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IN VITRO SYNTHESIS OF THE MYELIN BASIC PROTEINS IN THE DEVELOPING MOUSE BRAIN: PROPERTIES OF A HOMOGENATE SYSTEM
Author(s) -
Carey Gregory D.,
Campagi Anthony T.
Publication year - 1979
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.1471-4159.1979.tb11709.x
Subject(s) - puromycin , incubation , myelin basic protein , myelin , biochemistry , in vivo , leucine , in vitro , polyacrylamide gel electrophoresis , urea , incubation period , protein biosynthesis , chemistry , biology , chromatography , amino acid , central nervous system , enzyme , endocrinology , microbiology and biotechnology
—An in vitro system using mouse brain homogenates has been developed to study the synthesis of the myelin basic proteins. Incorporation of [ 3 H]leucine into protein in this system did not require additional energy sources. The system was slightly stimulated by glucose and strongly inhibited by puromycin. Myelin basic proteins were isolated from incubation mixtures by conventional techniques of solvent extraction and column chromatography, and finally separated into the large and small components by polyacrylamide gel electrophoresis in an acetic acid‐urea system. Gels were stained, sliced, dissolved, and counted, and relative rates of incorporation of label into the two basic proteins were determined at several ages. The ratio of radioactivity incorporated into the small (S) and large (L) basic proteins, over a 30 min incubation period, was found to increase from 0.97 at 10 days to 1.59 at 21 days and decline thereafter. These data generally agree with earlier studies on the in vivo synthesis of the myelin basic proteins in mice. An interesting feature of the time course was that incorporation of [ 3 H]leucine into the purified myelin basic proteins relative to incorporation into total protein in the homogenate increased almost 2‐fold during the course of the 30‐min incubation. This suggested that post‐translational processing of at least one of the two basic proteins was occurring. To examine this possibility further, experiments were conducted in which incorporation was allowed to proceed for 2–5 min, before being inhibited with puromycin; the incubation was then continued for up to 25 min longer. Although total incorporation was inhibited immediately after puromycin addition, label was found to continue to accumulate in the basic proteins to the extent of 30–100% above controls. These data support the notion that the MBPs are synthesized as precursors and then processed to yield authentic myelin basic proteins and that this processing can occur in vitro .