Trophic effect of a sciatic nerve extract on fast and slow myosin heavy chain synthesis.

Myosin heavy chain (MHC) synthesis in cultures from chick pectoralis muscle cells was determined by [35S]methionine incorporation. Two types of MHC, migrating as 200,000-dalton components on sodium dodecyl sulfate polyacrylamide gels, were distinguished with antibodies against adult fast and slow MHC. Their synthesis was revealed by autoradiography. The effect of a sciatic nerve extract on the synthesis of the two types of MHC was also determined. Control experiments show that fast MHC is primarily synthesized in 48-h cultures. At a later stage of development (5- to 7-day cultures), slow MHC is also produced. The nerve extract promotes muscle cell differentiation and stimulates the synthesis of the slow type of MHC at an earlier stage of development (i.e., at 48 h as compared with 5-7 day in controlled cultures). It is concluded therefore that presumptive fast muscle cells in culture synthesize initially fast MHC and later both types of MHC (slow and fast). These results also suggest that the sciatic nerve extract is capable either of activating the transcription of the structural gene for slow MHC or of activating the translation of preexisting messenger RNA coding for this protein.

Cytoplasmic utilization of liposome-encapsulated myosin heavy chain messenger ribonucleoprotein particles. During muscle cell differentiation.

Myosin heavy chain messenger ribonucleoprotein particles (MHC mRNPs) have been isolated. Characterization of the RNA components revealed an mRNA of approximately the same size as tobacco mosaic virus RNA and three low molecular weight components. The protein consists of 9-10 major bands ranging in molecular weight between 22,000 and 130,000. The messenger contained in these mRNPs was found to direct the synthesis of both fast-muscle and slow-muscle MHC in a cell-free system. When MHC [3H]mRNPs were encapsulated into liposomes and subsequently delivered to myoblasts and myotubes, the mRNPs were taken up by the cells at both stages of differentiation. However, the MHC [3H]mRNPs taken up by the myoblasts remained as free cytoplasmic particles (80-120S), whereas in myotubes the incorporated mRNP RA was associated with polysomes. The results indicate that MHC mRNPs contain a repressor molecule(s) and that myotubes possess a mechanism for activating these mRNPs that is absent from myoblasts.

Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.