Tumor glutaminase purification.
Two alternative purification schemes to obtain the glutaminase from Ehrlich tumor cells in a highly purified form have been developed. One experimental approach is based on conventional and high-performance liquid chromatography fractionation techniques, yielding a 37-fold higher purification than has been previously reported. The method comprises: isolation of mitochondria, solubilization with Triton X-100, ion-exchange and hydroxyapatite chromatography, ammonium sulfate precipitation, and hydrophobic interaction chromatography. A second purification schedule has been optimized employing native polyacrylamide gel electrophoresis, in situ activity staining, and electroelution of the protein band. This approach resulted in a simple and rapid isolation of a 10-fold higher purified glutaminase than before, minimizing also the potential for proteolytic inactivation of the enzyme. The apparent molecular weight of the protein in native form was determined by gel filtration and sucrose density gradient ultracentrifugation. Polyclonal antibodies raised against Ehrlich glutaminase were immunopurified against the pig kidney enzyme. Immunoblot analyses employing these antibodies as well as anti-rat kidney glutaminase antibodies revealed the same pattern of bands seen with the purified enzyme.
Segura
J A
JA
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Málaga, Spain.
Aledo
J C
JC
Gómez-Biedma
S
S
Núñez de Castro
I
I
Márquez
J
J
D000818:Animals; D015153:Blotting, Western; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis; D005972:Glutaminase / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D017830:Octoxynol / Q000494:pharmacology
D017830:Octoxynol; D010450:Endopeptidases; D005972:Glutaminase