0022-3M2 78 07014269 Kl2.02 0
MULTIPLE FORMS OF PROTEIN KINASES IN MYELIN A N D MICROSOMAL FRACTIONS OF BOVINE BRAIN E. MIYAMOTO, K. MIYAZAKI and R. HIROSE Department of Biochemistry, Kumamoto University Medical School, Kumamoto, Japan and A. KASHIBA The Research Division and Clinical Laboratory, Nakamiya Mental Hospital, Hirakata, Osaka, Japan ( R c ~ e i t v d28 September 1977. Riwsed 30 Novemher 1977. Accepted 5 December 1977)
Abstract-The activity profiles of the solubilized protein kinases from the microsomal and myelin fractions of bovine brain were examined by column chromatography and sucrose density gradient centrifugation. The main peak of adenosine 3’5’-monophosphate (cyclic AMP)-dependent activity with histone as substrate for each membrane enzyme was eluted with about 0.2hl-NaCl on a DEAE-cellulose column. A peak of activity stimulated with cyclic AMP was also eluted with about 0.1 M-N~CIfor the microsomal enzyme. A peak with prolamine and casein as substrate for the microsomal or myelin enzyme, respectively. was larger than that with histone as substrate for each enzyme, The first peak with histone as substrate on a DEAE-cellulose column appeared as two peaks on the Sepharose 68 column. The second peak with histone as substrate on DEAE-cellulose cnlumn was shown to be a holoenzyme consisting of regulatory and catalytic subunits. The holoenzyme and whunits were eluted at similar positions to each other between both membrane enzymes on Sepharose 6 8 column. The holoenzyme sedimented as two peaks of activity on sucrose density gradient centrifugation, both of which were stimulated with cyclic AMP. The preincubation of the holoenzyme with cyclic AMP resulted in shifting to a position of a smaller molecular size. The results indicate the occurrence of multiple forms of protein kinases in membrane fractions of brain with respect to substrate specificity and physical property.
BRAINhas been reported to contain protein kinase 1975; MCNAMARA & ABPEL,1977). The endogenous activity predominantly in the particulate fractions activity of protein kinase in the microsomal fraction of (MAENOet a/., 1971). The subcellular distribution of brain was first demonstrated by RODNIGHT& LAWN the enzyme is specific for brain, suggesting that the (1966), and WELLER& RODNIGHT(1970). I t was also functional significance of protein kinases and of studied by MAENO et a / . (1971) and SCHMIDT & phosphorylation of substrate proteins in the particu- SOKOLOFF(l973)With and without exogenous substrate. late fractions may be fairly important in brain. UNO et a/. (1976) have reported that the cyclic The occurrence of multiple forms of adenosine AMP-dependent enzymes solubilized from membrane 3’,5’-monophosphate (cyclic AMP)-dependent protein fractions of several tissues can be separated by DEAEkinases (ATP: protein phosphotransferase EC 2.7.1.37) cellulose column chromatography. Differences were in the cytosol fraction from a variety of mammalian reported in properties ofthe cytosol and the membraneet a/., bound protein kinases soluble in Triton X-100 that tissues is well known (TAOet al., 1970; REIMANN 1971; GILL& GARREN,1971; CHEN& WALSH. 1971; phosphorylate exogenous (UNO er a/., 1976) and YAMAMURA et a/., 1971; CORBINet a/., 1972; MENON. endogenous substrates (UNOet al., 1977). CORBIN& et a/., 1973). 1973; MIYAMOTO KEELY(1977) and CORBINet al. (1977) have recently The myelin fraction of brain contains protein kinase shown that protein kinases in Triton X-1Wtreated activity which is mainly responsible for the phos- particulate fraction from the heart show a peak of phorylation of myelin basic protein (CARNEGIE et a/., cyclic AMP-dependent activity with histone as sub1973, 1974; MIYAMOTO et al., 1974; MIYAMOTO & strate when eluted with a high ionic strength buffer KAKIUCHI, 1974; STECK& APPEL, 1974; MIYAMOTO, from a DEAE-cellulose column. These results indicate 1976) and may involved in its microheterogeneity that the protein kinases in the membrane fractions can ( D E I B L Ea/., R ~1975; ~ CHOUet al., 1976). The solubilized be purified o n column chromatography. However, no enzyme activity from the myelin fraction is stimulated attempt has been made to investigate multiple forms with cyclic AMP and has relatively more affinity for of protein kinases in myelin and microsomal fractions protamine and myelin basic protein (MIYAMOTO, 1975). of brain. The present communication deals with The occurrence of phosphoprotein phosphatase multiplicity of protein kinases from the myelin and responsible for dephosphorylation of myelin basic microsomal fractions of bovine brain, and their physiprotein was also reported (MNAMOTO & KAKIUCHI, cal properties, using exogenous substrates. 269
270
E. MIYAMOTO. K. MIYAZAKI, R. HIROSEand A. KASHIBA
with electron microscopy. Most of particles observed were found t o be myelin and, therefore. the myelin fraction Materials. Fresh bovine brains were obtained from a prepared was considered to be fairly homogenous. local slaughterhouse and transported on ice to the The preparations of the microsomal and myelin fractions laboratory. Cyclic AMP was obtained from Kohjin co. were immediately used or rapidly frozen and stored at DEAE-cellulose (DE 52) was purchased from Whatman. -2O'C until use for the following experiments. Sepharose 6 8 was obtained from Pharmacia Fine Chemicals. Sohtbilizatio~~ and purification of protein kinuse Irom the Calf thymus histone and [8-'H]cyclic AMP (27 Ci/mmol) microsomal and myidin fracfions. The microsomal (about were purchased from Scharq'Mann. Ptotamine and phos- 5.5mg of protein per ml) or myelin fraction (about 5.4mg vitin were obtained from Sigma Chemical Co. Casein and of protein per ml) was suspended in 0.2",, Triton X-100 and Triton X-100 were purchased from Wako Pure Chemical stirred for 40 min in an ice bath. The suspension was Industries, Ltd. ["P]Orthophosphate (carrier-free) was pur- centrifugedat 105,000gfor60min at 2.C. On treatment with chased from Japan Radioisotope Association. TritonX-100 the total activity of protein kinase increased by Assay for protein kinase actirity. The standard assay 87 and 364: for the microsomal and myelin fractions system for protein kinase contained, in a final volume of respectively. About 52 and 53"/;ofthe cyclic AMP stimulated 0.2ml. IOpmol of sodium acetate buffer. pH 6.0. 100pg of activity (histone as substiate) of the microsomal and myelin substrate protein as indicated, 1 nmol of [y-3ZP]ATP fractions, respectively, of the total activity were recovered in containing 2-5 x lo5c.p.m., 2 pnol of magnesium acetate. the supernatant. The clear supernatant solution of each 0.06 pnol of ethylene glycol bis-(8-aminoethy1ether)-N."fraction (165 and 113.9 mg of protein from the microsomal tetraacetic acid and the indicated amount of protein kinase and myelin fractions, respectively) was applied to a 2.6 x 3 1.O in the presence of absence of 0.2nmol of cyclic AMP. cm column of DEAE-cellulose (DE 52) which had been Incubation was carried out at 30°C for 5 min in a shaking equilibrated with 10mM-Tris-C1 buffer, pH 7.5, and 0.1:" water bath. The reaction was terminated and the degree of Triton X-100. The column was washed with 140ml of the protein phosphorylation measured as described previously buffer. Then enzyme protein was eluted with a linear et a/., 1973). A unit of enzyme activity was gradient of NaCl(0-0.4~)in lOm~-Tris-CI buffer, pH 7.5, (MIYAMOTO defined as that amount of enzyme which transferred 1 pmol and 0.1% Triton X-100 in a total volume of 100Oml. The of 3zP from [y-"P]ATP to protein in 5 min at 30°C 10-ml fractions were collected. As shown in Figs. 1 and 2, in the standard assay system. The assays were performed the main peak of cyclic AMP-dependent activity was eluted in duplicate. Each value represents the mean. with about 0 . 2 ~ - N a C lfor the microsomal or myelin Assaysforcyclic AMP-binding.The binding of cyclic AMP fraction, when assayed with histone as substrate. The active (1970). fractions of this cyclic AMP-dependent enzyme for each to protein was determined by the method of GILMAN except that 50mM-phosphate buffer, pH 7.0, was substituted fraction werecollected and concentrated to lOml by Amicon for 50m~-sodiumacetate buffer. pH 4.0. The concentration ultrafiltration apparatus with PM-10 membrane. The conof [3H]cyclic AMP added to reaction mixture was 18.5 nM. centrate (15.1 and 11.2mg of protein for microsomal and Preparation of microsomalfraction from bovine brain. The myelin enzymes, respectively) was applied to a 2.6 x 92cm microsomal fraction was prepared from the grey matter of column of Sepharose 6 8 which had been equilibrated and bovine cerebrum according to the method of UESUCIet al. eluted with IOmM-Tris+CI buffer, pH 7.5,0.l% Triton X-100 (1969)with some modifications. The grey matter of cerebrum and 0.3 M-NaCI. Then 5-ml fractions were collected and the which was separated from the white matter with scissors void volume of the column, determined with blue dextran was homogenized at a half speed for 1.5 min in 10 vol of 2000, was found t o be about 150ml. A peak of cyclic AMPice-cold 0.32~-sucrosein a Waring blendor. The homodependent activity for the microsomal or myelin enzyme was genate was centrifuged at 11,500g for 20 min. The superobserved in the eluate of about 265ml o n the column. natant was decanted to a beaker. care being taken to This enzyme preparation was designated peakll, and used leave the loosely precipitated layer behind. The supernatant for the following experiments. A smaller peak ofcyclic AMPwas further centrifuged at 105,000 g for 60 min in order to independent activity was eluted at a position of about 350 ml separate the microsomal and cytosol fractions. The preon the column. cipitates obtained was washed twice with 0.32~-sucrose. Sucrose density gradient centrjfugation. Ultracentrifugation centrifuged at 105,000g for 60 min each time and eventually of protein kinases was carried out at 37.500rev./min in a suspended in 10mM-Tris-C1 buffer, pH 7.5, at a concenHitachi SW 40 rotor for 16 h at 3-C. In addition to a tration of about lOmg of protein per ml. This was de5 to 20% sucrose gradient, the centrifuge tube contained signated microsomal fraction. The preparation of the microIOmM-Tris-CI buffer, pH 7.5, and 0.1% Triton X-100 in a soma1 fraction was examined with electron microscopy. In total volume of 5.0ml. At the termination of the centriview ofthe procedures used, the plasma membrane fragments fugation, the bottom of the tube was punched and 0.22-ml would be included in this fraction. Therefore, most of the fractions were collected. constituents observed were considered to be particles and Other methods. [y-32P]ATP was prepared by the method membranes which originated in endoplasmic reticulum and of POST & SEN (1967). Protein was determined by the plasma membrane. It was not possible to distinguish the method of LOWRYet al. (1951). with bovine serum albumin membranes of endoplasmic reticulum from those of plasma as the protein standard. membrane with electron microscopy. The slight contamination of small nerve ending particles and components RESULTS from other fractions was also observed in the preparation. Preparation of myelin fraction. The myelin fraction was DEAE-cellulose column chromatography w i t h various prepared from the white matter of bovine brain, which was substrates separated by scissors, according to the method of UYEMURA T h e activity profile was examined with 4 kinds of et al. (1972) with a slight modification (MIYAMOTO & KAKIUCHI, 1974). The purity of the fraction was examined substrates including histone, phosvitin, casein a n d EXPERIMENTAL PROCEDURES
Multiple forms of membrane protein kinases in brain
271
20 0 4
-
0 3 10 0 2 J
0
z
U
01
p
0
a CI N
10
0
Fraction number
FIG. 1. DEAE
MULTIPLE FORMS OF PROTEIN KINASES IN MYELIN A N D MICROSOMAL FRACTIONS OF BOVINE BRAIN E. MIYAMOTO, K. MIYAZAKI and R. HIROSE Department of Biochemistry, Kumamoto University Medical School, Kumamoto, Japan and A. KASHIBA The Research Division and Clinical Laboratory, Nakamiya Mental Hospital, Hirakata, Osaka, Japan ( R c ~ e i t v d28 September 1977. Riwsed 30 Novemher 1977. Accepted 5 December 1977)
Abstract-The activity profiles of the solubilized protein kinases from the microsomal and myelin fractions of bovine brain were examined by column chromatography and sucrose density gradient centrifugation. The main peak of adenosine 3’5’-monophosphate (cyclic AMP)-dependent activity with histone as substrate for each membrane enzyme was eluted with about 0.2hl-NaCl on a DEAE-cellulose column. A peak of activity stimulated with cyclic AMP was also eluted with about 0.1 M-N~CIfor the microsomal enzyme. A peak with prolamine and casein as substrate for the microsomal or myelin enzyme, respectively. was larger than that with histone as substrate for each enzyme, The first peak with histone as substrate on a DEAE-cellulose column appeared as two peaks on the Sepharose 68 column. The second peak with histone as substrate on DEAE-cellulose cnlumn was shown to be a holoenzyme consisting of regulatory and catalytic subunits. The holoenzyme and whunits were eluted at similar positions to each other between both membrane enzymes on Sepharose 6 8 column. The holoenzyme sedimented as two peaks of activity on sucrose density gradient centrifugation, both of which were stimulated with cyclic AMP. The preincubation of the holoenzyme with cyclic AMP resulted in shifting to a position of a smaller molecular size. The results indicate the occurrence of multiple forms of protein kinases in membrane fractions of brain with respect to substrate specificity and physical property.
BRAINhas been reported to contain protein kinase 1975; MCNAMARA & ABPEL,1977). The endogenous activity predominantly in the particulate fractions activity of protein kinase in the microsomal fraction of (MAENOet a/., 1971). The subcellular distribution of brain was first demonstrated by RODNIGHT& LAWN the enzyme is specific for brain, suggesting that the (1966), and WELLER& RODNIGHT(1970). I t was also functional significance of protein kinases and of studied by MAENO et a / . (1971) and SCHMIDT & phosphorylation of substrate proteins in the particu- SOKOLOFF(l973)With and without exogenous substrate. late fractions may be fairly important in brain. UNO et a/. (1976) have reported that the cyclic The occurrence of multiple forms of adenosine AMP-dependent enzymes solubilized from membrane 3’,5’-monophosphate (cyclic AMP)-dependent protein fractions of several tissues can be separated by DEAEkinases (ATP: protein phosphotransferase EC 2.7.1.37) cellulose column chromatography. Differences were in the cytosol fraction from a variety of mammalian reported in properties ofthe cytosol and the membraneet a/., bound protein kinases soluble in Triton X-100 that tissues is well known (TAOet al., 1970; REIMANN 1971; GILL& GARREN,1971; CHEN& WALSH. 1971; phosphorylate exogenous (UNO er a/., 1976) and YAMAMURA et a/., 1971; CORBINet a/., 1972; MENON. endogenous substrates (UNOet al., 1977). CORBIN& et a/., 1973). 1973; MIYAMOTO KEELY(1977) and CORBINet al. (1977) have recently The myelin fraction of brain contains protein kinase shown that protein kinases in Triton X-1Wtreated activity which is mainly responsible for the phos- particulate fraction from the heart show a peak of phorylation of myelin basic protein (CARNEGIE et a/., cyclic AMP-dependent activity with histone as sub1973, 1974; MIYAMOTO et al., 1974; MIYAMOTO & strate when eluted with a high ionic strength buffer KAKIUCHI, 1974; STECK& APPEL, 1974; MIYAMOTO, from a DEAE-cellulose column. These results indicate 1976) and may involved in its microheterogeneity that the protein kinases in the membrane fractions can ( D E I B L Ea/., R ~1975; ~ CHOUet al., 1976). The solubilized be purified o n column chromatography. However, no enzyme activity from the myelin fraction is stimulated attempt has been made to investigate multiple forms with cyclic AMP and has relatively more affinity for of protein kinases in myelin and microsomal fractions protamine and myelin basic protein (MIYAMOTO, 1975). of brain. The present communication deals with The occurrence of phosphoprotein phosphatase multiplicity of protein kinases from the myelin and responsible for dephosphorylation of myelin basic microsomal fractions of bovine brain, and their physiprotein was also reported (MNAMOTO & KAKIUCHI, cal properties, using exogenous substrates. 269
270
E. MIYAMOTO. K. MIYAZAKI, R. HIROSEand A. KASHIBA
with electron microscopy. Most of particles observed were found t o be myelin and, therefore. the myelin fraction Materials. Fresh bovine brains were obtained from a prepared was considered to be fairly homogenous. local slaughterhouse and transported on ice to the The preparations of the microsomal and myelin fractions laboratory. Cyclic AMP was obtained from Kohjin co. were immediately used or rapidly frozen and stored at DEAE-cellulose (DE 52) was purchased from Whatman. -2O'C until use for the following experiments. Sepharose 6 8 was obtained from Pharmacia Fine Chemicals. Sohtbilizatio~~ and purification of protein kinuse Irom the Calf thymus histone and [8-'H]cyclic AMP (27 Ci/mmol) microsomal and myidin fracfions. The microsomal (about were purchased from Scharq'Mann. Ptotamine and phos- 5.5mg of protein per ml) or myelin fraction (about 5.4mg vitin were obtained from Sigma Chemical Co. Casein and of protein per ml) was suspended in 0.2",, Triton X-100 and Triton X-100 were purchased from Wako Pure Chemical stirred for 40 min in an ice bath. The suspension was Industries, Ltd. ["P]Orthophosphate (carrier-free) was pur- centrifugedat 105,000gfor60min at 2.C. On treatment with chased from Japan Radioisotope Association. TritonX-100 the total activity of protein kinase increased by Assay for protein kinase actirity. The standard assay 87 and 364: for the microsomal and myelin fractions system for protein kinase contained, in a final volume of respectively. About 52 and 53"/;ofthe cyclic AMP stimulated 0.2ml. IOpmol of sodium acetate buffer. pH 6.0. 100pg of activity (histone as substiate) of the microsomal and myelin substrate protein as indicated, 1 nmol of [y-3ZP]ATP fractions, respectively, of the total activity were recovered in containing 2-5 x lo5c.p.m., 2 pnol of magnesium acetate. the supernatant. The clear supernatant solution of each 0.06 pnol of ethylene glycol bis-(8-aminoethy1ether)-N."fraction (165 and 113.9 mg of protein from the microsomal tetraacetic acid and the indicated amount of protein kinase and myelin fractions, respectively) was applied to a 2.6 x 3 1.O in the presence of absence of 0.2nmol of cyclic AMP. cm column of DEAE-cellulose (DE 52) which had been Incubation was carried out at 30°C for 5 min in a shaking equilibrated with 10mM-Tris-C1 buffer, pH 7.5, and 0.1:" water bath. The reaction was terminated and the degree of Triton X-100. The column was washed with 140ml of the protein phosphorylation measured as described previously buffer. Then enzyme protein was eluted with a linear et a/., 1973). A unit of enzyme activity was gradient of NaCl(0-0.4~)in lOm~-Tris-CI buffer, pH 7.5, (MIYAMOTO defined as that amount of enzyme which transferred 1 pmol and 0.1% Triton X-100 in a total volume of 100Oml. The of 3zP from [y-"P]ATP to protein in 5 min at 30°C 10-ml fractions were collected. As shown in Figs. 1 and 2, in the standard assay system. The assays were performed the main peak of cyclic AMP-dependent activity was eluted in duplicate. Each value represents the mean. with about 0 . 2 ~ - N a C lfor the microsomal or myelin Assaysforcyclic AMP-binding.The binding of cyclic AMP fraction, when assayed with histone as substrate. The active (1970). fractions of this cyclic AMP-dependent enzyme for each to protein was determined by the method of GILMAN except that 50mM-phosphate buffer, pH 7.0, was substituted fraction werecollected and concentrated to lOml by Amicon for 50m~-sodiumacetate buffer. pH 4.0. The concentration ultrafiltration apparatus with PM-10 membrane. The conof [3H]cyclic AMP added to reaction mixture was 18.5 nM. centrate (15.1 and 11.2mg of protein for microsomal and Preparation of microsomalfraction from bovine brain. The myelin enzymes, respectively) was applied to a 2.6 x 92cm microsomal fraction was prepared from the grey matter of column of Sepharose 6 8 which had been equilibrated and bovine cerebrum according to the method of UESUCIet al. eluted with IOmM-Tris+CI buffer, pH 7.5,0.l% Triton X-100 (1969)with some modifications. The grey matter of cerebrum and 0.3 M-NaCI. Then 5-ml fractions were collected and the which was separated from the white matter with scissors void volume of the column, determined with blue dextran was homogenized at a half speed for 1.5 min in 10 vol of 2000, was found t o be about 150ml. A peak of cyclic AMPice-cold 0.32~-sucrosein a Waring blendor. The homodependent activity for the microsomal or myelin enzyme was genate was centrifuged at 11,500g for 20 min. The superobserved in the eluate of about 265ml o n the column. natant was decanted to a beaker. care being taken to This enzyme preparation was designated peakll, and used leave the loosely precipitated layer behind. The supernatant for the following experiments. A smaller peak ofcyclic AMPwas further centrifuged at 105,000 g for 60 min in order to independent activity was eluted at a position of about 350 ml separate the microsomal and cytosol fractions. The preon the column. cipitates obtained was washed twice with 0.32~-sucrose. Sucrose density gradient centrjfugation. Ultracentrifugation centrifuged at 105,000g for 60 min each time and eventually of protein kinases was carried out at 37.500rev./min in a suspended in 10mM-Tris-C1 buffer, pH 7.5, at a concenHitachi SW 40 rotor for 16 h at 3-C. In addition to a tration of about lOmg of protein per ml. This was de5 to 20% sucrose gradient, the centrifuge tube contained signated microsomal fraction. The preparation of the microIOmM-Tris-CI buffer, pH 7.5, and 0.1% Triton X-100 in a soma1 fraction was examined with electron microscopy. In total volume of 5.0ml. At the termination of the centriview ofthe procedures used, the plasma membrane fragments fugation, the bottom of the tube was punched and 0.22-ml would be included in this fraction. Therefore, most of the fractions were collected. constituents observed were considered to be particles and Other methods. [y-32P]ATP was prepared by the method membranes which originated in endoplasmic reticulum and of POST & SEN (1967). Protein was determined by the plasma membrane. It was not possible to distinguish the method of LOWRYet al. (1951). with bovine serum albumin membranes of endoplasmic reticulum from those of plasma as the protein standard. membrane with electron microscopy. The slight contamination of small nerve ending particles and components RESULTS from other fractions was also observed in the preparation. Preparation of myelin fraction. The myelin fraction was DEAE-cellulose column chromatography w i t h various prepared from the white matter of bovine brain, which was substrates separated by scissors, according to the method of UYEMURA T h e activity profile was examined with 4 kinds of et al. (1972) with a slight modification (MIYAMOTO & KAKIUCHI, 1974). The purity of the fraction was examined substrates including histone, phosvitin, casein a n d EXPERIMENTAL PROCEDURES
Multiple forms of membrane protein kinases in brain
271
20 0 4
-
0 3 10 0 2 J
0
z
U
01
p
0
a CI N
10
0
Fraction number
FIG. 1. DEAE
