isolation and elucidation of some functional properties of the “mute” catalytic subunit of...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 222, No. 1, April 1, pp. 276-284, 1983

Isolation and Elucidation of Some Functional Properties of the “Mute” Catalytic Subunit of CAMP-Dependent Protein Kinase’

JENNIFER REED,2 MICHAEL GAGELMANN; AND VOLKER KINZEL

Institute of Experimental Pathology, German Cancer Research Center, Im Neuenheimer Feld 28O,D6900 Heidelberg, Federal Republic of Ge-rmany

Received August 9, 1982, and in revised form November 9, 1982

A mute isoenzyme of type II CAMP-dependent protein kinase from rat muscle has been reported that is released from the regulatory subunit by CAMP but remains inactive until combination with heat- and acid-stable modulator has occurred. This enzyme has now been obtained in isolation free of the normal catalytic subunit using affinity chromatography with both an ATP analog (Blue Dextran/Sepharose) and a protein substrate analog (Kemptide/CH-Sepharose). Separation can be effected in both cases before activation of the mute enzyme. Affinity of the mute enzyme for Blue Dextran-a ligand specific for the dinucleotide fold in this kinase-is somewhat higher than that of the normal enzyme. Conversely, before reaction with the modulatory protein the mute enzyme will not bind at all to Kemptide/CH-Sepharose, where the normal enzyme elutes at 50 mM KCl. When pretreated with the modulatory protein and so activated, mute enzyme binds to Kemptide with a very high affinity and can only be eluted using a natural substrate (phosphorylase kinase), up to 500 I’nM salt being ineffective. The modulator thus appears to act through alteration of the protein substrate binding site on the enzyme.

Cyclic 3X’-adenosine monophosphate- dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been shown by a number of workers to be the primary means by which cyclic AMP ex- erts its effects as a regulatory agent (1). This enzyme operates to activate or deac- tivate specific proteins by phosphorylation at critical sites. As the active catalytic subunit of CAMP-dependent protein kinase appears to be virtually identical over a wide range of tissues and species, and as it will phosphorylate a surprisingly large number of proteins in vitro, the question arises as to how it can be utilized to me-

1 This work was supported by the Deutsche For- schungsgemeinschaft.

‘To whom correspondence should be addressed. a Present address: University of Heidelberg, De-

partment of Physiology II, D-6900 Heidelberg, Fed- eral Republic of Germany.

diate highly specific processes in living or- ganisms. For this reason, any mechanism which might operate to control the free catalytic subunit is of especial interest.

This laboratory has recently reported the presence of a second inactive, or mute, isoenzyme of the catalytic subunit of CAMP-dependent protein kinase in rat muscle which must be activated by a low- molecular-weight heat- and acid-stable protein (2). The mute isoenzyme has subsequently been shown to occur as well in preparations of catalytic subunit extracted by a different method and from rabbit muscle (3).

In order to study the mechanism of mute enzyme activation and its possible interaction with the normal protein kinase in viva, it is necessary first to obtain both enzymes separate and in purified form. As the enzymes copurify and share such properties as molecular weight and isoelectric

0003-9861/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

2’76

ISOLATION AND FUNCTION OF MUTE PROTEIN KINASE 277

point within very close limits, their functional differences if any appear to afford the best basis for separation. By using techniques of affinity chromatography, it has been found possible to obtain both enzymes as homogenous isolates prior to mute enzyme activation. This has revealed fundamental differences in substrate affinity which help to explain both the opera- tional differences between the two enzymes and the mechanism of mute enzyme activation.

MATERIALS AND METHODS

Calf thymus histone (Cat. No. 9240) was purchased from Sigma. [y32P]ATP (>20 Ci/mmol) was obtained from New England Nuclear.

The purified catalytic subunit of CAMP-dependent protein kinase from rat muscle was prepared and

characterized as previously described (4,5). The heat- and acid-stable protein kinase modulator fraction

(PKM)4 was extracted from rat muscle according to the method of Walsh et al (6) through a Sephadex

G-100 step. Regulatory subunit (R) from CAMP-dependent protein kinase type II from bovine heart muscle was a kind gift from Dr. F. Hofmann, De-

partment of Pharmacology, University of Heidel- berg, and highly purified PK inhibitor (7) from Dr.

J. Demaille (Montpellier, France). We are also in- debted to Dr. U. Mwra for her donation of bovine

calmodulin. Assay of enzyme activity was carried out on iso-

electric focusing gels (8) or by the standard assay described in (4). Gel sections for pH gradient determination were incubated under nitrogen, as exposure

of Ampholine gels to air has been found to cause an artifactual lowering of pH values measured, espe-

cially in the region from pH 10 to 8. Samples were counted in Toluol/Liquifluor scintillator in a Packard

Tri-Carh 3360 liquid scintillation counter. Afinity chromatography on Blue Dextran/Sephar-

ose. 5 g of cyanogen bromide-activated Sepharose 4B

(Pharmacia Fine Chemicals AB, Uppsala, Sweden) was washed on a Biichner funnel equipped with filter for 15 min with 10e3 M HCI. It was then suspended

in 5 ml 0.4 M Na&Oa (brought to pH 10 with HCl) in which 0.1 g Blue Dextran 2000 (Pharmacia) had been dissolved. The suspension was rotated gently over-

night at 4°C. Columns (0.6 X 3 cm) were poured and

4 Abbreviations used: PKM, heat- and acid-stable protein kinase modulator fraction; MOPS, morpho-

linepropanesulfonic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; SDS, so- dium dodecyl sulfate.

washed with 600 ml 1 M KC1 before being equilibrated

with 10 mM Tris-HCI, pH 7.5 Catalytic subunit preparation was dialyzed against

10 mM Tris-HCI, pH 7.5 (exact protein concentrations are given in the figure legends). The dialyzed enzyme

was placed on the column and the immediate run- through collected. It was then eluted stepwise with 10 mM Tris-HCI pH 7.5 plus increasing concentra-

tions of KCI. All eluted fractions were tested for ac-

tivity with and without added PKM on isoelectric focusing gels.

ABnity chromatography on Kemptide/CH-Seph.ar-

ose. CH-Sepharose 4B (0.125 g) (Pharmacia) was al-

lowed to swell in excess 0.5 M NaCl, followed by 25 ml distilled water adjusted to pH 4.5.

Kemptide (9.5 mg) (Leu-Arg-Arg-Ala-Ser-Leu- Gly; Penninsula Laboratories Inc., San Carlos, Calif.) and 5 mg EDC (1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride; Fluka AG, Switzerland) were each dissolved in 5 ml distilled water adjusted to pH 4.5. The washed gel was suspended in the 5 ml

Kemptide solution and the EDC solution added a drop at a time under constant slow stirring on a vortex

mixer. The pH was immediately readjusted to 4.5 with 0.1 N NaOH. The mixture was rotated slowly

overnight at 30°C. The Kemptide/CH-Sepharose was poured in a 1.0

ml disposable hypodermic syringe modified for use as a chromatography column (final volume = 0.45 ml).

It was then washed sequentially with distilled water at pH 4.5, with 50 ml 100 mM phosphate buffer, pH

9.5-l M KCl, and 50 ml 100 mM phosphate buffer, pH 4.5-l M KCI. The column was stored under this last

buffer until immediately before use, when it was equilibrated with 5 mM Tris-HCl pH 7.5.

Catalytic subunit preparation (0.5 ml) was dialyzed

against 5 mM Tris-HCI, pH 7.5, before being loaded on the Kemptide/CH-Sepharose column. The im-

mediate run-through and the eluates from stepwise elution with 5 mM Tris-HCI, pH 7.5, plus increasing

concentrations of KC1 were tested for activity with and without added PKM on isoelectric focusing gels.

Peptide mapping. Protein digestion and peptide mapping were carried out by the method of Cleveland

et aL (9). V8 protease (2.5 mg) from Staphloccucus aureus (Miles Laboratories) was mixed with 2.5 ml

0.125 M Tris-HCl, pH 6.8-0.1% SDS-l mM EDTA-

10% glycerine. Five microliters of this mixture was added to 995 ~1 of the above buffer containing ca. 50

ng catalytic subunit. Digestion and electrophoresis of peptide fragments were as described in (9). Bands

were visualized through the silver staining procedure (10).

Activation of isolated mute enzyme. Isolated mute enzyme was activated by incubation in 0.5-ml aliquots with 30 pg PKM at 37°C for 15 min prior to

application to the Kemptide column. Phosphorylase

kinase for elution purposes (Sigma GmbH, Munich,

278 REED, GAGELMANN, AND KINZEL

West Germany) was dissolved in 5 mM Tris-HCl, pH 7.5, and dialyzed against the same buffer before use.

End concentration was approximately 1.3 mg protein/ml.

Eluticm of activated mute enzyme from focusing gels.

After focusing gels were equilibrated with 0.2 M MOPS

buffer, pH 6.25, as described (8) and cut into 2-mm slices. Each slice was transferred to a conical plastic

vial (Eppendorf), covered with 150 ~1 50 mM MOPS buffer, pH 6.8, containing 10 mM Mg-acetate, 1 mM

dithioerytritol, and 5 mg/ml histone, and homoge- nized with a conical Teflon pestle. After centrifuga-

tion for 1 min in an Eppendorf centrifuge, 20 nl of the supernatants was assayed for protein kinase activity as described (4) except that the histone con-

centration was doubled. The fractions containing the activated mute enzyme were pooled and character-

ized as described in Table II.

RESULTS

To establish firmly the independent identity of the mute enzyme, it is necessary to separate protein kinase preparations into the two enzyme fractions, mute enzyme and normal enzyme. An obvious way to achieve this is by use of affinity chromatography involving the ligand binding sites of the enzymes. These are the ATP binding site and the substrate binding site. Fortunately, appropriate affinity systems exist for both these cases. These are the Blue Dextran (on Sepharose) system for the ATP binding site and the Kemptide (on CH-Sepharose) system for the substrate binding site. Both systems are examined. The order of examination is critical to experimental understanding, as will be shown. In addition to achieving the desired separation and consequent proof of the independent existence of the mute enzyme, specific information about the functional differences of the mute and normal enzymes has been obtained.

As previously reported (2), the purified catalytic subunit preparation from rat muscle protein kinase contains two isoenzymes, one of which has a p1 value 0.3- 0.5 pH units lower than the other and dis- plays activity only after reaction with the heat- and acid-stable PKM preparation from the same tissue (Fig. la). The sen- sitivity of Ampholine gels in the high pH region to oxidative pH change means that

even after incubation under nitrogen there exists a certain variation in the absolute pH values registered. The ApI of ca. 0.4 pH units between the isoenzymes remains constant under all measuring conditions.

The activating component has been shown to be an acidic protein (p1 = 4.0) copurifying with but not identical to the standard inhibitor protein. The molecular weight is around 10 to 20k. The highly purified inhibitor fraction (7) will activate the mute enzyme very efficiently (data not shown), so the activating protein probably comprises part of the microheterogeneity reported by this group. The physical properties of the activator are therefore very close to those of the inhibitor. Calmodulin will not activate the mute enzyme either in the presence or the absence of Ca’+. Bo- vine serum albumin is also incapable of activating the mute enzyme.

When an enzyme preparation containing both isoenzymes (Fig. la) was loaded on a Blue Dextran/Sepharose column, the normal and the mute catalytic subunit eluted at different salt concentrations. The first enzyme activity was released from the column at around 100 mM KCl, little or no enzyme of either sort being recovered below this concentration. The early eluting enzyme focused at around pH 9.4 on isoelectric focusing gels (Fig. lb). No component focusing at pH 9.1 was found in the 100 mM eluate when tested either with or without PKM, and the activity peak at pH 9.4 was not significantly stimulated in its presence. The enzyme eluting at 100 mM

KC1 can thus be identified as the normal catalytic subunit essentially free of the mute component.

No further enzyme was released from the column until a KC1 concentration of at least 500 mM was reached. At this level although no activity whatsoever was seen in the absence of PKM, eluate mixed with PKM before focusing gave a small activity peak at a point about 0.4 pH units lower than the normal enzyme (Fig. lc). This fraction thus contains isolated mute enzyme. It should be noted that the PKM (p1 = 4.0) is separated from the enzyme by the focusing procedure before the assay takes place, and so is not acting as a substrate.


FIG. 1. Dextran Blue/Sepharose separation of mute and normal isoenzymes of protein kinase catalytic subunit from rat muscle. One milliliter of enzyme (ca. 100 pg protein/ml) in 10 mM Tris-

HCl, pH 7.5, was loaded on the column and washed with 4.0 ml of the same buffer. The column was then eluted with 4.0 ml of the same buffer. The column was then eluted with 4.0 ml each 50

mM, 100 mM, 250 mM, and 500 mM KC1 in 10 mM Tris-HCl, pH 7.5. Ninety-five-microliter aliquots of eluate were used for activity tests in the isoelectric focusing gel assay without (-) and with (- - -) 15 pg PKM. (a) Starting material (1:3 diluted); (b) 100 mM eluate; (c) 500 mM eluate.

For a detailed treatment of this problem, see (2).

A major problem with separation by Blue DextranLSepharose affinity chromatography is the very high ionic strength required to elute mute enzyme from the column. As the catalytic subunit has a ten- dency to adhere to glass, plastic, etc., di- alysis of anything but bulk volumes results in unacceptable loss of activity. Be- cause of this, the mute enzyme was loaded on focusing gels in high salt as eluted. It is interesting that under these conditions the level of activity recovered is much less than would be expected from the amount of mute enzyme present in the original preparation (Fig. la). That it is not due to

any fundamental change associated with separation from the normal enzyme can be seen from the excellent recovery of mute enzyme activity when separation was carried out on Kemptide columns (see below). The low activity can also not be ascribed to a simple salt inhibition of catalytic activity, since in the isoelectric focusing gel assay phosphorylation of histone takes place after the enzyme has (a) been focused, and (b) been equilibrated in 50 mM MOPS (8). Experiments with the protein kinase preparation before separation have shown that the usual stimulation of mute enzyme in the presence of PKM is elimi- nated when comparable KC1 concentrations are added to the sample mixture be-

REED, GAGELMANN, AND KINZEL

1 2346678 1 2345678 cm

FIG. 2. Kemptide/CH-Sepharose separation of mute and normal isoenzymes. Enzyme, 0.5 ml, in

5 mM Tris-HCI, pH 7.5, was loaded on the column. The column was eluted with five 2.0-ml aliquots of 5 mM Tris and subsequently with five 2.0-ml aliquots 50 mM KC1 in 5 mM Tris. The column void volume was 0.5 ml. Ninety-five-microliter aliquots of eluate were taken for activity tests without

(-) and with (- - -) 15 wg PKM. (a) 5 mM eluate; (b) 50 mM eluate.

fore focusing. It therefore seems most likely that it is the interaction between PKM and the mute enzyme which is inhibited at high salt concentrations. As a result, separation based on differing aflin- ities for Blue Dextran, while achievable, was not ideal. For these reasons affinity chromatography was attempted using Kemptide as a ligand.

CH-Sepharose with covalently bound Kemptide, a synthetic heptapeptide substrate analog (ll), might be expected to separate the isoenzymes on the basis of substrate affinity. CH-Sepharose was used for this purpose as the six-carbon spacer group acts to minimize steric interference arising when a small substrate is bound to a relatively large matrix. Kemptide bound in this manner can function quite effectively as substrate for the normal catalytic subunit (data not shown). This is in keeping with the finding of Kemp (12) that

substitution of the NHz-terminal leucine with various hydrophobic derivatives had no effect on the kinetics of Kemptide phosphorylation.

When the starting material (Fig. la) is loaded on a Kemptide/CH-Sepharose column, the pattern of elution is reversed. A loading buffer of very low ionic strength (5 mM) was used in an attempt to optimize binding conditions. Even so, the mute enzyme did not bind at all to the column, eluting only shortly after the void volumn (Fig. 2a). (Elution profiles for total protein, normal catalytic subunit and mute catalytic subunit are given in Fig. 3.) No trace of the normal enzyme is present in this fraction, and the enzyme activity on reaction with PKM is roughly that expected from the level of mute enzyme present in the starting material, allowing for the fourfold dilution attendent on elution. The mute enzyme fraction stored in 5 mM


).

i’

I’

1 .

. )p

)’

).

L

5mM 50mM a

FIG. 3. Kemptide/CH-Sepharose elution profiles.

(a) Total protein; (b) normal catalytic subunit; (c) mute catalytic subunit.

Tris-HCl, pH 7.5, at 4°C loses around 10% of its activity per week of storage, (pos- sibly due to adhesion), but retains its mute characteristics-that is, shows no activity in the absence of PKM treatment-for at least 5 weeks.

The normal enzyme first elutes from the Kemptide column at a KC1 concentration of 50 mM. No mute enzyme is present in this fraction, and the activity peak focuses at pH 9.3 (Fig. 2b). On storage of the pu-

rified normal enzyme, no PKM-inducible activity develops and the p1 does not change; the mute enzyme does not appear to be a spontaneous conversion product of the normal catalytic subunit.

One-dimensional peptide mapping of a V8 protease digest of mute and normal catalytic subunit showed no obvious difference between the two enzymes. The same pattern of 13 bands was seen in both cases. Any differences in primary structure between the two enzymes must therefore be fairly small. As molecular weight determination on SDS gels has a 10% margin of error, peptides differing by as much as 10 amino acids could comigrate under these conditions.

A certain percentage of “spontaneous” activity is sometimes seen in freshly isolated mute enzyme fractions. This disap- pears rapidly on storage, with the enzyme becoming completely mute. Experiments in isolating the mute enzyme in active form described below suggest that this is due to the presence in the isolate of a small amount of mute enzyme in the active form maintained in that state by bound substrate. As the substrate diffuses away in storage, the enzyme reverts to the inactive form.

When the mute enzyme fraction isolated on the Kemptide column is then activated by incubation with PKM and rerun on the same Kemptide column under identical conditions, its behavior is seen to have changed. The activated mute enzyme no longer elutes in the loading buffer even after repeated washings, or at any salt concentration up to 500 mM (Fig. 4a). The affinity of the enzyme for the column ligand is thus radically changed after activation with PKM. This change is directly con- cerned with the substrate binding affinity, as the enzyme is completely released even at 5 mM Tris-HCl when the natural substrate phosphorylase kinase is present in the elution buffer (Fig. 4b). The mute enzyme so released is still in the activated form. If reassayed on isoelectric focusing gels, there is no longer any difference between the curves obtained with and without PKM (Fig. 4b). Phosphorylase kinase focused alone gave no activity in the gel


8.

a 1 2 345678

4

1 2345678 cm

FIG. 4. Affinity of activated mute enzyme for Kemptide/CH-Sepharose. Isolated mute enzyme,

0.5 ml, was incubated for 15 min at 35°C with 30 pg PKM. It was then loaded on the Kemptide/ CH-Sepharose column and eluted with 5 mM Tris-HCl, pH 7.5, and 50, 100, 250, and 500 mM KC1 in 5 mM Tris as previously described. The column was then reequilibrated with 5 mrd Tris and

eluted with 2.0 ml phosphorylase kinase (1.3 mg/ml) in 5 mM Tris. Ninety-five-microliter aliquots of eluates and of the phosphorylase kinase solution were taken for activity tests without (0) and

with (+) 15 pg PKM. N.B. In comparing Fig. 4b with Fig. 2b, the fourfold dilution attendant on

reelution from the column should be taken into account. (a) 5 mM-500 mM eluates; (b) phosphorylase kinase eluate; (c) phosphorylase kinase.

assay (Fig. 4c) indicating that the com- mercial preparation used is free of protein kinase catalytic subunit.

Once activated and separated by the above procedure from the inhibitor also present in PKM, the mute enzyme can be assayed by the standard protein kinase test in solution with histone as substrate. Ta- ble I gives the results of the standard assay on activated mute enzyme with and without added purified regulatory subunit (R), CAMP, and PKM. Under these conditions, only the inhibitory factor in PKM is expressed. Phosphorylation of histone was inhibited 85-90% in the presence of

excess regulatory subunit. This inhibition was released when 5 PM CAMP was also present in the assay mixture. Mute enzyme activity in this test was also inhibited by the Walsh inhibitor present in the PKM preparation, which is why assay on isoelectric focusing gels has been necessary throughout these experiments. Electro- phoretic resolution of a partial acid hydrolysate of the phosphorylated histone under conditions giving maximal separation of phosphotyrosine and phospho- threonine showed that the radioactive phosphate had been incorporated into ser- ine and threonine residues. No detectable


TABLE I

CHARACTERIZATION OF THE ACTIVATED MUTE PK ELUTED FROMAFFINITYCOLUMNS

32P transfered to substrate from P~~]ATP in 5 min at

Additions/200 ~1 30°C (cpm)

None 7352 f 401 Regulatory subunit

(2.5 4 Regulatory subunit

905 f 63

(2.5 pg) and cyclic AMP (5 PM)

Heat- and acid-stable 11863 f 683

PKM ‘78’7 + 10

Note. 50 ~1 of the phosphorylase kinase eluate (activated mute enzyme) was assayed in solution using 40 pg histone IIa as substrate.

phosphotyrosine was present (data not shown). The same behavior with respect to the regulatory subunit was observed when mute enzyme in the activated form was obtained in the absence of added phosphorylase kinase (Table II). Here, mute enzyme was activated by exposure to PKM and then extracted from isoelectric focusing gels after the focusing procedure had removed the inhibitory component of PKM.

DISCUSSION

The activation of a previously inactive form of the catalytic subunit can most eas- ily be visualized as occurring through a modification of a ligand binding site, either that responsible for the binding of Mg-ATP or the point of attachment of protein substrate. This enzyme has been observed to exhibit a certain malleability (13) which supports the idea that conforma- tional alterations of this sort play a part in its regulation. Since the normal and mute isoenzyme copurify with one another and differ only marginally in their isoelectric points and migration rates on SDS gels, such modification also forms the most likely basis for their separation.

Blue Dextran, (the dextran-bound form of the sulfonated polyaromatic chromo- phore Cibacron Blue F3GA), has been found by Stellwagen and co-workers (14) to bind specifically to proteins containing

the supersecondary structure known as the dinucleotide fold, presumably by acting as an analog in its spatial configuration of the adenosine diphosphoryl portion of nu- cleotides. It has further been observed (15) that the catalytic subunit of CAMP-dependent protein kinase contains the dinucleotide fold as its ATP binding site. This would mean that any modification of this region in the mute enzyme should alter its affinity to a Blue Dextran/Sepharose column relative to that of the normal catalytic subunit.

Contrary to expectation, the mute enzyme not only bound to Blue Dextran/Se- pharose, but could not be eluted until salt concentrations 5- to lo-fold higher than those which released the normal enzyme were reached. Thus, the inactivity of the mute enzyme is not due to either steric blocking or structural alteration of the dinucleotide fold. In fact, judging from its binding properties to Blue Dextran, the affinity of the ATP binding site for its ligand may be considerably higher in the mute enzyme than in the normal catalytic subunit.

The synthetic heptapeptide Kemptide mimics the amino acid sequence typically found in the region of the phosphorylated site in natural substrates of CAMP-dependent protein kinase, and can itself act as a substrate with a Km of about 16 pM (11). The behavior of the mute and normal enzyme on Kemptide/CH-Sepharose affinity columns is exactly as would be expected were the former inactive through substrate binding site modification; the mute enzyme does not bind to the column, elut-

TABLE II

CHARACTERIZATION OF THE ACTIVATED MUTE PK ELUTED FROM FOCUSING GELS

Additions/200 ~1 Units of PK activity

None 1.93 f 0.19 Purified PK inhibitor (20 ng) 1.45 f 0.13 Heat- and acid-stable PKM

(4 PIT) 0.2 f 0.08 Regulatory subunit (2.5 rg) 0.06 + 0.03 Cyclic AMP (5 *M) and

regulatory subunit (2.5 pg) 1.64 + 0.31


ing in the 5 mM loading buffer, while the normal enzyme is first eluted at 50 mM

KCl. The affinity of the mute enzyme for substrate analog is thus extremely low. Due to the substantial difference in affinity, it is possible by this method to separate the mute enzyme in a highly purified form.

When the purified mute enzyme is incubated with PKM before being loaded onto the same affinity column, under identical conditions, the situation is changed dra- matically. The affinity of the activated mute enzyme for Kemptide is extremely high. The enzyme could only be recovered by eluting with a natural substrate of CAMP-dependent protein kinase, phosphorylase kinase. That it is then in fully activated form is shown by the superpo- sition of the activity peaks with and without PKM; in the mute form, where it does not bind to Kemptide, only the sample with PKM has significant activity.

This extreme change in the affinity of the mute enzyme for a substrate analog before and after activation indicates that PKM-induced activation proceeds through modification of the protein substrate binding site. Further studies using fluorescence and circular dichroism techniques will be needed to determine exactly how this modification is effected.

In summary, the mute isoenzyme of the catalytic subunit of CAMP-dependent protein kinase from rat muscle has now been isolated and shown conclusively to be a separate enzyme, maintaining after isolation both its mute characteristics and its ability to be activated by PKM. The activated enzyme can be assayed in solution under the conditions described and is inhibited by purified regulatory subunit of CAMP-dependent protein kinase. This inhibition is released in the presence of CAMP. It is therefore clear that the mute enzyme is a form of the catalytic subunit of CAMP-dependent protein kinase.

Activation proceeds through gross aBin- ity changes in the protein substrate binding site, which is of importance in eluci- dating the specific function of the mute enzyme in vivo. Once the protein binding site is activated, its affinity for substrate

is much higher than that of the normal enzyme. Given that mute and normal catalytic subunits phosphorylate the same substrate proteins in vivo, the mute isoenzyme once activated would rapidly out- compete the normal one. For this reason, one might expect the mute enzyme either to display different substrate specificity or to be activated only under conditions where the normal enzyme was relatively inactive.

ACKNOWLEDGMENTS

We should like to thank Dr. Dieter Kiibler for per-

forming the assay on the histone acid hydrolysate, and Herr Norbert KGnig for his excellent technical

assistance.

REFERENCES

1. LANGAN, T. A. (1967) in Advances in Cyclic Nu- cleotide Research (Greengard, P. and Robin-

son, G. A., eds.), Vol. 3, Raven Press, New York. 2. GAGELMANN, M., REED, J., K~BLER, D., PYERIN,

W., AND KINZEL, V. (1980) Proc. Nat. Acd Sci. USA 77, 2492-2496.

3. GAGELMANN, M., OBST, C., AND KINZEL, V. (1982) Biochem. Int. 5,317-324.

4. KOBLER, D., GAGELMANN, M., PYERIN, W., AND KINZEL, V. (1979) Hoppe-Seyler’s 2. Physiol. Chem. 360,1421-1423.

5. PYF,RIN, W., GAGELMANN, M., K~BI,ER, D., AND KINZEL, V. (1979) Z. Naturforseh. 34c, 1186-

1194.

6. WALSH, D. A., ASHBY, C. D., GONSALEZ, C., CALK- INS, D., FISCHER, E. H. AND KREBS, E. G. (1971) J. Biol Chem. 246, 1977-1985.

7. DEMAILLE, J. G., PETERS, K. A., STRANDJORD,

T. P., AND FISCHER, E. H. (1978) FEBS L&t. 86, 113-116.

8. GAGELMANN, M., PYERIN, W., KUBLER, D., AND KINZEL, V. (1979) Anal Biochem. 93,52-59.

9. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER,

M. W., AND LAEMMLI, U. K. (1977) J. Biol Chem 252, 1102-1106.

LO. OAKLEY, B. R., KIRSCH, D. R., AND MORRIS, N. R. (1980) Anal. Biochem. 105, 361-363.

11. KEMP, B. E., GRAVES, D. J., BENJAMIN, E., AND KREBS, E. G. (1977) J. Biol. Chem. 252, 4888- 4894.

12. KEMP, B. E. (1980) J. Biol. Chem 255,2914-2918.

13. KUPFER, A., JIMENEZ, J. S., AND SHAJXIEL, S. (1980) Biochem. Biophys. Res Commun. 96, 77-84.

14. THOMPSON, S. T., CASS, K. H., AND STELLWAGEN, E. (1975) Proc Nat. Acd Sci. USA 72, 669-

672. 15. WIT, J. J., AND ROSKOSKI, R. (1975) Biochemistry

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isolation and elucidation of some functional properties of the “mute” catalytic subunit of...

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