Proc. Nati. Acad. Sci. USA Vol. 76, No. 6, pp. 2561-2565, June 1979 Biochemistry
Cyclic nucleotide phosphodiesterase activity in 10-nm filaments and microtubule preparations from bovine brain (calcium-dependent regulator protein/neurofilaments/tubulin)
MARSCHALL S. RUNGE, PAULA B. IIEWGLEY, DAVID PUETT, AND ROBLEY C. WILLIAMS, JR. Biochemistry, Vanderbilt University, Nashville, Tennessee 37235 Communicated by Sidney P. Colowick, March 12, 1979 Departments of Molecular Biology and
ABSTRACT Cyclic nucleotide phosphodiesterase activity (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17), which is activatable by Ca2+-dependent regulator protein (CDR), has
been identified in cycled microtubule preparations from bovine brain. By using various methods to fractionate the microtubule preparation into subfractions (e.g., phosphocellulose chromatography to obtain purified 6S tubulin and soluble microtubule-associated proteins, and gel exclusion chromatography on Bio-Gel A-150m to obtain 10-nm filaments), we found that all the fractions exhibited some enzymic activity, but that most of the phosphodiesterase activity was localized in the 10-nm filament fraction. By using cyclic GMP as substrate, wa specific activity of 921 ± 168 pmol/mg of filament protein-min was determined. Also, 10-nm filaments were prepared directly from brain homogenates by differential centrifugation and gel exclusion chromatography. This fraction also contained phosphodiesterase activity but of slightly lower specific activity (752 + 9 pmol/mg of protein-min). The filament-associated enzymic activity was stable during storage (-700C) and to several salt extractions at moderate ionic strength (0.5 M); the latter finding indicates that the phosphodiesterase is not adsorbed to the filaments via nonspecific electrostatic interactions. Although a chelating agent was present in the initial homogenization buffer and generally in all buffers used in preparing fractions, an activator of a smooth muscle phosphodiesterase was released upon boiling the 10-nm filaments. This activator obtained in the boiled supernatant was Ca2+-sensitive, trifluoperazine-sensitive, and stimulated smooth muscle phosphodiesterase to nearly the same extent as purified (exogenous) CDR; thus, it probably represents filament-associated CDR.
homogenates, and the enzymic activity was retained after repeated salt extractions. Comparison of the morphology and protein composition of the 10-nm filaments (10) with those of mammalian brain neurofilaments (14, 15) strongly suggests that the 10-nm filaments are neurofilaments.
of other enzymes including adenylate cyclase (3). Moreover, CDR has been shown to be a component of the smooth and skeletal muscle Ca2+-dependent myosin light chain kinase (4, 5), a skeletal muscle protein kinase (6), and skeletal muscle phosphorylase kinase (7). It has recently been reported that CDR is present in the mitotic apparatus of eukaryotic cells (8) and inhibits microtubule assembly in vitro (9). Preliminary experiments suggested that crude bovine brain microtubule preparations contained a CDR-activatable cyclic nucleotide phosphodiesterase. Such microtubule preparations are known to contain various proteins and structures in addition to tubulin (10-13). In order to localize this enzymic activity further, microtubule preparations were fractionated. Enzymic activity was found in both tubulin and the soluble microtubule-associated proteins (MAPs); however, the 10-nm filament fraction contained the greatest amount of activity. The presence of phosphodiesterase in 10-nm filaments was also confirmed when these structures were prepared directly from bovine brain
MATERIALS AND METHODS Supplies. Cyclic [3HIGMP, with a specific activity of 8.28 Ci/mmol (1 Ci = 3.7 X 1010 becquerels), was purchased from New England Nuclear. Guanosine, GTP (type II-S), Crotalus atrox venom, and other standard reagents were from Sigma. Preparation of 10-nm Filaments Directly from Brain. Filaments were prepared exactly as described in ref. 24. Bovine brain was homogenized in a buffer (referred to hereafter as PM) of 0.1 M 1,4-piperazinediethanesulfonic acid (Pipes), 2 mM ethylene glycol bis(f-aminoethyl ether)-N,N,N'-N'-tetraacetic acid (EGTA), 1 mM MgSO4, and 2 mM dithioerythritol (pH 6.9). The homogenate was centrifuged at 6000 X g for 15 min, and the supernatant was centrifuged at 95,000 X g for 75 min. The resulting high-speed supernatant (10-15 ml) was applied directly to a 2.5 X 60 cm column of Bio-Gel A-150m, equilibrated, and developed with PM. The filament-containing fraction that emerged near the void volume of the column was recovered and centrifuged at 19,500 X g for 30 min. The supernatant was decanted and centrifuged at 95,500 X g for 120 min. The pellet contained the 10-nm filaments. In some preparations designated Ca2+-filaments, the high speed supernatant from the 95,500 X g spin was dialyzed against buffer containing 0.1 M Pipes, 1 mM MgSO4, 2 mM dithioerythritol, 0.1 mM CaCI2, 1 mM phenylmethylsulfonyl fluoride, and 5 mM tosyl-L-arginine methyl ester, and prepared as above in this Ca2+_containing buffer. Isolation of 10-nm Filaments from Microtubule Preparations. Twice-cycled microtubule protein was obtained by the assembly-disassembly procedure of Shelanski et al. (16) as modified by Berkowitz et al. (10), except that the two centrifugation steps employed at 4VC were of 15 min duration instead of 30 min. These preparations contain substantial amounts of the 10-nm filaments described by Berkowitz et al. (10). Depolymerized microtubule protein (5 ml containing approximately 60 mg of protein) thus prepared was applied to a 2.5 X 60 cm column of Bio-Gel A-150m and eluted with PM buffer. The filament-containing fraction that emerged near the void volume was collected and frozen dropwise in liquid N2. Fractionation of Microtubule Preparations. Tubulin and the MAPs were separately isolated from microtubule preparations by chromatography on phosphocellulose (PC) by the established method of Weingarten et al. (17) as modified by Detrich and Williams (18). The MAP fraction was further
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: CDR, Ca2+-dependent regulator (protein); EGTA, ethylene glycol bis (#-aminoethyl ether)-N,N,N',N'-tetraacetic acid; MAPs, microtubule-associated proteins; PC, phosphocellulose; Pipes, 1,4-piperazinediethanesulfonic acid.
It is known that bovine brain contains high levels of a Ca2+/ Mg2+-dependent cyclic nucleotide phosphodiesterase (3':5'cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) that can be activated by calcium-dependent regulator (CDR) protein (1). CDR, homologous to troponin C (2), also stimulates the activity
2561
2562
.~
Biochemistry: Runge et al.
Proc.
fractionated into two components by centrifugation at 106,000 X g and 4(C' for .30 min. The pelleted filaments were resuspended in PM buffer and dialyzed against 20 mM Tris-HCl, pH 7.5. The material so prepared is called PC-MAP pellet. The supernatant of this centrifugation, which contains soluble MAPs, was similarly dialyzed and is called PC-MAP supernatant. CDR Preparation and Assay. CDDR was prepared from fresh bovine brain (1.5 kg) by the method of Watterson et al. (19). Homogeneity was assayed by gel electrophoresis, amino acid analysis, and the ultraviolet absorption spectrum (the latter to ensure that no tryptophan-containing contaminants were present). Assays for CDR were based on the stimulation of a fraction of chicken gizzard phosphodiesterase using the conditions described in the following section. The activatable enzyme, generously provided by J. N. Wells (Vanderbilt University), was isolated according to established methods (20), and bovine serum albumin at 1 mg/ml was added to the appropriate fractions to improve the stability of the preparation. The stock enzyme solution was diluted 1:500, and 50 ,ul of this dilution was added to assay tubes containing 1 gM cyclic [3HIGMP in a final volume of 0.25 ml. This dilution of enzyme routinely gave a 2% breakdown of substrate during a 30 min incubation in the absence of exogenous (DR. Phosphodiesterase Assay. Phosphodiesterase activity was determined with 1 puM cyclic GMP by the tritiated substrate method (21) as described by Wells et al. (20). Enzymic activity
C
4,,
ALv.
J
Nati. Acad. Sci. USA 76 (1979)
was monitored by following the conversion of cyclic 13H IGMP to 5f-l3HIGMP, the latter being converted to 13H guanosine with the 5'-nucleotidase activity in snake (Crotalus atrox) venom. The tritiated guanosine was separated from the tritiated nu-
cleotides by chromatography with DEAE-Sephadex. For comparison of phosphodiesterase activities and activatibilitv among filamentous and microtubular preparations, dilutions were made such that the measured activity yielded less than 25% breakdown of cyclic [:3HIGMP. Product accumulation was linear over the 30-min assay time at enzyme concentrations yielding less than 25% breakdown of cyclic [3H]GMP. Other Methods. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was performed according to Laemmli (22) on 7.5% cylindrical or slab gels, which were subsequently stained with Coomassie brilliant blue R (18). Protein concentrations were determined by the method of Bradford (23) with bovine serum albumin as standard. Preparation of samples for electron microscopy was carried out as described (18). RESULTS Morphology and Protein Composition of the 10-nm Filaments. Fig. 1 A and B are electron micrographs of the microtubule preparation in its polymerized and depolymerized states, respectively. Both intact microtubules and 10-nm filaments are visible in the polymerized preparation, whereas ring structures and 10-nm filaments can be seen in depolymerized
Alnyts . . .4 f
t............ s ' '
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#K
Fu;. 1. Electron microscopy of microtubule preparations and of 10-nm filaments. (A) Microtubules prepared by two cycles of polymerization and depolymerization were polvmerized once more by incubation at, 340C, sedimented (106,000 X g for 60 min), resuspended in cold PM, depolymerized by incubation at. 40C for 30 min, centrifuged (106,000 X g for 15 min), and polvinerized at :34'C. Both intact microtubules and 10-nm filaments are present. Bar = 0.2 Am. (B) An aliquot of the same sample, taken just before the final polymerization. When depolymerized, ring structures and 10-nm filaments are the major morphological constituents. Bar = 0.1 um. (C) Filaments (10 nin) isolated from twice-cycled microtubtlle preparations. Bar = 0.1 pm. (D) Filaments (10 nm) isolated directly from brain. Bar = 0.1 pm.
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Runge et al.
I* ?,i,
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.--
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FI(G. 2. Protein com-
-68
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position of twice cycled microtubules and of 10-nm filaments. Gels: A, twice-cycled microtubule protein; B, 10-nm filaments isolated from twice-cycled microtubule preparations; C, 10-nm filaments isolated directly from brain. Aliquots of each sample were loaded on sodium dodecyl sulfate/7.5% polyacrylamide gels and electrophoresis was performed as described. Approximate apparent molecular weights are shown X 10-'.
material. Fig. IC shows the filaments isolated from a microtubule preparation by gel filtration. In all three cases, they appear indistinguishable from the filaments described by Berkowitz et al. (10). Fig. ID shows the filaments prepared directly from brain. They appear similar to those isolated from microtubule preparations, but they lack the characteristic "knobs" that decorate those structures. In Fig. 2 are shown sodium dodecyl sulfate/polyacrylamide gel electrophoretic patterns representing a microtubule preparation, a sample of filaments isolated from that preparation, and a sample of filaments isolated directly from brain. It is clear, first, that the filaments are distinct in their protein composition from the microtubule fraction, and second, that the filaments prepared by two separate procedures are quite similar to each other in their protein compositions. Phosphodiesterase Activity Associated With Microtubule Preparations and with 10-nm Filaments. Fig. 3 gives endogenous phosphodiesterase activity for several samples: a microtubule preparation, 10-nm filaments isolated from microtubule preparations, 10-nm filaments prepared directly from brain, and purified tubulin prepared by PC-chromatography. Both the microtubule preparations and the purified tubulin have levels of endogenous phosphodiesterase activity (per g of total protein) that are low in comparison to the levels shown by the 10-nm filaments. The data were compiled from various independent assays after different times of frozen storage at -70'C. The phosphodiesterase activity associated with the filaments was quite stable in the frozen state for at least several months. The average specific enzymic activity of the 10-nm filaments (Fig. 3) was 921 168 pmol/mng-min for the filaments isolated from microtubule preparations and 752 i 9 pmol/mg-min for the filaments prepared directly from brain. The difference may arise from a stabilizing effect of the 4 M glycerol in which microtubule proteins are suspended during polymerization steps in the course of their preparation. The observed specific phosphodiesterase activity of the filaments prepared directly from brain homogenates was found to be independent of protein concentration up to 100 og/ml. In contrast, the filaments isolated from microtubule preparations exhibited some concentration dependence of specific activity. +
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Fic.. 3. Phosphodiesterase activity (pmol/min) as a function of added protein. (Upper) Filaments (10 nm) prepared from twicecycled microtubules (*) and directly from brain homogenates (@). (Lower) Twice-cycled microtubules (-) and purified tubulin (A). (Note scale differences.) For the 10-nm-filament fractions the mean ± SD is given and n (for each protein concentration assayed) is denoted in parentheses. These data were obtained on freshly prepared material and on samples that had been stored (-700C) for several months; there was no detectable effect of storage at least up to 3 months. The data for the microtubule and tubulin preparations represent means from duplicates at each protein concentration; the range is indicated when it exceeds the size of the symbol. These particular fractions were stored for 6 weeks; as indicated in the text, somewhat higher activities are obtained with freshly prepared samples.
For example, at 5, 10, and 20 Ag/ml, the respective specific activities were 999 ± 199, 962 + 50, and 790 ± 85 pmol/mgmin.
Fresh microtubule preparations exhibited enzymic activities of 100-200 pmol/mg-min, and this figure decreased to ca. 40 pmol/mg-min after frozen storage for 2 months. PC-purified tubulin, freshly prepared, had a specific activity of about 50 pmol/mg-min, which decreased to 15-20 pmol/mg-min with storage. PC-MAP supernatant likewise exhibited only limited activity, i.e.,
Cyclic nucleotide phosphodiesterase activity in 10-nm filaments and microtubule preparations from bovine brain (calcium-dependent regulator protein/neurofilaments/tubulin)
MARSCHALL S. RUNGE, PAULA B. IIEWGLEY, DAVID PUETT, AND ROBLEY C. WILLIAMS, JR. Biochemistry, Vanderbilt University, Nashville, Tennessee 37235 Communicated by Sidney P. Colowick, March 12, 1979 Departments of Molecular Biology and
ABSTRACT Cyclic nucleotide phosphodiesterase activity (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17), which is activatable by Ca2+-dependent regulator protein (CDR), has
been identified in cycled microtubule preparations from bovine brain. By using various methods to fractionate the microtubule preparation into subfractions (e.g., phosphocellulose chromatography to obtain purified 6S tubulin and soluble microtubule-associated proteins, and gel exclusion chromatography on Bio-Gel A-150m to obtain 10-nm filaments), we found that all the fractions exhibited some enzymic activity, but that most of the phosphodiesterase activity was localized in the 10-nm filament fraction. By using cyclic GMP as substrate, wa specific activity of 921 ± 168 pmol/mg of filament protein-min was determined. Also, 10-nm filaments were prepared directly from brain homogenates by differential centrifugation and gel exclusion chromatography. This fraction also contained phosphodiesterase activity but of slightly lower specific activity (752 + 9 pmol/mg of protein-min). The filament-associated enzymic activity was stable during storage (-700C) and to several salt extractions at moderate ionic strength (0.5 M); the latter finding indicates that the phosphodiesterase is not adsorbed to the filaments via nonspecific electrostatic interactions. Although a chelating agent was present in the initial homogenization buffer and generally in all buffers used in preparing fractions, an activator of a smooth muscle phosphodiesterase was released upon boiling the 10-nm filaments. This activator obtained in the boiled supernatant was Ca2+-sensitive, trifluoperazine-sensitive, and stimulated smooth muscle phosphodiesterase to nearly the same extent as purified (exogenous) CDR; thus, it probably represents filament-associated CDR.
homogenates, and the enzymic activity was retained after repeated salt extractions. Comparison of the morphology and protein composition of the 10-nm filaments (10) with those of mammalian brain neurofilaments (14, 15) strongly suggests that the 10-nm filaments are neurofilaments.
of other enzymes including adenylate cyclase (3). Moreover, CDR has been shown to be a component of the smooth and skeletal muscle Ca2+-dependent myosin light chain kinase (4, 5), a skeletal muscle protein kinase (6), and skeletal muscle phosphorylase kinase (7). It has recently been reported that CDR is present in the mitotic apparatus of eukaryotic cells (8) and inhibits microtubule assembly in vitro (9). Preliminary experiments suggested that crude bovine brain microtubule preparations contained a CDR-activatable cyclic nucleotide phosphodiesterase. Such microtubule preparations are known to contain various proteins and structures in addition to tubulin (10-13). In order to localize this enzymic activity further, microtubule preparations were fractionated. Enzymic activity was found in both tubulin and the soluble microtubule-associated proteins (MAPs); however, the 10-nm filament fraction contained the greatest amount of activity. The presence of phosphodiesterase in 10-nm filaments was also confirmed when these structures were prepared directly from bovine brain
MATERIALS AND METHODS Supplies. Cyclic [3HIGMP, with a specific activity of 8.28 Ci/mmol (1 Ci = 3.7 X 1010 becquerels), was purchased from New England Nuclear. Guanosine, GTP (type II-S), Crotalus atrox venom, and other standard reagents were from Sigma. Preparation of 10-nm Filaments Directly from Brain. Filaments were prepared exactly as described in ref. 24. Bovine brain was homogenized in a buffer (referred to hereafter as PM) of 0.1 M 1,4-piperazinediethanesulfonic acid (Pipes), 2 mM ethylene glycol bis(f-aminoethyl ether)-N,N,N'-N'-tetraacetic acid (EGTA), 1 mM MgSO4, and 2 mM dithioerythritol (pH 6.9). The homogenate was centrifuged at 6000 X g for 15 min, and the supernatant was centrifuged at 95,000 X g for 75 min. The resulting high-speed supernatant (10-15 ml) was applied directly to a 2.5 X 60 cm column of Bio-Gel A-150m, equilibrated, and developed with PM. The filament-containing fraction that emerged near the void volume of the column was recovered and centrifuged at 19,500 X g for 30 min. The supernatant was decanted and centrifuged at 95,500 X g for 120 min. The pellet contained the 10-nm filaments. In some preparations designated Ca2+-filaments, the high speed supernatant from the 95,500 X g spin was dialyzed against buffer containing 0.1 M Pipes, 1 mM MgSO4, 2 mM dithioerythritol, 0.1 mM CaCI2, 1 mM phenylmethylsulfonyl fluoride, and 5 mM tosyl-L-arginine methyl ester, and prepared as above in this Ca2+_containing buffer. Isolation of 10-nm Filaments from Microtubule Preparations. Twice-cycled microtubule protein was obtained by the assembly-disassembly procedure of Shelanski et al. (16) as modified by Berkowitz et al. (10), except that the two centrifugation steps employed at 4VC were of 15 min duration instead of 30 min. These preparations contain substantial amounts of the 10-nm filaments described by Berkowitz et al. (10). Depolymerized microtubule protein (5 ml containing approximately 60 mg of protein) thus prepared was applied to a 2.5 X 60 cm column of Bio-Gel A-150m and eluted with PM buffer. The filament-containing fraction that emerged near the void volume was collected and frozen dropwise in liquid N2. Fractionation of Microtubule Preparations. Tubulin and the MAPs were separately isolated from microtubule preparations by chromatography on phosphocellulose (PC) by the established method of Weingarten et al. (17) as modified by Detrich and Williams (18). The MAP fraction was further
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: CDR, Ca2+-dependent regulator (protein); EGTA, ethylene glycol bis (#-aminoethyl ether)-N,N,N',N'-tetraacetic acid; MAPs, microtubule-associated proteins; PC, phosphocellulose; Pipes, 1,4-piperazinediethanesulfonic acid.
It is known that bovine brain contains high levels of a Ca2+/ Mg2+-dependent cyclic nucleotide phosphodiesterase (3':5'cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) that can be activated by calcium-dependent regulator (CDR) protein (1). CDR, homologous to troponin C (2), also stimulates the activity
2561
2562
.~
Biochemistry: Runge et al.
Proc.
fractionated into two components by centrifugation at 106,000 X g and 4(C' for .30 min. The pelleted filaments were resuspended in PM buffer and dialyzed against 20 mM Tris-HCl, pH 7.5. The material so prepared is called PC-MAP pellet. The supernatant of this centrifugation, which contains soluble MAPs, was similarly dialyzed and is called PC-MAP supernatant. CDR Preparation and Assay. CDDR was prepared from fresh bovine brain (1.5 kg) by the method of Watterson et al. (19). Homogeneity was assayed by gel electrophoresis, amino acid analysis, and the ultraviolet absorption spectrum (the latter to ensure that no tryptophan-containing contaminants were present). Assays for CDR were based on the stimulation of a fraction of chicken gizzard phosphodiesterase using the conditions described in the following section. The activatable enzyme, generously provided by J. N. Wells (Vanderbilt University), was isolated according to established methods (20), and bovine serum albumin at 1 mg/ml was added to the appropriate fractions to improve the stability of the preparation. The stock enzyme solution was diluted 1:500, and 50 ,ul of this dilution was added to assay tubes containing 1 gM cyclic [3HIGMP in a final volume of 0.25 ml. This dilution of enzyme routinely gave a 2% breakdown of substrate during a 30 min incubation in the absence of exogenous (DR. Phosphodiesterase Assay. Phosphodiesterase activity was determined with 1 puM cyclic GMP by the tritiated substrate method (21) as described by Wells et al. (20). Enzymic activity
C
4,,
ALv.
J
Nati. Acad. Sci. USA 76 (1979)
was monitored by following the conversion of cyclic 13H IGMP to 5f-l3HIGMP, the latter being converted to 13H guanosine with the 5'-nucleotidase activity in snake (Crotalus atrox) venom. The tritiated guanosine was separated from the tritiated nu-
cleotides by chromatography with DEAE-Sephadex. For comparison of phosphodiesterase activities and activatibilitv among filamentous and microtubular preparations, dilutions were made such that the measured activity yielded less than 25% breakdown of cyclic [:3HIGMP. Product accumulation was linear over the 30-min assay time at enzyme concentrations yielding less than 25% breakdown of cyclic [3H]GMP. Other Methods. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was performed according to Laemmli (22) on 7.5% cylindrical or slab gels, which were subsequently stained with Coomassie brilliant blue R (18). Protein concentrations were determined by the method of Bradford (23) with bovine serum albumin as standard. Preparation of samples for electron microscopy was carried out as described (18). RESULTS Morphology and Protein Composition of the 10-nm Filaments. Fig. 1 A and B are electron micrographs of the microtubule preparation in its polymerized and depolymerized states, respectively. Both intact microtubules and 10-nm filaments are visible in the polymerized preparation, whereas ring structures and 10-nm filaments can be seen in depolymerized
Alnyts . . .4 f
t............ s ' '
, 'aA -
#K
Fu;. 1. Electron microscopy of microtubule preparations and of 10-nm filaments. (A) Microtubules prepared by two cycles of polymerization and depolymerization were polvmerized once more by incubation at, 340C, sedimented (106,000 X g for 60 min), resuspended in cold PM, depolymerized by incubation at. 40C for 30 min, centrifuged (106,000 X g for 15 min), and polvinerized at :34'C. Both intact microtubules and 10-nm filaments are present. Bar = 0.2 Am. (B) An aliquot of the same sample, taken just before the final polymerization. When depolymerized, ring structures and 10-nm filaments are the major morphological constituents. Bar = 0.1 um. (C) Filaments (10 nin) isolated from twice-cycled microtubtlle preparations. Bar = 0.1 pm. (D) Filaments (10 nm) isolated directly from brain. Bar = 0.1 pm.
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Runge et al.
I* ?,i,
-210 -160
w--
.--
c
E
FI(G. 2. Protein com-
-68
-
eV -
A
2563
ID
B
_
-55
9
C
position of twice cycled microtubules and of 10-nm filaments. Gels: A, twice-cycled microtubule protein; B, 10-nm filaments isolated from twice-cycled microtubule preparations; C, 10-nm filaments isolated directly from brain. Aliquots of each sample were loaded on sodium dodecyl sulfate/7.5% polyacrylamide gels and electrophoresis was performed as described. Approximate apparent molecular weights are shown X 10-'.
material. Fig. IC shows the filaments isolated from a microtubule preparation by gel filtration. In all three cases, they appear indistinguishable from the filaments described by Berkowitz et al. (10). Fig. ID shows the filaments prepared directly from brain. They appear similar to those isolated from microtubule preparations, but they lack the characteristic "knobs" that decorate those structures. In Fig. 2 are shown sodium dodecyl sulfate/polyacrylamide gel electrophoretic patterns representing a microtubule preparation, a sample of filaments isolated from that preparation, and a sample of filaments isolated directly from brain. It is clear, first, that the filaments are distinct in their protein composition from the microtubule fraction, and second, that the filaments prepared by two separate procedures are quite similar to each other in their protein compositions. Phosphodiesterase Activity Associated With Microtubule Preparations and with 10-nm Filaments. Fig. 3 gives endogenous phosphodiesterase activity for several samples: a microtubule preparation, 10-nm filaments isolated from microtubule preparations, 10-nm filaments prepared directly from brain, and purified tubulin prepared by PC-chromatography. Both the microtubule preparations and the purified tubulin have levels of endogenous phosphodiesterase activity (per g of total protein) that are low in comparison to the levels shown by the 10-nm filaments. The data were compiled from various independent assays after different times of frozen storage at -70'C. The phosphodiesterase activity associated with the filaments was quite stable in the frozen state for at least several months. The average specific enzymic activity of the 10-nm filaments (Fig. 3) was 921 168 pmol/mng-min for the filaments isolated from microtubule preparations and 752 i 9 pmol/mg-min for the filaments prepared directly from brain. The difference may arise from a stabilizing effect of the 4 M glycerol in which microtubule proteins are suspended during polymerization steps in the course of their preparation. The observed specific phosphodiesterase activity of the filaments prepared directly from brain homogenates was found to be independent of protein concentration up to 100 og/ml. In contrast, the filaments isolated from microtubule preparations exhibited some concentration dependence of specific activity. +
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0.5[
-IA
4
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5
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15
20
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Protein, pg
Fic.. 3. Phosphodiesterase activity (pmol/min) as a function of added protein. (Upper) Filaments (10 nm) prepared from twicecycled microtubules (*) and directly from brain homogenates (@). (Lower) Twice-cycled microtubules (-) and purified tubulin (A). (Note scale differences.) For the 10-nm-filament fractions the mean ± SD is given and n (for each protein concentration assayed) is denoted in parentheses. These data were obtained on freshly prepared material and on samples that had been stored (-700C) for several months; there was no detectable effect of storage at least up to 3 months. The data for the microtubule and tubulin preparations represent means from duplicates at each protein concentration; the range is indicated when it exceeds the size of the symbol. These particular fractions were stored for 6 weeks; as indicated in the text, somewhat higher activities are obtained with freshly prepared samples.
For example, at 5, 10, and 20 Ag/ml, the respective specific activities were 999 ± 199, 962 + 50, and 790 ± 85 pmol/mgmin.
Fresh microtubule preparations exhibited enzymic activities of 100-200 pmol/mg-min, and this figure decreased to ca. 40 pmol/mg-min after frozen storage for 2 months. PC-purified tubulin, freshly prepared, had a specific activity of about 50 pmol/mg-min, which decreased to 15-20 pmol/mg-min with storage. PC-MAP supernatant likewise exhibited only limited activity, i.e.,
