Details

Autor(en) / Beteiligte

• Cortay, Jean-Claude
• Nègre, Didier
• Cozzone, Alain-Jean

Titel

[17] Analyzing protein phosphorylation in prokaryotes

Ist Teil von

• Methods in Enzymology, 1991, Vol.200, p.214-227

Ort / Verlag

San Diego, CA: Elsevier Science & Technology

Erscheinungsjahr

1991

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• This chapter focuses on the process of analyzing protein phosphorylation in prokaryotes. The detection of phosphorylated proteins necessitates their initial labeling either in vivo in a culture medium containing ortho-[32P]phosphate or in vitro at the expense of [γ-32P] adenosine triphosphate (ATP). The labeling of cells is achieved in the presence of carrier-free ortho-[32P]phosphate and double-labeling experiments are performed by adding simultaneously either [35S]sulfate or [35S]methionine. Some assays are carried out in the presence of 2–10 mM sodium fluoride, an inhibitor of certain phosphatases, or in the presence of molybdate or vanadate in the micromolar range. After labeling in vivo, cells are collected by low-speed centrifugation, suspended in a buffer made of 10 mM Tris-HCl, pH 7.4, 5 mM MgCl2, and 50 μg/ml pancreatic ribonuclease, then opened by alumina grinding or repeated ultrasonic disruption. In most cases, the analysis of phosphoproteins concerns total cellular preparations. However, experiments have been performed to study phosphoproteins from individual subcellular fractions. For this purpose, the conventional techniques of purification can be used. The techniques more frequently utilized to separate phosphoproteins are polyacrylamide gel electrophoresis and, to a lesser extent, chromatography. One important criterion in demonstrating protein phosphorylation in prokaryotes is showing that phosphoryl groups are covalently bound to certain amino acids of protein substrates and characterizing the nature of the linkage involved. The four major classes of phosphoamino acids (O-phosphomonoesters, phosphoramidates, acyl phosphates, and thiophosphates) are all present in bacterial proteins.