Identification of L-cell growth stimulating antibody as anti-actin.
Anti-L-cell antisera having potent cell growth stimulatory properties were shown by Western blotting to have predominant specificity toward a protein with a molecular weight of 42K which we identified as actin. Extractions of L cells, based upon the known insolubility of cytoskeletal proteins (including actin) in Triton X-100 and the solubility of actin in low ionic strength Ca2+ and ATP-containing buffer, led to actin-enriched preparations that retained immunoreactivity with the anti-L-cell antisera. The 42-kDa antigen binds to deoxyribonuclease I, has a pI = 5.2-5.4, and has an amino acid composition, including the presence of 3-methylhistidine, compatible with compositions determined for actins from other sources. Rabbit antiserum specific for this 42-kDa protein, isolated by SDS-PAGE, reproduced the cell growth stimulation by the anti-L-cell antisera and absorption of the antiserum with purified L-cell actin eliminated this stimulation. Moreover, these antibodies bind to the microfilaments of 3T3 fibroblasts. When purified actins were used as soluble antigen inhibitors of the immune reactivity of antiserum to 42-kDa protein with intact L cells, rabbit thymus actin competed with the surface molecules on L cells and reduced the stimulatory effect of the antiserum by 80% at an actin concentration of 150 micrograms/ml. Chicken muscle actin reduced the antibody stimulation effect by only 24% at the same protein concentration, and mouse muscle actin was ineffective as an inhibitor. The F(ab')2 fraction of anti-42K IgG was effective in stimulating L cells, thus documenting the immune nature of the actin-anti-42K interaction. We conclude that anti-actin antibodies, upon binding to actin-like cell surface determinants on L cells, stimulate cellular metabolism.
D000199:Actins / Q000276:immunology*; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification* / Q000502:physiology; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D002455:Cell Division; D005346:Fibroblast Growth Factors / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D005455:Fluorescent Antibody Technique; D007739:L Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D011817:Rabbits
D000199:Actins; D000906:Antibodies; D005346:Fibroblast Growth Factors