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  • PKG Substrate
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PKG Substrate

PKGSubstrate是一种cGMP依赖性的蛋白激酶(PKG)的选择性底物。

原价
¥1200-4175
价格
960-3340
PKG Substrate的二维码

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  • 货号: ajcx13190
  • CAS: N/A
  • 别名:
  • 分子式: C35H67N17O11
  • 分子量: 902.01
  • 纯度: >98%
  • 溶解度: Soluble in Water
  • 储存: Store at -20°C
  • 库存: 现货

Background

PKG Substrate is a selective substrate for cGMP-dependent protein kinase (PKG).


Incorporation of [33P]ATP into the synthetic peptide PKG substrate RKRSRAE is measured. N6-benzyl-ATP inhibits kinase activity of PKG Iα gatekeeper mutants but not WT. The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and PKG[1].


[1]. Wong A, et al. Cyclic GMP-dependent stimulation of serotonin transport does not involve direct transporter phosphorylation by cGMP-dependent protein kinase. J Biol Chem. 2012 Oct 19;287(43):36051-8.

Protocol

Kinase experiment:

Kinase activity is measured by determining the amount of 33P radioactivity incorporated from [33P]ATP or [33P]N6-benzyl-ATP into a PKG specific peptide substrate (RKRSRAE). The standard 75 μL assay mixture contains 0.15 μCi of [33P]ATP, 10 μM ATP, 15 μM PKG peptide substrate, 2 μM PKI (a synthetic peptide inhibitor of cAMP-dependent protein kinase), 1 μg of purified kinase, and 100 μM 8-Br-cGMP in 50 mM HEPES buffer, pH 7.4, containing 10 mM MgCl2, 0.1% Tween 20, and 1 mM DTT. After incubation at 30°C for 2 min, the reaction is immediately put on ice, and 20 μL of the assay mixture is spotted onto P81 phosphocellulose paper and then quenched in 0.42% H3PO4. The paper is further washed three times in 0.42% H3PO4 for 10 min with gentle agitation and rinsed once with acetone. After air drying, radioactivity on the paper is measured with a Beckman LS6500 liquid scintillation counter. For measuring the effect of N6-benzyl-ATP on the activity of PKG I utilizing ATP as a co-substrate, unlabeled N6-benzyl-ATP is added to each reaction at the indicated concentrations. Saturation kinetic analyses for Km and Vmax with ATP or N6-benzyl-ATP are performed over a concentration range (0.0015-100 μM) by adding unlabeled ATP or N6-benzyl-ATP to a given amount of [33P]ATP or [33P]N6-benzyl-ATP, respectively[1].

参考文献:

[1]. Wong A, et al. Cyclic GMP-dependent stimulation of serotonin transport does not involve direct transporter phosphorylation by cGMP-dependent protein kinase. J Biol Chem. 2012 Oct 19;287(43):36051-8.

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