In Vitro Synthesis of Brain Protein. II. Properties of the Complete System<sup>*</sup>

Stenzel, Kurt H.; Aronson, Ruth F.; Rubin, Albert L.

doi:10.1021/bi00867a019

Cited by 22 publications

(7 citation statements)

References 9 publications

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“…Stimulation of [14C]leucine incorporation by other amino acids has been reported in brain systems by several investigators (Clouet et ctl., 1966;Stenzel et al, 1966;Murthy and Rappoport, 1965). A mixture of 19 or 20 amino acids has proven to be stimulatory in our system also.…”

Section: Discussionmentioning

confidence: 60%

“…The requirements of this particular polyscmal system do not appear to depart widely in many respects from those reported for several other brain systems (Herriman and Hunter, 1965;Stenzel et al, 1966;Murthy and Rappoport, 1965;Clouet et at., 1966;Zomzely et al, 1964). The time course for leucine incorporation into TCA-precipitable material is very similar to that reported by Herriman and Hunter (1965) and Stenzel et al (1966), with maximal activity occurring after about 30 min. It was found that Mg2+ was maximally stimulatory at 10 mM but very high activity (>90%) extended over a wide range from 6 to 10 mM.…”

mentioning

confidence: 54%

“…We have not studied the whole Mg2+ was tested and incorporation was found to be considerably reduced compared to control values (Clouet et al, 1966;Zomzely et al, 1964). The system does not appear to be as sensitive to omission of GTP or ATP as are other brain systems (Stenzel et al, 1966;Murthy and Rappoport, 1965;Zomzely et al, 1964) , nor does it have a requirement for an ATPgenerating system (Herriman and Hunter, 1965;Clouet et al, 1966;Murthy and Rappoport, 1965;Stenzel et al, 1966;Zomzely etal., 1964).…”

mentioning

confidence: 89%

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Isolation and Properties of Polyribosomes from Cerebral Cortex^*

Campagnoni¹,

Mahler²

1967

Biochemistry

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Section: Discussionmentioning

confidence: 60%

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confidence: 54%

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confidence: 89%

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Isolation and Properties of Polyribosomes from Cerebral Cortex^*

Campagnoni¹,

Mahler²

1967

Biochemistry

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“…Potassium has been implicated in tRNA binding to ribosomes (36), in activation of peptidyl transferase by structural rearrangement (27), and in activation of pyruvate kinase (43). As observed quite markedly with the P. vulgaris system, increased concentrations of potassium above the optimum readily inhibit protein synthesis (15,36,37). This rapid decrease in incorporation at higher levels of potassium may be a result of ribosomal dissociation (4) that is due to competition for binding sites on the ribosomes between potassium and magnesium.…”

Section: Resultsmentioning

confidence: 98%

Protein synthesis by cell-free extracts of Proteus vulgaris

Backler¹,

Inniss²

1971

Can. J. Microbiol.

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BACKLER, K. L., and W. E. INNISS. 1971. Protein synthesis by cell-free extracts of Proteus vulgaris.Can. J. Microbial. 17: 1545-1551. A cell-free protein-synthesizing system from Proteus vrtlgaris was developed and characterized. Initially, a very low activity was found to occur; it was shown to be due to a deficiency in the endogenous transfer ribonucleic acid (tRNA) content of the cell extracts. When tRNA was added to the polyuridylic acid (poly U) directed system, incorporation of 14C-phenylalanine into polypeptide readily occurred. This incorporation was dependent on ribosomes and supernatant fraction and the optimun~ concentrations of the various components of the system were found to be as follows: 120 pglml tRNA, 30 m M magnesium, 0.5 m M spermidine, 60 m M potassium, 50 m M adenosine triphosphate (ATP), 5 m M guanosine triphosphate (GTP), 0.03 m M cytosine triphosphate (CTP) and uridine triphosphate (UTP), 5 m M phosphoenolpyruvate (PEP), 50 p g l n~l poly U, and 30 m M 2-mercaptoethanol. Un systen~e de synthese proteinique dtpourvu &elements cellulaires a ete realise et caracterise i~ partir d'une souche de Proterrs vrtlgaris. Au depart, I'activite du systeme s'est averee tres faible en raison d'une deficience dans le contenu en acide ribonucleique de transfert (ARN-t) endogene de I'extrait cellulaire. L'addition d'ARN-t au systeme dependant de l'acide polyuridylique (poly U) facilite la prolnpte incorporation de la phenylalanine 14C dans le polypeptide. Cette illcorporation etait sous la dependance des ribosomes et de la fraction surnageante. Les concentrations opti~num des divers composants du systeme ont Cte Ctablies colnme suit: ARN-t, 120 pglml; magnesium, 30 mM; spermidine, 0.5 mM; potassiunl, 60 mM; adenosine triphosphate (ATP), 50 mM; guanosine triphosphate (GTP), 5 mM; cytosine triphosphate (CTP) et uridine triphosphate (UTP), 0.03 mM; phosphoenolpyruvate (PEP), 5 mM; poly U, 50 pg/ml et 2-nlercaptoethanol, 30 n1M.

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“…Although cell-free systems dervied from brain are highly active in amino-acid incorporation (18)(19)(20)(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.…”

mentioning

confidence: 99%

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

Zomzely-Neurath

York

Moore

1972

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

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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...

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In Vitro Synthesis of Brain Protein. II. Properties of the Complete System^*

Cited by 22 publications

References 9 publications

Isolation and Properties of Polyribosomes from Cerebral Cortex^*

Isolation and Properties of Polyribosomes from Cerebral Cortex^*

Protein synthesis by cell-free extracts of Proteus vulgaris

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

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In Vitro Synthesis of Brain Protein. II. Properties of the Complete System*

Cited by 22 publications

References 9 publications

Isolation and Properties of Polyribosomes from Cerebral Cortex*

Isolation and Properties of Polyribosomes from Cerebral Cortex*

Protein synthesis by cell-free extracts of Proteus vulgaris

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

Contact Info

Product

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In Vitro Synthesis of Brain Protein. II. Properties of the Complete System^*

Isolation and Properties of Polyribosomes from Cerebral Cortex^*

Isolation and Properties of Polyribosomes from Cerebral Cortex^*