Nam Y, Madapallimattam G, Drzymala L, Bennick A
Department of Biochemistry, University of Toronto, Canada.
Arch Oral Biol. 1997 Aug;42(8):527-37. doi: 10.1016/s0003-9969(97)00051-4.
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 KM of 0.09 mM for ATP and the KM 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.
人类唾液中的磷蛋白包括富含脯氨酸的蛋白质、磷蛋白、组蛋白1和胱抑素SA-III。这些蛋白质中磷酸根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以及与羟基磷灰石的黏附。为了阐明这些蛋白质的磷酸化过程，研究了来自人类舌下腺的一种激酶对与酸性富含脯氨酸蛋白质中一个磷酸化位点氨基酸序列相同的肽（APRP8）的磷酸化作用。这种激酶高度不稳定，通过舌下腺匀浆分级分离和凝胶过滤纯化了58倍，但当尝试用常用于蛋白激酶的色谱技术进一步纯化时，该酶失活。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，进行了特性鉴定。该酶最适活性需要10 mM Mg2+，对ATP的KM为0.09 mM，对肽底物APRP8的KM为0.42 mM。它不受cAMP或钙调蛋白激活，这是与酪蛋白激酶和乳腺激酶共有的特性。舌下激酶以及酪蛋白激酶2都被肝素抑制，但在其他方面这两种激酶具有不同特性。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添加KCl抑制。此外，酪蛋白激酶2可以利用ATP和GTP作为磷酰供体，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，但与该激酶不同的是，它不能用Ca2+代替Mg2+。因此，舌下激酶虽然与酪蛋白激酶2和乳腺激酶都有一些共同特性，但也存在明显差异，与这些酶的关系仍有待确定。舌下激酶的特性鉴定将有助于其进一步纯化。
