Fatty acid acylated peroxidase as a model for the study of interactions of hydrophobically-modified proteins with mammalian cells.
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.
Slepnev
V I
VI
Laboratory of Biopolymer Chemistry, Russian Research Center of Molecular Diagnostics and Therapy (RCMDT), Moscow, Russia.
Phalente
L
L
Labrousse
H
H
Melik-Nubarov
N S
NS
Mayau
V
V
Goud
B
B
Buttin
G
G
Kabanov
A V
AV
D000215:Acylation; D000327:Adsorption; D000818:Animals; D001693:Biological Transport, Active; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004285:Dogs; D005227:Fatty Acids / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D008823:Micelles; D008954:Models, Biological; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D005227:Fatty Acids; D008823:Micelles; D011506:Proteins; D006735:Horseradish Peroxidase