 were added to thereaction mixture. The solution was mixed well,allowed to stand at 4 C for 15 min, and thencentrifuged at 1,000 x g for 20 min. Thesupernatant fluid was chromatographed on anAG50W x4 column. As shown in Fig. 3, a peak

C

(A'.

.'

L Iv}

IV

4

GotA

c.

15

FIG. 1. Adenyl cyclase activity of crude extractsobtained from mycelia (including fruiting bodies) ofvarious ages in four strains of Coprinus macrorhizus.The following three stages of fruiting body formationare indicated by arrows; cell aggregation (A), forma-tion of fruiting body primordia (B), and formation ofmature fruiting body (C). Symbols: O--O, fis'A8B7; 0----0, fis+ A8B7;Ai\ A, fis- A8B7; *----,dikaryon (A7B8 + A8B7).

10 20Fraction number

X.5

E

E-

0

a.

FIG. 2. Gel filtration of adenyl cyclase from Co-prinus macrorhizus. Crude extract from 8-day-oldmycelia of fisc A8B7 was precipitated with ammo-nium sulfate (10%o saturation); 2 ml of the enzymepreparation thus obtained was put onto a SephadexG-100 column (1.8 by 50 cm) and eluted with 0.05 MTris buffer, pH 7.4, into 3.5-ml fractions. Eachfraction was assayed for adenyl cyclase activity andprotein concentration. Elution position of Blue Dex-tran (BD) is shown by the arrow. Symbols: 0, adenylcyclase activity; *, protein concentration.

1250 J. BACTERIOL.

A

c

I.0 v

on August 8, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CYCLIC AMP AND FRUITING INDUCTION IN COPRINUS

6

EI

CI-)0

cAMP

A T P IADP

?

10

I n',l

I1Frcto nube

0.6

E0.4 O

(0C

0

d

C

InUI

cv0.2

20

FIG. 3. Chromatographic analysis of the reactionmixture incubated with the adenyl cyclase sample. A0.5-mi amount of Ba(OH2)-treated reaction mixturecombined with 0.1 ml of 10-3 M authentic cyclicAMP was applied to an AG50W x4 (hydrogen form)column (0.9 by 20 cm) and eluted with distilled waterinto 4.5-ml fractions. Ultraviolet absorption at 260nm and radioactivity of each fraction were deter-mined. Elution positions of ADP, ATP, and cyclicAMP (cAMP) are indicated by arrows. Symbols: 0,3H-radioactivity; 0, ultraviolet absorbance at 260nm.

of the radioactivity was observed at the peak ofultraviolet (UV) absoprtion corresponding tothe cyclic AMP position. The cyclic AMPfractions showing radioactivity were pooled,frozen, and dried. The resulting powder wasdissolved in 100 Mliters of distilled water andapplied to a cellulose thin-layer sheet. Asshown in Fig. 4, a significant amount of radi-oactivity was observed at the position of cyclicAMP. Through these purification procedures,the recovery of cyclic AMP was about 50%.From these results, it is concluded that thecell-free extract of the fisc strain contains theadenyl cyclase. The further characterization ofthis adenyl cyclase was extremely difficult,because this enzyme became inactive over ashort period.Detection and partial characterization of

phosphodiesterase that degrades cyclicAMP. Crude mycelial extracts of fisc A8B7,fis+ A8B7, fis-, A8B7 and the dikaryon(A7B8 + A8B7) were prepared at various stagesof culture and assayed for phosphodiesteraseactivity. As shown in Fig. 5, the phosphodies-terase activity was detected in strains whichare able to form fruiting bodies. The activity.was relatively high at the early stages of cul-

5 10cm from origin

FIG. 4. Detection of 'H-cyclic AMP involved inthe cyclic AMP fraction obtained after AG50W x4chromatography of the reaction mixture of adenylcyclase. The cyclic AMP fraction obtained afterAG50W x4 chromatography (Fig. 3) was chromato-graphed together with carrier cyclic AMP on acellulose thin-layer sheet (Eastman 6065) with thesolvent 1 M ammonium acetate-990o ethanol (30:75,vol/vol). The thin-layer sheet was separated by 1 cmand the radioactivity was determined. The elutionpositions of AMP, cyclic AMP (cAMP), and adeno-sine are indicated by arrows.

ture and decreased in the course of fruitingbody formation. The fis- strain showed nophosphodiesterase activity throughout theculture ages tested. The fis+ strain showed*about 10 times as much phosphodiesterase ac-tivity as the fisc strain.To purify the phosphodiesterase, 50 ml of the

crude extract of the fisc strain was preparedand precipitated with 35.2 g of ammoniumsulfate. After standing at 4 C for 2 h, thesupernatant fluid was removed by centrifuga-tion. The precipitate was dissolved in 8 ml of0.05 M Tris buffer, pH 7.4, and centrifuged toremove the precipitate. The supernatant fluidwag filtered through a Sephadex G-100 column.As shown in Fig. 6, a well-defined peak of thephosphodiesterase was observed at a positioncorresponding to a molecular weight of lessthan 150,000. The fractions showing phos-phodiesterase activity were pooled and used asthe enzyme preparation. The optimal pH ofthis phosphodiesterase was found to be 8.5 byassaying with cacodylate-HCl buffer and Trisbuffer. Of several divalent cations tested (Ta-

VOL. 113, 1973 1251

x2

on August 8, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

UNO AND ISHIKAWA

A

";

I5 10

Culture time (days )

15

FIG. 5. Phosphodiesterase activity of crude ex-

tracts obtained from mycelia (including fruitingbodies) of various ages in four strains of Coprinusmacrorhizus. See Fig. I for symbols.

.0-

E

CPE

).5 0a.

10 20 30Fraction number

FIG. 6. Gel filtration of phosphodiesterase. Crudeextract from 8-day-old mycelia of the fisc A8B7strainwas precipitated with ammonium sulfate (100%lo satu-ration); 2 ml of the enzyme preparation thus ob-tained was put onto a Sephadex G-100 column (1.8by 50 cm) and eluted with 0.05M Tris buffer, pH 7.4,into 3.5-ml fractions. Each fraction was assayed forphosphodiesterase activity and protein concentra-tion. Elution position of Blue Dextran (BD) is shownby the arrow, Symbols: 0, phosphodiesterase activ-ity; 0, protein concentration.

ble 1), Mg2+, Mn2+, and Ca2+ stimulated thephosphodiesterase activity. The enzyme activ-ity was completely abolished in the presence ofethylenediaminetetraacetate. Cu2+ and Zn 2+were potent inhibitors of this enzyme at theconcentration used, and Fe2+ and Ba2+ showed

TABLE 1. Effect of various compounds onphosphodiesterase activity

oncn Phosphodies-Addition terase activity

(MM) (% of control)

None ........................ 100MgCl2 ....................... 2 350MnSO4 ....................... 2 390BaCl2 ....................... 2 110FeSO4 ....................... 2 100CaCl2....................... 2 250CuSO4. ........................ 2 50ZnCl2....................... 2 38Ethylenediaminetetraacetate ... 2 0Theophylline .................. 2 0Caffeine ...................... 2 0ATP ....................... 2 100

20 73-AMPb ....................... 10 82

20 525'-AMP5 ...................... 20 1003-Guanosine monophosphate".. 20 965'-Guanosine monophosphate5.. 20 96

a One-tenth unit of phosphodiesterase was used.b Phosphodiesterase activity was assayed by a

method in which 3H-cyclic AMP was used to dif-ferentiate the reaction product from nucleotideadded in the reaction mixture. The reaction mixturecontained, in a final volume of 50 Mliters; 2.5 umol ofTris buffer, pH 8.0, 1 Amol of MgSO4, 5 nmol of3H-cyclic AMP (0.05 MCi), enzyme sample, and thenucleotide to be tested. The mixture was incubatedat 30 C for 30 min, and the reaction was terminatedby heating for 3 min in a boiling-water bath. Aftercentrifugation, 25 uliters of the supernatant fluidcontaining 50 nmol of carrier 5'-AMP was applied toa cellulose thin-layer sheet and developed with thesolvent 1 M ammonium acetate-99% ethanol (30:75,vol/vol). The spot corresponding to 5'-AMP was cutout, and its radioactivity was measured.

no effect. Theophylline, caffeine, and ATPwere effective inhibitors of the phosphodieste-rase of C. macrorhizus (Table 1), as has beenfound in many other organisms (3, 4, 16).Furthermore, this enzyme was inhibited by3-AMP, but not by 5'-AMP, 3'-guanosinemonophosphate, and 5'-guanosine monophos-phate (Table 1).

Reaction products of the phosphodiesterasethat degrades cyclic AMP were analyzed fromthe reaction mixture incubated at 30 C for 30min. After the reaction had been terminated byheating in a boiling-water bath for 3 min, theprecipitate was removed by centrifugation. Thesupernatant fluid was applied to an AG50W x4column (0.9 by 20 cm), and the column waseluted with distilled water into 4.5-ml frac-tions. Fractions corresponding to the elution

1.5

0.01E_ 1.0

u 0.5'._U

e,0.un

oiCL)

0

1252 J. BACTERIOL.

on August 8, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CYCLIC AMP AND FRUITING INDUCTION IN COPRINUS

position of AMP were pooled, concentrated,and chromatographed on a Dowex 1 x2 (for-mate form) column. As shown in Fig. 7, therewas a clear peak of 5'-AMP on the elutionprofile, but not of 3'-AMP. This result indi-cates that the reaction product of phos-phodiesterase of C. macrorhizus is 5'-AMP.

Incorporation of 3H-cyclic AMP into my-celial cells. Cyclic AMP induced monokaryoticfruiting bodies in the fis+ strain of C.macrorhizus (20). Therefore, mycelia whichhad incorporated 'H-cyclic AMP were frac-tionated to learn the role of cyclic AMP inmycelial cells. Cultures of the fisc, fis+, and fis-strains were incubated at 30 C for 8 days. A1-ml amount of 3H-cyclic AMP (20 uCi), steri-lized by filtration, was added to each flask. Thecultures were further incubated for 10 h underthe same conditions. The mycelia were har-vested on a filter paper, washed well withdistilled water to remove unincorporated 3H-cyclic AMP, and then suspended in 20 ml ofcold 5% trichloroacetic acid. The suspensionwas homogenized in a Waring blender for 15min, and the precipitate was removed bycentrifugation. A portion of the supernatantfluid was applied to an AG50W x4 (hydrogenform) column (0.9 by 20 cm) and eluted withdistilled water into 4.5-ml fractions. The radi-

40Fraction number

FIG. 7. Identification of AMP fraction obtainedafter AG50W x4 chromatography of reaction mixtureof phosphodiesterase. The AMP fraction obtainedafter AG50W x4 chromatography was concentrated,applied to a Dowex I x2 (formate form) column (0.9by 20 cm), and eluted, first with distilled water andthen with 0.1 Nformic acid. Ultraviolet absorption at260 nm of each fraction was determined. Elutionpositions of 5'-AMP and 3'-AMP are indicated byarrows.

oactivity of fractions corresponding to threepeaks (adenosine diphosphate [ADP] andATP, cyclic AMP, and AMP) was measured(Table 2). In addition, mycelia of each strainwere fractionated by the method of Schmidtand Thanhauser (22), and the radioactivity ofeach fraction was determined (Table 2). Theresults clearly indicated that mycelia of twostrains, fisc and fis+, were able to incorporate3H-cyclic AMP. A significant amount of the3H-cyclic AMP incorporated was transformedto ADP and ATP. An extremely small amountof AMP transformed from 3H-cyclic AMP wasfound in two strains. As shown in Table 2,mycelia of the fis- strain were unable toincorporate a significant amount of the 3H-cyclic AMP added to the culture medium,although the cyclic AMP fraction contained asmall amount of 3H radioactivity which may bederived from 3H-cyclic AMP adsorbed to my-celia.Induction of monokaryotic fruit bod-

ies by inhibitors of phosphodiesterase. Itwas found previously that 3T-AMP was one ofthe effective components of fruiting-inducingsubstances (20, 21). Since 3'-AMP is an inhibi-tor of phosphodiesterase, the other inhibitors ofthis enzyme, theophylline and caffeine, weretested for FIS activity. Various amounts ofinhibitors were added to MY media and thefis+ mycelia were inoculated. The cultures wereincubated at 30 C for 15 days under continuousillumination. As shown in Table 3, all of thesesubstances were effective in inducing monokar-yotic fruiting bodies at concentrations higherthan 2 x 10- 5 ,M. The highest concentration oftheophylline and caffeine used (2 x 10-1 ,uM)was inhibitory for induction of fruiting bodies.

TABLE 2. Summary offractionation of crude extractsprepared from mycelia of three strains of Coprinusmacrorhizus after incorporation of 'H-cyclic AMP

3H radioactivity(10' counts/min)

Fractionfisc fisj+ fis-A8B7 A8B7 A8B7

Incorporated 3H-cyclicAMP (total) ......... 85 75 1.7

Trichloroacetic acid-solu-ble fraction .......... 47.8 57.4 1.5

ATP + ADP ........... 32.9 35.1 0Cyclic AMP ........... 4.7 6.2 1.4AMP .................. 0.1 0.1 0

Trichloroaceticacid-insoluble fraction 12.5 18.5 0

Ribonucleic acid ....... 7.5 9.3 0Deoxyribonucleic acid 5.5 8.4 0

VOL. 113, 1973 1253

on August 8, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

UNO AND ISHIKAWA

TABLE 3. Effect of substances inhibitingphosphodiesterase on the induction of monokaryotic

fruiting bodies in Coprinus macrorhizusa

Concn (MuM)Addition 2x 2x 2x 2x *2x

10-1 10-2 10- 1-41 10-5

3'-AMP .......... 7 7 5 4 1Caffeine .......... 0 8 8 6 1Theophylline 0..... 8 8 4 1

aTen independent slant cultures of strain fis+A8B7 were made and incubated at 30 C for 15 daysunder constant illumination. The number of cultureswhich produced monokaryotic fruiting bodies is in-dicated.

DISCUSSIONMycelia of fisc and fis+ strains of C.

macrorhizus, which are able to form monokar-yotic fruiting bodies, showed an ability tosynthesize cyclic AMP from adenine, but thoseof the fis strain, which is unable to formfruiting bodies, did not (21). It was also foundthat cyclic AMP added to the fis+ culture waseffective in inducing monokaryotic fruiting bod-ies (20). The present results demonstrate theoccurrence of adenyl cyclase activity in mono-karyotic mycelia of fisc and fis+ strains and indikaryotic mycelia, but not in monokaryoticmycelia of the fis- strain. These results suggestthat cyclic AMP synthesized by adenyl cyclaseplays an important role in the formation offruiting bodies, particularly at the stage ofinduction of fruiting bodies.The molecular weight of adenyl cyclase was

estimated to be more than 150,000. There is apossibility that the adenyl cyclase obtained inthe present work may not be solubilized. Ade-nyl cyclase was first found in various verte-brates (11, 14, 15, 17). To our knowledge, thepresent report is the first to indicate theoccurrence of adenyl cyclase in a basidiomy-cete. The adenyl cyclase in some bacteria (8-10) and in higher plants (23) has been solu-bilized and partially purified.Yamamoto and Massey (24) reported that

phosphodiesterase that degrades cyclic AMPcould not be detected in several members of theplant kingdom, including mycelia of C.macrorhizus. In the present study, phos-phodiesterase was found in monokaryotic my-celia of fisc and fis+ strains and in dikaryoticmycelia of C. macrorhizus, but not in monokar-yotic mycelia of the fis- strain. The phospho-diestrase of C. macrorhizus degraded cyclicAMP to 5'-AMP. An enzyme of the samecharacteristics was found in animals and bac-

teria (2, 16), and an enzyme of different charac-teristics, which degrades cyclic AMP to 3'-AMP, was found in barley seeds and carrotleaves (C. J. Pollard and R. J. Venere, Fed.Proc. 29:670, 1970). Mycelia of the fis+ straincontained approximately 10 times as muchphosphodiesterase activity as mycelia of thefisc strain and the dikaryon. It may be specu-lated that mycelia of the fis+ strain containinga large amount of phosphodiesterase rapidlydegrade cyclic AMP synthesized by adenylcyclase, and therefore the fis+ strain requiresan external supply of cyclic AMP to formfruiting bodies. The addition of an extremelysmall amount of cyclic AMP (2 x 10-4 'qM)induced monokaryotic fruiting bodies in thefis+ strain (20). Therefore, it may be postulatedthat a cyclic AMP pool of a reasonable size ismaintained in fis+ and fisc cells by someunknown cellular mechanism which allows es-cape from the phosphodiesterase activity.As described previously (20, 21), 3'-AMP

could induce monokaryotic fruiting bodies inthe fis+ strain. The present results showed thattheophylline and caffeine added to the culturemedia were also effective in inducing monokar-yotic fruiting bodies in the fis+ strain. Ourfinding that 3'-AMP, theophylline, and caf-feine all inhibited phosphodiesterase activitysuggested a relationship between this charac-teristic and their induction of monokaryoticfruiting bodies. It is postulated that thesesubstances induce fruiting bodies in the fis+strain through some mechanism by which theiraddition to fis+ cultures leads to an increasedaccumulation of cyclic AMP by reducing theextraordinarily high level of phosphodiesteraseactivity of fis+ mycelia. Mycelia of the fis-strain failed to incorporate cyclic AMP intomycelial cells. This may be due either to a lackof enzymes metabolizing cyclic AMP or to theabsence of a permeation mechanism for cyclicAMP in this strain. This difference in the fis-mycelia explains why cyclic AMP added to theculture media was ineffective in inducing mon-okaryotic fruiting in the fis- strain, although itwas effective in the fis+ strain. Heidrick andRyan (7) indicated thiat the cyclic AMP incor-porated into animal cells was converted toAMP, ADP, ATP, and inosine. The presentresults indicate that an extremely smallamount of AMP was produced from cyclicAMP incorporated into mycelial cells of fisc andfis+ strains. Cyclic AMP which has been incor-porated may be converted by the action ofphosphodiesterase to 5'-AMP and may be usedimmediately to synthesize ADP, ATP, andnucleic acids. However, there is a possibility

1254 J. BACTERIOL.

on August 8, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CYCLIC AMP AND FRUITING INDUCTION IN COPRINUS

that cyclic AMP may be converted to ATP bythe reverse reaction catalyzed by adenyl cy-clase, since it has been shown that the purifiedadenyl cyclase from bacteria was capable ofsynthesizing ATP from cyclic AMP and pyro-phosphate (6, 10, 18; P. Greengard, 0. Hayai-shi, and S. P. Colowick, Fed Proc. 28:467,1969).

All of the results described in this and earlierreports (20, 21) indicate that adenyl cyclase,phosphodiesterase, and a protein which easilybinds with cyclic AMP are found in monokar-yotic mycelia of fis+ and fisc strains and indikaryotic mycelia, all of which are able toform fruiting bodies, but not in monokaryoticmycelia of the fis- strain, which are unable toform fruiting bodies. Cyclic AMP synthesizedby the action of adenyl cyclase may play animportant role in the induction of fruitingbodies by binding with a particular protein.The manner in which fruiting bodies are in-duced in the presence of a cyclic AMP-proteincomplex remains to be elucidated.

ACKNOWLEDGMENTSWe express our appreciation to T. Yanagita for his

encouragement and discussion during the course of this workand to K. Kimura for supplying the strains of C.macrorhizus.

This work was supported by a research grant from theMinistry of Education of Japan.

LITERATURE CITED1. Bray, G. A. 1960. A simple efficient liquid scintillator for

counting aqueous solutions in a liquid scintillationcounter. Anal. Biochem. 1:279-285.

2. Butcher, R. W., and E. W. Sutherland. 1962. Adenosine3',5'-phosphate in biological materials. I. Purificationand properties of cyclic 3', 5'-nucleotide phos-phodiesterase and use of this enzyme to characterizeadenosine 3',5'-phosphate in human urine. J. Biol.Chem. 237:1244-1250.

3. Cheung, W. Y. 1966. Inhibition of cyclic nucleotidephosphodiesterase by adenosine 5'-triphosphate andinorganic pyrophosphate. Biochem. Biophys. Res.Commun. 23:214-219.

4. Cheung, W. Y. 1967. Properties of cyclic 3', 5'-nucleotidephosphodiesterase from rat brain. Biochemistry6:1079-1087.

5. Greengard, P., and E. Costa. 1970. Role of cyclic AMP incell function. Raven Press, New York.

6. Hayaishi, O., P. Greengard, and S. P. Colowick. 1971.On the equilibrium of the adenylate cyclase reaction.J. Biol. Chem. 246:5840-5843.

7. Heidrick, M. L., and W. L. Ryan. 1971. Metabolism of3',5'-cyclic AMP by strain L cells. Biochim. Biophys.Acta 237:301-309.

8. Hirata, M., and 0. Hayaishi. 1965. Pyruvate dependentadenyl cyclase activity of Brevibacteriumliquefaciens. Biochem. Biophys. Res. Commun.21:361-365.

9. Hirata, M., and 0. Hayaishi. 1967. Adenyl cyclase ofBrevibacterium liquefaciens. Biochim. Biophys. Acta149:1-11.

10. Khandelwal, R. L., and I. R. Hamilton. 1971. Purificationand properties of adenyl cyclase from Streptococcussalivarius. J. Biol. Chem. 246:3297-3304.

11. Klainer, L. M., Y.-M. Chi, S. L. Freidberg. T. W. Rall,and E. W. Sutherland. 1962. Adenyl cyclase. IV. Theeffects of neurohormones on the formation of adeno-sine 3',5'-phosphate by preparations from brain andother tissues. J. Biol. Chem. 237:1239-1243.

12. Leloir, L. F., and C. E. Cardini. 1957. Characterizationof phosphorus compounds by acid lability, p.840-850.In S. P. Colowick, and N. 0. Kaplan (ed.), Methods inenzymology, vol. 3. Academic Press Inc., New York.

13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

14. Murad, F., Y.-M. Chi, T. W. Rall, and E. W. Suther-land. 1962. Adenyl cyclase. III. The effect of catechola-mines and choline esters on the formation of adenosine3', 5'-phosphate by preparations from cardiac muscleand liver. J. Biol. Chem. 237:1233-1238.

15. Rall, T. W., and E. W. Sutherland. 1962. Adenyl cyclase.II. The enzymatically catalyzed formation of adeno-sine 3',5'-phosphate and inorganic pyrophosphatefrom adenosine triphosphate. J. Biol. Chem.237:1228-1232.

16. Sutherland, E. W., and T. W. Rall. 1958. Fractionationand characterization of a cyclic adenosine ribonucleo-tide formed by tissue particles. J. Biol. Chem.232: 1077-1091.

17. Sutherland, E. W., T. W. Rall, and T. Menon. 1962.Adenyl cyclase. I. Distribution, preparation, and prop-erties. J. Biol. Chem. 237:1220-1227.

18. Takai, K., Y. Kurashina, C. Suzuki, H. Okamoto, A.Ueki, and 0. Hayaishi. 1971. The reversibility of theadenylate cyclase reaction. J. Biol. Chem.246:5843-5845.

19. Tao, M., and F. Lipmann. 1969. Isolation of adenylcyclase from Escherichia coli. Proc. Nat. Acad. Sci.U.S.A. 63:86-92.

20. Uno, I., and T. Ishikawa. 1971. Chemical and geneticalcontrol of induction of monokaryotic fruiting bodies inCoprinus macrorhizus. Mol. Gen. Genet. 113:228-239.

21. Uno, I., and T. Ishikawa. 1973. Purification and identifi-cation of the fruiting-inducing substances in Coprinusmacrorhizus. J. Bacteriol. 113:1240-1248.

22. Volkin, E., and W. E. Cohn. 1954. Estimation of nucleicacid. Methods Biochem. Anal. 1:287-303.

23. Wood, H. N., M. C. Lin, and A. C. Braun. 1972. Theinhibition of plant and animal adenosine 3',5'-cyclicmonophosphate phosphodiesterases by a cell-division-promoting substance from tissues of higher plantspecies. Proc. Nat. Acad. Sci. U.S.A. 69:403-406.

24. Yamamoto, M., and K. L. Massey. 1969. Cyclic 3',5'-nucleotide phosphodiesterase of fish (Salmogairdnerii) brain. Comp. Biochem. Physiol.30:941-954.

1255VOL. 113, 1973

on August 8, 2020 by guest

http://jb.asm.org/

Dow

nloaded from