Suc-Leu-Leu-Val-Tyr-AMC
(Synonyms: N-琥珀酰基-亮氨酰-亮氨酰-缬氨酰-酪氨酸-7-胺基-4-甲基香豆素) 目录号 : GC44962
A fluorescent substrate of the 20S proteasome and calpain
Cas No.:94367-21-2
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
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Kinase experiment: |
Immunoprecipitation is carried out for the two sets of samples, using the same amount of protein. The 20 S and 26 S proteasome immunoprecipitates are washed with 50 mM Hepes/KOH (pH 7.5), and 50 mM Hepes/KOH (pH 7.5) containing 2 mMATP, respectively, prior to the determination of peptidase activity using 50 μM suc-Leu- Leu-Val-Tyr-AMC as substrate in these buffers[3]. |
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References: [1]. Roberta L. Debiasi, et al. Reovirus-Induced Apoptosis Is Preceded by Increased Cellular Calpain Activity and Is Blocked by Calpain Inhibitors. J Virol. 1999 Jan; 73(1): 695–701. |
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Suc-Leu-Leu-Val-Tyr-AMC is a fluorogenic substrate.
Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY) is a membrane-permeable calpain-specific fluorogenic substrate, pteolytic hydrolysis of the peptidyl-7-amino bond liberates the highly fluorescent 7-amino-4-methylcoumarin (AMC) moiety[1]. The effectof TGF-β on hydrolysis of these substrates (e.g Suc-Leu-Leu-Val-Tyr-AMC) are assessed. Biliary epithelial H69 cells are incubated with 10, 1, 0.1, or 0 ng/mL TGF-β for 24 h. Substrate hydrolysis is then fluorometrically assessed in cytosolic extracts. Basal activity is 1.12, 8.33, and 14.52 nmol AMC/mg protein/min for suc-LLVY-AMC, z-LLE-AMC, and z-LLL-AMC hydrolysis, respectively[2].
References:
[1]. Roberta L. Debiasi, et al. Reovirus-Induced Apoptosis Is Preceded by Increased Cellular Calpain Activity and Is Blocked by Calpain Inhibitors. J Virol. 1999 Jan; 73(1): 695–701.
[2]. Tadlock L, et al. Transforming growth factor-beta inhibition of proteasomal activity: a potential mechanism of growth arrest. Am J Physiol Cell Physiol. 2003 Aug;285(2):C277-85. Epub 2003 Mar 19.
[3]. Gardner RC, et al. Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells. Biochem J. 2000 Mar, 2:447-54.
| Cas No. | 94367-21-2 | SDF | |
| 别名 | N-琥珀酰基-亮氨酰-亮氨酰-缬氨酰-酪氨酸-7-胺基-4-甲基香豆素 | ||
| Canonical SMILES | CC(C1=CC=C(C=C1O2)NC([C@H](CC3=CC=C(C=C3)O)NC([C@@H](NC([C@@H](NC([C@@H](NC(CCC(O)=O)=O)CC(C)C)=O)CC(C)C)=O)C(C)C)=O)=O)=CC2=O | ||
| 分子式 | C40H53N5O10 | 分子量 | 763.9 |
| 溶解度 | DMF: 30 mg/ml,DMSO: 30 mg/ml,DMSO:PBS(pH7.2) (1:1): 0.5 mg/ml,Ethanol: 20 mg/ml | 储存条件 | Store at -20°C, protect from light, stored under nitrogen |
| 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 | 1.3091 mL | 6.5454 mL | 13.0907 mL |
| 5 mM | 0.2618 mL | 1.3091 mL | 2.6181 mL |
| 10 mM | 0.1309 mL | 0.6545 mL | 1.3091 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 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % 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。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Kinetic characterization of the chymotryptic activity of the 20S proteasome
Biochemistry 1996 Apr 2;35(13):3899-908.PMID:8672420DOI:10.1021/bi952262x.
In this paper, we report kinetic studies for the chymotryptic activity of the 20S proteasome. Major observations include the following: (1) Reaction progress curves that are recorded at concentrations of Suc-Leu-Leu-Val-Tyr-AMC greater than about 40 microM are biphasic and characterized by initial velocities that decay by a first-order process to final, steady-state velocities. (2) Also at [Suc-Leu-Leu-Val-Tyr-AMC] > 40 microM, initial and steady-state velocities are smaller than predicted from simple, Michaelis-Menten kinetics. (3) The first-order rate constant for the approach to steady-state has a complex dependence on substrate concentration and decreases sigmoidally as substrate concentration increases. These results indicate that the 20S proteasome is a hysteretic enzyme and is subject to substrate inhibition. To explain these observations we propose a minimal kinetic model with two critical mechanistic features: (1) the 20S proteasome has two cooperative active sites for Suc-Leu-Leu-Val-Tyr-AMC and (2) there are two interconvertible conformers of active 20S proteasome. To probe this mechanism in greater detail, we explored the kinetic mechanism of inhibition of the 20S proteasome-catalyzed hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC by the peptide aldehyde, Ac-Leu-Leu-Nle-H. Our studies reveal a nonlinear dependence of reciprocal steady-state velocity on inhibitor concentration (i.e., parabolic inhibition) as well as a nonlinear dependence of the apparent inhibitor dissociation constant on substrate concentration. Both of these observations are explained by binding of inhibitor at multiple sites on the enzyme. Taken together, the results of this study indicate that the 20S proteasome is a conformationally flexible protein that can adjust to the binding of ligands and that has multiple and cooperative active sites. These results support a view of the proteasome's substrate specificity in which (1) substrates are recognized and hydrolyzed by more than one active site; (2) each active site can bind substrates that possess a variety of P1 residues; and (3) the P1 residue plays a relatively minor role as a specificity determinant. Finally, we interpret the results of this study to suggest that, in vivo, the 20S proteasome requires conformational plasticity for its interactions with regulatory complexes and, after it has combined with appropriate regulatory complexes, to catalyze hydrolysis of proteins.
Synthesis and inhibition study on tripeptide inhibitor modified poly(L-lysine) dendrimers
J Biomater Appl 2012 Jul;27(1):17-26.PMID:21123282DOI:10.1177/0885328210386821.
Peptide dendrimers are attractive nonviral gene vectors. But a biological barrier for their application in gene delivery is the fast degradation catalyzed by proteasomes. Proteasome inhibitors are efficient at prohibiting the degradation of peptide nonviral vectors, thus enhancing gene transfection efficiency. In this study, N(α)-Boc-protected leucine vinyl ester proteasome inhibitor Boc-Leu-Leu-Leu-ve was synthesized by the liquid-phase method and was then immobilized onto poly(L-lysine) dendrimers. Suc-Leu-Leu-Val-Tyr-AMC was used as fluorimetric substrate and the inhibition capacity of Boc-Leu-Leu-Leu-ve immobilized onto G(3) and G(6) poly(L-lysine) dendrimers for the chymotrypsin-like activity of ACHN cell proteasome was tested. The results indicated that both Boc-Leu-Leu-Leu-ve peptide and the peptide immobilized on G(3) dendrimer showed low inhibition capacity when the concentration was below 0.2 μM. When the inhibitor concentrations were increased to 5.0 μM, however, the percentage inhibition of Boc-Leu-Leu-Leu-ve peptide and the peptide immobilized on G(3) dendrimer became about 50% and 25%, and that of peptide immobilized on the G(6) dendrimer was 7.5% only. These results indicated that dendritic structure and linker length could be the main factors affecting proteasome inhibition capacity. The cytotoxicity of the dendritic inhibitors was found to be low. Thus, whilst the synthetic production of poly(L-lysine) dendrimers immobilized with peptide inhibitors was successful and these modified dendrimers could work to inhibit proteasome activity, further studies will need to be carried out to improve inhibition capacity.
Transforming growth factor-beta inhibition of proteasomal activity: a potential mechanism of growth arrest
Am J Physiol Cell Physiol 2003 Aug;285(2):C277-85.PMID:12646415DOI:10.1152/ajpcell.00550.2002.
Although the proteasome plays a critical role in the controlled degradation of proteins involved in cell cycle control, the direct modulation of proteasomal function by growth regulatory signaling has not yet been demonstrated. We assessed the effect of transforming growth factor (TGF)-beta, a potent inhibitor of cell growth, on proteasomal function. TGF-beta selectively decreased hydrolysis of the proteasomal substrate Cbz-Leu-Leu-Leu-7-amido-4-methyl-coumarin (z-LLL-AMC) in a concentration-dependent manner but did not inhibit hydrolysis of other substrates Suc-Leu-Leu-Val-Tyr-AMC (suc-LLVY-AMC) or Cbz-Leu-Leu-Glu-AMC (z-LLE-AMC). An increase in intracellular oxidative injury occurred during incubation with TGF-beta. Furthermore, in vitro hydrolysis of z-LLL-AMC, but not suc-LLVY-AMC, was decreased by hydrogen peroxide. TGF-beta did not increase cellular expression of heat shock protein (HSP)90, a potent inhibitor of z-LLL-AMC hydrolysis in vitro. The physiological relevance of TGF-beta inhibition of proteasomal activity was studied by assessing the role of z-LLL-AMC hydrolysis on cyclin-dependent kinase inhibitor expression and cell growth. TGF-beta increased expression of p27KIP1 but did not alter expression of p21WAF1 or p16INK4A. The peptide aldehyde Cbz-Leu-Leu-leucinal (LLL-CHO or MG132) potently inhibited z-LLL-AMC hydrolysis in cell extracts as well as increasing p27KIP1 and decreasing cell proliferation. Thus growth inhibition by TGF-beta decreases a specific proteasomal activity via an HSP90-independent mechanism that may involve oxidative inactivation or modulation of proteasomal subunit composition and results in altered cellular expression of key cell cycle regulatory proteins such as p27KIP1.
Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells
Biochem J 2000 Mar 1;346 Pt 2(Pt 2):447-54.PMID:10677365doi
Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (Suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with Suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with Suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.
Proteasomal activity in mammalian spermatozoa
Mol Reprod Dev 2004 Sep;69(1):87-93.PMID:15278908DOI:10.1002/mrd.20152.
The proteasome is a multicatalytic cellular complex, which possess three different enzymatic activities, trypsin-like, chymotrypsin-like, and peptidylglutamyl peptidase. Its function is to remove abnormal or aged proteins. Recently, it has been suggested the participation of the sperm proteasome during mammalian fertilization. In this study, we present evidence that indicates that sperm extracts from several mammalian species, including hamster, mice, rats, bovine, rabbits, and humans all possess proteasome activity. We characterized the three specific activities of the proteasome using specific synthetic substrates and specific proteasome inhibitors. The results indicates that the highest specific activity detected was in mouse sperm toward the trypsin substrates and it was 1,114% of the activity of human sperm toward the chymotrypsin substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC, which was considered as 100%). In all cases, the lowest activity was toward substrates for the peptidylglutamyl peptidase hydrolyzing activity, and it was lowest for rabbit sperm (1.7% of the activity of human sperm toward the chymotrypsin substrate SLLVY-AMC). In addition, specific proteasome inhibitors were able to block all proteasome activities almost 100%, with the exception of clasto-Lactacystin beta-lactone upon rat sperm. All sperm extracts tested evidenced bands of about 29-32 kDa by Western blots using a monoclonal antibody against proteasome subunits alpha 1, 2, 3, 5, 6, and 7. In conclusion, sperm from several mammals possess enzymatic activities that correspond to the proteasome. The proteasome from the different species hold similar but distinctive enzymatic characteristics.