CHARACTERIZATION OF HUMAN SUBLINGUAL-GLAND PROTEIN-KINASE BY PHOSPHORYLATION OF A PEPTIDE RELATED TO SECRETED PROTEINS

Phosphoproteins in human saliva include proline-rich proteins, stather ins, histatin 1 and cystatin SA-III. The presence of phosphate in thes e proteins is necessary for various functions in the mouth including c alcium binding, inhibition of precipitation of calcium phosphate, inhi bition of growth of hydroxyapatite crystals and adherence to hydroxyap atite. To elucidate the process of phosphorylation of these proteins, the phosphorylation of a peptide (APRP8) with an amino acid sequence i dentical to one of the phosphorylated sites in acidic proline-rich pro teins by a kinase from the human sublingual gland was investigated. Th e kinase, which was highly labile, was purified 58-fold by fractionati on of sublingual gland homogenate and gel filtration, but the enzyme w as inactivated when further purification by chromatographic techniques commonly used for protein kinases was attempted. To compare the enzym e with other kinases, and to obtain information that could be used in its further purification, a characterization was undertaken. The enzym e required 10 mM Mg2+ for optimum activity, it had a K-M of 0.09 mM fo r 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 w ith casein kinases and mammary gland kinase. The sublingual kinase as well as casein kinase 2 were inhibited by heparin, but in other respec ts the two kinases had different properties. While casein kinase 2 is activated by polylysine and has optimal activity in 150 mM KCl, sublin gual kinase was inhibited by polylysine and the addition of KCl. Moreo ver, casein kinase 2 can utilize both ATP and GTP as phosphoryl donors , but GTP was not a substrate for sublingual kinase. The sublingual ki nase 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 case in kinase 2 and mammary gland kinase, distinct differences were also s een and the relationship to these enzymes remains to be determined. Th e characterization of the sublingual kinase will be useful in its furt her purification. (C) 1997 Elsevier Science Ltd.