Plant Science Letters, 15 (1979) 373--385 © Elsevier/North-Holland Scientific Publishers Ltd.

373

MEMBRANE-BOUND PROTEIN KINASE ACTIVITY IN JERUSALEM ARTICHOKE RHIZOME TISSUES

M. GIANNATTASIO, G.F. TUCCI, G. CARRATI~ and PONZI

lstituto Botanico, Facoltd di Agraria, Universitd di Napoli, 80055 Portici, Napoli (Italia)

(Received February 19th, 1979) (Revision received March 28th, 1979) (Accepted March 28th, 1979)

SUMMARY

A membrane-bound protein kinase, which catalyses the phosphorylation of endogenous substrates, has been isolated from h o m o ~ . ~ s of Jerusalem artichoke rhizome tissues by differential and disconi~muous sucrose-gradient centrifugation. Electron microscopic analysis revealed that the membrane fractions showing endogenous protein kinase activity contained vesicles of various sizes and that most of these structures were stained by phospho- tungstic acid/chromic acid, a stain that is known to be almost specific for plant plasma membranes. The enzyme has an almost absolute requirement for Mg 2.. It is unaffected by cyclic AMP, indoleacetic acid, gibberellic acid and fusicoccin. Adenosine and its cytokinin derivative isopentenyladenosine were inhibitory. When acidic (casein and phosvitin) or basic (histone and protamine) proteins were used as exogenous substrates only acidic proteins are phosphorylated by the enzyme. The endogenous phosphorylated sub- strates have been characterised by SDS-polyacrylarnide gel electrophoresis.

INTRODUCTION

The phosphorylation of specific proteins by cyclic AMP dependent kinases is a well-known regulatory mechanism of metabolism in animal cells [1 ]. In addition, it has been recently shown that some protein components of the membranes are also phosphorylated by kinases. The possibility that this latter event affects the structure and functions of the animal membranes is currently under active consideration. [2].

Although protein kinase activity has been found in higher plant tissues [3], its physiological significance is still obscure. In fact, the plant protein kinases tested to date do not show sensitivity to cyclic A~vIP, whereas most of the animal protein kinases do. Further, no endogenous substrate for the plant kinases has yet been identified.

37~

We have previously reported that a soluble fraction from Jerusalem arti- choke rhizome tissues contains protein kinase activity towards exogenous substrates such as histone, phosvitin and casein [4]. Although this activity was associated with cyclic AMP binding, it was unn~ected by cyclic AMP.

In this paper we shall show that membrane preparation from the above tissues also exhibits protein kinase activity and that, in addition to exogen- ous acid proteins, some protein components of membrane serve as sub- strates for it. The effect of cyclic AMP as well as adenosine and some plant hormones on the membrane-bound protein kinsse will be also reported.

MATERIALS AND METHODS

Materials Dormant rhizomes of Jerusalem artichoke (Helianthus tuberosus L.) were

harvested in November ~-ld stored at 4°C for 4--6 months before experi- mental use.

l~eparation o f membranes by differential centrifugation In a typical experiment, 50 g of peeled rhizomes were cut into small

pieces and homogenised in an Omni Mixer set at high speed for five 30 s periods with cooling between each homogenisation. The homogenising medium (medium A) consisted of 0.25 M sucrose, 3 mM EDTA, 1 mM mercaptoethanol and 10 mM Tris--HCl, (pH 7.6). The homogenate was filtered through 2 layers of cheesecloth; the resulting filtrate was centri- fuged at 3000 g for 30 rain and the supernatant at 80 000 g for 30 min. The 3000--80 000 g pellet suspended in fresh homogenising medium lacking in EDTA (medium B) was used for the experiments described below.

Partial purification of membranes by discontinuous sucrose gradient The suspension of the 3000--80 000 g pellet was layered onto a discon-

tinuous sucrose gradient according to Hodges and Leonard [5] and centri- fuged for 3 h at 24 000 rev./min in a Spinco SW 25 rotor. Visible mem- brane bands occurred at all sucrose interfaces. They were removed with a Pasteur pipette, diluted with medium B, pelleted by centrifugation and re- suspended in fresh medium B.

Determination of protein kinase activity The protein kinase activity was measured by a modification of the method

previously described [4]. Each reaction mixture contained in a final volume of 0.2 ml, 40 mM Tris--HCl (pH 7.6), 10 mM MgCI2, 5 mM mercaptoethanol, 50 mM NaF and 20 ~M [7-32P]ATP (50--100 cpm]/~mol). Usually 100 ~g of membrane protein were added to each reaction mixture. The incubation was carried out for 2 min at 30 ° C. The reaction was stopped by addition of 5 ml of cold 5% TCA containing 10-4M ATP. The suspension was heated at 90 ° C for 20 rain to hydrolyse the nucleic ~cids. The precipitate was collected on

375

Whatman GF/C glass fibre papers, washed serially with 25 ml of 5% TCA, 25 ml of ethanol/ether/chloroform (2 : 2 : I by vol.) and counted for radio- activity in a toluene/0.5% diphenyloxazone/0.005% 1,4-bis-(2-(5-phenylox- azole)benzene scintillation liquid.

Determination of A TPase activity ATPase activity was determined according to the conventional method

described by Hodges and Leonard [5]. The final concentration of the re- action components was 3.0 mM ATP, 1.5 mM MgSO4, 33 mM Tris--MES (pH 6) and, when added, 50 mM KCI in a final volume of I ml. The reaction was initiated by the addition of 100/~g of membrane protein.

Acid hydrolysis of 32P-labelled membranes Samples of membranes were self-phosphorylated in the standard protein

kinase assay mixture and processed as described under 'Determination of protein kinase activity'. After the washing with ethanol/ether/chloroform mixture, the membranes were suspended in 0.4 ml of 6 N HCI and hydroly- seal in sealed tubes for 5 h at 105 ° C. The hydrolysed samples were dried in vacuo, redissolved in water and applied onto Whatman No. 3 MM paper, and the components were separated by high voltage electrophoresis in 2.5% for- mic acid/7.8% acetic acid (pH 1.85) at 2 kV for 2 h. [7-32P]ATP, [32P]ortho- phosphate, phosphoserine and phosphothreonine were run simultaneously as markers. Amino acids were detected by spraying with ninhydrin. To mea- sure the radioactivity, the paper strips were cut in I cm sections and put in toluene scintillation liquid.

8DS-po lyacry lamide gel electropho resis Membrane protein (160/~g) were incubated in a total volume of 0.1 ml of

the above reaction mixture. The protein kinase reaction was stopped by the addition of 0.1 ml of solution containing 2% SDS, 0.05 M Na carbonate, 10% mercaptoethanol, 20% sucrose and 0.001% bromophenol blue. The mix- ture was incubated for 15 min at 37°C and then layered on the top of SDS polyacrylamide gels (100 m m × 8 ram) prepared as described by Weber and Osborn [6]. Final concentrations in the gel were 5.6% acrylamide/0.1% SDS in 0.1 sodium phosphate buffer (pH 7.1). The electrode buffer (pH 7.1) con- tained 0.1 M sodium phosphate at pH 7.1 and 0.1% SDS. Electrophoresis was carried out at a constant voltage giving 8 mA per gel for 6 h. After the run, gels were stained with 0.01% Coomassie brilliant blue in 50% methanol and 10% acetic acid for 12 h. After removal of excess stain by a few changes of 20% methanol and 7% acetic acid mixture, the gels were scanned at 575 nm For 32p radioactivity determination, the gels were frozen laterally in 2 mm- thick sections by a mechanical slicer. The slices were place~ in separate scintillation vials and the radioactivity counted.

Electron microscopy Membranes were fixed for electron microscopy in 2% buffered glutaralde-

376

hyde for 2 h at room temperature and post-fixed in 1% buffered osmic acid for I h. After dehydration in alcohol and treatment with propylene oxide, the material was embedded in an Epon-Araldite-DDSA mi~-ture before sect- ioning. Thin sections were stained either with uranyl acetate/lead citrate or with 1% phos~;hotungstic/chromic acid which has been reported to stain preferentially the plant plasma membranes [7 ].

Protein determination Protein was determined by the method of Lowry et al. [8] in the pres-

ence of 0.4% SDS using bovine serum albumin as standard.

RESULTS

Evidence for the presence of membrane-bound protein kinase phosphory. lating endogenous substrate

As shown in Fig. 1, when membrane fragments isolated from Jerusalem artichoke rhizome tissues by differential centrifugation (3000--80 000 g) were incubated in the presence of [7-32P]ATP and Mg 2., incorporation of radioactive phosphate groups into membrane components took place. This incorporation was linear with the amount of membrane protein incubated. In order to have information on the membrane components being phospho-

to

10- C) E

W t -

n- O §° Q.. n- O U Z I.-I

II.

0 I I

0 50 100 MEMBRANE PROTEIN (pg)

Fig. 1. Self-phosphorylation of membrane as a function c f varying concentrations of membrane protein. Membranes, at yarying concentrations, were incubated in the reaction mixture described under Determination of protein kinase activity. After 2 rain incub- ation, phosphorylatioi~ was stopped by addition o f 5 ml of cold 5% trichloroacetic acid containing 10 -4 M ATP and the suspension was heated at 90°C for 20 rain. The precip- itate was collected on Whatman GF/C glass fibre papers, washed with 5% trichloroacetic acid and counted for radioactivity.

377

rylated, the phosphorylated membranes were washed serially as reported in Table I. Abou t 40% of original cold 5% trichlorOacetic acid precipitable radioactivity appeared to be bound to nucleic acids since i td i sappeared after nucleic acid-hydrolysing t reatment with ho t trichloroacetic acid (Table I, procedure 2). A small amoun t of radioactivity (less than 10%) was bound to lipids (Table I, procedure 3). Fur ther t rea tment of the phosphorylated membranes with hydroxylamine, which is known to cleave acyl phosphate bonds bu t to leave phosphoester bonds intact [ 9], was carried out to deter- mine whether the observed phosphorylat ion involved formation of acyl bonds or of ester bonds. In fact, it has been shown that ATPase activity is associated with the format ion of an acyl phosphate intermediate [9], where- as protein kinases have been shown to catalyse the formation of phosphoes- ter bonds [10]. It was found (Table I, procedure 4) that no acyl phosphate bonds were present in the washed precipitate. The radioactivity remaining associated with the precipitate after the above treatments should be incor- porated in membrane proteins.

In order to obtain more direct evidence that phosphate incorporation was into protein and identify the amino acid residues being phosphorylated, the membranes were phosphorylated, treated with hot tricbloroacetic acid and chloroform/methanol mixture and then hydroysed and subjected to high

TABLE I

SOLUBILITY CHARACTERISTIC OF PHOSPHORYLATED MEMBRANE COMPONENTS

Membranes (100//g) were incubated in the reaction mixture described under Determina- tion of protein kinase activity, precipitated with 5 ml of cold 5% trichloroacetic acid, washed two times with 5 ml of cold 5% trichloroacetic acid, and the radioactivity counted (Procedure 1). In Procedure 2, the samples precipitated with cold trichioroacetic acid as above were heated at 90 ° C for 15 min in trichloracetic acid. After chilling the tubes at 0°C for 15 min, the precipitate was counted for radioactivity. In Procedure 3, the precip- Rate obtained after heating samples at 90°C in 5% trichloroacetic acid was extracted with ethanol/ether/chloroform (2 : 2 " 1 v/v) to remove lipids befqre counting. In Pro- cedure 4, the precipitate after the extraction with' ethanol/ether/chloroform was suspen- ded in 3 ml of 0.8 M hydroxylamine in 0.1 M sodium acetate (pH 5.3) and incubated for 10 min at 30°C. After incubation, 1.3 ml of cold 50% trichloroacetic acid were added and radioactivity of the precipitate counted.

Solubilisation procedure Percentage of original cold 5% trichloroacetic acid-pre- cipitable radioactivity

1 -- Cold 5% trichloroacetic acid 2 -- 5% trichloroacetic acid

heated at 90 ° C for 15 min 3 -- Ethanol/ether/chloroform

( 2 : 2 : 1 v/v) 4 -- Hydroxylamine

100 63

52

52

378

TABLE II

MEMBRANF~BOUND PROTEIN KINASE AND K*-STIMULATED ATPase DISTRIBU- TIONS ON DISCONTINUOUS GRADIENT

The suspension of the 3000--80 000 g pellet was layered onto a discontinuous sucrose gradient prepared according to Hodges and Leonard [5 ]. Protein kinase and K+-stimulated ATPase activity was measured as reported under Materials and Methods, The ATPase data represent the component activated by 50 mM KCI (i.e. the activity in the presence of 1.5 mM MgSO4has been subtracted).

Membrane fraction K+-stimulated ATPase Membrane-bound protein kinase

Spec. act. (#moi/mg Purif. Spec. act. (/~mol/mg Purif protein/30 min) (fold) protein/2 min (fold)

3000--80 000 g supernatant 0.6 1.00 0.10 1.0

Band Sucrose interfaces (% w/w)

1 20--25 0.1 0.16 0.10 1.0 2 25--30 1.3 2.16 0.11 1.1 3 30--34 1.5 2.50 0.12 1.2 4 34--38 2.0 3.33 0.21 2.1 5 38--45 1.9 3.16 0.20 2.0

voltage paper electrophoresis. More than 80% of the radioactivity was in- corporated into phosphoserine. On the basis of the above results we con- clude that protein kinase activity which phosphorylates the endogenous substrate is associated with the membrane fragments.

Membrane fractionation by discontinuous sucrose gradient The 3000--80 000 g preparation was resolved on discontinuous sucrose

gradient similar to that described by Hodges and Leonard [5] and the iso- lated membrane bands were tested for protein kinase activity. As Table II shows, although all of fractions exhibited protein kinase activity, the highest specific activity was found to be present in the membrane fractions of the bands 4 and 5. These fractions were also found to be enriched in ion-stimul-

Fig. 2. Electron mierographs of intact tissues and pellets of isolated membrane fractions of Jerusalem artichoke rhizome. ( 1,2 ): cells stained with uranyl acetate/lead citrate (1) and with phosphotungstic acid/chromic acid (2), x 20 000o (3,4): Membrane fraction 3000--80 000 g) stained with uranyi acetate/lead citrate (3) and with phosphotungstic acid/chromic acid (4), X 16 000. (5,6): purified membrane fraction (band 4 from discon- tinuous sucrose gradient) stained with uranyl acetate/lead citrate (5) and with phosphot- ungstic acid/chromic acid (6); X 16 000. W=cell wall; NfNucleus; pffiplastids; pmffiplasma membrane; erfendoplasmic reticulum; mfmitochondria; tftonoplast; V and Vlfmem- brane vesicles that stain with uranyl acetate/lead citrate and with phosphotungstic acid/ chromic acid respectively.

379

380

ated ATPase, an enzyme which has been supposed to be located in higher plant plasma membranes [5,11]. Electron microscopy (Fig. 2) revealed that the above fractions contained vesicles of various sizes and that most of these structures (above 80%) were stained by phosphotungstic acid/chromic acid, a stain that is known to be almost specific for plant plasma membranes [7]. We used membranes of the band 4 for all of the experiments described below.

Time course of protein kirmse activity As reported in Fig. 3, incorporation of radioactive phosphate groups into

membrane protein was linear as a function of time for 2 rain and reached a plateau after 4 min. No radioactive inorganic phosphate could be detected in the reaction mixture during the first 2 rain of incubation indicating that neither protein phosphatase nor ATPase occurred in the experimental condit- ions used for the protein kinase assay (data not shown).

Effects of ions, cyclic AMP and other solutes on the protein kinase activity As Table HI reports, protein kinase has an absolute requirement for Mg 2÷.

At concentrations varying between 10 -9 and 10 -4 M, cyclic AMP was in- effective. The addition of [3H]cyclic AMP to reaction mixtures as an internal standard, revealed that no significant degradation of cyclic AMP occurred in the experimental conditions used for protein kainase assay (data not shown). The other cyclic nucleotides, GMP and IMP were ineffective, as were also the plant hormones indoleacetic acid and gibberellic acid and fungal toxin fusi- coccin. Adenosine and its cytokinin derivative, isopentenyladenosine, were strongly inhibitory at a concentration of 10- 4 M.

+,o- +--I Q

E r.!

o W

20.

0

o U Z

0 I w ' m O 3 6

TIME (min)

Fig. 3. Time course of protein kinase activity. Each reaction mixture contained 100 ~g of • . . . . . . O

membrane protem. Protein kmase actzvlty was assayed under standard conditions at 30 C for the indicated times.

TABLE III

EFFECTS OF SOLUTES ON THE MEMBRANE-BOUND PROTEIN KINASE

The assay was performed as described under Determination of protein kinase activity except that MgCI2 concentration was varied as reported.

381

~ubstance added Protein kinase activity (pmol~P incorp./mg prot./2 min)

None 0 MgCI2( 1 raM) 20 MgCI2(3 mM) 90 MgCI2( 10 mM) 200 MgCls( 10-~ M) + adenosine 90 MgCI2(10" M) + isopentenyladenosine 95 MgCl2(20 mM) 280

Effect Of exogenous substrates on protein kinase activity As Table IV shows, acidic proteins such as casein and phosvitin were good

substrates for protein kinase. Histone was poorly phosphorylated whereas protarnine and albumin were not phosphorylated at all. Cyclic AMP was also ineffective in stimulating protein kinase activity towards exogenous sub- strates.

Extraction of protein kin~e and phosphorylated endogenous substrate with solutions of high" strength and Triton X-1 O0

As reported in Table V, treatment of the membrane fraction either with buffer of high ionic strength or with the detergent Triton X-100, did not re- move significant amounts of protein kinase activity. The same treatment to membranes after their phosphorylation failed to remove the phosphorylated

TABL~IV

SUBSTRATE SPECIFICITY OF THE PLASMA MEMBRANE-ASSOCIATED PROTEIN KINASE

The assay was performed as described under Determination of protein kinase activity. Exogenous substrate was added to the reaction at a concentration of I mg/ml.

Substrate added Protein kinase activity (% of control)

None 100 Casein 210

. Phosvitin 205 Histone 130 Protamine 100 Albumin 100

382

TABLE V

EXTRACTION OF PROTEIN KINASE AND PHOSPHORYLATED MEMBRANE PROTEINS WITH SOLUTIONS OF HIGH IONIC STRENGTH AND WITH THE DETERGENT TRITON X-100

(1) E z ~ c t i o n of protein kinase: Membranes (100/Jg) were incubated for I h at 0°C in 40 mM Tris--HCl (pH 7.6) 10 mM MgCI~ and I mM mercaptoethanol containing the in- dicated concentration of ~ or Trixon X-100 before centrifugation at 105 000 g for I h and dialysis of the supernatant and pellet against the same buffer. (2) Extraction of phosphorylated endogenous substrate: Meml~anes were incubated as reported under Determination of protein klnn~e activity, chilled in ice after addition I ml of cold 40 mM Tris--HC! (pH 7.6) containing 10 ~ M ATP and centrifuged at 105 000 g for 30 min. The pellets were then suspended in 0.1 ml of 40 mM Tris--HC! (pH 7.6)containing either 0.5 M NH4C! or 0.5% Triton X-100 before centrifugation at 105 000 g for 30 rain. The pellets and supematants were precipitated separately with cold 5% trichloracetic acid and processed for measurement of incorporated radioactivity.

Treatment

None 0.5 M NH4CI 0.5% Triton X-100

1 -- Protein kinase activity (pmol Y~P incorporated/100/zg protein)

(a) Membrane-bound protein 90 kinase (b) Solubilised protein kinase 1

18 16

4 not tested

2 -- Extraction of phosphorylated endogenous substrate (tricholoroacetic-precipitable radioactivity, cpm)

(a) Membrane pellet 6200 5800 (b) Supernatant 460 750

5600 not tested

endogenous substrate. Cyclic AMP, tested on the protein kinase activity of Triton X-100 treated membranes, was also ineffective (data not shown).

8DS-polyacrylamide gel electrophoresis of the membrane protein components SDS-polyacrylamide gel electrophoresis of the membrane protein com-

ponents (Fig. 4) shown a consistent pattern of 18 bands, at least 9 of which were phosphorylated by endogenous kinase. Phosphorylation in the presence of cyclic AMP did not affect the qualitative and quantitative pattern of the phosphorylated proteins (data not shown).

DISCUSSION

We have previously r e p o ~ the presence of protein kinase activity in the soluble fraction of Jerusalem artichoke rhizome tissues [4]. The above data indicate that these tissues also contain a membrane-hound protein kinase

re lo t i ve mob i l i t y ,

! " I ._

! I I I

I I I ! I I I / I

VI I # I

| | i ' v I A l;If~

V H | ~ v , t , . ,, I l' '4 Ii'~

i', VII II I ~ • , I I f I I l I I I . I

30011 ', -, , 8 I I " ,=- 1,v:"l I / v.,

/1 1 I I i " =" / I v I / I f g 200 II III

III I I "" 100 . . . . . . .

i i i . i • • II • II I I I I I I i i • • • I i II i I i | I I I II ~I | lllg | | I | I •

0 10 20 30 40

.1A

,1.2

.1.0

0.8

,0.6

,04

-0.2

s l i c e n u m b e r

Fig. 4. Polyacrylamide gel electrophoresis of solubilised membrane protein. Phosphory- lation, electro]~horesis, staining and slicing were performed as described under Materials and Methods. 2p incorporated ( - - - - - ) ; AsTs ( . . . . . . )

383

that phosphorylates endogenous substrate. The enzyme does not exhibit substrate-specificity as it is revealed by the complex pattern of phosphory- lated proteins on SDS disc-gel electrophoresis and by its ability to phos- phorylate exogenous acidic proteins. However, we found that, when phos- phorylation of membrane components occurred in vivo, only two mem- brane protein components were rapidly phosphorylated (Giannattasio et al., in preparation) suggesting that the a factor(s) controlling the protein kinase activity in vivo was lost during the isolation of the membranes.

Both protein kinase and endogenous substrate appear to be firmly bound to the membranes, since they were not released in significant amounts from the membranes in a solution of high strength or following the Triton X-100 treatment. Thus, the possibility that kinase and its relative substrate are strnctural membrane components can be envisaged.

384

As for the soluble protein kinase previously reported [4], the membrane- bound enzyme is not regulated by cyclic AMP. 3~e following data support this conclusion: (a) at concentrations varying between l f f 9 and 10 -4 M, cy- clic AMP was ineffective; (b) cyclic AMP did not affect protein kinase activ- ity after membrane treatment with Triton X-100, this treatment being able to unmask latent cyclic AMP dependent protein kinase in mammalian cells [12--13]; (c) cyclic AMP did not affect the electrophoretic pattern of phos- phorylated membrane proteins; (d) in the experimental conditions described for the protein kinase assay, no cyclic AMP degradation occurred. Therefore if the membrane-bound protein kinase is susceptible to some regulation, this latter could be different from that involving cyclic AMP. Examples for regulation of protein kinase by ions and protein modulators have now been found in animal systems [14].

When the membrane f~agments were fractioned by discontinuous sucrose gradient, a membrane fraction enriched in protein kinase was obtained. This fraction was also enriched in ion-stim-!~ted ATPase, an enzyme that has been found to be associated with the plasma membrane of plant tissues [5--11]. Further, most of the vesicles present in this fraction stained with phosphotungstic acid/osmic acid, a stain that is known to be specific for plant plasma membrane [7]. These preliminmT data suggest an association of protein kinase to the plasma membrane.

The physiological significance of the presence of protein kinase and its relative substrate in higher plant membranes is currently unknown. In this respect, it seems worthwhile emphasising that in animal systems phosphory- lation and dephosphorylation of membrane protein have been suggested as a mechanism for structural rearrangement of membrane components [15] and that the phosphorylation of specific plasma membrane proteins appears to be involved in the flux of ions, water and other solutes across the mem- brane [2,16].

ACKNOWLEDGEMENTS

This research was supported by C.N.R. Grants No. 76.01575.0(; and No. 77.01289.06, awarded to M. Giannattasio and P. Pizzologo, respectively.

The authors thank Prof. E. Brown. for critical review of the manuscript and Prof. A. BA, io'for a gift of fusicoccin.

REFERENCES

1 T.A. Laugan, Adv. Cyclic Nueleotide Res., 3 (1973) 99. 2 C.S. Rubin and O.M. Rosen, Annu. Rev. Bioehem., 44(1975) 903. 3 A. Trewavas; Annu. Rev. Pla~t Physiol., 27 (1976) 349. 4 M. Giarmattasio, G. Carrat~ and G.F. Tueei, FEBS Lett., 49 (1974) 249.

• ,5 ~ T.K. Hodges and R.T. Leonard, Methods Enzymol. 32 (1974) 392. 6 K. Weber and M. Osborn, BioL Chem., 244 (1964) 4406.

385

7 J.C. Roland, C.A. Lembi and D.J. MorrO, Stain Technology, 47 (1972) 195. 8 0 ~ H . Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J.Biol. Chem., 193

(1951) 265. 9 L.E. Hokin, P.S. Sastry, P.S. Galsworthy and A. Yoda, Proc. Natl. Acad. ScL U.S.A.,

54 (1965) 177. 10 T.A. Langan, Science, 162 (1968) 579. 11 R.T. Leonard and T.K. Hodges, Plant Physiol., 52 (1973) 6. 12 H. Macho, E.M. Johnson and P. Greengard, J. Biol. Chem., 246 (1971) 134. 13 A. Lemay, M. Deschenes, S. Lemaire, G. Poirier, L. Poulin and F. Labrie, J. Biol.

Chem., 249 (1974) 323. 14 D. McMahon, Science, 185 (1974) 1012. 15 Y. Gazitt, I. Ohad and A. Loyter, Biochim. Biophys. Acta, 436 (1976) 1. 16 G.J. Strewler and J. Orloff, Adv. Cyclic Nucleotide Res., 8 (1977) 311.