Synthesis of a Brain-Specific Protein (S100 Protein) in a Homologous Cell-Free System Programmed with Cerebral Polysomal Messenger RNA

Abstract

Polyribosomes, carrying nascent polypeptide chains, were prepared from whole brain, cortex, and hindbrain-medullary white matter of young adult rats. In a homologous cell-free system, a brain-specific protein (S100 protein) was identified in the mixture of polypeptides released from the polyribosomes during incubation for 1 hr at 37°. De novo synthesis of the S100 protein was achieved in a reconstituted cerebral cell-free system containing polysome-derived mRNA and 40S + 60S subunits. The radioactively labeled S100 protein synthesized in vitro was identified by precipitation with antibody to S100 after addition of purified S100 as a carrier, and migration of the solubilized precipitate on acrylamide gels in the presence of sodium dodecyl sulfate. In vitro synthesis of the S100 protein did not occur in analogous cell-free systems derived from hepatic tissue or in a heterologous system containing liver polyribosomes and cerebral enzymes.One of the active areas of research on mammalian protein biosynthesis has been the identification of products formed during cell-free incorporation of amino acids by polyribosomal preparations. The demonstration of active incorporation into globin chains by particles from reticulocytes was the first example of clear-cut evidence for an identifiable product formed in vitro (1-3). Some of the more recent and numerous examples include the demonstration of active incorporation into serum proteins and nonserum liver proteins by particulate preparations from hepatic tissue (4-11), into myosin by polyribosomes from embryonic-chick muscle (12) and myoglobin by polysomes from duck skeletal muscle (13), into heavy and light chains of antibodies by polysomes from rabbit lymph nodes (14, 15) and ovalbumin by chick-oviduct polysomes (16), and into B-lactoglobulin by particles from ewe mammary gland (17).Although cell-free systems dervied from brain are highly active in amino-acid incorporation (18-21), identification of products formed in vitro is difficult because of the complexity and heterogeneity of cerebral tissue. Nevertheless, Rubin and Stenzel (22) reported the in vitro synthesis of a soluble acidic protein (S100 protein) by a ribosomal system from rabbit cortical gray matter. Mahler and Brown (21) later reported that in a polysomal system from rat brain, about 33% of the counts released into the soluble portion during cell-free protein synthesis were found among several acidic proteins, but identification of a specific protein was not attempted in their studies. The isolation, purification, and characterization of two brain-specific low molecular weight proteins by Moore et al., as well as preparation of antisera to these proteins, has made possible a study of the in vitro synthesis of specific brain proteins by cerebral polyribosomes. The S100 protein (23) is primarily of glial origin (24-26), and represents 0.2% of the total soluble protein of brain. The other purified protein (14-3-2) is localized primarily in neurons (27). The function of these proteins is unknown, but t...

In Vitro Synthesis of the Myelin Basic Proteins: Subcellular Site of Synthesis

“…Earlier attempts in our laboratory to observe the synthesis of myelin basic proteins using purified polyribosomal systems similar to those employed by others for the synthesis of brain proteins (Zomzely-Neurath et al, 1972;1973) were unsuccessful. Although these in v i m systems were highly active, no evidence for the synthesis of the MBPs could be obtained using the identification criteria of co-chromatography and co-electrophoresis employed in this work.…”

Regulation of protein synthesis at the translational level in neuroblastoma cells.

“…For measurement of the activity of the soluble factors involved in protein synthesis, the incubation mixture contained 30 ,ug of heterologous, deoxycholate-treated ribosomes from rat brain cortex (10) and increasing amounts of neuroblastoma cell sap. Incubation and further processing were as described for the standard cellfree protein synthesis procedure.…”

“…[3H]Leucine (30-40 Ci/mmol) was purchased from New England Nuclear; [mH]methionine (7. Tris-HCl buffer (pH 7.4), 25 mM KCl, 4 mM MgCI2, and 1 mM dithiothreitol], homogenized in a Dounce homogenizer, and centrifuged at 15,000 X g for 10 min at 4°. The supernatant ("lysate") was dialyzed in a cold room (40) against 500 volumes of Medium A for 3 hr, with the medium changed every hour.…”

Homologous Mammalian Brain Cell Lysate System for the Initiation and Translation of Exogenous mRNAs

“…To study the regulatory events associated with changes in mammalian brain protein synthesis, investigators in the past have relied on two general approaches: measurement of the synthesis of polypeptides directed by an artificial template such as polyuridylic acid (poly U) and the synthesis of natural proteins by polyribosomes preprogrammed with endogenous mRNAs (Zomzely-Neurath et al, 1972;1973;Wiche et al, 1974;Gilbert, 1974;Johnson, 1976;Gilmore-Herbert and Heywood, 1976;Cooper et al, 1977;Sato et al, 1978;Marotta et al, 1979;Cary and Campagnoni, 1979;Gilbert, 1980). The advantages of utilizing an artificial template like poly U or of measuring "run-off'' synthesis have been outweighed by the disadvantages of such systems when one wanted to study the regulation of initiation or the fidelity of the translational process under a variety of natural or experimental conditions.…”