D-Ala-Lys-AMCA
目录号 : GC34896
D-Ala-Lys-AMCA是一种已知的质子偶联寡肽转运蛋白1(pept1)底物，可发出蓝色荧光。D-Ala-Lys-AMCA可能被转运到肝癌细胞和Caco-2细胞中。
Cas No.:375822-19-8
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
D-Ala-Lys-AMCA is a known proton-coupled oligopeptide transporter 1 (PEPT1) substrate that emits blue fluorescence. D-Ala-Lys-AMCA may be transported into liver cancer cells and Caco-2 cells based on fluorescence analysis[1].
[1]. Gong Y, et al. Specific expression of proton-coupled oligopeptide transporter 1 in primary hepatocarcinoma-anovel strategy for tumor-targeted therapy. Oncol Lett. 2017 Oct;14(4):4158-4166.
| Cas No. | 375822-19-8 | SDF | |
| Canonical SMILES | NC1=CC=C(C(C)=C(CC(NCCCC[C@H](NC([C@@H](C)N)=O)C(O)=O)=O)C(O2)=O)C2=C1 | ||
| 分子式 | C21H28N4O6 | 分子量 | 432.47 |
| 溶解度 | Soluble in DMSO | 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 | 2.3123 mL | 11.5615 mL | 23.123 mL |
| 5 mM | 0.4625 mL | 2.3123 mL | 4.6246 mL |
| 10 mM | 0.2312 mL | 1.1561 mL | 2.3123 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。
Intestinal peptide transport: ex vivo uptake studies and localization of peptide carrier PEPT1
Am J Physiol Gastrointest Liver Physiol 2001 Sep;281(3):G697-704.PMID:11518682DOI:10.1152/ajpgi.2001.281.3.G697.
The nature of protein breakdown products and peptidomimetic drugs such as beta-lactams is crucial for their transmembrane transport across apical enterocyte membranes, which is accomplished by the pH-dependent high-capacity oligopeptide transporter PEPT1. To visualize oligopeptide transporter-mediated uptake of oligopeptides, an ex vivo assay using the fluorophore-conjugated dipeptide derivative D-Ala-Lys-N(epsilon)-7-amino-4-methylcoumarin-3-acetic acid (D-Ala-Lys-AMCA) was established in the murine small intestine and compared with immunohistochemistry for PEPT1 in murine and human small intestine. D-Ala-Lys-AMCA was accumulated by enterocytes throughout all segments of the murine small intestine, with decreasing intensity from the top to the base of the villi. Goblet cells did not show specific uptake. Inhibition studies revealed competitive inhibition by the beta-lactam cefadroxil, the angiotensin-converting enzyme inhibitor captopril, and the dipeptide glycyl-glutamine. Controls were performed using either the inhibitor diethylpyrocarbonate or an incubation temperature of 4 degrees C to exclude unspecific uptake. Immunohistochemistry for PEPT1 localized immunoreactivity to the enterocytes, with the highest intensity at the apical membrane. This is the first study that visualizes dipeptide transport across the mammalian intestine and indicates that uptake assays using D-Ala-Lys-AMCA might be useful for characterizing PEPT1-specific substrates or inhibitors.
Specific expression of proton-coupled oligopeptide transporter 1 in primary hepatocarcinoma-a novel strategy for tumor-targeted therapy
Oncol Lett 2017 Oct;14(4):4158-4166.PMID:28943923DOI:10.3892/ol.2017.6724.
Proton-coupled oligopeptide transporter 1 (PEPT1) is a membrane protein which expressed predominantly in intestine and recognized as the target of dietary nutrients (di/tripeptide) or peptidomimetic drug for delivery. The information on the existence of PEPT1 in carcinomas were limited. Our study aimed to investigate the expression profile and transport activity of PEPT1 both in human hepatocarcinoma tissues and cell lines. Western blotting and an immunofluorescence assay revealed the high level of PEPT1 protein expression in hepatocarcinoma Bel-7402, SMMC-7721, HepG2, HEP3B, SK-HEP-1 cell lines. Quantitative real time PCR showed the mRNA expression of PEPT1 in Bel-7402, SMMC-7721, HepG2, HEP3B, SK-HEP-1 cells. High level PEPT1 expression in hepatocarcinoma patient samples were observed by Immunohistology and showed a significant correlation between protein level and pathological grade. Functional activities were also studied using D-Ala-Lys-AMCA (a substrate of peptide transporter) in above five hepatocarcinoma cell lines. The uptake tests performed by fluorescent microscopy suggested that PEPT1 can transport both D-Ala-Lys-AMCA into the hepatocarcinoma cells and the uptake can be competitively inhibited by three PEPT1 substrates (Gly-sar, Gly-gln and Glyglygly). In conclusion, our findings provided the novel information on the expression and function of PEPT1 in human hepatocarcinoma and expanded the potential values for tumor specific drug delivery.
Expression profile and functional activity of peptide transporters in prostate cancer cells
Mol Pharm 2013 Feb 4;10(2):477-87.PMID:22950754DOI:10.1021/mp300364k.
Peptide transporters are expressed predominantly in intestinal and renal epithelial cells. The functional expression of peptide transporters is also identified in other types of tissues, such as glia cells, macrophages, and the epithelia of the bile duct, the lungs, and the mammary glands. However, their presence and role are poorly understood in carcinomas. We explored the expression profile and functional activity of peptide transporters in the prostate cancer cell lines LNCaP, PC-3, and DU145. Quantitative real time RT-PCR (qRT-PCR) and Western blot were used to evaluate the expression profile of peptide transporter 1 (PEPT1), peptide transporter 2 (PEPT2), peptide histidine transporter 1 (PHT1), and peptide histidine transporter 2 (PHT2) in these cells. LNCaP expresses high levels of PEPT2 and PHT1, while PC-3 demonstrates strong expression of PEPT1 and PHT1. DU145 shows only weak expression of PEPT1 and PHT1. Functional activities were studied in these cell lines using radiolabeled glycylsarcosine ([(3)H]Gly-Sar) and l-histidine ([(3)H]-l-histidine). The uptake of [(3)H]Gly-Sar and [(3)H]-l-histidine was time- and pH-dependent. A kinetic study showed that the uptake of Gly-Sar and l-histidine is saturable over the tested concentration range. The binding affinity (K(m)) and the maximal velocity (V(max)) exhibited in the three cell lines were consistent with the expression profiles we observed in qRT-PCR and Western blot analysis. A competitive inhibition study revealed that peptide transporters in prostate cancer cells exhibited broad substrate specificity with a preference for hydrophobic dipeptides, such as Leu-Leu. Fluorescence microscopy study revealed that the fluorescent dipeptide probe D-Ala-Lys-AMCA (a substrate of peptide transporters) specifically accumulated in the cytoplasm of LNCaP and PC-3, but not DU145 cells. Inhibiting the peptide transporter activity by Gly-Sar suppressed the growth of LNCaP and PC-3 cells. Our study indicated that PC-3 cells can be established as a new cell culture model for PEPT1 study, and LNCaP can be used as a model for PEPT2 study. Moreover, our results suggested that peptide transporters are overexpressed in prostate cancer cells and can be adopted as a promising target for tumor-specific drug delivery.
Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells
Am J Physiol 1998 Dec;275(6):C1573-9.PMID:9843719DOI:10.1152/ajpcell.1998.275.6.C1573.
The reabsorption of filtered di- and tripeptides as well as certain peptide mimetics from the tubular lumen into renal epithelial cells is mediated by an H+-coupled high-affinity transport process. Here we demonstrate for the first time H+-coupled uptake of dipeptides into the renal proximal tubule cell line LLC-PK1. Transport was assessed 1) by uptake studies using the radiolabeled dipeptide D-[3H]Phe-L-Ala, 2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and 3) by measurement of intracellular pH (pHi) changes as a consequence of H+-coupled dipeptide transport. Uptake of D-Phe-L-Ala increased linearly over 11 days postconfluency and showed all the characteristics of the kidney cortex high-affinity peptide transporter, e.g., a pH optimum for transport of D-Phe-L-Ala of 6.0, an apparent Km value for influx of 25.8 +/- 3. 6 microM, and affinities of differently charged dipeptides or the beta-lactam antibiotic cefadroxil to the binding site in the range of 20-80 microM. pHi measurements established the peptide transporter to induce pronounced intracellular acidification in LLC-PK1 cells and confirm its postulated role as a cellular acid loader.
Renal assimilation of short chain peptides: visualization of tubular peptide uptake
Pharm Res 2002 Aug;19(8):1209-14.PMID:12240948DOI:10.1023/a:1019810512519.
Purpose: Renal assimilation of short chain peptides plays an important role in systemic protein metabolism and amino acid homeostasis. The transepithelial peptide transport across the apical membrane of tubular cells is mediated almost exclusively by pH-dependent H(+)-peptide symport pathways. The current study was designed to identify by visualization functional peptide transport activity along the nephron structures. Methods: Visualization of peptide uptake was achieved by using the fluorescent dipeptide derivative D-Ala-Lys-AMCA and unlabelled cefadroxil and glycylglutamine as transport competitors to demonstrate specificity. To confirm these assays, rat specific cRNA probes were synthesized and non-isotopic high-resolution in-situ-hybridization and northern blot analysis were carried out to demonstrate the expression of the high-affinity peptide transporter PEPT2. Results: The reporter molecule was accumulated by cells of the proximal tubulus but not in glomerular or endothelial cells. Inhibition studies revealed competitive inhibition of D-Ala-Lys-AMCA uptake by the betalactam cefadroxil and the dipeptide glycylglutamine. The control organs intestine and spleen did not show uptake of the systemically administered molecule. Non-isotopic mRNA in-situ-hybridization, using an antisense probe for rat PEPT2 confirmed up-take assays by identifying PEPT2 expression throughout segments of the straight proximal tubule at the inner cortex and outer stripe. Conclusions: We demonstrate for the first time renal in vivo transport activity of a dipeptide that allows cells that participate in peptide reabsorption to be visualized. This functional assay may be used to investigate renal peptide transport mechanisms and test new compounds that are transported via proton-driven peptide transporters.