In Vivo and In Vitro Phosphorylation Regions of Bone Sialoprotein
In Vivo and In Vitro Phosphorylation Regions of Bone Sialoprotein
复制标题
骨唾液蛋白的体内和体外磷酸化区域
作者：
E. Salih
本研究首次采用多种方法和技术对骨涎蛋白（bone saloprotein， BSP）的体内和体外磷酸化区域进行了评价。体外磷酸化位点采用纯化酪蛋白激酶II （CKII）对天然BSP进行32p标记，然后进行肽定位和固相n端序列分析。体内磷酸化位点通过(i)与BSP蛋白水解酶的1-S-[14 C]羧甲基二硫苏糖醇（[14 C] CM-DTT）衍生化确定，然后进行分离和n端肽序列分析；（ii）利用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）分析天然BSP的蛋白水解肽。原生BSP通过CKII吸收了~2.5 mol /mol的磷酸，分布在肽图中磷酸化程度不同的4个主肽峰和3个肩峰上。使用[14 C] CM-DTT硫醇试剂进一步研究表明，天然和去糖基化的BSP分别吸收了5.84和5.80 mol的14 C/mol的BSP。这证实了BSP自然（体内）磷酸化位点约为5.8 mol P-Ser/mol，磷酸化位点和糖基化位点之间没有重叠。5.8 mol P-Ser/mol BSP反映了自然发生磷酸化的总摩尔数，在体内被CKII （4.1 mol）、蛋白激酶C （0.9 mol）和cgmp依赖性激酶（0.8 mol）磷酸化。体外（32 P）和体内（14 C）磷酸化肽的肽n端序列分析表明，磷酸化残基主要位于蛋白质的n端一半，包括CKII的识别序列，例如LESDEENGVFK（残基12-22）。
The present study for the first time evaluated both the in vitro and in vivo phosphorylation regions of bone sialoprotein (BSP) by utilizing multiple approaches and techniques. The in vitro phosphorylation sites were determined by 32 P-labeling of native BSP using purified casein kinase II (CKII), followed by peptide mapping and solid-phase N-terminal sequence analyses. The in vivo phosphorylation sites were determined by (i) derivatization with 1-S-[ 14 C]carboxymethyl-dithiothreitol ([ 14 C] CM-DTT) of the proteolytic digests of BSP, followed by isolation and N-terminal peptide sequence analysis; and (ii) analyzing the proteolytic peptides of native BSP using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Native BSP incorporated ~2.5 mol of phosphate/mol of BSP by CKII, which were distributed over four major peptide peaks and three shoulder peaks within the peptide map with varying degrees of phosphorylation. Further studies using the [ 14 C] CM-DTT thiol reagent indicated that native and deglycosylated BSP incorporated 5.84 and 5.80 mol of 14 C/mol of BSP, respectively. This confirmed that there were ~5.8 mol P-Ser/mol of BSP naturally (in vivo) occurring phosphorylation sites and that there was no overlap between the phosphorylation and glycosylation sites. The 5.8 mol P-Ser/mol BSP reflects the total number of mols of naturally occurring phosphorylation, phosphorylated in vivo by CKII (4.1 mol), protein kinase C (0.9 mol), and cGMP-dependent kinase (0.8 mol). Peptide N-terminal sequence analyses of both in vitro ( 32 P) and in vivo ( 14 C) phosphorylated peptides indicated that the phosphorylated residues were predominantly on the N-terminal half of the protein that included recognition sequences for CKII, e.g., LESDEENGVFK (residues 12-22).
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影响因子：
4.1
作者：
Wang, J;Glimcher, MJ;Salih, E
通讯作者：
Salih, E
DOI：
10.1098/rstb.1984.0041
发表时间：
1984-01-01
影响因子：
6.3
作者：
GLIMCHER, MJ
通讯作者：
GLIMCHER, MJ
骨磷蛋白磷酸化程度的自然变化是软组织和骨环境中脱矿骨基质诱导的体内新骨形成的函数。
DOI：
10.1042/bj20011272
发表时间：
2002
期刊：
The Biochemical journal
影响因子：
--
作者：
Salih,Erdjan;Wang,Jinxi;Mah,James;Fluckiger,Rudolf
通讯作者：
Fluckiger,Rudolf
影响因子：
2.8
作者：
Rapuano, BE;Wu, C;MacDonald, DE
通讯作者：
MacDonald, DE