[42] UMP-CMP kinase from Tetrahymena pyriformis

[42]

UMP-CMP K1NASE

331

a reduction in molecular weight from approximately 53,000 to 17,000, as measured by molecular sieve chromatography. This low-molecularweight form is partially active in the presence of 5 mM 2-mercaptoethanol but becomes inactive upon removal of the reducing agent. Furthermore, higher concentrations of 2-mercaptoethanol (50 mM) fully reactivate the CMP(ATP) kinase activity, followed by dCMP(ATP) and CMP(dCTP) kinase activities in a sequential manner, without further change in molecular weight. Alkylation by iodoacetamide of the enzyme at different stages of reactivation in dithiothreitol suggests an ordered appearance of the various enzyme activities. Furthermore, iodoacetamide inactivates the fully active enzyme. Thioredoxin was found to activate the enzyme in a manner similar to 2-mercaptoethanol and dithiothreitol. 8

Inhibitors and Activators Fluoride was found to inhibit enzymic activity completely at a concentration of approximately 25 mM. Inhibition was also observed with NaSCN and NaC104, although at significantly higher concentrations (250 mM) NaCzH302, NaCI, and Na2SO4 stimulated CMP-kinase activity in the latter concentration range.

Feedback Regulation The enzyme does not appear to be subject to inhibition by CTP, dCTP, UTP, or dTTP (1.0 raM), regardless of the phosphate acceptor. It is possible that the activity of the enzyme in vivo is regulated by the concentration of thiols in the cell. r M. L o m b a r d i and A. Orengo, u n p u b l i s h e d results. s p. M a n e s s and A. Orengo, Biochirn. Biophys. Acta 429, 182 (1976).

[42] UMP-CMP K i n a s e f r o m T e t r a h y r n e n a p y r i f o r m i s By

ELIZABETH P. ANDERSON

Mg2÷ UMP + ATP ~ UDP + ADP Mg2+ CMP + ATP ) CDP + A D P

As purified from Tetrahymena pyriformis, this kinase phosphorylates both UMP and CMP, and the ratio of these two activities remains M E T H O D S IN E N Z Y M O L O G Y , VOL. L I

Copyright© 1978by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5

332

PYRIMIDINE METABOLIZING ENZYMES

[42]

constant during the purification. 1 The purified enzyme also retains appreciable kinase activity for dCMP. ~It is therefore presently classified as ATP:(d)CMP phosphotransferase, EC 2.7.4.14, 2 which has also been purified from other sources, sometimes for activity with different substrates. For example, it may be the same as the dCMP kinase purified from calf thymus. 3"4 The present enzyme preparation was purified for phosphorylation of UMP and, as a corollary, of CMP. In contrast to this substrate specificity, CMP-dCMP and UMP kinase activities can be separated in Escherichia coli extracts, ~ and in Salmonella typhimurium, UMP and CMP kinases do not appear to be encoded by the same structural gene.4"6 Assay Method

Principle. The assay measures the conversion of radioactive UMP to labeled UDP (or UDP plus UTP), with chromatographic isolation of the product(s) formed. It is specific enough for use in either crude or purified fractions of the enzyme. Reagents Potassium phosphate buffer, 0.5 M, pH 7.5 UMP, 0.1 M, or CMP, 0.1 M [2-14C]UMP (25 mCi/mmole), or [2-14C]CMP (25 mCi/mmole) ATP, disodium salt, 0.1 M, adjusted to pH 7.5 MgC12, 0.2 M

Procedure. 1,7 The standard reaction mixture contains 3/.d potassium phosphate buffer, 3 /zl UMP or CMP, 100 nCi [2-14C]UMP or [214C]CMP, 3 /xl ATP, 2 /zl MgC12, enzyme protein (in 5-20 /xl 0.1 M potassium phosphate buffer, pH 7.5), and water to give a final volume of 32/~l. For best linearity, the enzyme protein concentration is kept below 0.15 k~g. The assay is normally run in duplicate or triplicate. The i B. W. Ruffner, Jr. and E. P. Anderson, J. Biol. Chem. 244, 5994 (1969). 2 "Enzyme Nomenclature," Recommendations (1972) of the Commission of Biochemical Nomenclature of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry on the Nomenclature and Classification of Enzymes. Elsevier, Amsterdam, 1973. 3 y. Sugino, H. Teraoka, and H. Shimono, J. Biol. Chem. 241, 961 (1966). 4 E. P. Anderson, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 9, p. 49. Academic Press, New York, 1973. 5 S. Hiraga and Y. Sugino, Biochim. Biophys. Acta 114, 416 (1966). J. L. Ingraham and J. Neuhard, J. Biol. Chem. 247, 6259 (1972). 7 T. Q. Garvey, III, F. K. Millar, and E. P. Anderson, Biochim. Biophys. Acta 302, 38 (1973).

[42]

UMP-CMP K1NASE

333

reaction is initiated by the addition of enzyme and carried out at 25 ° for periods of time up to 10 min, 15 t~l being removed at each of two time points. The period of linearity with time should be confirmed for the preparation being assayed. The aliquot removed is pipetted into a tube preheated in boiling water, and the reaction is terminated by heating in boiling water for 1 min. Labeled substrate and products are then separated by ascending chromatography for 2.5 hr in 1 M LiC1 on thinlayer PEI-cellulose on plastic sheets (1-inch or 0.5-inch channels). The PEI-cellulose sheets s are stored at 0-4 °. Before use they are washed at least twice by ascending chromatography in water to remove any degraded PEI. To assist in localization, 0.1/zmole each of the nucleoside mono-, di-, and triphosphate are applied to the origin of every channel and cochromatographed with the sample. The pyrimidine compounds are localized by ultraviolet fluorescence and counted in a scintillation counter. (Rss: UMP, 0.80; UDP, 0.65; UTP, 0.37; CMP, 0.63; CDP, 0.36; and CTP, 0.12.) Separate chromatography of the starting substrate can indicate any necessary corrections for radioactive contamination of the substrate as supplied. The ultraviolet localization has been checked against more time-consuming localization by radioautography of the chromatogram on X-ray film, with excellent agreement; the radioautogram can be useful if additional or unknown by-products may be expected. Definition of Unit and Specific Activity. A unit of enzymic activity is defined as the amount phosphorylating 1 /xmole of UMP in 1 rain. Specific activity is expressed as units per milligram of protein. Protein concentration is estimated by the method of Lowry et al. 9 with bovine serum albumin as standard. Alternative Assay Procedures. The enzymic activity can alternatively be assayed as conversion of radioactive ATP to ADP using 14C-labeled ATP and unlabeled nucleoside monophosphate. In double-label experiments, [2-~4C]UMP and [3H]ATP have both been included in the incubation. The incubation mixture is then chromatographed in duplicate, once with the set of uridine nucleotide standards and once with adenosine nucleotide standards. In addition, better resolution is attained by using serial ascents (without drying between changes of solvent) in 0.5 M, 1.0 M, and 1.5 M LiCI, for 5, 50, and 40 min, respectively. An additional assay that couples ADP formation to the pyruvate kinase-lactate dehydrogenase system is especially useful for initial 8

Available from J. T . Baker Chemical Co., Phillipsburg, New Jersey. O. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

334

PYRIMIDINE METABOLIZINGENZYMES

[42]

velocity studies since it follows the reaction continuously, spectrophotometrically monitoring the oxidation of N A D H at 340 nm. This has proven feasible for studies of CMP phosphorylation, since CDP is a very poor substrate of pyruvate kinasel°; it is less satisfactory for the assay of UMP phosphorylation, at least for initial velocity studies, because UDP is an appreciable substrate and the Vmax with UDP is very different from that with ADP. Since this assay removes ADP product as it is formed, it has the added advantage of eliminating inhibition seen in the radioactivity assay at moderate to high levels of ATP. r

Purification P r o c e d u r e 1

Step 1. Crude Lysate. The enzyme is purified from late log-phase cultures of T. pyriformis. All operations are performed at 0--4 ° unless otherwise stated. Cells are harvested on the fifth day of culture growth by centrifugation at 400 g at 4°; the usual yield is 10 ml of packed cells per 4 liters of culture. After most of the medium is decanted, the cells are resuspended and then packed by centrifugation at 10,000 g for 10 min. The packed cells are resuspended in an equal volume of 0.1 M potassium phosphate buffer, pH 7.5, and alternately frozen in Dry Iceacetone and thawed in ice water three times. The frozen-thawed lysate is centrifuged at 100,000 g for 90 min, and the precipitate is discarded. Step 2. Fractionation by Acid Precipitation. To the supernatant solution from the crude lysate, sufficient 1 M acetic acid is added drop by drop, with stirring, to lower the pH to 5.0. This preparation is allowed to stand at 0 ° for 20 rain, the precipitate is removed by centrifugationat 10,000 g for 10 rain, and the supernatant solution is neutralized to pH 7.5 with 1 M ammonia. Step 3. Ammonium Sulfate Fractionation. Saturated ammonium sulfate solution (4 °) is added to the preparation over a period of 1-2 min with stirring, in sufficient quantity to bring the final concentration to 60% saturation. After 20 rain of equilibration, the precipitate is removed by centrifugation at 10,000 g for 10 min. This is discarded, and a volume of saturated ammonium sulfate solution equal to that of the ammonium sulfate supernatant solution is added over a period of 1-2 rain to bring the preparation to 80% saturation. After standing for 20 min, the mixture is again centrifuged at 10,000 g for 10 min to collect the precipitated protein; the supernatant fraction is discarded. A minimal amount (us10T. Q. Garvey, III and E. P. Anderson, unpublished results.

[42]

UMP-CMP KINASE

335

ually 4 ml) of potassium phosphate buffer, pH 7.5, is added to dissolve the orange pellet. Step 4. Fractionation on Sephadex G-75. The ammonium sulfate fraction (60-80%) is applied to a column (2.2 × 43.0 cm) of Sephadex G75 (superfine, previously allowed to swell overnight at room temperature in 0.1 M potassium phosphate buffer, pH 7.5, and then cooled to 4°). The column is then eluted with the same phosphate buffer at a flow rate of 4 ml/hr. All fractions are assayed for protein and enzyme activity. The enzyme is eluted from the column immediately ahead of a prominent, yellow-orange band which serves as a convenient marker. The active enzyme as eluted from the column is generally contained in approximately 16 ml of buffer (the peak activity tubes typically contain a total of 4 ml). The protein concentration of these tubes is approximately 0.2 mg/ ml. Experimental studies on the enzyme are usually carded out with preparations of maximal activity from the Sephadex columns. The pooled fractions are stored at 4 ° for further use. When stored in this way, the enzyme loses approximately 20% of its activity in 2 weeks. Experiments are therefore carried out, if possible, within 1 week after preparation of the enzyme. A typical purification procedure (see the table) achieved approxiPURIFICATION OF UMP KINASE FROM Tetrahymena pyriforrnis a

Fraction Frozen-thawed lysate Supernatant from 100,000 g pH 5 supernatant Ammonium sulfate fractionation (60-80% saturation) Sephadex G-75 Pooled fractions Peak fractions

Total protein (rag)

Specific Total units activity (/zmole/min) (units/mg)

Yield (%)

Ratio of UMP kinase to CMP kinase activity

1315

118

0.090

--

0.72

620 416

121 Ill

0.195 0.266

100 94

0.75 0.78

51

54

1.06

46

0.77

32 14

0.68

3.04 0.60

38 16.5

12.5 27.5

Enzymic activity was assayed with either [lq2]UMP or [14C]CMP as substrate. The purification was based on assays with UMP as substrate, but the ratio of UMP kinase activity to CMP kinase remained approximately constant throughout the purification (last column). Each fraction was assayed at several protein concentrations, and specific activity was calculated from tubes containing approximately the same amount of enzyme activity (0.01 unit).

336

PYRIMIDINE METABOLIZING ENZYMES

[42]

mately 300-fold purification over the crude lysate with 14% yield in the tubes of peak activity from Sephadex G-75, or about 140-fold purification with 32% yield in the total preparation after gel filtration. 11 The fractions from Sephadex are essentially free of UDP kinase, adenosine triphosphatase, and uridine diphosphatase; all of these activities are present in the original lysate. In the presence of equimolar UDP and ADP, however, the purified preparation catalyzes the reverse of the UMP kinase reaction, at a velocity about one-third that of the forward reaction.

Stability. The purified enzyme is very labile to dilution; 3-fold dilution with either water or phosphate buffer results in 50% loss of activity in 1 hr. Loss of CMP kinase activity parallels that of UMP kinase. Bovine serum albumin (100 ~g/ml) prevents this loss of activity in diluted preparations. The enzyme is also labile to elution from Sephadex in 0.1 M Tris.HC1 (pH 7.5) instead of potassium phosphate; under these conditions the enzyme is eluted with a partition coefficient twice that observed in the phosphate buffer. If albumin (I00/xg/ml) or NaC1 (2 M) is added to the Tris buffer, the elution pattern is like that observed in phosphate. Properties 1

Optimum pH. The enzyme exhibits a broad optimum between pH 7.0 and 8.0 (fraction E; 0.1 M buffers: Tris-maleate, pH 4.2-8.0; potassium phosphate, pH 6.0-7.8; Tris'HC1, pH 7.2-9.0). Specificity of the Phosphate Donor. Of the nucleoside triphosphates tested, only dATP can substitute for ATP as phosphate donor in the UMP kinase reaction, and it is only about one-tenth as active as ATP under standard assay conditions. GTP, UTP, CTP, dCTP, and dTTP are essentially inactive in this respect (fraction E). Specificity of the Phosphate Acceptor. With [8-14C]ATP and unlabeled nucleoside monophosphates as substrates, the enzyme phosphorylates CMP, UMP, and dCMP, in order of decreasing activity. AMP, GMP, dGMP, and dTMP are not phosphorylated. CMP kinase activity parallels UMP kinase activity throughout purification (see the table), and also through further fractionation on ion-exchange cellulose, as well as during inactivation. The activity with dCMP has not been followed through the purification procedure. 11 Preliminary experiments have indicated that it is possible to purify UMP kinase activity further from the Sephadex G-75 fraction using hydroxyapatite columns. This procedure gave about 2-fold purification over that already achieved, r

[43]

337

DEOXYCYTIDINE KINASE FROM CALF THYMUS

Kinetic Constants and Reaction Mechanism. The initial velocity pattern observed with CMP as substrate (using the coupled spectrophotometric assay) is one of intersecting rather than parallel lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex. ~° With UMP as substrate, a similar conclusion is indicated by data on isotope exchange in the partial reactions. The enzyme catalyzes neither half-reaction, i.e., exchange of phosphate between one substrate-product pair in the absence of the second substrate, again ruling out a ping-pong mechanism of addition of one substrate and release of its product prior to addition of the second substrate, r The enzyme has a Km for ATP of 0.5 mM, for CMP of 0.8 mM, and for U M P near 1.25 mM. CDP inhibition is competitive with CMP. The initial velocity pattern for the reverse reaction likewise indicates a sequential reaction mechanism.

[43]

Deoxycytidine

Kinase

from Calf Thymus

1

B y D A V I D H . IVES a n d S U E - M A Y W A N G

2'-Deoxycytidine + ATP ~ 5'-dCMP + ADP

This enzyme from the cytosol of calf thymus is so named because deoxycytidine (dCyd) is the preferred substrate at low concentrations, 2 although its specificity is rather broad. 3 It has been partially purified in several laboratories 4-6 through the use of very similar procedures. The method described here is a modification of the technique used in this laboratory. 5 Assay

Method

Principle. The assay method used is based on the ability of anionexchange paper to retain the labeled deoxynucleotide product, while ~This work was supported in part by Public Health Service Grant CA-06913 from the National Cancer Institute. 2 j. p. Durham and D. H. Ives, Mol. Pharmacol. 5, 358 (1%9). 3 T. Krenitsky, J. Tuttle, G. Koszalka, I. Chen, L. Beacham, III, J. Rideout, and G. Elion, J. Biol. Chem. 251, 4055 (1976). 4 R. Momparler and G. A. Fischer, J. Biol. Chem. 243, 4298 (1%8). 5 j. p. Durham and D. H. Ives, J. Biol. Chem. 245, 2276 (1970). 6 y . Kozai, S. Sonoda, S, Kobayashi, and Y. Sugino, J. Biochem. (Tokyo) 71,485 (1972). METHODS

IN ENZYMOLOGY,

VOL. LI

Copyright ~) 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5