Identification of cytosolic protein tyrosine kinases of human prostate by renaturation after SDS/PAGE.
The identification of protein tyrosine kinases (PTKs) was successfully achieved by renaturation in gels after SDS/PAGE. To this effect, samples were mixed with a PTK substrate, namely the polydispersed co-polymer of glutamic acid and tyrosine [poly(Glu, Tyr), M(r) from 30,000 to 94,000], and were simultaneously submitted to electrophoresis. Following guanidine hydrochloride denaturation, renaturation and phosphorylation with [gamma-32P]ATP, kinase activity was detected by autoradiography. When applied to cytosol from human hyperplastic prostate, eleven protein kinases were detected, among which one major (M(r) 50,000) and two minor proteins (M(r) 40,000 and 38,000) were identified as PTKs by the presence of phosphotyrosine. Incubation of the gel in hot alkali after glutaraldehyde cross-linking almost completely eliminated the detection of non-PTK enzymes. On the other hand, in the absence of poly(Glu,Tyr), no PTK activity was detected. Partial purification of cytosolic PTKs indicates that the native M(r) of the major phosphotransferase was 44,000, as estimated by gel filtration following ammonium sulphate precipitation and anion-exchange chromatography. Upon renaturation after electrophoresis, this fraction showed only one major band active on poly(Glu,Tyr) which was associated with the polypeptide of M(r) 50,000. This enzyme was also identified following two-dimensional electrophoresis and renaturation in the presence of poly(Glu,Tyr), allowing the determination of a pI in the range 7.5-7.8. Thus PTKs can be easily renatured following electrophoresis and rapidly identified on the basis of their M(r) and pI in both crude or partially purified preparations. With the crucial role played by PTKs in the activation of cell function and carcinogenesis, this procedure could be useful in the identification of such enzymes and in distinguishing them from their substrates in gels.
Durocher
Y
Y
Department of Biochemistry, University of Montréal, Québec, Canada.
Chapdelaine
A
A
Chevalier
S
S
D000596:Amino Acids / Q000032:analysis; D002458:Cell Fractionation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D011467:Prostate / Q000201:enzymology*; D011489:Protein Denaturation; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases