554 BIOCHIMICA ET BIOPHYSICA ACTA

Preliminary Notes

Formation of deoxycytidine 5'-phosphate from cytidine 5'-phosphate with enzymes from Escherichia coli

Deoxyribonucleotides can directly be formed through a reduction of ribonucleotides in different types of cells (see e.g. ref. I). Most of the evidence for such a type of reaction comes from experiments with intact cells. We have now found that a "par- ticulate-free" extract from E. cell B carries out the transformation of CMP to deoxy- CMP, and that this reaction depends on the presence of ATP, Mg ++ and TPNH.

The requirements for the overall reaction are demonstrated in Table I. Deoxy- CMP was identified as the product of the reaction by the following criteria: (I) identi- cal amounts of product were obtained with either 3H- or 3~P-labelled CMP; (2) on paper chromatography 1 the product obtained from either 3H- or asP-labelled CMP moved as deoxy-CMP; (3) after dephosphorylation with prostatic phosphatase the product obtained from [sH]CMP behaved as deoxycytidine on Dowex-5 ol or on paper chromatography.

A strong stimulation of deoxy-CMP formation with ATP + Mg ++ appears directly with the crude extract. The dependence on TPNH, however, emerges first after treatment of the enzyme with Dowex-2. DPNH stimulated much less.

Negligable amounts of deoxy-CMP (and deoxycytidine) were formed from cytidine 2'(3')-phosphate or cytidine.

Table II demonstrates results of three experiments during which incubation was

TABLE I

REQUIREMENTS FOR THE FORMATION OF DEOxY-CMP

The complete system contained: o.05 #,mole of 8H- or 32P-labelled substrate, I /*mole ATP, 5/,moles MgC1 v io / ,moles Tris buffer, pH 7.4. Incubation for io min at 37 ° with 3.4 mg enzyme, final vol. 0.25 ml. The reaction was stopped with I ml i M HC104. After centrifugation the supernatant was boiled for IO min, and CMP and deoxy-CMP were separated on Dowex 5 ox.

The amounts of deoxy compounds formed were calculated from the radioactivity.

Radioaaive substrate mlJmole deoxy. CMP /ormed

Complete system [sH]CMP o.55 Complete system [*~P] CMP 0.53 Minus ATP and Mg ++ [82P]CMP o.o 3 Complete system [*~P]cytidine 2'(3')-phosphate o.oi Complete system [3H]cytidine 0.04 Complete system* [82P]CMP o.o 4 Complete system*

+ TPNH (3 ° m/~moles) [a2P]CMP 0.35 Complete system

+ DPNH (3 ° m/~moles) [3~P]CMP o.io

* Dowex-2-treated enzyme.

Abbreviations: CMP, CDP and CTP, cytidine 5'-mono-, di- and triphosphate, respectively; ATP, adenosine 5'-triphosphate; DPNH and TPNH, reduced di- and triphosphopyridine nucleo- tide, respectively; Tris, tris(hydroxymethyl)aminomethane.

Biochim. Biophys. Acta, 37 (196o) 554-555

PRELIMINARY NOTES 555

carried out in the presence of large amounts of non-labelled deoxy-CMP, deoxy-CDP and deoxy-CTP, respectively. In each case the specific activities of the three deoxy- nucleotides were determined at different time intervals. Only one early-time point is given for each experiment in Table II. I t is evident that in all three experiments the highest specific activity was found in deoxy-CDP, demonstrating that this nucleotide was the first product of the reaction.

TABLE II DEOxY-CDP AS THE FIRST PRODUCT OF THE REACTION

[a2P]CMP (I .7 / ,moles , 23- IO e counts/min]/ ,mole) , i o / , m o l e s ATP, 5 ° / , m o l e s MgC1 v 3 / ,moles deoxy-nucleotide and 31 mg enzyme, p H 7.4, were incubated for 4 min at 37 ° (vol., 2. 5 ml). Mono-, di- and t r iphosphates were sepaxated on Dowex-2, the ribo- and deoxyribonucleotides within each group were then separated on Dowex-5o and their specific activities were determined.

Deoxynucleotids a d d e d Conms/min#tmole a/ttr 4 rain at start o/incubation deoxy -CMP deoxy-CDP deoxy-GTP

deoxy-CMP 13,000 77,000 75,000 deoxy-CDP 9,000 27,ooo 14,ooo deoxy-CTP 5,000 2o,ooo i i ,ooo

These experiments thus lead us to the following formulation of the reaction sequence during the reduction of CMP to deoxy-CMP:

CMP ATP Mg++ TPNH ' ~, CDP > deoxy-CDP > deoxy-CMP.

With CDP as substrate the formation of deoxy compounds was still strongly stimulated by ATP and Mg ++. This might indicate that the actual reduction step involves a more complicated course than that shown in the above scheme and that it might involve the synthesis of an intermediate which requires ATP and Mg++.

Our results are not in agreement with those reported earher by GROSSMAN AND HAWKINS 2. I t was reported that deoxyribosyl compounds could be formed in a rather unspecific way from ribonucleotides or ribonucleosides with an extract of Salmonella typhimurium. The reaction did not require ATP or TPNH, but was strongly stimulated by dithiopropanol. Besides, considerably larger amounts of deoxyribosyl compounds were formed than in our experiments.

Using the techniques reported by GROSSMAN AND HAWKINS we have to a large extent been able to confirm the results of these authors with S. typhimurium. However, when comparing the amounts of deoxyribosides formed as measured by deoxyribose analysis with the results of isotopic assays, it became evident that only a very small fraction of the deoxyribosyl compounds conceivably could have arisen through a direct reduction of ribosyl compounds. Instead, it seems likely that the results with S. typhimurium can be best explained on the basis of the action of deoxyribosyl- transferring enzymes.

Departments of Chemistry I and of Bacteriology, PETER REICHARD Karolinska Institutet, Stockholm (Sweden) LARS RUTBERG

1 p. REICHARD, J . Biol. Chem., 234 (1959) 1244. 2 L. GROSSMAN AND G. R. HAWKINS, Biochim. Biophys. Acta, 26 (1957) 657, and Federatlon Pro*.,

17 (1958 ) 235.

Received November 26th, 1959

Biochim. Biophys. Acta, 37 (196o) 554-555