Expression and Purification of the Extracellular Ligand-Binding Domain of the Atrial Natriuretic Peptide (ANP) Receptor: Monovalent Binding with ANP Induces 2:2 Complexes

心钠素 化学 受体 跨膜结构域 2尼泊尔卢比 细胞外 配体(生物化学) 生物化学 结合位点 利钠肽 生物 内科学 内分泌学 医学 心力衰竭
作者
Kunio S. Misono,Natarajan Sivasubramanian,Kathleen L. Berkner,Xiaolun Zhang
出处
期刊:Biochemistry [American Chemical Society]
卷期号:38 (2): 516-523 被引量:40
标识
DOI:10.1021/bi982127v
摘要

The receptor for atrial natriuretic peptide (ANP) is a type-I transmembrane protein containing an extracellular ligand-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. Binding of ANP to the extracellular domain causes activation of the GCase domain by an as yet unknown mechanism. To facilitate studies of the receptor structure and signaling mechanism, we have expressed the extracellular ANP-binding domain of rat ANP receptor (NPR-ECD) in a water-soluble form. NPR-ECD was purified to homogeneity by ANP-affinity chromatography. SDS−PAGE gave a single 61-kDa band, which coincided with a radioactive band obtained by photoaffinity-labeling with N4α-azidobenzoyl-125I-ANP(4−28). Edman degradation gave a single amino-terminal sequence expected for the mature protein. Both trifluoromethanesulfonic acid and peptide-N-glycosidase F treatments yielded a 50-kDa band, indicating N-glycosylation. The molecular mass of 57 725 Da determined by mass spectrometry indicates the carbohydrate content at 16%. NPR-ECD bound ANP with an affinity comparable to that of the full-length receptor. The ligand selectivity of NPR-ECD (in the order ANP > brain natriuretic peptide ≫ C-type natriuretic peptide) was also similar to that of the full-length receptor. HPLC gel filtration of NPR-ECD gave a peak with an apparent mass of 74 kDa. Preincubation with ANP generated a new 150-kDa peak with a concomitant decrease of the 74-kDa peak. This shift in peak positions was ANP concentration-dependent and was complete at the NPR-ECD-to-ANP molar ratio of 1:1, indicating equimolar binding. The change in the apparent native molecular weight from 74 to 150 kDa suggests that binding causes dimerization of the NPR-ECD:ANP complex to yield an [NPR-ECD:ANP]2 complex.
最长约 10秒,即可获得该文献文件

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
PDF的下载单位、IP信息已删除 (2025-6-4)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
xixi发布了新的文献求助10
刚刚
1秒前
研友_LjbjzL发布了新的文献求助10
1秒前
2秒前
2秒前
研友_ZzwoR8完成签到 ,获得积分10
2秒前
卡卡西应助小小小何77采纳,获得20
3秒前
王杏利发布了新的文献求助10
3秒前
1234发布了新的文献求助10
3秒前
筱晓完成签到,获得积分0
4秒前
4秒前
Cala洛~完成签到 ,获得积分10
5秒前
车访枫完成签到 ,获得积分10
5秒前
小王爱学习给小王爱学习的求助进行了留言
5秒前
量子星尘发布了新的文献求助10
6秒前
YYT发布了新的文献求助10
6秒前
留白完成签到,获得积分10
6秒前
8秒前
韦凌青发布了新的文献求助10
8秒前
1111jfdasfkdanf完成签到 ,获得积分10
8秒前
XZZ发布了新的文献求助10
9秒前
Alan完成签到 ,获得积分10
10秒前
心肝宝贝甜蜜饯完成签到,获得积分10
11秒前
慕青应助Cici采纳,获得10
11秒前
BP完成签到,获得积分10
11秒前
13秒前
李二牛发布了新的文献求助10
14秒前
韦凌青完成签到,获得积分10
14秒前
研友_VZG7GZ应助堵门洞采纳,获得10
15秒前
16秒前
17秒前
岩追研完成签到,获得积分10
18秒前
18秒前
YYT完成签到,获得积分10
19秒前
奥特曼发布了新的文献求助10
19秒前
20秒前
21秒前
21秒前
阳佟半仙发布了新的文献求助10
21秒前
21秒前
高分求助中
A new approach to the extrapolation of accelerated life test data 1000
Cognitive Neuroscience: The Biology of the Mind 1000
Technical Brochure TB 814: LPIT applications in HV gas insulated switchgear 1000
Toward a Combinatorial Approach for the Prediction of IgG Half-Life and Clearance 500
ACSM’s Guidelines for Exercise Testing and Prescription, 12th edition 500
Picture Books with Same-sex Parented Families: Unintentional Censorship 500
Nucleophilic substitution in azasydnone-modified dinitroanisoles 500
热门求助领域 (近24小时)
化学 材料科学 医学 生物 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 遗传学 基因 物理化学 催化作用 冶金 细胞生物学 免疫学
热门帖子
关注 科研通微信公众号,转发送积分 3969940
求助须知:如何正确求助?哪些是违规求助? 3514642
关于积分的说明 11175298
捐赠科研通 3249947
什么是DOI,文献DOI怎么找? 1795178
邀请新用户注册赠送积分活动 875617
科研通“疑难数据库(出版商)”最低求助积分说明 804891