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THE CELL‐FREE SYNTHESIS OF ALKALOID‐BINDING PROTEINS IN IMMATURE RAT BRAIN 1
Author(s) -
Raeburn S.,
Twomey S. L.,
Baxter C. F.
Publication year - 1976
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.1471-4159.1976.tb01557.x
Subject(s) - amino acid , vinblastine , biochemistry , sephadex , in vivo , colchicine , in vitro , chemistry , protein biosynthesis , cell free system , biology , enzyme , genetics , microbiology and biotechnology , chemotherapy
— cell‐free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [ 14 C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble 14 C‐labeled proteins formed in the cell‐free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of 14 C‐labeled amino acids. Criteria for the formation of vinblastine‐binding, 14 C‐labeled proteins were: (1) aggregation of 14 C‐labeled soluble protein by one m m ‐vinblastine sulfate and (2) immunoprecipitation of 14 C‐labeled soluble protein by an antiserum against vinblastine sulfate‐precipitable material. Criteria for the formation of [ 3 H]colchicine‐binding, 14 C‐labeled protein were based upon: (1) co‐precipitation of the 3 H‐and 14 C‐labeled materials by vinblastine sulfate and (2) the coincidence of 3 H‐ and 14 C‐labeled elution peaks from columns of Sephadex G‐200, DEAE‐Sephadex A‐50 and isoelectric focusing. Both in the in vitro and in the in vivo system, 14 C‐labeled amino acids were incorporated into soluble proteins of the post‐microsomal supernatant fraction. Proteins labeled with 14 C‐labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, 14 C‐labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G‐200 which is identical to [ 3 H]colchicine‐binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine‐binding and colchicine‐binding proteins in the in vitro cell‐free system.