Several small GTP-binding proteins are strongly down-regulated in simian virus 40 (SV40) transformed human keratinocytes and may be required for the maintenance of the normal phenotype.
High resolution two-dimensional (2-D) gel electrophoresis in combination with the blot overlay nucleotide binding assay was used to reveal low molecular weight GTP-binding proteins expressed by primary cultured, normal human keratinocytes. Forty one small GTP-binding proteins (30 isoelectric focusing, IEF; and 11 nonequilibrium pH gradient electrophoresis, NEPHGE) ranging in molecular weights from 18,000 to 30,000 and isoelectric points from 4.4 to 8.0 were detected and mapped in the master human keratinocyte database. Four GTP-binding proteins were identified by 2-D gel immunoblotting and these correspond to rap1 and 2 and two forms of rab6. ras-Proteins are most likely present in the [alpha 32P]GTP 2-D gel blots but their levels may be too low to be detected by immunoblotting. Quantitative changes in the relative expression levels of [alpha 32P]GTP-binding proteins in normal proliferating and simian virus 40 (SV40) transformed human keratinocytes (K 14) were determined by scintillation counting of the radioactive spots excised from the nitrocellulose blots. The results showed that thirteen of these proteins were not expressed in transformed K14 keratinocytes, implying that they may play a role in the maintenance of the normal cell phenotype.
Gromov
P S
PS
Institute of Medical Biochemistry, Aarhus University, Denmark.
Celis
J E
JE
D001345:Autoradiography / Q000379:methods; D002454:Cell Differentiation; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010641:Phenotype; D010761:Phosphorus Radioisotopes; D013539:Simian virus 40 / Q000235:genetics*; D013462:Sulfur Radioisotopes
D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D006160:Guanosine Triphosphate; D008715:Methionine; D019204:GTP-Binding Proteins