22 MOLECULES, VIRUSES, AND BACTERIA 



as a substrate, only extracts from infected cells were capable of fixing 

 C ^^-formaldehyde in an acid-stable form to yield hydroxymethyl deoxy- 

 cytidylate (dHMP), as in Figure 2 (Flaks and Cohen, 1957, 1959a). 



Although thymidylate synthetase was indeed present in uninfected 

 E. coli strain B, it was shown to be markedly increased in phage- 

 inf ected bacteria ( Flaks and Cohen, 1957, 1959b ) . The mechanism of 

 the reaction forming thymidylate does not appear to involve a free hy- 

 droxymethyl derivative, unlike the reaction which produces dHMP 

 (Friedkin, 1959; Flaks and Cohen, 1959b). However, it was observed 

 that hydroxymethyl deoxyuridylate and thymidylate were generated to 

 a small extent during the conversion of dCMP to dHMP (Flaks and 

 Cohen, 1959b). This has been shown to be due to the appearance of a 

 third enzyme, dCMP deaminase, in infected cells ( Flaks, unpublished 

 results) which not only deaminated dCMP but also deaminated 

 dHMP. The appearance of this enzyme in virus infection has also been 

 described recently in another laboratory ( Keck et al., 1960 ) . 



Properties of the dCMP hydroxymethylase. Three assays have been 

 developed for the enzyme, based on the fixation of C^^-HCHO to 

 dCMP to form radioactive dHMP (Pizer and Cohen, 1960a). The ion- 

 exchange method developed by Somerville et al. ( 1959 ) is the simplest 

 and most accurate of the three and is the method of choice at present 

 in following purification and other properties of the enzyme. The hy- 

 droxymethylase is completely inactive on cytosine ribonucleotide; how- 

 ever, cytosine arabinonucleotide appears to possess of the order of 0.6 

 per cent of the substrate activity of dCMP ( Pizer and Cohen, 1960b ) . 

 Using a purified enzyme preparation, the following Km values have 

 been determined: 6 x 10"^ M for dCMP, 1.5 x 10"^ M for formaldehyde, 

 and 1 x 10-4 M for THFA. 



The separation of the hydroxymethylase and the thymidylate syn- 

 thetase may be effected easily; the former enzyme is far more stable 

 ( Flaks and Cohen, 1959b ) . A much greater degree of purification of the 

 hydroxymethylase was accomplished on a diethylaminoethyl ( DEAE ) 

 cellulose column, as described by Kornberg et al. (1959) and in some 

 greater detail by Pizer and Cohen ( 1960a ) . Figure 3 presents the elu- 

 tion from the column of the protein and enzyme derived from infected 

 cells in 40 liters of Biogen culture containing 10^ infected bacteria per 

 ml. Phage multiplication was stopped at 15 minutes by addition of 

 chloramphenicol to the culture, which was expelled onto ice, and the 

 cells were collected by centrifugation. The infected cells (40 to 50 gms. 

 wet weight) were frozen and thawed and extracted twice in the 

 Waring Blendor for one minute in 250 ml. 0.05 glycylglycine buffer pH 

 7.2 (0.001 M with respect to glutathione). The extract, containing 

 about four grams of protein, was centrifuged at 30,000 rpm for 30 min- 

 utes, and after removal of nucleic acids by precipitation with strepto- 



