The protein subunit of calf brain neurofilament

133

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...

Section: Distribution and Biochemical Purity Of Synaptosomalmentioning

confidence: 56%

Muscle-Like Contractile Proteins and Tubulin in Synaptosomes

Blitz

Fine

1974

133

“…Both smooth muscle and nervous tissue contain heterogeneous cell populations which impose the time-consuming steps of fractionation to obtain homogeneous cell types as in brain (1,16,21,25), or to obtain complex organelles such as the dense bodies of gizzards (17,18,26,28) prior to application of procedures to enrich 10-nm filaments. All techniques used on mammalian cells to date require anywhere from a few to 15 days to attain significant enrichment (1, 16-18, 21, 26, 28) of morphologically distinguishable filaments.…”

mentioning

confidence: 99%

Isolation and preliminary characterization of 10-nm filaments from baby hamster kidney (BHK-21) cells.

Starger

Goldman

1977

124

A procedure for isolating 10-nm filaments as juxtanuclear caps from normal and colchicine-treated BHK-21 cells is described. These aggregates of 10-nm filaments retain their birefringence and their structural integrity. The major proteins comprisin the filament preparations can be resolved as two bands on sodium dodecyl sulfate gels, corresponding to approximate molecular weights of 54,000 and 55,000.

“…In neuroblastoma cells the bundles of 100 A filaments are located in the perinuclear region and extend out into the cell processes, where they are oriented parallel to the long axis of the process (9, 10). Neurofilaments approximately 100 A in diameter have been isolated and partially characterized from the giant axon of squid (11) and from axons of calf brain (12)(13)(14).Very little is known about the function of these filaments, though it has been suggested that they play a role in axoplasmic transport (15, 16). The major reason for this lack of understanding is that no procedure has been discovered that specifically interferes with the integrity of these filaments.…”

mentioning

confidence: 99%

Localization of the neurofilament protein in neuroblastoma cells by immunofluorescent staining.

Jorgensen¹,

Subrahmanyan²,

Turnbull³

et al. 1976

(9,10). In neuroblastoma cells the bundles of 100 A filaments are located in the perinuclear region and extend out into the cell processes, where they are oriented parallel to the long axis of the process (9, 10). Neurofilaments approximately 100 A in diameter have been isolated and partially characterized from the giant axon of squid (11) and from axons of calf brain (12)(13)(14).Very little is known about the function of these filaments, though it has been suggested that they play a role in axoplasmic transport (15, 16). The major reason for this lack of understanding is that no procedure has been discovered that specifically interferes with the integrity of these filaments. In addition, no specific characteristics that could be used for an assay of the filaments or filament subunits have been identified. To obtain the major protein, the isolated neurofilaments were solubilized in sodium dodecyl sulfate (NaDodSO4) and their components separated by NaDodSO4-acrylamide gel electrophoresis (17). The major protein band (54,000-56,000 daltons) was then cut out from the NaDodSO4-gel (14)