Journal of Neurochemisfry. 1977. Vol. 28, pp. 543-547. Pergamon Press. Printed in Great Britain
SUBCELLULAR DISTRIBUTION OF A HEAT-STABLE PROTEIN INHIBITOR OF CYCLIC AMP-DEPENDENT PROTEIN KINASE IN RAT BRAIN R. ROSKOSKI, JR. and C. E. FREDERICK The Department of Biochemistry, The University of Iowa, Iowa City, IA 52242, U.S.A. (Received 23 July 1976. Revised 4 October 1976. Accepted 5 October 1976)
Abstract-Cyclic AMP (CAMP)-dependent protein kinase catalyzes the phosphorylation of polypeptidic serine and threonine residues according to the following chemical equation: ATP + protein -+ phosphoprotein + ADP. A heat stable, trypsin labile factor present in brain, skeletal muscle and other tissues inhibits enzymatic phosphorylation of some proteins and enhances that of others. Since brain is one of the richest sources of adenylate cyclase, CAMP,CAMP-dependent protein kinase and the heat stable protein kinase inhibitor and because they may play a role in neurotransmission, an investigation of the subcellular distribution of the heat stable factor in rat brain was undertaken. Although present in the nuclear, mitochondrial and microsomal fractions, the highest activity of protein kinase inhibitor is in the soluble fraction: its activity parallels that of the cytoplasmic enzyme marker, lactate dehydrogenase. The inhibitory activity is also found in the synaptosome or pinched-off nerve ending fraction. Following osmotic lysis of this fraction, about 90% of the factor occurs in the soluble fraction. On the other hand, only 40% of the CAMP-dependent protein kinase is solubilized and 60% remains membrane-bound. Using this membrane-bound protein kinase, phosphorylation of endogenous substrate is unaltered by inhibitor, but phosphorylation of added histone substrate is decreased.
A MAJOR class of protein kinase requires adenosine 3',5'-cyclic monophosphate (CAMP) for activity. The mechanism of activation is expressed by the equation RC cAMP$C + R.cAMP in which RC represents the holoenzyme and R and C are the regulatory and catalytic subunits, respectively (GILL & GARREN,1970; TAOet al., 1970; KUMONet al., 1970; REIMANN et al., 1971). cAMP promotes the dissociation of the RC complex producing a free catalytic subunit and a CAMP-regulatory subunit complex. The freed catalytic subunit is active and cAMP independent. A heat stable protein which inhibits CAMPdependent protein kinase is present in many tissues (APPLEMAN et al., 1966; ASHBY & WALSH, 1972). ASHBY& WALSH(1972) reported that skeletal muscle and brain contain the highest inhibitory activity. They also reported (ASHBY& WALSH,1973) that the inhibitor complexes with the free catalytic subunit (C), but not holoenzyme (RC). The inhibitor-catalytic subunit complex, which is catalytically inactive, is incapable et of binding to the regulatory subunit. DONNELLY al. (1973~1,b) reported that a protein factor, purified to homogeneity from lobster muscle, inhibits histone phosphorylation but stimulates protamine phosphorylation and suggest that the factor functions as a modulator. The modulator, furthermore, alters the substrate specificity of CAMP- and cGMP-dependent
+
protein kinases prepared from various invertebrate et al., 1973b). and vertebrate tissues (DONNELLY Changes in modulator activity under physiologic and pathologic conditions suggest a regulatory role. For example, modulator activity in rat brown adipose tissue varies during development (SKALAet at., 1974) and Kuo (1975) reports that it is decreased in epididyma1 fat pads in alloxan-diabetic rats. As a first step in establishing and correlating possible physiologic roles for the inhibitor, its subcellular distribution was determined in rat brain cerebral cortex, a rich source of adenylate cyclase (SUTHERLAND et aL, 1962) CAMP-dependent protein kinase (Kuo & GREENGARD, 1969) and the protein kinase inhibitor (ASHBY& WALSH,1972). We find that inhibitory activity parallels that of lactate dehydrogenase, a cytoplasmic enzyme marker. Although 30% of the inhibitory activity is found with the crude mitochondria1 fraction containing synaptosomes (pinched off nerve endings), ,osmotic lysis solubilizes about 90% of this activity, but only 40% of the protein kinase activity. Using membrane-bound protein kinase, the inhibitor fails to alter phosphorylation of endogenous substrate, but it inhibits phosphorylation of exogenous histone. A preliminary account of this work has et al., 1976). appeared (ROSKOSKI
MATERIALS AND METHODS This investigation was Supported by Grant NS-11310 of the US. Public Health Service. Subcellular fractionation. Using male Sprague-Dawley Abbreviation used: CAMP, cyclic AMP. rats (200-250 g), primary subcellular fractions (nuclei, 543
R. ROSKOSKI,JR. and C. E. FREDERICK
544
crude mitochondria, microsomes, and cytosol) from cere- or usually a suitable dilution to give 14% inhibition was bral cortex were prepared by the methodology of MAENO assayed for inhibitory activity. The reaction was initiated and co-workers (1971). Using discontinuous Ficoll gra- by adding 5p1 of bovine brain protein kinase with the dients, synaptosome-enriched fractions were prepared from standard activity (2.59 pmol 32Pincorporation per min at the crude mitochondrial suspension by the procedure of 30°C) to 45 pl of solution containing inhibitor, buffer, ATP, et al. (1973) and hypotonic lysis of the crude histone and CAMP (lo-' M) as previously described (WITT MCGOVERN (1975~)).Incubations were for 30 min at 30°C mitochondrial fraction or the synaptosome-enriched frac- & ROSKOSKI, & during which time incorporation of 32Pinto histone was tion was performed using the method of DEROBERTIS co-workers (1962, 1966). To release possible sequestered linear. The labeled phosphoprotein was resolved from enzyme activity after subcellular fractionation, one-ninth [y-32P]ATP and metabolites by the phosphocellulose volume of 10% Triton X-100 (v/v) was added to each frac- paper procedure as previously described (WITT & ROStion at least 5min prior to each of the enzyme assays. KOSKI,1975a,b). Protein kinase activity was measured in 20p1 aliquots Lactate dehydrogenase, fumarase, acetylcholinesterase, and choline acetyltransferase (enzyme markers for the soluble of the designated fractions for 2min at 30°C by the pro1975~). cytoplasm, mitochondria, external synaptosomal mem- cedure previously documented (WITT& ROSKOSKI, brane and occluded synaptosomal cytoplasm (WHITTAKER Materials. Sources of other materials are previously 1975a, b). Bovine brain & BARKER, 1972) were measured by the methodology de- documented (WITT & ROSKOSKI, scribed by HSIEH & VESTLING (1966), WHITTAKER& protein kinase was prepared as previously described BARKER (1972), ROSKOSKI(1973) and ROSKOSKI(1974), re- through the Sephadex G-200 step (WITT & ROSKOSKI, spectively. The first three spectrophotometric assays were 1975a). The enzyme fraction contained insignificant inhibicarried out at ambient temperature in a final vol of 300 pl tory activity. Other enzymes were obtained from Worthusing 5 or lop1 of the designated fraction. Protein was ington Biochemical Corp. measured by the method of LOWRYet al. (1951). Measurement of protein kinase inhibitor activity. As RESULTS ASHBY & WALSHreported (1972), the degree of protein kinase inhibition is dependent upon the respective concen- Subcellular distribution of' protein kinase inhibitory trations of inhibitor, protein kinase and the protein sub- activity strate. We adopted their definition of 1 unit of protein The distribution of protein kinase inhibitor in rat kinase inhibitor as that amount which decreases phosphorbrain cerebral cortex parallels that of lactate dehydroylation of substrate from 7 pmol min-' to 6 pmol min-' genase, a cytoplasmic enzyme marker (Table 1). The in 0.13 ml or, by proportion, 2.59 pmol min-' to nuclear, mitochondrial, microsomal and cytoplasmic 2.31 pmol min-' in 0.05 ml. The units of activity differ, fractions contain 10, 34, 18 and 38"/,, respectively, of however, since we used bovine brain protein kinase as the enzyme source and histone as substrate (contrast with rab- the total activity. This distribution also parallels that bit skeletal muscle and casein). In this study, only inhibi- of protein kinase. The crude mitochondrial fraction tion of phosphorylation (not modulation) was measured. contains the synaptosome or pinched-off nerve ending In early studies we found that when 30 times the kinase fraction (DEROBERTIS & RODRIGUEZ DE LORRES activity was used in the assay, modulator activity was un- ARNAIS,1969). About 75% of the activity of choline detectable in bovine heart extracts but detectable in bovine acetyltransferase, a marker for the synaptosomal cytoand rat brain. After decreasing enzyme activity to plasm (WHITTAKER8z BARKER,1972), is present in 2.59 pmol min-', modulator activity in heart was detect. the mitochondrial fraction. able, indicating the increased sensitivity with lower enzyme The crude mitochondrial fraction was subfracactivity. To measure the activity of the inhibitor, portions (lo0 pl) of the specified fractions were placed in a boiling tionated into myelin, synaptosomes, and mitochonwater bath for 10 min. After cooling, one-ninth volume of dria by discontinuous Ficoll gradient centrifugation. 10% Triton X-100 (v/v) was added to release possibly Again, the activity of the protein kinase inhibitor parsequestered inhibitory activity. Then 20 pl of the fraction alleled that of lactate dehydrogenase, protein kinase TABLE1.
Protein (mg/g tissue)
Fraction ~
~
Inhibitor (unitsig)
Protein kinase activity +CAMP -CAMP (units/g)
BRAIN*
Lactate dehydroFumarase genase Inhibitor
Choline acetyltransferase
~
Nuclei Crude mitochondrial fraction Microsomes Cytosol Homogenate Recovery (%) ~
* The
PROTEIN KINASE INHIBITOR DISTRIBUTION IN SUBCELLULAR FRACTIONS OF RAT
~~
15.6
780
42.2
2690
27.8
25.1 23.9 120 89
1400 2990 9140 86
16.8 19 77.4 93
~
8.41
2.33
0.5
0.7
0.7
0.2
16.9
1.8
0.8
0.9
1.8
2.9 2.2 27.1 88
0.5 0.0
0.4 1.8
0.7 1.7
0.3 0.2
~~~~
fractions were prepared by differential centrifugation; enzymes, protein kinase inhibitor and protein were measured by methods documented in the Materials and Methods section. The results are expressed per g of rat cerebral cortex. One unit of protein kinase is defined as one nmol of 3zP incorporation per min at 30°C under standard conditions. The activity of fumarase, lactate dehydrogenase, protein kinase inhibitor, and choline acetyltransferase is expressed as the ratio of the percentage recovered activity to the per cent recovered protein (relative specific activity).
Protein kinase inhibitor TABLE 2. PROTEIN KINASE INHIBITOR
DISTRIBUTTON IN SUBMITOCHONDRIAL FRACTIONS RESOLVED BY DISCONTINUOUS GRADIENT CENTRIFUGATION*
Protein Fraction
tissue)
Inhibitor (units/g)
Myelin Synaptosomes Mitochondria Recovery (%)
11.7 20.1 6.3
1570 330
(mg/g
90
545
632 94
Protein kinase activity +CAMP -CAMP (units/g) 6.9 21.6 2.3 91
FICOLL
Lactate Choline dehydroacetylFumarase genase Inhibitor transferase
3.1 9.4
0.0
1.2 87
1.1
0.4
0.6 1.4 0.6
0.8
1.2 0.8
0.4 1.5 0.7
* A crude mitochondrial fraction, prepared as that in Table 1, was resolved into the three components by the methodology of MCGOVERN & co-workers (1973). Activities are expressed as described in Table 1. and choline acetyltransferase (Table 2). Fumarase, a mitochondrial marker, is found in the mitochondrial and synaptosomal subfractions. The latter activity is presumably associated with intrasynaptosomal mitochondria as well as inevitable cross-contamination from the mitochondrial fraction. To lyse osmotically sensitive components, 9 volumes of distilled water were added to a preparation of the crude mitochondrial fraction. The solution was centrifuged at l0,OOOg for 20min to pellet the M-1 fraction (myelin, nerve endings, free mitochondria). The resulting supernatant was centrifuged at 100,OOO g (30 min) forming a pellet (M-2) consisting of membranes and synaptic vesicles and soluble portion (M-3). About 60% of the protein kinase activity is found in the M-1 fraction and 30% is in the soluble M-3 fraction (Table 3). Nearly all of the inhibitor, however, is found in the soluble portion and little is present in the membrane fractions M-1 or M-2. These results were obtained in four different experiments. The synaptosome enriched fraction prepared by the discontinuous Ficoll gradient method (McGovERN et a/., 1973) was osmotically lysed and used to prepare similar M-1, M-2, and M-3 fractions. Again, most of the protein kinase activity (60%) is membrane associated and nearly all the heat stable, trypsin-labile inhibitor is in the soluble fraction (not shown). To exclude the possibility that the 32P incorporation in the protein kinase assay might represent the formation of the acyl-phosphate intermediate of the Na+/K+ ATPase the assays were repeated by quenching the reaction in 25mm potassium phosphate (pH 7.5). The acyl-phosphate bond of Na+/KC KINASE TABLE 3. PROTEIN
Fraction
tissue)
Inactivation of the protein kinase inhibitor by trypsin and chymotrypsin
To substantiate the supposition that the observed inhibition is related to a heat stable protein and not to other factors, the boiled fractions were incubated with several hydrolytic enzymes. Trypsin and chymotrypsin, but not DNase, RNase, or neuraminidase, completely abolished the inhibitory activity (Table 4). In similar experiments, the inhibitory activity of each of the subfractions in Tables 2 and 3 is similarly inactivated by treatment with the proteolytic enzymes (not shown). Characteristics of inhibition of membrane-associated protein kinase by the heat stable protein
The protein kinase activity associated with M-1 fraction (l0,OOO g pellet from the osmotically lysed synaptosome-enriched fraction) catalyses the phosphorylation of endogenous protein and exogenous histone (Table 5). In contrast, the enzyme derived from the cytoplasm is more dependent upon exogenous substrate. The protein inhibitor from rat brain inhibits histone phosphorylation catalyzed by the membrane-associated or soluble protein kinase. In contrast, it fails to inhibit the phosphorylation of endogenous substrate by the membrane-associated enzyme. These studies with inhibitor were carried out
INHIBITOR DISTRIBUTION IN SYNAPTOSOME SUBFRACTIONS PREPARED AFTER OSMOTIC LYSIS*
Protein (mg/g
ATPase which is unstable under these conditions (HOKINet al., 1965) would be cleaved. The results obtained by this procedure agreed within 10%so that 32Pincorporation related to formation of an acylphosphate linkage is thereby excluded.
Inhibitor (units/g)
Protein kinase activity +CAMP -CAMP (units/gtissue)
Lactate dehydroFumarase genase Inhibitor
Acetyl-
cholinesterase
M-1 Nerve endings, 14.6 myelin,
90
13.1
6.0
2.1
0.0
0.0
1.2
mitochondria M-2 Membrane synaptic vesicles
0.9
40
1.o
0.6
0
0.3
0.5
0.4
4.4
1500 112
6.0 93
2.4
0
3.3
4.2
0.1
M-3 Soluble fraction Recovery (%)
99
88
* From a crude mitochondrial fraction, the M-1, M-2, and M-3 subfractions were prepared as documented in the Materials and Methods section. Activities are expressed as described in Table 1.
R. ROSKOSKI,JR. and C. E. Far OERICK
546
TABLE 4. TRYPSIN AND CHYMOTRYPSIN
INACTIVATION OF HEAT STABLE PROTEIN KINASE INHIBITOR FROM THE PRIMARY SUBCELLULAR BRAIN FRACTIONS*
~~
Inhibitor units/mg protein Homogenate Nuclei Mitochondria Microsomes Cytosol
Treatment No enzyme Trypsin Chymotrypsin DNAse RNAse Neuraminidase
76 3 0 74 71 75
47 0 1 42 47 43
63 0 1 60 66 60
56 1 0 58 59 57
115 3 0 119 121 I12
* The fractions were prepared as described in the Materials and Methods section and aliquots were placed in a boiling water bath 10 min. After cooling, enzyme (100 pg) was added to 90 p1 samples of protein kinase inhibitor containing 50 mM-morpholinopropane sulfonic acid (pH 7.0) and incubated 30 min at 30°C. After placing the treated fractions in a boiling water bath 10min to inactivate the enzymes, 20pI samples were assayed for inhibitor activity as described in the Materials and Methods section. A control was treated identically except that no enzyme was added during the incubation. with a 10s incubation since UEDA and coworkers (1973) report that membrane phosphorylation occurred rapidly. Under these conditions, where enzyme activity is considerably greater than 2.59 pmol min-' 32Pincorporation, more inhibitor is required to decrease phosphorylation since inhibitory activity depends on the ratio of inhibitor and protein kinase activity as well as the substrate (ASHBY& WALSH,1972). DISCUSSION Adenylate cyclase is chiefly located in membranes with highest specific activity in the microsomal fraction (DEROBERTISet al., 1969). This fraction is heterogeneous consisting of fragments of plasma membrane, endoplasmic reticulum as well as small synaptosomes. Adenylate cyclase is also associated with the synaptic membrane fractions. Our studies on the subcellular localization of protein kinase activity confirm those TABLE5. CHARACTERISTICS OF THE INHIBITION OF THE MEMBRANE-BOUND AND SOLUBLE CAMP-DEPENDENT PROTEIN KINASE BY THE HEAT STABLE PROTEIN*
Addition
Protein kinase activity (pmol "P incorporated/min) Membrane-bound Cytoplasmic +CAMP -CAMP +CAMP -CAMP ( +histone)
None Inhibitor
76.9 25.1
31.2 17.3
None Inhibitor
23.1 23.2
10.3 11.0
38.1 12.7
8.5 9.1
(- histone)
6.2 7.4
3.7 3.9
* The membrane-bound and cytoplasmic protein kinase were obtained from the M-1 and cytoplasmic fractions as described in the Materials and Methods section. Samples (10 pl) containing 45 p g and 12 pg protein were assayed for protein kinase activity for 10s at 25°C (fhistone f CAMP)as previously described (WITT& ROSKOSKI, 1975~). The inhibitor was obtained by boiling the M-3 fraction 10 min. It contained 460 protein kinase inhibitor units and 1.24 mg protein per ml. Portions of 20 pl were used where specified in the 50 pl assay medium.
of MAENOand co-workers (1971). These workers measured activity in the supernatant and precipitates Triton X-100. We measured activity in the whole fraction in the presence of Triton X-100 and compared our activity with the total activity reported by these investigators. We found somewhat more of the total protein kinase activity in the cytoplasm and crude mitochondrial fraction and less in the microsomal fraction. Our present studies show that the protein kinase inhibitor, on the other hand, is chiefly a soluble component and is readily released from particulate fractions by osmotic lysis. The activity associated with particulate fractions parallels that of lactate dehydrogenase, a soluble enzyme marker, and is consistent with the hypothesis that inhibitor activity associated with the sedimentable fractions represents cytoplasmic contamination. The amount of measured protein kinase inhibitor varies among the various experiments by factors of two-to-four. Whether this represents differences among animals or variations in methodology is presently unknown. The proportion of inhibitor in the various fractions in four different experiments varied less than 8%. Osmotic lysis of the crude mitochondrial fraction dissociates inhibitor and protein kinase activity. About 90% of the inhibitor and 40% of the protein kinase activity is present in the supernatant fraction. In agreement with MAENOet a!. (1971) salt or p H changes fail to desorb the kinase activity. In agreement with UNO et al. (1976) Triton X-100 liberates protein kinase activity from membrane fractions derived from bovine brain, but we find that it fails to desorb the activity from membranes derived from rat brain synaptosome fractions. Dissociation of the membrane-associated protein kinase from the inhibitor provides evidence that the latter is not derived from the CAMP-dependent protein kinase by the vigorous denaturing conditions : the inhibitor is resolved from the enzyme by centrifugation at 4°C and neutral pH prior to heating. To test the effect
of the various subfractions
Protein kinase inhibitor
547
DONNELLY T. E., Kuo J. F., MIYAMOTO E. & GREENGARD P. (1973a) J . biol. Chem. 248, 199-203. DONNELLY T. E., Kuo J. F., REYESP. L., LIU U.-P. & GREENGARD P. (19736) J. bid. Chem. 248, 19G-198. GILLG. N. & GARRENL. D. (1970) Biochem. Biophys. Res. Commun. 39, 335-343. HOKINL. E., SASTRYP. S., GALSWORTHY P. R. & YODA A. (1965) Proc. natn. Acad. Sci., U.S.A. 54, 177-184. HSIEHW. T. & VESTLINGC. S. (1966) in Biochemical PrepA. C., ed.) pp. 69-75. John Wiley, New arations (MAEKLY York. Y. (1970) Biochem. KUMON A,, YAMAMURA H. & NISHIZUKA Biophys. Res. Commun. 41, 129G1297. Kuo J. F. (1975) Metabolism 24, 321-329. Kuo J. F. & GREENGARD P. (1969) Proc. natn. Acad. Sci., U.S.A. 64, 1349-1355. LOWRY0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) J. bid. Chem. 193, 265-275. MAENOH., JOHNSON E. M. & GREENGARD P. (1971) J. bid. Chem. 246, 134-142. MCGOVERN S., MAGUIRE M. E., GURDR. S., MAHLERH. R. & MOOREW. J. (1973) FEBS Lett. 31, 193-198. REIMANN E. M., BROSTROM C. O., CORBINJ. D., KINGC. A. & KREBSE. G. (1971) Biochem. Biophys. Res. Commun. 42, 187-194. ROSKOSKIR., JR. (1973) Biochemistry, Easton 12, 3709-3713. ROSKOSKIR., JR. (1974) Biochemistry, Easton 13, 5 141-5144. ROSKOSKI R., JR., FREDERICK C. E., GREIFB. J. & GARVEY M. L. (1976) Fedn Proc. Fedn Am. SOCSexp. Biol. 35, 1384. SKALAJ. P., DRUMMOND G. I. & HAHNP. A. (1974) Biochem. J. 130, 195-199. SUTHERLAND E. W., RALLR. W. & MENONT. (1962) J . bid. Chem. 237, 122G1227. TAO M., SALASM. L. & LIPMANNF. (1970) Proc. natn. REFERENCES Acad. Sci., U.S.A. 67, 408414. APPLEMANM. M., BIRNBAUMER L. & TORRESH. N. (1966) UEDAT., MAENOH. & GREENGARD P. (1973) J. b i d . Chem. Archs Biochem. Biophys. 116, 39-43. 248, 8295-8305. ASHBY C. D. & WALSHD. A. (1972) J. bid. Chem. 247, UNO I., UEDAT. & GREENGARD P. (1976) J. bid. Chem. 66374642. 251, 2192-2195. ASHBY C. D. & WALSHD. A. (1973) J. b i d . Chem. 248, WHITTAKER V. P. (1969) in Handbook of Neurochemistry 1255-1 261. (LAJTHAA., ed.) Vol. 2, pp. 327-366, Plenum Press, New DEROBERTIS E., PELLEGRINO DE IRALDIA,, RODRIGUEZ DE York. LORESARNAIZG. & SALGANICOFF L. (1962) J . Neuro- WHITTAKER V. P. & BARKERL. A. (1972) in Methods of chem. 9, 23-35. Neurochemistry (FRIED R., ed.) Vol. 2, pp. 1-52. Dekker, DEROBERTIS E., ALBERICIM., RODRIGUEZ DE LORES ARNAIZ New York. G. & AZAIRRAJ. M. (1966) Lije Sci. 5, 577-582. R., JR. (1975a) Analyt. Biochem. WITT J. J. & ROSKOSKI DEROBERTIS E. & RODRIGUEZ DE LORESARNAIZG. (1969) 66,253-259. in Handbook of Neurochemistry (LAJTHAA., ed.) Vol. 2, WITT J. J. & ROSKOSKI R., JR. (1975b) Biochemistry 14, pp. 365-392. Plenum Press, New York. 45034507.
of the source of the CAMP-dependent protein kinase on the assay of inhibitor activity, the experiments in Table 1 and 2 were repeated with rat brain and bovine heart protein kinase purified through the DEAE-cellulose chromatography step, and the results were the same. In addition to heat stability and inactivation by trypsin and chymotrypsin, inhibitor isolated from the rat brain is non-dialyzable, and trichloracetic acid and (NH4)2S04 precipitable. The factor also binds to DEAE-cellulose from which it is eluted by salt. These experiments are consistent with the alleged protein nature of the heat stable factor. UEDAet al. (1973) proposed that CAMP-dependent protein kinase catalyzed phosphorylation of membrane-associated substrates is important in neurotransmission. Although the inhibitor may play a role in the regulation of the phosphorylation of soluble substrates, in agreement with UEDAet al. (1973), our studies suggest that the heat stable, trypsin labile factor fails to alter the phosphorylation of membrane-associated components. Identification of biochemically and physiologically important substrates, however, is required to test this proposal. Two other aspects of this work merit comment. Although we have no evidence to the contrary, the inhibitory activity measured in the various fractions may not represent the same chemical species. Furthermore, in the preparation of inhibitor for biochemical studies, it is convenient to resolve the soluble fraction from membrane fractions, especially in brain, prior to heat treatment. The yield is identical and the fractions are easier to manipulate.
SUBCELLULAR DISTRIBUTION OF A HEAT-STABLE PROTEIN INHIBITOR OF CYCLIC AMP-DEPENDENT PROTEIN KINASE IN RAT BRAIN R. ROSKOSKI, JR. and C. E. FREDERICK The Department of Biochemistry, The University of Iowa, Iowa City, IA 52242, U.S.A. (Received 23 July 1976. Revised 4 October 1976. Accepted 5 October 1976)
Abstract-Cyclic AMP (CAMP)-dependent protein kinase catalyzes the phosphorylation of polypeptidic serine and threonine residues according to the following chemical equation: ATP + protein -+ phosphoprotein + ADP. A heat stable, trypsin labile factor present in brain, skeletal muscle and other tissues inhibits enzymatic phosphorylation of some proteins and enhances that of others. Since brain is one of the richest sources of adenylate cyclase, CAMP,CAMP-dependent protein kinase and the heat stable protein kinase inhibitor and because they may play a role in neurotransmission, an investigation of the subcellular distribution of the heat stable factor in rat brain was undertaken. Although present in the nuclear, mitochondrial and microsomal fractions, the highest activity of protein kinase inhibitor is in the soluble fraction: its activity parallels that of the cytoplasmic enzyme marker, lactate dehydrogenase. The inhibitory activity is also found in the synaptosome or pinched-off nerve ending fraction. Following osmotic lysis of this fraction, about 90% of the factor occurs in the soluble fraction. On the other hand, only 40% of the CAMP-dependent protein kinase is solubilized and 60% remains membrane-bound. Using this membrane-bound protein kinase, phosphorylation of endogenous substrate is unaltered by inhibitor, but phosphorylation of added histone substrate is decreased.
A MAJOR class of protein kinase requires adenosine 3',5'-cyclic monophosphate (CAMP) for activity. The mechanism of activation is expressed by the equation RC cAMP$C + R.cAMP in which RC represents the holoenzyme and R and C are the regulatory and catalytic subunits, respectively (GILL & GARREN,1970; TAOet al., 1970; KUMONet al., 1970; REIMANN et al., 1971). cAMP promotes the dissociation of the RC complex producing a free catalytic subunit and a CAMP-regulatory subunit complex. The freed catalytic subunit is active and cAMP independent. A heat stable protein which inhibits CAMPdependent protein kinase is present in many tissues (APPLEMAN et al., 1966; ASHBY & WALSH, 1972). ASHBY& WALSH(1972) reported that skeletal muscle and brain contain the highest inhibitory activity. They also reported (ASHBY& WALSH,1973) that the inhibitor complexes with the free catalytic subunit (C), but not holoenzyme (RC). The inhibitor-catalytic subunit complex, which is catalytically inactive, is incapable et of binding to the regulatory subunit. DONNELLY al. (1973~1,b) reported that a protein factor, purified to homogeneity from lobster muscle, inhibits histone phosphorylation but stimulates protamine phosphorylation and suggest that the factor functions as a modulator. The modulator, furthermore, alters the substrate specificity of CAMP- and cGMP-dependent
+
protein kinases prepared from various invertebrate et al., 1973b). and vertebrate tissues (DONNELLY Changes in modulator activity under physiologic and pathologic conditions suggest a regulatory role. For example, modulator activity in rat brown adipose tissue varies during development (SKALAet at., 1974) and Kuo (1975) reports that it is decreased in epididyma1 fat pads in alloxan-diabetic rats. As a first step in establishing and correlating possible physiologic roles for the inhibitor, its subcellular distribution was determined in rat brain cerebral cortex, a rich source of adenylate cyclase (SUTHERLAND et aL, 1962) CAMP-dependent protein kinase (Kuo & GREENGARD, 1969) and the protein kinase inhibitor (ASHBY& WALSH,1972). We find that inhibitory activity parallels that of lactate dehydrogenase, a cytoplasmic enzyme marker. Although 30% of the inhibitory activity is found with the crude mitochondria1 fraction containing synaptosomes (pinched off nerve endings), ,osmotic lysis solubilizes about 90% of this activity, but only 40% of the protein kinase activity. Using membrane-bound protein kinase, the inhibitor fails to alter phosphorylation of endogenous substrate, but it inhibits phosphorylation of exogenous histone. A preliminary account of this work has et al., 1976). appeared (ROSKOSKI
MATERIALS AND METHODS This investigation was Supported by Grant NS-11310 of the US. Public Health Service. Subcellular fractionation. Using male Sprague-Dawley Abbreviation used: CAMP, cyclic AMP. rats (200-250 g), primary subcellular fractions (nuclei, 543
R. ROSKOSKI,JR. and C. E. FREDERICK
544
crude mitochondria, microsomes, and cytosol) from cere- or usually a suitable dilution to give 14% inhibition was bral cortex were prepared by the methodology of MAENO assayed for inhibitory activity. The reaction was initiated and co-workers (1971). Using discontinuous Ficoll gra- by adding 5p1 of bovine brain protein kinase with the dients, synaptosome-enriched fractions were prepared from standard activity (2.59 pmol 32Pincorporation per min at the crude mitochondrial suspension by the procedure of 30°C) to 45 pl of solution containing inhibitor, buffer, ATP, et al. (1973) and hypotonic lysis of the crude histone and CAMP (lo-' M) as previously described (WITT MCGOVERN (1975~)).Incubations were for 30 min at 30°C mitochondrial fraction or the synaptosome-enriched frac- & ROSKOSKI, & during which time incorporation of 32Pinto histone was tion was performed using the method of DEROBERTIS co-workers (1962, 1966). To release possible sequestered linear. The labeled phosphoprotein was resolved from enzyme activity after subcellular fractionation, one-ninth [y-32P]ATP and metabolites by the phosphocellulose volume of 10% Triton X-100 (v/v) was added to each frac- paper procedure as previously described (WITT & ROStion at least 5min prior to each of the enzyme assays. KOSKI,1975a,b). Protein kinase activity was measured in 20p1 aliquots Lactate dehydrogenase, fumarase, acetylcholinesterase, and choline acetyltransferase (enzyme markers for the soluble of the designated fractions for 2min at 30°C by the pro1975~). cytoplasm, mitochondria, external synaptosomal mem- cedure previously documented (WITT& ROSKOSKI, brane and occluded synaptosomal cytoplasm (WHITTAKER Materials. Sources of other materials are previously 1975a, b). Bovine brain & BARKER, 1972) were measured by the methodology de- documented (WITT & ROSKOSKI, scribed by HSIEH & VESTLING (1966), WHITTAKER& protein kinase was prepared as previously described BARKER (1972), ROSKOSKI(1973) and ROSKOSKI(1974), re- through the Sephadex G-200 step (WITT & ROSKOSKI, spectively. The first three spectrophotometric assays were 1975a). The enzyme fraction contained insignificant inhibicarried out at ambient temperature in a final vol of 300 pl tory activity. Other enzymes were obtained from Worthusing 5 or lop1 of the designated fraction. Protein was ington Biochemical Corp. measured by the method of LOWRYet al. (1951). Measurement of protein kinase inhibitor activity. As RESULTS ASHBY & WALSHreported (1972), the degree of protein kinase inhibition is dependent upon the respective concen- Subcellular distribution of' protein kinase inhibitory trations of inhibitor, protein kinase and the protein sub- activity strate. We adopted their definition of 1 unit of protein The distribution of protein kinase inhibitor in rat kinase inhibitor as that amount which decreases phosphorbrain cerebral cortex parallels that of lactate dehydroylation of substrate from 7 pmol min-' to 6 pmol min-' genase, a cytoplasmic enzyme marker (Table 1). The in 0.13 ml or, by proportion, 2.59 pmol min-' to nuclear, mitochondrial, microsomal and cytoplasmic 2.31 pmol min-' in 0.05 ml. The units of activity differ, fractions contain 10, 34, 18 and 38"/,, respectively, of however, since we used bovine brain protein kinase as the enzyme source and histone as substrate (contrast with rab- the total activity. This distribution also parallels that bit skeletal muscle and casein). In this study, only inhibi- of protein kinase. The crude mitochondrial fraction tion of phosphorylation (not modulation) was measured. contains the synaptosome or pinched-off nerve ending In early studies we found that when 30 times the kinase fraction (DEROBERTIS & RODRIGUEZ DE LORRES activity was used in the assay, modulator activity was un- ARNAIS,1969). About 75% of the activity of choline detectable in bovine heart extracts but detectable in bovine acetyltransferase, a marker for the synaptosomal cytoand rat brain. After decreasing enzyme activity to plasm (WHITTAKER8z BARKER,1972), is present in 2.59 pmol min-', modulator activity in heart was detect. the mitochondrial fraction. able, indicating the increased sensitivity with lower enzyme The crude mitochondrial fraction was subfracactivity. To measure the activity of the inhibitor, portions (lo0 pl) of the specified fractions were placed in a boiling tionated into myelin, synaptosomes, and mitochonwater bath for 10 min. After cooling, one-ninth volume of dria by discontinuous Ficoll gradient centrifugation. 10% Triton X-100 (v/v) was added to release possibly Again, the activity of the protein kinase inhibitor parsequestered inhibitory activity. Then 20 pl of the fraction alleled that of lactate dehydrogenase, protein kinase TABLE1.
Protein (mg/g tissue)
Fraction ~
~
Inhibitor (unitsig)
Protein kinase activity +CAMP -CAMP (units/g)
BRAIN*
Lactate dehydroFumarase genase Inhibitor
Choline acetyltransferase
~
Nuclei Crude mitochondrial fraction Microsomes Cytosol Homogenate Recovery (%) ~
* The
PROTEIN KINASE INHIBITOR DISTRIBUTION IN SUBCELLULAR FRACTIONS OF RAT
~~
15.6
780
42.2
2690
27.8
25.1 23.9 120 89
1400 2990 9140 86
16.8 19 77.4 93
~
8.41
2.33
0.5
0.7
0.7
0.2
16.9
1.8
0.8
0.9
1.8
2.9 2.2 27.1 88
0.5 0.0
0.4 1.8
0.7 1.7
0.3 0.2
~~~~
fractions were prepared by differential centrifugation; enzymes, protein kinase inhibitor and protein were measured by methods documented in the Materials and Methods section. The results are expressed per g of rat cerebral cortex. One unit of protein kinase is defined as one nmol of 3zP incorporation per min at 30°C under standard conditions. The activity of fumarase, lactate dehydrogenase, protein kinase inhibitor, and choline acetyltransferase is expressed as the ratio of the percentage recovered activity to the per cent recovered protein (relative specific activity).
Protein kinase inhibitor TABLE 2. PROTEIN KINASE INHIBITOR
DISTRIBUTTON IN SUBMITOCHONDRIAL FRACTIONS RESOLVED BY DISCONTINUOUS GRADIENT CENTRIFUGATION*
Protein Fraction
tissue)
Inhibitor (units/g)
Myelin Synaptosomes Mitochondria Recovery (%)
11.7 20.1 6.3
1570 330
(mg/g
90
545
632 94
Protein kinase activity +CAMP -CAMP (units/g) 6.9 21.6 2.3 91
FICOLL
Lactate Choline dehydroacetylFumarase genase Inhibitor transferase
3.1 9.4
0.0
1.2 87
1.1
0.4
0.6 1.4 0.6
0.8
1.2 0.8
0.4 1.5 0.7
* A crude mitochondrial fraction, prepared as that in Table 1, was resolved into the three components by the methodology of MCGOVERN & co-workers (1973). Activities are expressed as described in Table 1. and choline acetyltransferase (Table 2). Fumarase, a mitochondrial marker, is found in the mitochondrial and synaptosomal subfractions. The latter activity is presumably associated with intrasynaptosomal mitochondria as well as inevitable cross-contamination from the mitochondrial fraction. To lyse osmotically sensitive components, 9 volumes of distilled water were added to a preparation of the crude mitochondrial fraction. The solution was centrifuged at l0,OOOg for 20min to pellet the M-1 fraction (myelin, nerve endings, free mitochondria). The resulting supernatant was centrifuged at 100,OOO g (30 min) forming a pellet (M-2) consisting of membranes and synaptic vesicles and soluble portion (M-3). About 60% of the protein kinase activity is found in the M-1 fraction and 30% is in the soluble M-3 fraction (Table 3). Nearly all of the inhibitor, however, is found in the soluble portion and little is present in the membrane fractions M-1 or M-2. These results were obtained in four different experiments. The synaptosome enriched fraction prepared by the discontinuous Ficoll gradient method (McGovERN et a/., 1973) was osmotically lysed and used to prepare similar M-1, M-2, and M-3 fractions. Again, most of the protein kinase activity (60%) is membrane associated and nearly all the heat stable, trypsin-labile inhibitor is in the soluble fraction (not shown). To exclude the possibility that the 32P incorporation in the protein kinase assay might represent the formation of the acyl-phosphate intermediate of the Na+/K+ ATPase the assays were repeated by quenching the reaction in 25mm potassium phosphate (pH 7.5). The acyl-phosphate bond of Na+/KC KINASE TABLE 3. PROTEIN
Fraction
tissue)
Inactivation of the protein kinase inhibitor by trypsin and chymotrypsin
To substantiate the supposition that the observed inhibition is related to a heat stable protein and not to other factors, the boiled fractions were incubated with several hydrolytic enzymes. Trypsin and chymotrypsin, but not DNase, RNase, or neuraminidase, completely abolished the inhibitory activity (Table 4). In similar experiments, the inhibitory activity of each of the subfractions in Tables 2 and 3 is similarly inactivated by treatment with the proteolytic enzymes (not shown). Characteristics of inhibition of membrane-associated protein kinase by the heat stable protein
The protein kinase activity associated with M-1 fraction (l0,OOO g pellet from the osmotically lysed synaptosome-enriched fraction) catalyses the phosphorylation of endogenous protein and exogenous histone (Table 5). In contrast, the enzyme derived from the cytoplasm is more dependent upon exogenous substrate. The protein inhibitor from rat brain inhibits histone phosphorylation catalyzed by the membrane-associated or soluble protein kinase. In contrast, it fails to inhibit the phosphorylation of endogenous substrate by the membrane-associated enzyme. These studies with inhibitor were carried out
INHIBITOR DISTRIBUTION IN SYNAPTOSOME SUBFRACTIONS PREPARED AFTER OSMOTIC LYSIS*
Protein (mg/g
ATPase which is unstable under these conditions (HOKINet al., 1965) would be cleaved. The results obtained by this procedure agreed within 10%so that 32Pincorporation related to formation of an acylphosphate linkage is thereby excluded.
Inhibitor (units/g)
Protein kinase activity +CAMP -CAMP (units/gtissue)
Lactate dehydroFumarase genase Inhibitor
Acetyl-
cholinesterase
M-1 Nerve endings, 14.6 myelin,
90
13.1
6.0
2.1
0.0
0.0
1.2
mitochondria M-2 Membrane synaptic vesicles
0.9
40
1.o
0.6
0
0.3
0.5
0.4
4.4
1500 112
6.0 93
2.4
0
3.3
4.2
0.1
M-3 Soluble fraction Recovery (%)
99
88
* From a crude mitochondrial fraction, the M-1, M-2, and M-3 subfractions were prepared as documented in the Materials and Methods section. Activities are expressed as described in Table 1.
R. ROSKOSKI,JR. and C. E. Far OERICK
546
TABLE 4. TRYPSIN AND CHYMOTRYPSIN
INACTIVATION OF HEAT STABLE PROTEIN KINASE INHIBITOR FROM THE PRIMARY SUBCELLULAR BRAIN FRACTIONS*
~~
Inhibitor units/mg protein Homogenate Nuclei Mitochondria Microsomes Cytosol
Treatment No enzyme Trypsin Chymotrypsin DNAse RNAse Neuraminidase
76 3 0 74 71 75
47 0 1 42 47 43
63 0 1 60 66 60
56 1 0 58 59 57
115 3 0 119 121 I12
* The fractions were prepared as described in the Materials and Methods section and aliquots were placed in a boiling water bath 10 min. After cooling, enzyme (100 pg) was added to 90 p1 samples of protein kinase inhibitor containing 50 mM-morpholinopropane sulfonic acid (pH 7.0) and incubated 30 min at 30°C. After placing the treated fractions in a boiling water bath 10min to inactivate the enzymes, 20pI samples were assayed for inhibitor activity as described in the Materials and Methods section. A control was treated identically except that no enzyme was added during the incubation. with a 10s incubation since UEDA and coworkers (1973) report that membrane phosphorylation occurred rapidly. Under these conditions, where enzyme activity is considerably greater than 2.59 pmol min-' 32Pincorporation, more inhibitor is required to decrease phosphorylation since inhibitory activity depends on the ratio of inhibitor and protein kinase activity as well as the substrate (ASHBY& WALSH,1972). DISCUSSION Adenylate cyclase is chiefly located in membranes with highest specific activity in the microsomal fraction (DEROBERTISet al., 1969). This fraction is heterogeneous consisting of fragments of plasma membrane, endoplasmic reticulum as well as small synaptosomes. Adenylate cyclase is also associated with the synaptic membrane fractions. Our studies on the subcellular localization of protein kinase activity confirm those TABLE5. CHARACTERISTICS OF THE INHIBITION OF THE MEMBRANE-BOUND AND SOLUBLE CAMP-DEPENDENT PROTEIN KINASE BY THE HEAT STABLE PROTEIN*
Addition
Protein kinase activity (pmol "P incorporated/min) Membrane-bound Cytoplasmic +CAMP -CAMP +CAMP -CAMP ( +histone)
None Inhibitor
76.9 25.1
31.2 17.3
None Inhibitor
23.1 23.2
10.3 11.0
38.1 12.7
8.5 9.1
(- histone)
6.2 7.4
3.7 3.9
* The membrane-bound and cytoplasmic protein kinase were obtained from the M-1 and cytoplasmic fractions as described in the Materials and Methods section. Samples (10 pl) containing 45 p g and 12 pg protein were assayed for protein kinase activity for 10s at 25°C (fhistone f CAMP)as previously described (WITT& ROSKOSKI, 1975~). The inhibitor was obtained by boiling the M-3 fraction 10 min. It contained 460 protein kinase inhibitor units and 1.24 mg protein per ml. Portions of 20 pl were used where specified in the 50 pl assay medium.
of MAENOand co-workers (1971). These workers measured activity in the supernatant and precipitates Triton X-100. We measured activity in the whole fraction in the presence of Triton X-100 and compared our activity with the total activity reported by these investigators. We found somewhat more of the total protein kinase activity in the cytoplasm and crude mitochondrial fraction and less in the microsomal fraction. Our present studies show that the protein kinase inhibitor, on the other hand, is chiefly a soluble component and is readily released from particulate fractions by osmotic lysis. The activity associated with particulate fractions parallels that of lactate dehydrogenase, a soluble enzyme marker, and is consistent with the hypothesis that inhibitor activity associated with the sedimentable fractions represents cytoplasmic contamination. The amount of measured protein kinase inhibitor varies among the various experiments by factors of two-to-four. Whether this represents differences among animals or variations in methodology is presently unknown. The proportion of inhibitor in the various fractions in four different experiments varied less than 8%. Osmotic lysis of the crude mitochondrial fraction dissociates inhibitor and protein kinase activity. About 90% of the inhibitor and 40% of the protein kinase activity is present in the supernatant fraction. In agreement with MAENOet a!. (1971) salt or p H changes fail to desorb the kinase activity. In agreement with UNO et al. (1976) Triton X-100 liberates protein kinase activity from membrane fractions derived from bovine brain, but we find that it fails to desorb the activity from membranes derived from rat brain synaptosome fractions. Dissociation of the membrane-associated protein kinase from the inhibitor provides evidence that the latter is not derived from the CAMP-dependent protein kinase by the vigorous denaturing conditions : the inhibitor is resolved from the enzyme by centrifugation at 4°C and neutral pH prior to heating. To test the effect
of the various subfractions
Protein kinase inhibitor
547
DONNELLY T. E., Kuo J. F., MIYAMOTO E. & GREENGARD P. (1973a) J . biol. Chem. 248, 199-203. DONNELLY T. E., Kuo J. F., REYESP. L., LIU U.-P. & GREENGARD P. (19736) J. bid. Chem. 248, 19G-198. GILLG. N. & GARRENL. D. (1970) Biochem. Biophys. Res. Commun. 39, 335-343. HOKINL. E., SASTRYP. S., GALSWORTHY P. R. & YODA A. (1965) Proc. natn. Acad. Sci., U.S.A. 54, 177-184. HSIEHW. T. & VESTLINGC. S. (1966) in Biochemical PrepA. C., ed.) pp. 69-75. John Wiley, New arations (MAEKLY York. Y. (1970) Biochem. KUMON A,, YAMAMURA H. & NISHIZUKA Biophys. Res. Commun. 41, 129G1297. Kuo J. F. (1975) Metabolism 24, 321-329. Kuo J. F. & GREENGARD P. (1969) Proc. natn. Acad. Sci., U.S.A. 64, 1349-1355. LOWRY0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) J. bid. Chem. 193, 265-275. MAENOH., JOHNSON E. M. & GREENGARD P. (1971) J. bid. Chem. 246, 134-142. MCGOVERN S., MAGUIRE M. E., GURDR. S., MAHLERH. R. & MOOREW. J. (1973) FEBS Lett. 31, 193-198. REIMANN E. M., BROSTROM C. O., CORBINJ. D., KINGC. A. & KREBSE. G. (1971) Biochem. Biophys. Res. Commun. 42, 187-194. ROSKOSKIR., JR. (1973) Biochemistry, Easton 12, 3709-3713. ROSKOSKIR., JR. (1974) Biochemistry, Easton 13, 5 141-5144. ROSKOSKI R., JR., FREDERICK C. E., GREIFB. J. & GARVEY M. L. (1976) Fedn Proc. Fedn Am. SOCSexp. Biol. 35, 1384. SKALAJ. P., DRUMMOND G. I. & HAHNP. A. (1974) Biochem. J. 130, 195-199. SUTHERLAND E. W., RALLR. W. & MENONT. (1962) J . bid. Chem. 237, 122G1227. TAO M., SALASM. L. & LIPMANNF. (1970) Proc. natn. REFERENCES Acad. Sci., U.S.A. 67, 408414. APPLEMANM. M., BIRNBAUMER L. & TORRESH. N. (1966) UEDAT., MAENOH. & GREENGARD P. (1973) J. b i d . Chem. Archs Biochem. Biophys. 116, 39-43. 248, 8295-8305. ASHBY C. D. & WALSHD. A. (1972) J. bid. Chem. 247, UNO I., UEDAT. & GREENGARD P. (1976) J. bid. Chem. 66374642. 251, 2192-2195. ASHBY C. D. & WALSHD. A. (1973) J. b i d . Chem. 248, WHITTAKER V. P. (1969) in Handbook of Neurochemistry 1255-1 261. (LAJTHAA., ed.) Vol. 2, pp. 327-366, Plenum Press, New DEROBERTIS E., PELLEGRINO DE IRALDIA,, RODRIGUEZ DE York. LORESARNAIZG. & SALGANICOFF L. (1962) J . Neuro- WHITTAKER V. P. & BARKERL. A. (1972) in Methods of chem. 9, 23-35. Neurochemistry (FRIED R., ed.) Vol. 2, pp. 1-52. Dekker, DEROBERTIS E., ALBERICIM., RODRIGUEZ DE LORES ARNAIZ New York. G. & AZAIRRAJ. M. (1966) Lije Sci. 5, 577-582. R., JR. (1975a) Analyt. Biochem. WITT J. J. & ROSKOSKI DEROBERTIS E. & RODRIGUEZ DE LORESARNAIZG. (1969) 66,253-259. in Handbook of Neurochemistry (LAJTHAA., ed.) Vol. 2, WITT J. J. & ROSKOSKI R., JR. (1975b) Biochemistry 14, pp. 365-392. Plenum Press, New York. 45034507.
of the source of the CAMP-dependent protein kinase on the assay of inhibitor activity, the experiments in Table 1 and 2 were repeated with rat brain and bovine heart protein kinase purified through the DEAE-cellulose chromatography step, and the results were the same. In addition to heat stability and inactivation by trypsin and chymotrypsin, inhibitor isolated from the rat brain is non-dialyzable, and trichloracetic acid and (NH4)2S04 precipitable. The factor also binds to DEAE-cellulose from which it is eluted by salt. These experiments are consistent with the alleged protein nature of the heat stable factor. UEDAet al. (1973) proposed that CAMP-dependent protein kinase catalyzed phosphorylation of membrane-associated substrates is important in neurotransmission. Although the inhibitor may play a role in the regulation of the phosphorylation of soluble substrates, in agreement with UEDAet al. (1973), our studies suggest that the heat stable, trypsin labile factor fails to alter the phosphorylation of membrane-associated components. Identification of biochemically and physiologically important substrates, however, is required to test this proposal. Two other aspects of this work merit comment. Although we have no evidence to the contrary, the inhibitory activity measured in the various fractions may not represent the same chemical species. Furthermore, in the preparation of inhibitor for biochemical studies, it is convenient to resolve the soluble fraction from membrane fractions, especially in brain, prior to heat treatment. The yield is identical and the fractions are easier to manipulate.
