Characterization of human sublingual-gland protein kinase by phosphorylation of a peptide related to secreted proteins
Characterization of human sublingual-gland protein kinase by phosphorylation of a peptide related to secreted proteins
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DOI：
10.1016/s0003-9969(97)00051-4
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发表时间：
1997-08-01
影响因子：
3
通讯作者：
Bennick, A
中科院分区：
文献类型：
--
作者：
Nam, Y;Madapallimattam, G;Bennick, A
人类唾液中的磷蛋白包括富含脯氨酸的蛋白质、statherins、histatin 1和cystatin SA-III。这些蛋白质中磷酸盐的存在对于口腔中的各种功能是必需的，包括钙结合、抑制磷酸钙沉淀、抑制羟基磷灰石晶体生长和粘附于羟基磷灰石。为了阐明这些蛋白质的磷酸化过程，研究了来自人舌下腺的激酶对具有与酸性富含脯氨酸的蛋白质中的磷酸化位点之一相同的氨基酸序列的肽（APRP 8）的磷酸化。该激酶，这是高度不稳定的，纯化58倍，通过分级分离的舌下腺匀浆和凝胶过滤，但酶被灭活时，通常用于蛋白激酶的色谱技术进一步纯化尝试。为了将该酶与其他激酶进行比较，并获得可用于其进一步纯化的信息，进行了表征。该酶需要10 mM Mg 2+的最佳活性，它有一个K-M为0.09 mM的ATP和肽底物APRP 8的K-M为0.42 mM。它不被激活的cAMP或钙调蛋白，与酪蛋白激酶和乳腺激酶共享的特性。肝素对舌下激酶和酪蛋白激酶2均有抑制作用，但在其他方面，这两种激酶具有不同的性质。虽然酪蛋白激酶2被多聚赖氨酸激活，并在150 mM KCl中具有最佳活性，但舌下激酶被多聚赖氨酸和加入KCl抑制。此外，酪蛋白激酶2可以利用ATP和GTP作为磷酰基供体，但GTP不是舌下激酶的底物。舌下激酶与乳腺激酶共享底物识别序列，但与该激酶不同，它不能利用Ca 2+代替Mg 2+。虽然舌下激酶因此与酪蛋白激酶2和乳腺激酶共享一些特性，但也观察到明显的差异，并且与这些酶的关系仍有待确定。舌下激酶的特性将有助于其进一步纯化。(C)1997 Elsevier Science Ltd.
Phosphoproteins in human saliva include proline-rich proteins, statherins, histatin 1 and cystatin SA-III. The presence of phosphate in these proteins is necessary for various functions in the mouth including calcium binding, inhibition of precipitation of calcium phosphate, inhibition of growth of hydroxyapatite crystals and adherence to hydroxyapatite. To elucidate the process of phosphorylation of these proteins, the phosphorylation of a peptide (APRP8) with an amino acid sequence identical to one of the phosphorylated sites in acidic proline-rich proteins by a kinase from the human sublingual gland was investigated. The kinase, which was highly labile, was purified 58-fold by fractionation of sublingual gland homogenate and gel filtration, but the enzyme was inactivated when further purification by chromatographic techniques commonly used for protein kinases was attempted. To compare the enzyme with other kinases, and to obtain information that could be used in its further purification, a characterization was undertaken. The enzyme required 10 mM Mg2+ for optimum activity, it had a K-M of 0.09 mM for ATP and the K-M for the peptide substrate APRP8 was 0.42 mM. It was not activated by cAMP or calmodulin, characteristics that are shared with casein kinases and mammary gland kinase. The sublingual kinase as well as casein kinase 2 were inhibited by heparin, but in other respects the two kinases had different properties. While casein kinase 2 is activated by polylysine and has optimal activity in 150 mM KCl, sublingual kinase was inhibited by polylysine and the addition of KCl. Moreover, casein kinase 2 can utilize both ATP and GTP as phosphoryl donors, but GTP was not a substrate for sublingual kinase. The sublingual kinase shared a substrate recognition sequence with mammary gland kinase, but, unlike that kinase, it could not utilize Ca2+ instead of Mg2+. While the sublingual kinase thus shared some properties with both casein kinase 2 and mammary gland kinase, distinct differences were also seen and the relationship to these enzymes remains to be determined. The characterization of the sublingual kinase will be useful in its further purification. (C) 1997 Elsevier Science Ltd.