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Tubulin Synthesis in Rat Forebrain: Studies with Free and Membrane‐Bound Polysomes
Author(s) -
Gilbert Jeffrey M.,
Strocchi Paola,
Brown Beverly A.,
Marotta Charles A.
Publication year - 1981
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1981.tb01670.x
Subject(s) - polysome , tubulin , biology , biochemistry , isoelectric point , reticulocyte , cytoplasm , peptide , protein biosynthesis , microbiology and biotechnology , ribosome , microtubule , rna , gene , enzyme
Free and membrane‐bound polysomes were prepared from rat forebrain and added to a cell‐free system containing rabbit reticulocyte factors and L‐[ 35 S]methionine. The translation products were analyzed by two‐dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (α 1 , α 2 , β 1 , and α 2 ) that are found in rat forebrain cytoplasm. The membrane‐bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two‐dimensional gel. MB has a molecular weight and isoelectric point similar to α‐tubulin. Only trace amounts of α‐ and β‐tubulin and actin were synthesized by the membrane‐bound polysomes. MB co‐purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane‐bound polysomes) and α‐ and β‐tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of α‐ and β‐tubulin; MB yielded peptides not found in tubulin. We conclude that membrane‐bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane‐bound polysomes and is similar, but not identical, to α‐tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.