Uv 3101pc uv visible spectrophotometer

The overall experimental procedure was the same as that used for assessing the Michaelis-Menten kinetics, with the addition of 2.4 μM p97 at the start of the reaction. When ASPL was included in the assay, 7.2 μM of ASPL was added into the mixture along with p97. The reaction was monitored at 265 nm and 310 K using a UV-3101PC UV-Visible spectrophotometer (Shimadzu, Japan). Three independent measurements were conducted.

The in vitro methyltransferase activity of VCPKMT was assessed by measuring the decrease in absorbance at 265 nm in a reaction coupled with SAH nucleosidase (EC 3.2.2.9) and adenine deaminase (EC 3.5.4.2), as previously described in the literature.²² (link) In this assay, the SAH, which is produced by VCPKMT, is enzymatically hydrolyzed by SAH nucleosidase, resulting in the formation of S-ribosylhomocysteine and adenine. Furthermore, recombinant adenine deaminase converts the adenine generated from SAH to hypoxanthine and ammonia through a deamination reaction. This deamination process leads to a measurable decrease in absorbance at 265 nm, which can be continuously monitored during the assay. The reaction mixture contained 60 μM s-adenosyl-L-methionine (SAM), 2.76 μM VCPKMT, 208 μM SAH nucleosidase, 0.1 μM adenine deaminase, and 1050 μM manganese sulfate, along with eight different concentrations of p97 N/D1 (N21 - Q458) in 50 mM Tris-HCl (pH 7.5), 5 mM magnesium chloride, 50 mM potassium chloride, and 10 % (v/v) glycerol. The reaction was initiated by adding p97 to the mixture and was monitored at 265 nm and 310 K using a UV-3101PC UV-Visible spectrophotometer (Shimadzu, Japan) in thermostatted 1-cm quartz cuvette for 90 seconds. Three independent measurements were carried out.