Motoi Miyazu

Oxidatively induced Cu for Mn exchange in protein phosphatase 1γ: A new method for active site analysis

Protein phosphatase 1gamma, a serine/threonine phosphatase, is a metalloprotein that coordinates two Mn(2+) in the active site when expressed in Escherichia coli in a buffer containing MnCl(2). Herein, we report on the oxidatively induced copper for manganese exchange in protein phosphatase 1gamma, thus enabling firm confirmation of the four histidine (His) amino acid residues (His66, His125, His173, and His248) involved in metal coordination. By exchanging manganese with copper the oxidation yields for the peptides increased dramatically, thus simplifying detection of the oxidized peptides and analysis of the oxidation sites within the oxidized peptides. We also found that when copper was added during the oxidation process a new metal coordination center was formed at cysteine 39, 105, 140, and 155.

Examination of signalling pathways involved in muscarinic responses in bovine ciliary muscle using YM-254890, an inhibitor of the Gq/11 protein

In the ciliary muscle, the tonic component of the contraction produced by cholinergic agonists is highly dependent on Ca2+ provided by influx through non‐selective cation channels (NSCCs) opened by stimulation of M3 muscarinic receptors. We examined effects of YM‐254890 (YM), a Gq/11‐specific inhibitor, on contraction, NSCC currents and [Ca2+]i elevation induced by carbachol (CCh).