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Turnover of actin in rat brain
Brain Research, 57 (1973) 259-260 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
259
Turnover of actin ini rat brain
KARI HEMMINKI
Department of Medical Chemistry, University of Helsinki, SF-O0170 Helsink i 17 (Finland) (Accepted April 5th, 1973)
An actin-like protein has been isolated from the central nervous system resembling muscle actin by peptide maps 5 and binding to muscle myosinL This protein appears to be enriched in the synaptosomal fraction of the brain 1 and it is a major protein synthesized in sympathetic ganglia at the time of axonization5. It has been
F T[rne (days) w
1
~
~,~i
2
Fig. l. SDS-polyacrylamide gels of actin isolated from rat brain and muscle as described by Fine and Bray 5. a, 50 #g of brain actin; b, coelectrophoresis of 25 #g of brain and muscle actin. Fig. 2. Turnover of purified actin. Rats received an intracerebral injection of 10 #Ci of [SH]leucine and were killed at time points indicated. The labelling of the total homogenate ( 0 ) , and the purified actin (O) was determined. Each point is the mean of 2-3 experiments.
260
SHORT COMMUNICATIONS
considered a component of the cytochalasin-sensitive microfilaments8, while some workers relate it to the release of transmitter substances ~. In this study we have examined the turnover of the actin-like protein in maturing rat brain (2-3 weeks old). The animals received an intracerebral injection of [aH]leucine (10 #Ci, spec. act. 38 Ci/mmole) and were killed after 1 h, l, 3, 6 and 12 days. Their cortices were washed in 0.25 M sucrose to remove external blood and actin was isolated as described by Fine and Bray ~. The protein purified produced a single band at a molecular weight of 46,000 on an SDS acrylamide gel v (Fig. la). Its similarity with muscle actin was confirmed by coelectrophoresis (Fig. lb). The labelling of the purified actin was determined by liquid scintillation counting after washing the sample twice with CCIaCOOH and once with ethanol; protein was determined according to Lowry et al. 6. The labelqng of actin was less active than that of the total homogenate (Fig. 2), but their turnover appeared almost identical with an apparent half-life of 4.4 days. These data show that actin is not labelled particularly actively in maturing rat brain even though it may be a major constituent of the growing axons. Its half-life is rather similar to that of tubulin in mouse brain 4 but is much shorter than that of S-100 (see ref. 3), another purified brain protein with a known half-life. This study was supported by the Sigrid Jus61ius Foundation, and the National Research Council for Medical Sciences, Finland. The skilled technical assistance of Mrs. Kirsti Salmela is appreciated.
1 BERL,S., PUSZKIN,S., AND NICKLAS,W. J., Actomyosin-like protein in brain, Science, 179 (1973) 441-446. 2 BURTON,P. R., AND KIRKLAND, W. L., Actin detected in mouse neuroblastoma cells by binding of heavy meromyosin, Nature New Biol., 239 (1972) 244-246. 3 CICERO, Y. J., AND MOORE, B.W., Turnover of the brain specific protein, S-100, Science, 169 (1970) 1333-1334. 4 DUTTON,G. R., ANDBARONDES,S., Microtubular protein: synthesis and metabolism in developing brain, Science, 166 (1969) 1637-1638. 5 FINE, R. E., AND BRAY, O., Actin in growing nerve cells, Nature New Biol., 234 (1971) 115-1t8. 6 LOWRY,O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 7 WEBER, K., AND OSBORN, M., The reliability of molecular weight determinations by dodecyl sulfate-polyactylamide gel electrophoresis, J. biol. Chem., 244 (1969) 4406-4412. 8 WESSELLS,N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. D., LUDUENA,M. A., TAYLOR,E. L., WRENS, J.Z., AND YAMADA, K.M., Microfilaments in cellular and developmental processes, Science, 171 (1971) 135-143.
259
Turnover of actin ini rat brain
KARI HEMMINKI
Department of Medical Chemistry, University of Helsinki, SF-O0170 Helsink i 17 (Finland) (Accepted April 5th, 1973)
An actin-like protein has been isolated from the central nervous system resembling muscle actin by peptide maps 5 and binding to muscle myosinL This protein appears to be enriched in the synaptosomal fraction of the brain 1 and it is a major protein synthesized in sympathetic ganglia at the time of axonization5. It has been
F T[rne (days) w
1
~
~,~i
2
Fig. l. SDS-polyacrylamide gels of actin isolated from rat brain and muscle as described by Fine and Bray 5. a, 50 #g of brain actin; b, coelectrophoresis of 25 #g of brain and muscle actin. Fig. 2. Turnover of purified actin. Rats received an intracerebral injection of 10 #Ci of [SH]leucine and were killed at time points indicated. The labelling of the total homogenate ( 0 ) , and the purified actin (O) was determined. Each point is the mean of 2-3 experiments.
260
SHORT COMMUNICATIONS
considered a component of the cytochalasin-sensitive microfilaments8, while some workers relate it to the release of transmitter substances ~. In this study we have examined the turnover of the actin-like protein in maturing rat brain (2-3 weeks old). The animals received an intracerebral injection of [aH]leucine (10 #Ci, spec. act. 38 Ci/mmole) and were killed after 1 h, l, 3, 6 and 12 days. Their cortices were washed in 0.25 M sucrose to remove external blood and actin was isolated as described by Fine and Bray ~. The protein purified produced a single band at a molecular weight of 46,000 on an SDS acrylamide gel v (Fig. la). Its similarity with muscle actin was confirmed by coelectrophoresis (Fig. lb). The labelling of the purified actin was determined by liquid scintillation counting after washing the sample twice with CCIaCOOH and once with ethanol; protein was determined according to Lowry et al. 6. The labelqng of actin was less active than that of the total homogenate (Fig. 2), but their turnover appeared almost identical with an apparent half-life of 4.4 days. These data show that actin is not labelled particularly actively in maturing rat brain even though it may be a major constituent of the growing axons. Its half-life is rather similar to that of tubulin in mouse brain 4 but is much shorter than that of S-100 (see ref. 3), another purified brain protein with a known half-life. This study was supported by the Sigrid Jus61ius Foundation, and the National Research Council for Medical Sciences, Finland. The skilled technical assistance of Mrs. Kirsti Salmela is appreciated.
1 BERL,S., PUSZKIN,S., AND NICKLAS,W. J., Actomyosin-like protein in brain, Science, 179 (1973) 441-446. 2 BURTON,P. R., AND KIRKLAND, W. L., Actin detected in mouse neuroblastoma cells by binding of heavy meromyosin, Nature New Biol., 239 (1972) 244-246. 3 CICERO, Y. J., AND MOORE, B.W., Turnover of the brain specific protein, S-100, Science, 169 (1970) 1333-1334. 4 DUTTON,G. R., ANDBARONDES,S., Microtubular protein: synthesis and metabolism in developing brain, Science, 166 (1969) 1637-1638. 5 FINE, R. E., AND BRAY, O., Actin in growing nerve cells, Nature New Biol., 234 (1971) 115-1t8. 6 LOWRY,O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 7 WEBER, K., AND OSBORN, M., The reliability of molecular weight determinations by dodecyl sulfate-polyactylamide gel electrophoresis, J. biol. Chem., 244 (1969) 4406-4412. 8 WESSELLS,N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. D., LUDUENA,M. A., TAYLOR,E. L., WRENS, J.Z., AND YAMADA, K.M., Microfilaments in cellular and developmental processes, Science, 171 (1971) 135-143.