Eur J Biocheni 130. 599-603 (1983) ( FEBS 1983

Characterization of Phosphorylated and Native cCMP-Dependent Protein Kinase

Franz HOFMANN and Veit F'LOCKERZI

Pharmakologisches Institut der Universitat Heidelberg

(Received July 4,'October 13, 1982) - EJB 5777

Pure preparations of cGMP-dependent protein kinase are autophosphorylated in the presence of CAMP and cGMP. The stoichiometry and functional significance of this reaction has been studied. Pure preparations of cCMP-dependent protein kinase contained 1.4 0.1 (7) mol phosphate/mol subunit as determined by chemical phosphate analysis. The protein-bound phosphate was hydrolyzed by 1 M NaOH, but was stable in 0.1 M HCI or 0.8 M NH40H or hot 16";, trichloroacetic acid. In the presence of CAMP and [./-j2P]ATP, Mg another 2.5 0.06 (5) mol phosphate/mol subunit. cGMP also stimulated the phosphorylation of the enzyme although to a lower extent than CAMP. The autophosphorylation reaction was half-maximally stimulated at 0.12 pM and 7.0 pM cGMP and CAMP, respectively. CAMP-dependent autophosphorylation of the native enzyme did not change the apparent Kn for cGMP in a phosphotransferase assay ( K A = 0.15 pM) but decreased the K A for CAMP from 19.6 pM to 1.75 pM. Phosphorylation did not affect the apparent K, for the substrate peptide but doubled the apparent V of the phosphotransferase reaction determined in the presence of saturating concentrations of cGMP or CAMP. Phosphorylation did not change the affinity of the enzyme for cGMP, when cGMP binding was determined in the absence of ATP (apparent & = 18 nM). ATP . Mg decreased the affinity of the native enzyme for cGMP about threefold to an apparent KCi of 54 nM ; in contrast, ATP . Mg had only a minimal effect on the binding kinetics of the phosphorylated enzyme. These results support the notion that autophosphorylation of cGMP-dependent protein kinase considerably influences the kinetic parameter of the enzyme.

0.2 (5) mol phosphate/mol subunit were incorporated into the enzyme yielding a total of 4.2

cGMP-dependent protein kinase has been purified to homogeneity from bovine lung [l -51 and heart muscle [6] . The enzyme exists as a dimer of two identical subunits ( M , between 75000 and 81 000). The purified enzyme resembles CAMP-dependent protein kinase in a number of properties including substrate specificity and the ability to undergo self- phosphorylation [7- 101. The stoichiometry of this reaction is not clear, since incorporation of one [ I l , 121 or two 1131 moles phosphate/mole subunit has been obtained. The re- action is stimulated by addition of CAMP and this stimulation is inhibited by cGMP [4]. Lincoln et al. [9] suggested that (a) autophosphorylation occurs in the absence of cGMP or CAMP, (b) autophosphorylation does not occur in the pres- ence of cGMP and (c) CAMP stimulates this reaction by promoting dissociation of cGMP from the purified enzyme. These conclusions were supported by Walter et al. [I41 who did not observe stimulation of the autophosphorylation by CAMP. In contrast, Foster et al. [I21 found that the rate of autophosphorylation in the absence of added cyclic nu- cleotides was equal to that in the presence of cGMP and that cAMP stimulated the initial rate of phosphorylation sixfold. Results similar to those of Foster et al. were obtained by Gill and McCune [IS].

So far, most groups have not observed that the properties of cGMP-dependent protein kinase change when the enzyme

Ahhroiu/ ion.\. Native cGMP-dependent protein kinase refcrs always to the isolated pure enzyme which has not been incubated in the presence of CAMP and Mg . ATP; phosphorylated cGMP-dependent protein kinase refers always to the pure enzyme which has been phosphorylated in the presenceofcAMPand Mg.ATP; Tes, 2-{[2-hydroxy-l,l-bis(hydroxy- methyI)ethyl]-amino}ethanesulfonic acid.

Enzymrs. cGMP-dependent protein kinase (EC 2.7.1.37); acid phos- phatase (EC 3.1.3.2); alkaline phosphatase (EC' 3.1.3.1).

is autophosphorylated. More recently, Foster et al. [12] reported that autophosphorylation decreased the concen- tration of CAMP required for half-maximal stimulation of protein kinase activity from 1.46 pM to 0.79 pM. This small increase in the affinity was only observed in the presence of histone substrates. No change in the affinity for cGMP or in the affinity constant for binding of cGMP or cAMP has been reported. This raised the possibility that the small change observed was specific to the particular substrate used, especially since inactivation of cGMP-dependent protein kinase by positively charged proteins has been reported [16,17].

The reason for the differences observed by different groups with respect to the autophosphorylation reaction, its stoichiometry, and its possible functional role are unclear. They are presumably not related to species or tissue differences since all groups used the enzyme purified from bovine lung. On the other hand, most if not all differences could be ex- plained by differences in the amount of cyclic nucleotides contaminating the purified enzyme. It is well known that it is difficult to remove bound cGMP or CAMP from the enzyme at low temperature. In order to reconcile some of these dif- ferences the autophosphorylation of the enzyme has been reinvestigated using an enzyme from which CAMP was removed by gel filtration at room temperature.

MATERIALS AND METHODS

Matcriuls

Alkaline phosphatase (Esclievichiu coli 20 - 30 units/mg) and benzamidine were obtained from Sigma. The substrate peptide (Leu-Arg-Arg-Ala-Ser-Gly) was from Peninsula La-

600

boratories. Acid phosphatase (potato, 56 unitsjmg) was from Calbiochem Behring Corp. Bovine serum albumin was purchased from Miles Laboratory. [3H]cGMP and 32P, were obtained from NEN Chemicals. ATP, CAMP, cGMP were from Waldhof, Mannheim. All other chemicals were of the highest purity available. X-ray films (X-Omat AR) for auto- radiograms were purchased from Kodak. Poly(ethy1eneimine)- cellulose, sodium dodecylsulfate and acrylamide were from Serva, Heidelberg. AcA-34 was from LKB ; nitrocellulose filters were from Schleicher & Schull.

Enzynw Assays

The bindig of cGMP by cGMP-dependent protein kinase was determined by the filter technique at pH 7.0 in a 50 mM Tes buffer containing 0.5 mg/ml bovine serum albumin, 0.5 mM EDTA, 5 mM magnesium acetate and [3H]cCMP. If added, the concentration of ATP was 1 mM and that of sodium chloride was 150 mM. Reactions were started by addition of aliquots of the enzyme. Samples were incubated at 2°C for 90 min in a total volume of 100 pl. Aliquots of 80 p1 were spotted on nitrocellulose filters which were rinsed with 3 ml ice-cold 30 mM potassium phosphate pH 6.8. Filters were dissolved and counted in a toluene/Triton X-100 based scintillator.

Phosphotransferase activity of cGMP-dependent protein kinase was determined at 30°C and pH 7.0 in a 50 mM Tes buffer containing 1 mM EDTA, 3 mM magnesium acetate, 0.5 mg/ml bovine serum albumin, 0.2 mM [p3'P]ATP and 0.1 mg/ml substrate peptide. The reactions were started by addition of diluted enzyme and terminated after 2 min by addition of glacial acetic acid. Samples were further processed as described in [18].

Phosphate Determination

Inorganic phosphate was determined by the malachite green method [I 91 as described previously [20]. Protein-bound phosphate was hydrolysed by incubation of aliquots in 1 M sodium hydroxide at 60°C for 18 h.

Preparation of Proteins

cGMP-dependent protein kinase was purified from bovine lung as described for the enzyme from heart muscle [6]. Elution from the cGMP-affinity column [6] was carried out at room temperature using 60 mM CAMP. The eluted enzyme was concentrated at 4 "C by vacuum dialysis and then chroma- tographed at 22°C on an AcA-34 column (I x 120 cm) equili- brated in buffer A ( 5 mM Tes. pH 7.0, 0.2 mM benzamidine, 0.2 mM EDTA, 100 mM NaCl and 0.5 mM dithiothreitol). Elution time was between 6 h and 12 h. Fractions containing the enzyme, which were well separated from fractions con- taining CAMP, were pooled and dialyzed at 4°C against buffer A containing 50 glycerol.

Phosphorylation of the enzyme was carried out at 30 "C for 2 h adding 1 mM [y3'P]ATP and 5 mM magnesium acetate to a large aliquot of the enzyme taken directly after the vacuum dialysis step. cAMP was not added since the enzyme preparation contained millimolar concentrations of CAMP. The phosphorylation was stopped by adding EDTA in a final concentration of 10mM. The enzyme was then chromatographed at 22°C on the same AcA-34 column as described for the native enzyme. The heat-stable inhibitor

protein of CAMP-dependent protein kinase was purified through the DEAE-cellulose step as described in [21].

Other Methods

acrylamide) in the presence of 0.1 sodium dodecylsulfate was carried out according to Laemmli [22]. Autoradiography was carried out at - 70 'C for 1 - 3 days using intensifying screens. Protein was determined by the Lowry [23] or Coomassie blue procedure [24] using bovine serum albumin as standard. [j-32P]ATP was prepared according to [25] [3H]cGMP was repurified on a poly(ethy1eneimine)-cellulose column [26]. The concentrations of all nucleotide solutions were determined spectrophotometrically at 260 nm. The molecular weight of the subunit of cGMP-dependent protein kinase was taken as 75000. All enzyme preparations were stored at -20 C.

Polyacrylamide slab gel electrophoreses (1 0

RESULTS

Phosplzorylution of cGMP-Dependent Protein Kinusc

Previously, several groups reported that cGMP-dependent protein kinase incorporates, in an apparent autophosphoryla- tion reaction, 1 rnol phosphate/mol subunit [I 1,121. The incorporation of slightly more than 2 rnol phosphatejmol subunit has also been observed [13]. Reinvestigation of the stoichiometry indicates that, in the presence of cAMP and cGMP, 2.5 and 0.75 rnol phosphate/mol suhunit are incor- porated (Fig.1). In the absence of cyclic nucleotides an extremely slow phosphorylation of the enzyme was apparent, the rate of which is stimulated by either cGMP or CAMP. In agreement with earlier reports 14,121, phosphorylation in the presence of cAMP was not suppressed by the addition of the heat-stable inhibitor protein of CAMP-dependent protein kinase. Addition of cGMP and cAMP together resulted in phosphorylation values similar to those obtained in the presence of cGMP alone. Gel electrophoresis in the presence of sodium dodecylsulfate of an enzyme phosphorylated in the presence of cAMP indicated that all the radioactivity present in the gel comigrated with cGMP-dependent protein kinase (Fig. 2). No radioactivity was found in that region of the gel which corresponds to the mobility of the regulatory subunits of CAMP-dependent protein kinase I and 11. Phosphorylation of cGMP-dependent protein kinase was half-maximally stimulated at concentrations of 7.0 pM and 0.15 pM of CAMP and cGMP, respectively (Fig. 3). The results of these experi- ments are in line with the idea [4] that phosphorylation of cGMP-dependent protein kinase is due to the enzyme itself and is not the result of a contaminating enzyme such as CAMP-dependent protein kinase or a cyclic-nucleotide-in- dependent protein kinase.

The nature of the phosphoester formed was investigated by using chemical phosphate measurements. As shown in Table 2 , these determinations indicated that the native enzyme already contained 1.4 rnol phosphate/mol subunit. This value was increased to 4.2 rnol phosphate/mol subunit when phos- phorylation was carried out in the presence of CAMP. Slightly more than 2 rnol phosphate/mol were determined after cGMP-dependent phosphorylation. The phosphate bound to the enzyme before and after CAMP-dependent auto- phosphorylation was not hydrolyzed by 0.1 M HCI or 0.8 M hydroxylamine (Table 2). Incubation in the presence of 0.1 M NaOH liberated approximately 3 mol phosphate. The last mole of phosphate was liberated after incubation in 1 M

Incubation time I h) Fig. 1. Autopho.cphor}Jlution of pure CGMP-dtyendeirnt protein kinase. Pure cGMP-dependent protein kinase (1.6 pg) was incubated at 30 "C and pH 7.0 in a buffer containing 50 mM Mes, 0.3 mM EGTA, 150 mM NaCI, 10 mM magnesium acetate, 0.1 pg/ml bovine serum albumin, 0.2 mM [y3*P]ATP for the time indicated. The reaction was stopped by the addition of 1 ml 20% trichloroacetic acid. Tubes were centrifuged and the pellets washed twice with 1 ml of 20% (w/v) trichloroacetic acid and counted. Each value represents the mean of duplicate incubations from which the appropriate blank value has been substracted. Phos- phorylation was carried out in the absence ( x ) and presence of 3 pM cGMP (0) or 0.3 m M CAMP (0)

Fig. 2. Polyacrylamidc> gel electrophore.vis of phosphorylufed 1GMP- dependent protein kinase. Pure cGMP-dependent protein kinase (4 pg), containing 3.2 mol [12P]phosphate/75000 g, was chromatographed on a 10% acrylamide gel in the presence of 0.1 "/, sodium dodccylsulfate. The upper part of the figure shows the Cooniasaie bluc staining, the lower part the corresponding autoradiogram

lag CcGMP 1 or I CAMP J Fig. 3. Concentration of cCMP or C A M P reyuired,for stimulation of' the autopho.rphorylation reaction. Pure cGMP-dependent protein kinase (1.6 pg) was incubated for 2 h at 30 "C in the presence of various concen- trations of cGMP (0) or CAMP (0). Values shown are the mean of duplicate incubations. For further details see legend to Fig. 1

NaOH. These results indicated that the phosphate was present as an alkali-labile ester, and that the one mole of the phosphate which is already present in the native enzyme (Table 2) was relatively resistant to the alkali treatment. Incubation of the native enzyme containing 1.5 mol phosphate/mol subunit with alkaline or acid phosphatase liberated approximately 0.4 mol phosphate/mol subunit. The remaining mole of

Tablc I . Alkali-labile phosphate content of pure cGMP-dependent protein k ina.w The phosphate content o f various enzyme preparations was determined before and after CAMP-stimulated phosphorylation. In addition, the amount of radioactive phosphate incorporated in the presence of CAMP is also shown. n.d., not determined

Enzyme Phosphate content preparation ~ ~ ~ . ~ ~ ~~ ~ ~~- ~ ~. ~ ~ ~~~ ~ ~

before [3ZP]phosphate after phosphorylation phosphorylation

mo1/75000 g

1 2 3 4 5 6 I

P k SEM ( M )

I .5 1.6 I .5 1.8 1.6 0.9 1.2

1.4 k 0.1 (7) ~ ~~ ~~~

2.0 n.d. 3.2 n.d. 2.0 2.6 2.8 - ~~~ ~~-~ ~

2.5 * 0.2 ( 5 )

4 3 n d 4 3 n d 4 1 4 0 4 3

4 2 +_ 0 06 ( 5 ) ~ ~ _ _

Table 2. Stability characteristics of phosphate present in nufivo and phosphorjdated CG MP-dependent protein kinase Aliquots of native cGMP-dependent protein kinase (65 pg) and enzyme phosphorylated in the presence of CAMP (50 pg) were incubated under the indicated conditions. Reactions were stopped by addition of 1 ml 20% trichloroacetic acid. Samples were thcn processed for determination of chemical phosphate as described under Materials and Methods. In- cubalions in the presence of freshly prepared hydroxylamine were carried out a t pH 5.3 [27]

~ ~ ~~~

Addition Concn Temp Tinic Phosphate content of

native phoaphor- enzyme yldted

- _ _

eniyme

M "C min mo1/75000 g -~ ~ ~ ~- ~

HzO - 100 30 1 . I 4.1 HCI 0.1 100 30 1 .o 4.1 NaOH 0.1 100 30 0.9 1.2 NHiOH 0.8 37 30 1.2 4.2

phosphate could not be hydrolyzed by these enzymes although incubation periods for up to 24 h at 30°C were used. Similar results were obtained with the phosphorylated enzyme. Treatment of an enzyme containing 4 mol phosphate/mol subunit with alkaline or acid phosphatase removed about 3 mol phosphate/mol. This indicates that three moles of the phosphate are present as relatively alkali-labile phospho- esters and one mole of phosphate is present as a relatively alkali-stable phosphoester. Recent amino acid sequence data indicate that cGMP-dependent protein kinase contains a threoninc phosphate ester. The relative stability of threonine phosphoesters to hydrolysis is known [27].

Functional Significance of Phosphate Incorporation

The data shown so far indicate that cGMP-dependent protein kinase contains about four sites per subunit which can be phosphorylated and that slightly more than one mole of phosphate is incorporated in vivo into one mole of the enzyme. To test the possible functional role of the autophosphorylation

I I

A

10 -

5 -

lOg IcGMPlf,,,

Fig.4. E f f k r of M g . A T P oii / / i r c~yuilihriirm hindin:: of' c G M P . The bindin_e ofcGMP by native ( A ) and phosphorylated (B) enzyme (0.5 pg) w a s determined in the absence (0 ) and presence (0) of I mM ATP. Magnesium acetate ( 5 m M ) and sodium chloride (150 niM) were present i n each tube

;" "p ;,y* ;- L - 0

2

,o-- O +ASP

* ATP - al o.o-o 0

5 5 10 20 0 5 10 x) go

Incubation Time (min)

- 3 0 -5 - 3 OL4

0 - 5 log IMgATPI

Fig. 5. &ffic.t of Mg . A TP oii ci.s.sr~c~iu/ion of ( G M P mu' c,onccw/rutiou o f ' / M g . ATP wquircd fhr rdiihition of cGMP hincling. Aliquots of native cGMP-dependent protein kinase (left half of the figure) and phos- phorylated enzyme (right half of the ligure) were incubated in the presence o f X.5 nM [3H]cCMP and iii the absence (0) and presence (0) of I i1iM ATP for the time indicated at 2 C (upper half of the figure). In the lower half of the figure the effect of various concentrations of Mg . ATP on the binding of 8.5 nM [31-I]cGMP is shown. The amount of each en/yme was 17.5 ng and incubation was carried out at 2 C fbr 5 min. Magnesium acetate ( 5 mM) was present in each tube

reaction, the properties of enzymes containing about 4.1 mol phosphate/mol subunit and native enzymes containing about 1.4 mol phosphate/mol subunit were studied. Before use, these enzymes were chromatographed at 22'C on a gel filtration column to remove bound cyclic nucleotides. As shown in Fig.4, both enzymes bound 15 nmol cGMP/mg enzyme. Comparison of the binding isotherms for native and phos-

1 7++*',- , , 1 O O -5 -3

l og I CAMP 1 Fig. 6. Aclivarion of cC;MP-tkcpe~iitIent piitc,in k i m w h.1. C A M P . Aliquots of native (0) and phosphorylated (0) cGMP-dependent protein kinase (40 ng) was incubated in the presence of various concentrations ofcAMP for 2 min. Phosphorylation was stopped by addition 3f acetic acid. For further details see Materials and Methods

Table 3. Kinrtic i'owstniits for. cGMP-drprwrJent pruluiii kinuse Kinetic constants were derived froln Lineweaver-Burk plots a hich were a lways linear. Native and phosphorylated cGMP-dependent protein kinasc (32 ng) were incubated for 2 inin at 30 C; with \arious conceii- trations of substrate peptide in the presence of 1.2 pM cGMP or 0.12 mM CAMP. The molecular weight of the substrate peptide was taken iis 772. The values shown are .Y & SEM obtained with three differen1 preparations of each form of the enzyme. The tot;il concentration of magnesium acetate was 3 mM and incubation time was 2 min

Enzyme Aclivator K , 1.

Native cGMP 75 F 12 4.0 & 0.4 3.4 * 0.6 CAMP

Phosphorylated cGMP 87 _+ 16 7.6 * 0.4 CAMP 7 9 + 8 8.6 0.4

129 * 30

phorylated enzymes indicated that addition of Mg . ATP caused a significant shift in the apparent KCI for cGMP (about threefold from 18 nM to 54 nM) when the native en- zyme was used (Fig.4). Interestingly this effect of Mg . ATP was not observed when the phosphorylated enzyme was used. This differential elTect of Mg . ATP on the binding of cGMP to native and phosphorylated enzyme could be exaggerated by using low concentrations of enzymes and cGMP in a concentration corresponding to the apparent Kd value (Fig. 5 ) . Under this condition, ATP . Mg lowered the amount of cCMP bound to the native enzyme by a factor of five and had only a minimal effect on the phosphorylated enzyme. Mg ATP inhibited the binding of cGMP half-maximally at a concen- tration of about 50 pM. This concentration of Mg . ATP is similar to the K , for Mg . ATP determined in a phospho- transferase assay. Incubation of the enzyme under the con- ditions used for the experiments shown in Fig.4 and 5 in the presence of [~I-~'P]ATP instead of ATP did not reveal signifi- cant incorporation of phosphate into either form of the enzyme. As reported previously [28], the addition of Mg . ATP to cGMP binding assays had no effect when the incubations were carried out at pH 8.0. These results suggested that not only the binding of cGMP to the enzyme but also the acti- vation of the enzyme by cyclic nucleotides might be affected by autophosphorylation. Determination of the apparent

activation constant ( K A ) for cGMP did not reveal any signifi- cant difl'erence between both forms of the enzyme (apparent K A 0.15 pM). However, under the same conditions an over tenfold decrease in the apparent K A value for CAMP was observed (Fig. 6). The phosphorylated and native enzyme were activated at a concentrations of 1.75 pM and 19.6 pM CAMP (average for three different preparations), respectively. As is apparent froin Fig. 6, the phosphorylated enzyme had a higher specific activity than the native enzyme. Further in- vestigation of this phenomenon indicated that phosphoryla- tion of the enzyme had only a small if any effect on the K,, for the substrate peptide (Table 3). On the other hand, phos- phorylation increased the maximal velocity of the phospho- transferase rcaction approximately twofold. This change in V was observed regxdless of whether the enzyme was activated by cGMP or by CAMP.

DISCUSSION I t has bccn shown that cGMP-dependent protein kinase

incorporates phosphate in an apparently autocatalytic process [4]. The results obtained in this study confirm these obser- vations and extend them, in so far as the stoichiometry of this rcaction has been determined to be 4 mol phosphatc/niol subunit. I t is not quite clear why this higher number has not been detected previously. One reason may be that most groups used a CAMP-affinity column and eluted the enzyme from this column with cGMP. As noted in this as well as in other studies [I 1,151, cGMP inhibits the CAMP-dependent phosphory- lation, but in contrast to others, cGMP promotes the incor- poration of approximately 1 mol phosphate/mole subunit. I t is conceivable that part of the cGMP used during elution of the enzyme remained bound to the enzyme and thereby inhibited the incorporation of more than 1 mol phosphate. This interpretation is supported by the recent finding that cG MP-dependent protein kinase binds 4 mol cGMP/niol holoenzyme [29]. In order to obtain complete phosphorylation of the enzyme it may be necessary for all four binding sites to be occupied by CAMP. On the other hand, it cannot be ex- cluded that the enzyme used by other investigators already con- tained 3 inol phosphate/mol subunit, which would allow the further incorporation of only 1 mol phosphate. Acid/alkali- lability studies of the incorporated phosphate indicated that it is present as an alkali-labile ester, but the 1 mol phosphate present already in the native enzyme was only released by 1 M NaOH suggesting that this phosphate is present a s a rather stable phosphate ester. The occurrence of such stable phosphate esters has been documented [27] and it is possible that this phosphate is identical to the threonine phosphate identified in pure preparations of cGMP-dependent protein kinase. Autophosphorylation induced a significant functional change in several properties of cGMP-dependent protein kinase. Phosphorylation lowered the concentration of CAMP necessary for activation of the enzyme over tenfold and increased the apparent velocity of the transferase rcaction twofold. At present it is not clear if the effect on V represents a true change in velocity. It is more likely that the phosphory- lated enzyme is more resistant than the native enzyme against the inactivating properties of positivc charges [16,17]. The change in the activation constant indicates that phosphoryla- tion might also affect the binding of cyclic nucleotides to the enzyme. This latter effect of phosphorylation has bccn observed for cGMP binding.

The findings obtained so far strongly support the notion that autophosphorylation affects several properties of cCMP- dependent protein kinase in vitro. I t is not known if similar changes a s observed in vitro occur iri vivo, since the extent of the phosphorylation of the enzyme in vivo is unknown. The isolated enzyme already contains phosphate indicating that phosphorylation of the enzyme occurs in vivo. However, the stoichiometry of this reaction might be different froin that observed in vitro. since the high concentrations of CAMP, necessary to obtain complete autophosphorylation, are not prevalent in vivo. Therefore, it will be necessary to determine the stoichiometry of the phosphoi-ylation of the enzyme in vivo before a physiological significance can be attributed to the observed functional changes.

We thank Hans-Peter Gensheinier for excellent technical a and Frau Karin Brandt for typing the manuscript. This work was supported by grants Crom Deut.si~lie For.re~z~~ngs~~eniein,sc/~u~~ and Fonds t l ~ r cl.ieniist.lrm Intfusii.ir.

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F. Hofmann and V. Flockerzi, Pharmakologisches Institut der Ruprecht-Karl-Univcrsi t~t Heidelberg, l m Neuenheitner Fcld 366, D-6900 Heidelberg 1, Federal Republic of Germany