Synthesis of membrane protein in slices of rat cerebral cortex. Source of proteins of the synpatic plasma membranes

Larry Jones, H. R. Mahler, W. J. Moore

Research output: Contribution to journalArticle

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Abstract

Protein synthesis was studied in slices from rat cerebral cortex and localized in various purified subcellular membrane fractions isolated after incubation with l leucine. A synaptosomal fraction isolated from a several times washed crude mitochondrial pellet showed very little contamination by free membranes, and the synaptic membrane fraction isolated from it was estimated to contain about 50% of this component. Leucine incorporation into all fractions was highly sensitive ( > 95%) to emetine except for the cell and synaptic mitochondrial subfractions. They were only 60 to 70% inhibited by emetine and showed 10 to 20% inhibition by chloramphenicol which probably was due to those proteins synthesized in situ by the mitochondria. The net rate of incorporation of labeled protein into the synaptosomal and synaptic membrane subfractions was low, and axonal flow was excluded as a significant source of this label. On the basis of autoradiography it was concluded that the majority of the protein label was contributed by free and membrane enclosed ribosome containing contaminants in these fractions. Unlabeled and labeled subcellular fractions were also analyzed by electrophoresis in the presence of sodium dodecyl sulfate. The staining profiles of the microsomal and synaptic membrane subfractions were nearly identical, whereas those of synaptic mitochondria and the soluble proteins of the cell bodies were unique. The labeling of all proteins was blocked by emetine, except for the synaptic mitochondria (cell mitochondria were not examined). These products of mitochondrial protein synthesis exhibited 3 labeling peaks, the major one with a molecular weight of approximately 38,000. In the absence of emetine, slices incorporated amino acids into soluble and microsomal proteins with high specific activity. The labeled proteins of the soluble fraction were more highly concentrated in the range of molecular weights smaller than 50,000 than were those in the microsomal fraction, and it was concluded that a considerable portion of the protein synthesizing machinery of the brain is geared to form soluble proteins. The gel patterns of labeled microsomal and synaptic membranes were consistent with contamination by the former contributing most of the protein label in the latter. In contrast to many of the opinions expressed in the current literature it was concluded that presynaptic nerve endings do not synthesize protein by any mechanism other than one due to their mitochondria.

Original languageEnglish
Pages (from-to)973-983
Number of pages11
JournalJournal of Biological Chemistry
Volume250
Issue number3
StatePublished - 1975

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Cell membranes
Cerebral Cortex
Rats
Membrane Proteins
Cell Membrane
Mitochondria
Emetine
Synaptic Membranes
Proteins
Membranes
Labels
Subcellular Fractions
Leucine
Labeling
Contamination
Molecular Weight
Molecular weight
Mitochondrial Proteins
Presynaptic Terminals
Chloramphenicol

ASJC Scopus subject areas

  • Biochemistry

Cite this

Synthesis of membrane protein in slices of rat cerebral cortex. Source of proteins of the synpatic plasma membranes. / Jones, Larry; Mahler, H. R.; Moore, W. J.

In: Journal of Biological Chemistry, Vol. 250, No. 3, 1975, p. 973-983.

Research output: Contribution to journalArticle

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abstract = "Protein synthesis was studied in slices from rat cerebral cortex and localized in various purified subcellular membrane fractions isolated after incubation with l leucine. A synaptosomal fraction isolated from a several times washed crude mitochondrial pellet showed very little contamination by free membranes, and the synaptic membrane fraction isolated from it was estimated to contain about 50{\%} of this component. Leucine incorporation into all fractions was highly sensitive ( > 95{\%}) to emetine except for the cell and synaptic mitochondrial subfractions. They were only 60 to 70{\%} inhibited by emetine and showed 10 to 20{\%} inhibition by chloramphenicol which probably was due to those proteins synthesized in situ by the mitochondria. The net rate of incorporation of labeled protein into the synaptosomal and synaptic membrane subfractions was low, and axonal flow was excluded as a significant source of this label. On the basis of autoradiography it was concluded that the majority of the protein label was contributed by free and membrane enclosed ribosome containing contaminants in these fractions. Unlabeled and labeled subcellular fractions were also analyzed by electrophoresis in the presence of sodium dodecyl sulfate. The staining profiles of the microsomal and synaptic membrane subfractions were nearly identical, whereas those of synaptic mitochondria and the soluble proteins of the cell bodies were unique. The labeling of all proteins was blocked by emetine, except for the synaptic mitochondria (cell mitochondria were not examined). These products of mitochondrial protein synthesis exhibited 3 labeling peaks, the major one with a molecular weight of approximately 38,000. In the absence of emetine, slices incorporated amino acids into soluble and microsomal proteins with high specific activity. The labeled proteins of the soluble fraction were more highly concentrated in the range of molecular weights smaller than 50,000 than were those in the microsomal fraction, and it was concluded that a considerable portion of the protein synthesizing machinery of the brain is geared to form soluble proteins. The gel patterns of labeled microsomal and synaptic membranes were consistent with contamination by the former contributing most of the protein label in the latter. In contrast to many of the opinions expressed in the current literature it was concluded that presynaptic nerve endings do not synthesize protein by any mechanism other than one due to their mitochondria.",
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