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| Targets |
cAMP-Dependent Protein Kinase Inhibitor-α (5-24) (human, mouse, rabbit, rat) (CAS#: 99534-03-9) targets:
cAMP-dependent protein kinase catalytic subunit (competitive inhibition versus protein substrate): Ki = 2.3 nM (synthetic IP20-amide) [1]; cGMP-dependent protein kinase type I (wild-type): Ki = 160 ± 7 μM [2]; cGMP-dependent protein kinase mutant (Ser555Tyr/Ser559Phe): Ki = 42 ± 0.3 μM [2]. |
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| ln Vitro |
PKI (5-24) is a synthetic peptide of 20 residues that matches the myostatin active site [1]. PKI (5-24) prevents phosphosome cGMP activation at its active site [1]. stronger, with corresponding Ki values of 42 μM and 160 μM [2].
In vitro activities of cAMP-Dependent Protein Kinase Inhibitor-α (5-24) (human, mouse, rabbit, rat) (CAS#: 99534-03-9): • Inhibits cAMP-dependent protein kinase catalytic subunit competitively with respect to protein substrate (Kemptide). The synthetic IP20-amide (identical sequence) has a Ki of 2.3 nM. At 1–5 μM concentration, it does not inhibit casein kinase I, casein kinase II, proteolytically activated protein kinase I, Ca2+-phospholipid-dependent protein kinase, cGMP-dependent protein kinase, Ca2+-calmodulin-dependent protein kinase, or myosin light chain kinase. [1] • Inhibits wild-type cGMP-dependent protein kinase with Ki values of 147, 164, and 171 μM for substrates Kemptide, histone H2B peptide, and PKI(14-22) peptide, respectively (mean Ki = 160 ± 7 μM). Inhibits a mutant cGMP kinase (Ser555Tyr/Ser559Phe) with a mean Ki of 42 ± 0.3 μM. The inhibitory effect is competitive with respect to peptide substrates. [2] • A modified peptide with Phe10 replaced by alanine [PKI(Phe10Ala)] inhibits wild-type cGMP kinase with Ki = 169 ± 27 μM and mutant cGMP kinase with Ki = 160 ± 11 μM, indicating the importance of Phe10 for the increased affinity observed with the mutant kinase. [2] |
| Enzyme Assay |
Enzyme assays for cAMP-Dependent Protein Kinase Inhibitor-α (5-24) (human, mouse, rabbit, rat) (CAS#: 99534-03-9):
[1] Inhibition of cAMP-dependent protein kinase: The reaction mixture contained 25 mM MES buffer (pH 6.8), 5 mM magnesium chloride, 0.25 mg/ml bovine serum albumin, 3.75 mM 2-mercaptoethanol, 125 μM [γ-32P]ATP, 32.5 μM Kemptide, and 3.5 nM bovine heart protein kinase catalytic subunit. The reaction was initiated by adding Kemptide after a 10 min preincubation at 30°C of all other components. Reaction time was 10 min. [32P]phospho-Kemptide was measured. None of the inhibitory peptides were phosphorylated under these conditions. Ki values for tight-binding inhibitors (Ki <150 nM) were determined by Henderson analysis; for others, Lineweaver-Burk reciprocal plots were used. For synthetic IP20-amide, Ki = 2.3 nM via Henderson analysis. [1][2] Inhibition of cGMP-dependent protein kinase: The assay mixture contained 50 mM MES buffer (pH 6.9), 1 mM magnesium acetate, 10 mM NaCl, 10 mM dithiothreitol (DTT), 0.1 mM [γ-32P]ATP (50–100 cpm/pmol), 300 nM PKI(5-24)amide (to inhibit endogenous cAMP kinase in cell extracts), varying concentrations of substrate peptides (Kemptide, histone H2B substrate, or PKI(14-22) substrate), and 5 μg cytosolic extract protein from transfected COS-7 cells expressing wild-type or mutant cGMP kinase. Incubation was for 15 min (Kemptide and H2B substrate) or 3 min (PKI(14-22) substrate) at 30°C. Less than 20% of substrate was phosphorylated. Blank values were obtained using extract from mock-transfected cells. Ki values were determined from Dixon plot intersections of titration curves. For wild-type cGMP kinase, Ki of PKI(5-24) was 160 ± 7 μM; for mutant (Ser555Tyr/Ser559Phe), Ki = 42 ± 0.3 μM. [2] |
| References | |
| Additional Infomation |
Additional information on cAMP-Dependent Protein Kinase Inhibitor-α (5-24) (human, mouse, rabbit, rat) (CAS#: 99534-03-9):
• The peptide corresponds to residues 5–24 of the heat-stable inhibitor protein (PKI). Its high-affinity binding to cAMP-dependent protein kinase is due to two critical domains: an arginine-cluster basic subsite (positions 11-15: Arg-Arg-Asn-Ala) and the N-terminal hydrophobic region (first 6 residues, especially Thr-Phe). The synthetic peptide IP20-amide has the sequence Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp. [1] • The peptide is a specific inhibitor of cAMP-dependent protein kinase and does not affect several other serine/threonine kinases at concentrations up to 5 μM. It is not phosphorylated by the kinase under assay conditions. [1] • Potential applications include: use as a chemically defined reagent for specifically inhibiting cAMP kinase in crude extracts; defining interactive domains of the catalytic subunit; microinjection to block cAMP-mediated events; providing a strongly bound substrate analog for x-ray crystallography; and generating fluorescent or biotinylated derivatives for histochemical and electron microscopic studies of hormonal regulation. [1] • Structural insights: The hydrophobic pocket formed by Tyr235 and Phe239 in the cAMP kinase catalytic subunit is essential for high-affinity binding of PKI(5-24) via Phe10 of the inhibitor. In cGMP kinase, the corresponding residues are Ser555 and Ser559, which account for its weak inhibition. Mutation of these serines to tyrosine and phenylalanine increased PKI affinity 4-fold but did not achieve nanomolar binding, suggesting additional structural differences between the two kinases. [2] |
| Molecular Formula |
C94H148N32O31
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| Molecular Weight |
2222.37672
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| Exact Mass |
2221.1
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| CAS # |
99534-03-9
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| Related CAS # |
PKI(5-24) TFA
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| Appearance |
White to off-white solid powder
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| LogP |
-12
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| SMILES |
CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)CNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](CC3=CC=C(C=C3)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)N
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| InChi Key |
AXOXZJJMUVSZQY-OCDBTFLZSA-N
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| InChi Code |
InChI=1S/C94H148N32O31/c1-11-42(3)70(124-85(150)58(31-50-19-14-13-15-20-50)117-84(149)61(35-67(135)136)116-74(139)44(5)110-81(146)57(32-51-24-26-53(131)27-25-51)119-90(155)73(49(10)130)126-86(151)69(96)47(8)128)88(153)112-45(6)75(140)122-63(40-127)77(142)107-38-65(133)114-55(22-17-29-105-93(99)100)80(145)125-72(48(9)129)87(152)108-39-66(134)113-54(21-16-28-104-92(97)98)78(143)115-56(23-18-30-106-94(101)102)79(144)118-60(34-64(95)132)82(147)111-46(7)76(141)123-71(43(4)12-2)89(154)120-59(33-52-37-103-41-109-52)83(148)121-62(91(156)157)36-68(137)138/h13-15,19-20,24-27,37,41-49,54-63,69-73,127-131H,11-12,16-18,21-23,28-36,38-40,96H2,1-10H3,(H2,95,132)(H,103,109)(H,107,142)(H,108,152)(H,110,146)(H,111,147)(H,112,153)(H,113,134)(H,114,133)(H,115,143)(H,116,139)(H,117,149)(H,118,144)(H,119,155)(H,120,154)(H,121,148)(H,122,140)(H,123,141)(H,124,150)(H,125,145)(H,126,151)(H,135,136)(H,137,138)(H,156,157)(H4,97,98,104)(H4,99,100,105)(H4,101,102,106)/t42-,43-,44-,45-,46-,47+,48+,49+,54-,55-,56-,57-,58-,59-,60-,61-,62-,63-,69-,70-,71-,72-,73-/m0/s1
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| Chemical Name |
(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S,3R)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S,3R)-2-amino-3-hydroxybutanoyl]amino]-3-hydroxybutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]propanoyl]amino]-3-carboxypropanoyl]amino]-3-phenylpropanoyl]amino]-3-methylpentanoyl]amino]propanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]amino]acetyl]amino]-5-carbamimidamidopentanoyl]amino]-5-carbamimidamidopentanoyl]amino]-4-oxobutanoyl]amino]propanoyl]amino]-3-methylpentanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]butanedioic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O : ~25 mg/mL (~11.25 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.4500 mL | 2.2498 mL | 4.4997 mL | |
| 5 mM | 0.0900 mL | 0.4500 mL | 0.8999 mL | |
| 10 mM | 0.0450 mL | 0.2250 mL | 0.4500 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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