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Muscle-Like Contractile Proteins and Tubulin in Synaptosomes (Brain/Peptide Mapping/Actin, Myosin, Tropomyosin/Membrane-Associated Protein) A

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Muscle-Like Contractile Proteins and Tubulin in Synaptosomes (Brain/Peptide Mapping/Actin, Myosin, Tropomyosin/Membrane-Associated Protein) A Proc. Nat. Acad. Sci. USA - Vol. 71, No. 11, pp. 4472-4476, November 1974 Muscle-Like Contractile Proteins and Tubulin in Synaptosomes (brain/peptide mapping/actin, myosin, tropomyosin/membrane-associated protein) A. L. BLITZ AND R. E. FINE Department of Physiology, Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts 02118 Communicated by Frances 0. Schmitt, August 16, 1974 ABSTRACT Material in major bands with molecular MATERIALS AND METHODS weights corresponding to those of actin, brain tropo- myosin, and myosin is present in purified rat synaptosomes Isolation and Fractionation of Synaptosomes. Rat cortical dissolved in sodium dodecyl sulfate and subjected to synaptosomes were purified from washed brain mitochondrial electrophoresis on dodecyl sulfate-acrylamide gels. A band pellets by centrifugation through sucrose gradients (10) or corresponding to tubulin appears to be the major con- stituent of synaptosomes, confirming the work of Feit and Ficoll gradients (11), or by flotation on Ficoll (12). his coworkers. We have demonstrated by peptide mapping Synaptosomal membranes were obtained by lysis of synapto- that the proteins in these bands have strong chemical somes in distilled water (13) or in 1 mM sodium phosphate similarities to actin, brain tropomyosin, myosin, and containing 0.1 mM EDTA (12) and centrifugation through tubulin. We have prepared synaptic membrane, vesicle, and soluble fractions from synaptosomes. The polypeptide discontinuous sucrose gradients or through continuous sucrose composition of synaptic membranes, as determined by gradients (14). dodecyl sulfate-acrylamide gel electrophoresis, is similar Crude synaptic vesicles were obtained from the upper phase to that of synaptosomes, with tubulin, actin, and tropo- of the continuous sucrose gradients used to purify membranes. myosin being major constituents. Synaptic vesicles have Purer vesicles were obtained by lysis of a brain mitochondrial as their major polypeptide an unidentified protein with a molecular weight of 50,000; they also have many bands in pellet followed by centrifugation through a discontinuous common with synaptosomes. The soluble fraction pre- gradient (13). dominantly contains actin and tubulln. The possibility that the muscle-like contractile proteins and tubulin are Electron Microscopy. Pellets were fixed in Karnovsky's membrane-associated in various cell types is discussed, as fixative (15), post-fixed in Millonig's buffered 1% osmium is their possible role in neurotransmitter release. tetroxide (16), dehydrated in graded ethanol solutions, and Increasingly, reports have appeared of muscle-like proteins embedded in Epon-araldite. identified in or isolated from brain. Fine and Bray (1) have Electro- and Sodium Dodecyl Sulfate (NaDodSO4)-Acrylamide shown an actin-like protein in embryonic chick brain, phoresis of Synaptosomal Proteins. Pellets containing synapto- more recently, Fine et al. (2) have isolated a tropomyosin-like somes, membranes, or vesicles were dissolved in 2% NaDod- protein from the same source. The isolation from brain of a at 1000 for biochem- S04 containing 5% 2-mercaptoethanol by heating calcium-activated adenosinetriphosphatase similar 2-5 min. The samples were subjected to electrophoresis at pH ically to muscle actomyosin has also been reported (3). 7.2 on 7.5% acrylamide gels containing 0.8% N,N'-methylene- Tubulin, the microtubule subunit protein, exists in highest bisacrylamide (17). concentration in whole brain (4, 5) and has also been reported in nerve endings (6). Recovery of Proteins from NaDodSO4-Acrylamide Gels. The Since both muscle contraction and release of neurotrans- desired bands were cut from the gels, divided into small cubes, mitter from axon termini are dependent on calcium ion (7, 8), and extracted for 2 days at 370 with 1-2 ml of 50 mM sodium it is of interest to search for a role for brain contractile proteins phosphate (pH 7.2) containing 0.1% NaDodSO4 (18). The in synaptic transmission. These considerations led Berl et al. resulting blue extracts were dialyzed against methanol con- (9) to the identification of a calcium-stimulated, myosin-like taining 5% acetic acid until colorless and then further dialyzed adenosinetriphosphatase activity associated with synaptic against 0.1 M ammonium bicarbonate overnight. The dialyzed vesicles which is stimulated by synaptic membrane compo- solutions were lyophilized, and the residues were dissolved in nents. a minimum volume of distilled water and lyophilized again. We offer here more rigorous criteria for the presence and distribution of proteins similar to actin, myosin, and tropo- Proteolytic Digestion of Proteins and Peptide Mapping. The myosin in rat cortical synaptosomes and in the vesicle, mem- lyophilized proteins were digested with either chymotrypsin brane, and soluble fractions of lysed synaptosomes. We also or iA-tosylamido-2-phenylethyl chloromethyl ketone offer evidence that confirms an earlier report of tubulin in (TPCK)-trypsin by standard procedures. For peptide map- nerve endings (6), as well as evidence concerning its localiza- ping, 30-50 .g of protein, estimated with flourescamine (19), tion. were applied to 20 X 20-cm silica gel G thin-layer chroma- tography plates (250 Mm in thickness) and subjected to chro- Abbreviation: NaDodSO4, sodium dodecyl sulfate. matography followed by electrophoresis at pH 6.5 (2). 4472 Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Contractile Proteins in Synaptosomes 4473 Preparation of Protein Standards. Brain myosin was iso- lated from calf brain by the method of Adelstein and coworkers (20) used for the preparation of myosin from fibroblasts. This method was first applied to brain by Dennis Bray and co- workers (unpublished). This preparation was resolved by NaDodSO4-acrylamide electrophoresis into two bands con- taining material of high molecular weight (200,000 and 170,000) and one lower molecular weight component. It displayed a potassium-EDTA ATPase activity of 0.7 ,omol/- min per mg of protein. Chick brain tropomysin was isolated by the method of Fine and coworkers (2). Rat brain myelin was prepared by the method of Norton and Poduslo (21). Rabbit muscle actin and rat brain tubulin were the generous gifts of Dr. William Lehman and Dr. Clara Asnes, respectively. Rat brain actin and tubulin were also obtained from a brain homogenate by pre- cipitation with vinblastine sulfate (1). RESULTS Morphological Purity of Synaptosomes and Their Subfrac- tions. The electron micrographs of the synaptosome pellet (Fig. la) displays a nearly homogeneous preparation of well- preserved nerve-ending particles. There is little myelin, a frequent contaminant of synaptosome preparations, few free mitochondria or synaptic vesicles, and a few empty syn- aptosomes. The most prevalent contaminant is a structure that appears to derive from post-synaptic material, either dendrites or axons. The membrane fraction (Fig. lb) shows mostly empty membranes, which vary widely in diameter. Some membranes have vesicles still attached, and some free vesicles can be seen. The vesicle fraction (Fig. ic) is perhaps not so pure as the other two fractions. It appears to be contaminated with small (about 1500 A in diameter) mem- branes. These structures may, however, be large synaptic vesicles. They are only about 50% larger than the vesicles isolated from the Torpedo electroplax (22). Presence in Synaptosomes and Subfractions of Proteimns Similar to Myosin, Tubulin, Actin, and Tropomyosin. The photograph in Fig. 2a shows the banding patterns of NaDod- S04-acrylamide gels of NaDodSO4-soluble proteins of synaptosomes, synaptic membranes, synaptic vesicles, and the soluble proteins, for synaptosomes obtained after lysis and centrifugation to remove particulate material. A diagram of the gels in Fig. 2a is also presented (Fig. 2b) to clarify the poor contrast of the photographs*. There are major compo- nents in some or all of these fractions that migrate with actin, tubulin, and brain tropomyosin, and less intense bands that migrate with the two high-molecular-weight components of brain myosin. Two components are also present in platelet myosin (23). These gel patterns are highly reproducible from FIG. 1. Electron micrographs of synaptosomes and synapto- one preparation to the next. Also, synaptosomes prepared by some subfractions. (a) Synaptosomes prepared by flotation on three different procedures all yield nearly identical gel pat- Ficoll (12). X23,500. (b) Synaptic membrane fraction prepared terns. by lysis of synaptosomes followed by purification on discontinuous The gels of membranes prepared by two different procedures sucrose gradients (13). X 19,300. (c) Synaptic vesicle fraction are nearly identical to those of whole synaptosomes except for prepared by lysis of a rat brain mitochondrial pellet followed by the absence of a few minor bands which may represent vesicle purification on discontinuous sucrose gradients (13). X26,900. or mitrochondrial proteins. Membranes prepared by lysis of synaptosomes in phosphate buffer containing EDTA (12) con- tain less actin and tropomyosin. * The original photograph of the purified vesicle fraction is not Most of the material in the bands in the gels of the crude shown because of its poor contrast. vesicle preparation migrate with those of whole synaptosomes Downloaded by guest on September 26, 2021 4474 Biochemistry: Blitz and Fine Proc. Nat. Acad. Sci. USA 71 (1974) b MYI 4 200,000 MY2 4 170,000 a MY 1 MY 2 I TUB 4 55,000 * TUB ACT 4 45,000 ACT I I I BTM 4 29,000 ! BTM 4 I m a pp. 4 i S-T mol. wt. SYN MEM VES SOL STD SYN MEM VESc VESp SOL STD FIG. 2 (a). NaDodSO4-acrylamide gels of synaptosomes and subfractions. (b) Diagram of NaDodSO4 gels shown in (a). NaDodSO4- acrylamide gels containing 7.5% acrylamide, 0.8% N,N'-methylene-bisacrylamide, and 0.1% NaDodSO4 were subjected to electropho- resis at pH 7.2 (17) and stained with Coomassie blue R. SYN, synaptosomes prepared on Ficoll gradients (11). MEM, synaptic mem- branes obtained by lysing synaptosomes in distilled water followed by purification on continuous sucrose gradients (14). VESc, a crude synaptic vesicle preparation obtained from the gradients used to purify MEM.
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