Adenosine Monophosphate → Camp
Reaction equation
[CH:1]1[N:6]=[C:5]([NH2:7])[C:4]2[N:8]=[CH:9][N:10]([C@@H:11]3[O:15][C@H:14]([CH2:16][O:17][P:18]([OH:21])([OH:20])=[O:19])[C@@H:13](O)[C@H:12]3[OH:23])[C:3]=2[N:2]=1.N1C(N)=C2C(=NC=N2)NC=1>>[CH:1]1[N:6]=[C:5]([NH2:7])[C:4]2[N:8]=[CH:9][N:10]([C@@H:11]3[O:15][C@@H:14]4[CH2:16][O:17][P:18]([OH:20])([O:21][C@H:13]4[C@H:12]3[OH:23])=[O:19])[C:3]=2[N:2]=1Participants
Procedure
The adenylate cyclase activity induced by each of the tested polypeptides of the invention was also measured in SaOS-2/B 10 cells as described previously (Rodain et al., J. Clin. Invest. (1983) 72,1511; Goldman, et al., Endocrinology (1988) 123, 1468). Conflúnt SaOS-2/B10 cells in 24 wells plates were incubated with 0.5 μCi [3H]adenine (26.9 Ci/mmol, New England Nuclear, Boston, Mass. in fresh medium at 37° C. for 2 hrs. and washed twice with Hank's solution. The cells were treated with 1 mM IBMX (isobutylmethylxanthine, Sigma, St. Louis, Mo.) in fresh medium for 15 min. and a tested PTH analog was added to the medium to incubate for 5 min. The reaction was stopped by the addition of 1.2M TCA followed by sample neutralization with 4N KOH. Cyclic AMP was isolated by the two-column chromatographic method (Salomon, et al., Anal. Biochem. (1974) 58, 541). The radioactivity was counted in a scintillation counter (Liquid scintillation counter 2200CA, PACKARD, Downers Grove, Ill.). The EC50s (half maximal stimulation of adenylate cyclase) were calculated for the tested PTH analogs and are shown in Table 4 below:
Workup
- washand washed twice with Hank's solution
- additionThe cells were treated with 1 mM IBMX (isobutylmethylxanthine, Sigma, St. Louis, Mo.) in fresh medium for 15 min.
- additiona tested PTH analog was added to the medium
- waitto incubate for 5 min
- additionThe reaction was stopped by the addition of 1.2M TCA
Sources & evidence
- ORD record
- ord-f65a4716835240ba91ec6cbb46df1a58
- Source dataset
- uspto-grants-2010_09
- Patent number
- US07799757B2
Reaction notes
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