Protein Phosphorus and Phosphokinases In Connective Tissue 169 



Although the peptide fractions of dental enamel which we obtained after partial 

 acid hydrolysis, column chromatography and electrophoresis may not have been 

 homogeneous, it is of interest to compare their amino acid compositions to the phos- 

 phorus-containing peptides from collagens and phosphopeptides derived from other 

 proteins (Table 1). The similarity of the amino acid composition of these fractions of 

 different proteins is indeed intriguing. 



Table 1. Predominant amino acids of phosphorus-containing peptides of various 

 phosphoproteins 



Source Aiiiinn Acids lU'lVrunce 



Enamel proteins ,Vr, Asp, Glu, Gly, Leu Glimcher and Krane, 



(calf embryo) 1964 b. 



Gelatin Ser, Asp, Glu, Gly, Ala Glimcher et a!., 1964 



(guinea pig skin) 



Fibrinogen (human) ' Ser, Asp, Glu, Gly, Ala, Leu Blomback ef al., 1963 



Ovalbumin Ser, Asp, Glu, lieu, Ala i Flavin, 1954 



Ovalbumin Ser, Asp, Glu, Leu, Ala, Val | Perlmann, 1952 



Casein (human) I Ser, Glu, Thr, lieu ! Mellander, 1963 



When it was shown that the enamel matrix proteins contained phosphoserine, we 

 considered the possible pathways of synthesis of the protein-phosphorus linkages. 

 Burnett and Kennedy (1954) had demonstrated the presence of enzymatic activity 

 in liver mitochondria which catalyzed the transfer of the terminal phosphoryl from 

 ATP to protein-bound serine of casein. Rabinowitz and Lipmann (1960) purified 

 protein phosphokinases from yeast and calf brain and further characterized the reac- 

 tion. These protein phosphokinases catalyze the phosphorylation of protein or poly- 

 peptide-bound serine but not of the free amino acid. The observations of Sanger and 

 HocQUARD (1962) using hen oviduct as an ovalbumin-synthesizing system are also 

 consistent with the concept that phosphorylation of the serine residues of phospho- 

 proteins occurs after incorporation of serine into the polypeptide chain. 



We, therefore, have assayed several connective tissues for protein phosphokinase 

 activity. Enzymatic activity which catalyzed the phosphorylation of casein and 

 partially dephosphorylated phosvitin was found in all of the tissues examined includ- 

 ing bone, cartilage, skin, tendon, muscle and enamel organ (Krane et al., 1965). It 

 was possible to purify the enzymatic activity from acetone powders of several of 

 these tissues approximately 45-fold by ammonium sulfate fractionation, Sephadex 

 and DEAE resin column chromatography. These enzymes, as well as one purified 

 from hog kidney by similar procedures, catalyzed the transfer of the terminal phos- 

 phoryl from ATP to enamel matrix protein, whereas the enzyme purified from 

 brewers' yeast was inactive towards this substrate. The reaction was measured using 

 the transfer of label from ATP-y'-P. At the end of the reaction, 900/o of the ^-P 

 which was transferred to the protein was released as inorganic orthophosphate in 

 1 N NaOH, 100° for 1 hour whereas only 50/o of the ^^p was liberated in 1 N HCl, 

 100° for 10 minutes. The alkali lability and acid stability are characteristic of poly- 

 peptide-bound phosphoserine. Furthermore, "'-'P-labeled phosphoserine was identified 

 in partial acid hydrolysates. 



More recently we have tested this reaction using collagens and gelatins purified 

 from several sources as substrates in the phosphokinase reaction. The results of 



