PKG Substrate
目录号 : GC34227
PKGSubstrate是一种cGMP依赖性的蛋白激酶(PKG)的选择性底物。
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
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]. |
References: [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. | |
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.
| Cas No. | SDF | ||
| Canonical SMILES | Arg-Lys-Arg-Ser-Arg-Ala-Glu | ||
| 分子式 | C35H67N17O11 | 分子量 | 902.01 |
| 溶解度 | Soluble in Water | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 1.1086 mL | 5.5432 mL | 11.0864 mL |
| 5 mM | 0.2217 mL | 1.1086 mL | 2.2173 mL |
| 10 mM | 0.1109 mL | 0.5543 mL | 1.1086 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
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| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
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Role of G-Substrate in the NO/cGMP/PKG Signal Transduction Pathway for Photic Entrainment of the Hamster Circadian Clock
The mammalian circadian clock at the hypothalamic suprachiasmatic nuclei (SCN) entrains biological rhythms to the 24-h cyclic environment, by encoding light-dark transitions in SCN neurons. Light pulses induce phase shifts in the clock and in circadian rhythms; photic signaling for circadian phase advances involves a nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) pathway, increasing the expression of Period (Per) genes. Effectors downstream of PKG remain unknown. Here we investigate the role of G-substrate (GS), a PKG substrate, in the hamster SCN. GS and phosphorylated G-substrate (p-GS) were present in a subset of SCN cells. Moreover, GS phosphorylation (p-GS/GS ratio) increased in SCN homogenates after light pulses delivered at circadian time (CT) 18 and intraperitoneal treatment with sildenafil, an inhibitor of phosphodiesterase 5 (a cGMP-specific phosphodiesterase). On the other hand, intracerebroventricular treatment with the PKG inhibitor KT5823, reduced photic phosphorylation of GS to basal levels. Since p-GS could act as a protein phosphatase 2 A (PP2A) inhibitor, we demonstrated physical interaction between p-GS and PP2A in SCN homogenates, and also a light-pulse dependent decrease of PP2A activity. Intracerebroventricular treatment with okadaic acid, a PP2A inhibitor, increased the magnitude of light-induced phase advances of locomotor rhythms. We provide evidence on the physiological phosphorylation of GS as a new downstream effector in the NO/cGMP/PKG photic pathway in the hamster SCN, including its role as a PP2A inhibitor.
G-substrate: the cerebellum and beyond
The discovery of nitric oxide (NO) as an activator of soluble guanylate cyclase (sGC) has stimulated extensive research on the NO-sGC-3':5'-cyclic guanosine monophosphate (cGMP)-cGMP-dependent protein kinase (PKG) pathway. However, the restricted localization of pathway components and the lack of information on PKG substrates have hindered research seeking to examine the physiological roles of the NO-sGC-cGMP-PKG pathway. An excellent substrate for PKG is the G-substrate, which was originally discovered in the cerebellum. The role of G-substrate in the cerebellum and other brain structures has been revealed in recent years. This review discusses the relationship between the G-substrate and other components of the NO-sGC-cGMP-PKG pathway and describes the characteristics of the G-substrate gene and protein related to diseases. Finally, we discuss the physiological role of G-substrate in the cerebellum, where it regulates cerebellum-dependent long-term memory, and its role in the ventral tegmental area and retina, where it acts as an effective neuroprotectant.
The Role of NO/sGC/cGMP/PKG Signaling Pathway in Regulation of Platelet Function
Circulating blood platelets are controlled by stimulatory and inhibitory factors, and a tightly regulated equilibrium between these two opposing processes is essential for normal platelet and vascular function. NO/cGMP/ Protein Kinase G (PKG) pathways play a highly significant role in platelet inhibition, which is supported by a large body of studies and data. This review focused on inconsistent and controversial data of NO/sGC/cGMP/PKG signaling in platelets including sources of NO that activate sGC in platelets, the role of sGC/PKG in platelet inhibition/activation, and the complexity of the regulation of platelet inhibitory mechanisms by cGMP/PKG pathways. In conclusion, we suggest that the recently developed quantitative phosphoproteomic method will be a powerful tool for the analysis of PKG-mediated effects. Analysis of phosphoproteins in PKG-activated platelets will reveal many new PKG substrates. A future detailed analysis of these substrates and their involvement in different platelet inhibitory pathways could be a basis for the development of new antiplatelet drugs that may target only specific aspects of platelet functions.
cGMP-binding prepares PKG for substrate binding by disclosing the C-terminal domain
Type I cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) is involved in the nitric oxide/cGMP signaling pathway. PKG has been identified in many different species, ranging from unicel?lular organisms to mammals. The enzyme serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses, ranging from smooth-muscle relaxation to neuronal synaptic plasticity. In the absence of a crystal structure, the three-dimensional structure of the homodimeric 152-kDa kinase PKG is unknown; however, there is evidence that the kinase adopts a distinct cGMP-dependent active conformation when compared to the inactive conformation. We performed mass-spectrometry-based hydrogen/deuterium exchange experiments to obtain detailed information on the structural changes in PKG I alpha induced by cGMP activation. Site-specific exchange measurements confirmed that the autoinhibitory domain and the hinge region become more solvent exposed, whereas the cGMP-binding domains become more protected in holo-PKG (dimeric PKG saturated with four cGMP molecules bound). More surprisingly, our data revealed a specific disclosure of the substrate-binding region of holo-PKG, shedding new light into the kinase-activation process of PKG.
Retinal G-substrate, potential downstream component of NO/cGMP/PKG pathway, is located in subtype of retinal ganglion cells and amacrine cells with protein phosphatases
The aim of this study was to determine the distribution and function of G-substrate, a specific substrate of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-cGMP-dependent protein kinase (PKG) signaling pathway, in normal rat retina and in G-substrate knockout mice. The retinas of adult wild-type rats and mice and G-substrate knockout mice were studied immunohistologically to characterize the upstream and downstream components of the NO-cGMP-PKG pathway. Immunoblot analysis showed that the molecular weight of retinal G-substrate was similar to that of cerebellar G-substrate. In adult rats and mice, retinal G-substrate was located in a subpopulation of amacrine cells and in C38-positive retinal ganglion cells (RGCs) but not in alpha RGCs. In addition, retinal G-substrate was co-expressed with other upstream and downstream signaling components of the NO-cGMP-PKG-G-substrate-phosphatase pathway in the adult retina. Electroretinographic (ERG) analysis demonstrated that there was no significant difference between the ERGs of wild-type and G-substrate knockout mice. These results suggest that retinal G-substrate plays a role as a downstream component of the NO-cGMP-PKG pathway. The co-localization of retinal G-substrate with protein Ser/Thr phosphatases suggests that it acts as an endogenous protein phosphatase inhibitor as in the cerebellum.