Artificial fatty acylation of proteins has attracted significant attention during the last decade as a method for modification of protein specificity and efficacy of action on mammalian cells (A. V. Kabanov and V. Yu. Alakhov (1994) J. Contr. Release 28, 15-35). Horse radish peroxidase (HRP) is used in this work to study the interaction of a fatty acylated protein with various mammalian cells. The HRP is modified with the chloranhydride of the stearic acid in the reversed micelles of sodium bis-(2-ethylhexyl)sulfosuccinate (Aerosol OT) in octane, a convenient protocol allowing production of protein molecules with a controlled, low modification degree (A.V. Kabanov et al. (1987) Ann. N. Y. Acad. Sci. 501, 63-66). The influence of the hydrophobic group on the binding and internalization of HRP in MDCK, P3-X63-Ag8, CHO, and HepG2 cells is examined. The major results are as follows: (i) the fatty acylation of a protein significantly enhances its binding to all tested mammalian cell lines, with a line-specific efficiency; (ii) the binding efficiency can be modified by changing growth conditions in a defined medium; (iii) along with the enhancement of protein adsorption on the plasma membrane, fatty acylation increases internalization of the protein during incubations at 37 degrees C; (iv) internalized protein was observed in endocytic vesicles; no evidence was obtained for a cytoplasmic distribution. These results are discussed in connection with previously observed effects of the fatty acylated proteins on cell activity.
Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot‐immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot‐immunodetection, was found to be markedly biased towards a few RNA polymerase subunits: A135, A49, A43, and A14.5. A different monoclonal antibody directed against the largest subunit, A190, was obtained by immunizing a mouse with RNA polymerase A dissociated into its subunits with SDS. Two antibodies, which probably recognized the same antigenic determinant on subunit A135, inhibited in vitro RNA synthesis. Inhibition was prevented by preincubation of the enzyme with DNA, suggesting a role for the A135 subunit in template binding. The antibody directed against A14.5 interacted with the A14.5 kd subunit present in all three forms of the yeast nuclear RNA polymerases but did not interfere with RNA polymerase activity. These antibody probes will be useful to study subunit function in reconstituted transcription systems.
Anti-peroxidase antibody (Ab)-secreting hybrids have been produced by fusion of peroxidase (PO)-immunized mouse lymph node cells and immunoglobulin (Ig)-secreting P3-X63-Ag8 (X63) myeloma cells. Identification of Ab-secreting hybrids can be performed as early as day 5 after cell fusion by the hemolytic plaque assay. Immediately after identification, hybrids were directly isolated, by means of a micropipette, into Terasaki microchambers containing nutrient medium and a thymocyte filler layer. The yield of secreting hybrids is improved by using this procedure. All the cells of the PO 772 C2 clone show the same ultrastructural pattern and immunocytological properties; they are proplasmocytes, as are the parental X63 cells; they present intracisternae Ab and show no Ig or Fc receptors at the cell surface. Over 90% of viable PO 772 C2 cells form specific plaques. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the cells of this clone secrete Ab; the secreted Ig are formed with chi and gamma 1 chains from the parental X63 cells and specific L and H chains from the lymphoid parent. These biological investigations demonstrate the relative stability of the PO 772 C2 clone secreting anti-peroxidase antibody.
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