In vitro synthesis of two brain-specific proteins (S100 and 14-3-2) by polyribosomes from rat brain

Zomzely-Neurath, Claire; York, Curtis; Moore, Blake W.

doi:10.1016/s0003-9861(73)80009-8

Cited by 49 publications

(11 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, the present report presents the first evidence for axoplasmic transport of not merely a specific macromolecule, but of a soluble protein which is unique to nervous tissue. Furthermore, since the 14-3-2 protein is made primarily on free polysomes [23] and travels by slow axonal flow, it is possible that proteins transported rapidly along the axon are synthesized primarily on membrane-bound polysomes. Since the levels of the 14-3-2 protein in nervous tissue appear to be correlated with functional rather than morphological development [ 48], it would be possible, with present techniques, to determine whether axoplasmic flow begins at the time of appearance of the 14-3-2 protein in the nerve cell body.…”

Section: Discussionmentioning

confidence: 99%

“…At the present time, the function of the 14-3-2 protein is unknown, although it has been shown to be primarily of neuronal localization [19,20] and to be correlated with functional rather than morphological development [21,22]. Since previous studies from our laboratory had demonstrated that the 14-3-2 protein was synthesized primarily on free rather than membrane-bound polysomes of rat cerebral cortex [23], we posed the question of transport of this protein from the site of synthesis in the neuronal perikaryon to the axon terminals. In other tissues, such as liver and pancreas, proteins destined for export are synthesized by membrane-bound polysomes [24][25][26].…”

Section: Introductionmentioning

confidence: 94%

“…After dialysis, 20-µl aliquots were taken for measurement of total radioactivity and 10-µl aliquots for Lowry protein determination [35]. Hot trichloroacetic acid-insoluble radioactivity was measured using the Millipore filter assay method with bovine serum albumin as carrier protein [23]. After aliquots were taken, the samples were lyophilized, dissolved in 0.5 ml of Tris/phosphate (pH 7.2) and subjected to Sephadex G-150 (superfine) chromatography in order to eliminate a large proportion of non-(14-3-2)-labeled material.…”

Section: Identification Of the 14-3-2 Protein In Retinae And Optic Lobesmentioning

confidence: 99%

See 2 more Smart Citations

Axoplasmic transport of a brain-specific soluble protein☆

Marangos¹,

Zomzely-Neurath²,

York³

et al. 1975

Biochimica et Biophysica Acta (BBA) - General Subjects

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

Section: Identification Of the 14-3-2 Protein In Retinae And Optic Lobesmentioning

confidence: 99%

See 1 more Smart Citation

Axoplasmic transport of a brain-specific soluble protein☆

Marangos¹,

Zomzely-Neurath²,

York³

et al. 1975

Biochimica et Biophysica Acta (BBA) - General Subjects

View full text Add to dashboard Cite

“…This system included various rat brain components which have already been demonstrated to support in vitro protein synthesis directed by ratbrain polyribosomes (8). The fidelity of this in vitro protein system will be demonstrated by the identification of tubulin as one of the synthesized proteins.…”

mentioning

confidence: 99%

“…Ribosomal pellets were stored at -70°and showed no loss of activity over a period of 6 months. For analysis, the ribosomal pellets were suspended in lysis buffer, then layered on 15 (8). The concentration of total protein in the complete reaction was approximately 6.5 mg/ml with the polyribosomal material accounting for about 15% of the protein input.…”

mentioning

confidence: 99%

In Vitro Synthesis of Mouse Neuroblastoma Tubulin

Wiche

Zomzely-Neurath

Blume

1974

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Polyribosomes were isolated from a clonal line of mouse neuroblastoma grown in culture. In a heterologous in vitro system containing rat brain components, these polyribosomes were shown to direct the synthesis of neuroblastoma tubulin. Identification of the tubulin synthesized in vitro was achieved by coelectrophoresis with native neuroblastoma tubulin on sodium dodecyl sulfate polyacrylamide gels, immunoprecipitation, and demonstration of specific aggregation. Tubulin accounted for 2% of the total proteins synthesized. This in vitro protein synthesizing system offers a model for studying possible translational control mechanisms regulating the synthesis of proteins involved in nerve cell function.The existence of specific translational and(or) transcriptional mechanisms regulating the synthesis of various proteins playing major roles in the function of nerve cells has been postulated (1-3). A model system for the investigation of these regulatory mechanisms at a molecular level would be an in vitro protein synthesizing system directed by nerve cell components. Mouse neuroblastoma (NB) tissue culture cells which exhibit many of the properties of mature nerve cells, including cell morphology (4, 5), electrical excitability (6), and the expression of several nerve specific enzyme activities (5, 7), seemed to be a prime candidate for the development of such a system. Since homogeneous cloned lines of NB can be easily grown in culture, large quantities of NB cell constituents involved in protein synthesis can be isolated without concern about other neuronal or glial contaminants.In the present report, we describe the isolation of NB polyribosomes and their ability to direct the synthesis of NB protein in a heterologous cell-free system. This system included various rat brain components which have already been demonstrated to support in vitro protein synthesis directed by ratbrain polyribosomes (8). The fidelity of this in vitro protein system will be demonstrated by the identification of tubulin as one of the synthesized proteins. Although tubulin is not unique to cells of neuronal origin (9), it is found in relatively high concentration in NB cells (10) and appears to play a role in the outgrowth and maintenance (3,(11)(12)(13) Cells. Mouse NB, clone NS20 (17), was grown in Dulbecco's modified Eagle medium supplemented with 10 units/ml of penicillin, 10 iug/ml of streptomycin sulfate, and 10% fetalcalf serum (growth medium). Stock cultures were maintained in falcon T flasks and cells used for experiments were grown in roller bottles containing 200 ml of growth medium.Preparation and Analysis of Polyribosomes. Cells grown to stationary phase were washed in modified saline Di solution (7) and then detached and lysed in a solution containing 50 mM Tris* HCl (pH 7.4), 100 mM KCl, and 5 mM MgCl2 (lysis buffer) plus 0.5% NP40. Cell lysates were centrifuged at 12,000 X g for 10 min at 40, the supernatants were layered on discontinuous sucrose gradients (0.5 and 2.0 M sucrose in lysis buffer), and the ribosomal ma...

show abstract

Poly(A)‐ and nonpoly(A)‐RNA associated with rat brain microsomal fractions: In vivo labelling studies

Czosnek

Soifer

Gal

et al. 1980

J of Neuroscience Research

View full text Add to dashboard Cite

The time course of incorporation of radiolabelled precursor into RNA associated with rat brain free polyribosomes, rough membranes, and smooth membranes was measured following a single intracranial injection of [3H] orotic acid. Polyadenylated RNAs were separated from nonpolyadenylated RNAs by affinity chromatography on oligo (dT)-cellulose columns. Poly(A)-RNA associated with each of the microsomal fractions became more rapidly labelled than did the nonpoly(A)-RNA of the same fractions. While the labelling profiles of the nonpoly(A)-RNA isolated from the polyribosomes and rough membranes are similar from one fraction to another, the specific radioactivity of the poly(A)-RNA isolated from free polyribosomes increased much more drastically than that of the poly(A)-RNA associated with rough membranes. The labelling profiles of RNA species isolated from smooth membranes were very different in this respect from the two ribosomal fractions. There was a lag of more than four hours before significant label appeared in the RNA associated with the smooth membrane fraction. These studies demonstrate that the different populations of brain microsomal RNA are labelled at different rates, perhaps reflecting differences in the turnover of these RNAs and differences in their function.

show abstract

In vitro synthesis of two brain-specific proteins (S100 and 14-3-2) by polyribosomes from rat brain

Cited by 49 publications

References 36 publications

Axoplasmic transport of a brain-specific soluble protein☆

Axoplasmic transport of a brain-specific soluble protein☆

In Vitro Synthesis of Mouse Neuroblastoma Tubulin

Poly(A)‐ and nonpoly(A)‐RNA associated with rat brain microsomal fractions: In vivo labelling studies

Contact Info

Product

Resources

About