170 Krane/Glimcher: Protein Phosphorus and Phosphokinases in Connective Tissue 



representative experiments are shown in Tab. 2. In these experiments, the reaction 

 was stopped by heating at 65 °C for 15 minutes and the reaction mixture transferred 

 to a 30X2 cm column of Sephadex G-25 equilibrated with a pyridine-acetic acid 



Table 2. Gelatins as phosphoryl acceptors in protein phospbokinase reaction 



Incubation: 37', pH 8.0, 30 min. 

 The enzymes used in these experiments were different preparations from hog kidney and 

 contained variable amounts of acceptor protein activity. Therefore each experiment must be 

 considered in relation to the control values obtained. 



buffer, pH 4.4, and eluted with the same buffer. The fractions containing the protein 

 were pooled and the protein precipitated by the addition of trichloroacetic acid to a 

 final concentration of 13Vo. The protein was collected by centrifugation, dissolved in 

 water and reprecipitated twice with trichloroacetic acid. It is seen that small amounts 

 of ^-P were transferred to the protein in the presence of protein phospbokinase. Since 

 the amounts transferred are small the labeled site has not yet been identified. It is 

 also not certain whether the phosphoryl group is transferred to previously unphos- 

 phorylated residues or if an exchange reaction between ATP and protein-bound 

 phosphorus occurs. 



The data reported here further support our hypothesis that matrix-bound organic 

 phosphorus plays an important role in the initial phases of the calcification process. 



Acknozi'ledgments 

 Supported by research grants of the USPHS, N.I.H. (AM-3564, AM-06375). The 

 John A. Hartford Foundation, Inc., the Easter Seal Research Foundation and the 

 USAEC (AT(30-1)2183). This is publication number 387 of the Robert W. Lovett 

 Memorial for the Study of Diseases Causing Deformities. S. M. K. is an Established 

 Investigator of the Helen Hay Whitney Foundation. 



References 



Blomback, B., M. Blomback, R. F. Doolittle, B. Hessel, and P. Edman: On the proper- 

 ties of a new human fibrinopeptide. Blochlm. blophys. Acta 78, 563 (1963). 



Burnett, G., and E. P. Kennedy: The enzymatic phosphorylation of proteins. |. biol. 

 Chem. 211, 969 (1954). 



Flavin, M.: The linkage of phosphate to protein In pepsin and ovalbumin. J. biol. Chem. 

 210, 771 (1954). 



