Muscle-Like Contractile Proteins and Tubulin in Synaptosomes

Abstract

Material in major bands with molecular weights corresponding to those of actin, brain tropomyosin, and myosin is present in purified rat synaptosomes dissolved in sodium dodecyl sulfate and subjected to electrophoresis on dodecyl sulfate-acrylamide gels. A band corresponding to tubulin appears to be the major constituent of synaptosomes, confirming the work of Feit and his coworkers. We have demonstrated by peptide mapping that the proteins in these bands have strong chemical similarities to actin, brain tropomyosin, myosin, and tubulin. We have prepared synaptic membrane, vesicle, and soluble fractions from synaptosomes. The polypeptide composition of synaptic membranes, as determined by dodecyl sulfate-acrylamide gel electrophoresis, is similar to that of synaptosomes, with tubulin, actin, and tropomyosin being major constituents. Synaptic vesicles have as their major polypeptide an unidentified protein with a molecular weight of 50,000; they also have many bands in common with synaptosomes. The soluble fraction predominantly contains actin and tubulln. The possibility that the muscle-like contractile proteins and tubulin are membrane-associated in various cell types is discussed, as is their possible role in neurotransmitter release.Increasingly, reports have appeared of muscle-like proteins identified in or isolated from brain. Fine and Bray (1) have shown an actin-like protein in embryonic chick brain, and more recently, Fine et al. (2) have isolated a tropomyosin-like protein from the same source. The isolation from brain of a calcium-activated adenosinetriphosphatase similar biochemically to muscle actomyosin has also been reported (3).Tubulin, the microtubule subunit protein, exists in highest concentration in whole brain (4, 5) and has also been reported in nerve endings (6).Since both muscle contraction and release of neurotransmitter from axon termini are dependent on calcium ion (7,8), it is of interest to search for a role for brain contractile proteins in synaptic transmission. These considerations led Berl et al. (9) to the identification of a calcium-stimulated, myosin-like adenosinetriphosphatase activity associated with synaptic vesicles which is stimulated by synaptic membrane components.We offer here more rigorous criteria for the presence and distribution of proteins similar to actin, myosin, and tropomyosin in rat cortical synaptosomes and in the vesicle, membrane, and soluble fractions of lysed synaptosomes. We also offer evidence that confirms an earlier report of tubulin in nerve endings (6), as well as evidence concerning its localization. MATERIALS AND METHODSIsolation and Fractionation of Synaptosomes. Rat cortical synaptosomes were purified from washed brain mitochondrial pellets by centrifugation through sucrose gradients (10) or Ficoll gradients (11), or by flotation on Ficoll (12).Synaptosomal membranes were obtained by lysis of synaptosomes in distilled water (13) or in 1 mM sodium phosphate containing 0.1 mM EDTA (12) and centrifugation through discontinuous sucrose gra...

Actin associated with purified lymphocyte plasma membrane

“…As two independent methods of disrupting pig lymphocytes (see Materials and methods) gave rise to plasma membrane with similar actin contents, it appears unlikely that the association results from a particular method of cell rupture. In a similar study of synaptosomal membrane, Blitz and Fine [22] approached the problem of non-specific association by adding radiolabelled actin to the cell homogenate. They found that the purified membrane possessed essentially no radioactivity and concluded that the membraneassociated actin was 'intrinsic to the synaptosomes'.…”

Interaction of microtubule proteins with phospholipid vesicles.

“…On the other hand, there is increasing evidence for a direct association of tubulin (the major subunit component of microtubules) with membranes. For example, there is specific colchicine binding to isolated membrane fractions from brain (6, 10); and tubulin has been identified in synaptic vesicles by polyacrylamide gel electrophoresis (7, 11,19), by reaction with specific antibodies (33), and recently by analysis on two-dimensional electropheretograms and by tryptic peptide mapping (17). Stephens' (30) analyses of membranes of the scallop have also supported the existence of membrane-bound tubulin: it was clearly shown that tubulin is associated with ciliary but not flageUar membranes.…”

Serotonin binds specifically and saturably to an actin-like protein isolated from rat brain synaptosomes.

“…The similarity between Ca2l-stimulated actomyosin interactions and neurotransmitter release mechanisms has prompted many investigators to suggest that actin and myosin may be directly involved in the release of some neurotransmitters (2)(3)(4)(5)(6)(7)(8). Contractile processes in smooth muscle and nonmuscle cells are regulated by phosphorylation of the light chain of myosin by a Ca2+-calmodulin-dependent kinase (for review, see refs.…”

“…Synaptosomes contain both actin and myosin (3,4) as well as a Ca2+-stimulated myosin-like ATPase activity, which is associated with synaptic vesicles (3). Actin has been identified in highly purified synaptic vesicle preparations (12) and is also present, mainly as unpolymerized Gactin, within neuronal cytoplasm (13).…”