Characterization of human sublingual-gland protein kinase by phosphorylation of a peptide related to secreted proteins.
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.
Nam
Y
Y
Department of Biochemistry, University of Toronto, Canada.
Madapallimattam
G
G
Drzymala
L
L
Bennick
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000268:Adhesiveness; D001940:Breast / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D002130:Calcium Phosphates / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D047388:Casein Kinases; D011232:Chemical Precipitation; D002845:Chromatography; D002850:Chromatography, Gel; D015891:Cystatins / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D017886:Durapatite / Q000037:antagonists & inhibitors; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D054808:Histatins; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008274:Magnesium / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011107:Polylysine / Q000494:pharmacology; D055232:Proline-Rich Protein Domains; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides / Q000378:metabolism*; D013361:Sublingual Gland / Q000201:enzymology* / Q000378:metabolism
C525172:CST2 protein, human; D002130:Calcium Phosphates; D002135:Calcium-Binding Proteins; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D054808:Histatins; D007527:Isoenzymes; D010455:Peptides; D010748:Phosphopeptides; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; C018149:STATH protein, human; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides; C068074:HTN1 protein, human; D011107:Polylysine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006493:Heparin; D017886:Durapatite; D011494:Protein Kinases; D047388:Casein Kinases; D008274:Magnesium; D002118:Calcium