Nucleotide pyrophosphatase from yeast. The presence of bound zinc

ARCHIVES

OF

BIOCHEMISTRY

Nucleotide

AND

BIOPHYSICS

Pyrophosphatase

184, 249-266 (1977)

from Yeast. The Presence Zinc’

J. S. TWU,2 R. K. HAROZ,3 Department

of Chemistry,

Biochemistry

AND

R. K. BRETTHAUER4

and Biophysics Program, Indiana 46556 Received

of Bound

University

of Notre Dame, Notre Dame,

May 19, 1977

Nucleotide pyrophosphatase from yeast was inhibited by thiols, o-phenanthroline, 8 hydroxyquinoline, EDTA, and 8-hydroxyquinoline-5-sulfonic acid. The inhibition by chelating agents was time and concentration dependent. Inhibition by EDTA was decreased by complexing the EDTA with metal ions before addition to the enzyme. The effectiveness of the metal ions in preventing inhibition by EDTA paralleled the stability constants of the EDTA-metal complexes. Partial recovery of EDTA-inhibited enzyme activity was achieved with Zn2+, Co*+, Fez+, and Mn*+. Analyses for zinc in the purified enzyme by atomic absorption spectroscopy and by titration with &%hydroxyquinoline-5-sulfonic acid revealed the presence of approximately 1 g atom/m01 of enzyme (M, 65,000). The data indicate that yeast nucleotide pyrophosphatase is a metalloenzyme in which the zinc plays some role in activity.

A nucleotide pyrophosphatase (EC 3.6.1.9) from yeast was previously shown to catalyze hydrolysis of the pyrophosphate bond of several disubstituted pyrophosphates such as sugar nucleotides and pyridine nucleotide coenzymes (1). The purified enzyme exhibited no requirement for monovalent or divalent cations, regardless of the substrate utilized. However, it was observed that addition of thiols or EDTA to the enzyme assay mixture resulted in significant inhibition of activity, suggesting that the enzyme, as isolated, may contain tightly bound functional metal ions. This communication provides evidence for the presence of zinc in the native enzyme and suggests a requirement of the metal ion for enzyme activity. 1 This work was supported by Grants GB 38356 and PCM 73-06961 from the National Science Foundation and by a grant from Miles Laboratories, Inc. * Present address: Veterans Administration Center, Los Angeles, California 90073. 3Present address: Battele Research Center, Geneva, Switzerland. 4To whom correspondence should be addressed at the Department of Chemistry, University of Notre Dame, Notre Dame, Indiana 46556.

MATERIALS

AND

METHODS

Preparation of enzyme. The isolation procedure as previously described (1) for a hybrid Saccharomyces yeast was applied to commercial pressed bakers’ yeast so that larger quantities of enzyme could be obtained. Modifications of the original procedure included: the use of 5 mM phenylmethylsulfonyl fluoride as a protease inhibitor in all buffers through the DEAE-cellulose5 steps; omission of the centrifugation step to remove ribosomes, as nucleic acids were effectively removed in the following heat and acid steps; elution of the hydroxylapatite column with a linear concentration gradient of 0.005-0.20 M potassium phosphate buffer, pH 6.5; and substitution of the DEAE-cellulose, pH 9.4, step by chromatography on a 1 x 15-cm column of GE-cellulose (Cl), using a linear gradient formed pH 7.38, and 300 ml from 300 ml of 0.02 M Tris-Cl, of 0.05 M Tris-acetate, pH 6.4, containing 0.2 M NaCl. The enzyme from commercial bakers’ yeast exhibited physical properties, substrate specificity, and inactivation by chelating agents similar to those of the hybrid yeast enzyme previously described (1). For studies involving the preparation and reacti’ Abbreviations used: DEAE-, diethylaminoethyl; nitrophenyl-pdT, deoxythymidine 5’-p-nitrophenyl phosphate; QSA, 8-hydroxyquinoline-5-sulfonic acid; GE-, guanidoethyl.

249 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISEN

0003-9861

250

TWU,

HAROZ,

AND

vation of apoenzyme and for metal analysis by atomic absorption spectroscopy, precautions were taken to avoid extraneous metal ion contamination. All reagents were prepared from glass-distilled deionized water and were stored in polyethylene bottles. The Sephadex G-ZOO column used in the final purification step was washed with 5 mM EDTA before equilibrating with the buffer. Schwarz/Mann Ultra Pure Tris base was used in all Tris buffers. Dialysis tubing was boiled in 0.05 M EDTA solution and was rinsed exhaustively with deionized water. Glassware utilized in the final purification step was soaked in concentrated hydrochloric acid and was rinsed with deionized water. Metal analysis. Prior to analysis by atomic absorption spectrometry, the protein samples were dialyzed against 5 mM Tris-HCl, pH 7.6. The analyses were performed with a Beckman Model 97900 atomic absorption system according to procedures outlined in the Beckman instruction manual. Duplicate aliquots of the protein solutions were aspirated directly into an air-acetylene flame of the laminar flow burner assembly. The dialysis buffers were also analyzed, and the absorbances were subtracted from the sample values. Sufficient concentrations of protein samples were analyzed so that at least a 10% absorbance above the buffer level was obtained. Assay procedures. Enzyme assays have been described in detail previously (1). The hydrolysis of deoxythymidine 5’-p-nitrophenyl phosphate (nitrophenyl-pdT) was followed by measuring the absorption at 400 nm of the liberated p-nitrophenolate. The production of glucose-l-P from UDP-glucose was measured spectrophotometrically at 340 nm with phosphoglucomutase and glucose-6-P dehydrogenase. The liberation of [‘4C1mannose-1-P from GDP-[14Clmannose was followed by radiochromatography. Amino acid and carbohydrate analyses. For amino acid analysis, protein was hydrolyzed in 6 N HCl at 110°C for 24 h. The analyses were carried out on a Beckman Model 117 amino acid analyzer using single column methodology. Tryptophan was determined by the spectrophotometric method of Goodwin and Morton (2). The tyrosine content derived from the spectrophotometric method was 15% higher than that from amino acid analyses of acid hydrolysates. Neutral carbohydrate was determined by the phenol-sulfuric acid method (31, using mannose as a standard. The results of these analyses are shown in Table I. The sum of the products of (residue molecular weights x quantities shown per 0.125 A,,,) was used to calculate the relationship of 1 A.&O.63 mg of enzyme/ml. This extinction coefficient was utilized in calculations concerning enzyme concentration. Chemicals. EDTA, 8-hydroxyquinoline, and ophenanthroline were obtained from Fisher. The 8

BRETTHAUER TABLE

I

COMPOSITION OF NUCLEOTIDE PYROPHOSPHATASE Component

Noa;o&es/ 280

Aspartate Threonine Serine Glutamate Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan Neutral carbohydrate

Males/65,000 &!

73.84 36.55 64.95 66.83 18.20 60.07 58.73 49.15 27.78 45.59 13.24 26.26 13.91 50.78 13.97 16.50 63.6

60.8 30.0 53.4 54.9 15.0 49.4 48.3 40.4 22.8 37.5 10.9 21.6 11.5 41.8 11.5 13.6 52.4

hydroxyquinoline-5-sulfonic acid (QSA) from Eastman was recrystallized from hot water before use. A sample of m-phenanthroline was obtained from Dr. Thomas Nowak. Atomic absorption reference standard metal solutions were from Fisher. Bovine liver catalase was obtained from Worthington. Other chemicals were from sources previously described (1). RESULTS

Znhibition Binding

by Thiols Agents

and

Other

Metal-

It was previously observed (1) that addition of various thiols as possible activators or stabilizers of enzyme activity routinely resulted in inhibition. A comparison of these effects is shown in Table II. After 1 h in the presence of 4 mM L-cysteine, the enzyme was completely inactivated. Similar treatment with 4 mM dithiothreitol resulted in recovery of only 8% activity. The enzyme was also inhibited by p-mercaptoethanol, but higher concentrations (16 mM) were required to achieve better than 90% inhibition. Ascorbic acid, a good reducing agent, had very little effect on enzyme activity at concentrations up to 16 mM. The inhibition observed with either 4 mM L-cysteine or dithiothreitol could be prevented by inclusion of 4 mM ZnCl, in the incubation mixtures, which suggested that the thiol inhibition may be due to binding of an essential metal ion rather

ZINC TABLE

IN YEAST

NUCLEOTIDE

II

EFFECT OF METAL-BINDING AGENTS ON NUCLEOTIDE PYROPHOSPHATASEACTIVITY” Addition None L-Cysteine (4 mM) L-Cysteine (4 mkf) + ZnCl, (4 mM) Dithiothreitol (4 mM) Dithiothreitol (4 IIIM) + ZnCl, (4 mM) /3-Mercaptoethanol (4 mM) P-Mercaptoethanol (8 mM) P-Mercaptoethanol (16 mM) L-Ascorbic acid (4 mM) L-Ascorbic acid (16 mM) EDTA (2 mM) o-Phenanthroline (2 mM) m-Phenanthroline (2 mM)

Activity

(%)

100 0 95 8 92 81 52 6 99 92 0 0 100

LI Enzyme (1 pg) was incubated for 1 h at 30°C in 0.02 ml containing 0.1 M Tris-HCl, pH 8.5, and additives as indicated. Residual enzyme activity was determined with 0.5 mM nitrophenyl-pdT.

than reduction of a disulfide bond. A further initial observation which supported the idea that the yeast nucleotide pyrophosphatase is a metalloenzyme was that incubation of the enzyme for 1 h with either 2 mM EDTA or o-phenanthroline resulted in complete inactivation. Enzyme activity was unaffected by m-phenanthroline, indicating that the observed inhibition by o-phenanthroline was not due to nonspecific binding or other effects of the phenanthroline ring. Furthermore, addition of mono- or divalent metal ions (2-5 mM) to an enzyme preparation which had been extensively dialyzed or passed through a Sephadex G-100 column did not significantly enhance activity (<5%), indicating that the metal ion, if present, is tightly bound to the protein. Cysteine and other thiols are known to form stable chelates with divalent metal ions (4-61, and several zinc metalloenzymes have been shown to be inhibited by thiols (7-10). However, it has been reported that dithiothreitol can inhibit, through an indirect manner, the utilization of glutamate by carbamyl-phosphate synthase fromEscherichia coli (11). In the presence of air, dithiothreitol was shown to be oxidized with the formation of hydrogen peroxide, which then oxidized some critical residue(s) of the enzyme. Since

251

PYROPHOSPHATASE

inactivation of the synthase could be prevented by exclusion of air or by the presence of catalase, experiments were carried out with the yeast nucleotide pyrophosphatase to see if a nitrogen atmosphere or the presence of catalase would prevent the observed inhibition by dithiothreitol. The experiments were carried out at relatively low concentrations of dithiothreitol so that the kinetics of inhibition could be followed. As shown in Fig. 1, the enzyme is very sensitive to preincubation with dithiothreitol, a 0.5 mM concentration resulting in 95% inhibition in 30 min at 30°C. When catalase was included with dithiothreitol in the preincubation mixture, the same extent of inhibition was observed. The effect of excluding air from the preincuba-

IO TIME

20

30

(min)

FIG. 1. Effect of dithiothreitol, catalase, and nitrogen on nucleotide pyrophosphatase activity. Enzyme (5 pg of protein) was incubated at 30°C in 1.0 ml of 1.0 M Tris-HCl buffer, pH 8.5, containing 0, 0.25, or 0.50 mM dithiothreitol. At the times indicated, enzyme activity was measured after adding 0.02 ml of 50 mM nitrophenyl-pdT. Catalase was added to the preincubation mixtures at a final concentration of 0.016 mg/ml; the controls for this experiment contained catalase without dithiothreitol. The experiment with nitrogen was carried out by flushing the buffer and dithiothreitol solution with nitrogen for 50 min before addition of enzyme and then incubating for the indicated times in a sealed tube. The controls for this experiment were carried out in an identical manner without dithiothreitol. Enzyme activity is expressed as a percentage of the control incubation for each time point. 0.25 mM dithiothrei(0-O) Controls; (A-A) tol; (U-0) 0.50 mM dithiothreitol; (A) 0.25 mM dithiothreitol under nitrogen; (m) 0.50 mM dithiothreitol with catalase.

252

TWU, HAROZ, AND BRETTHAUER

tion mixture was examined at 0.25 mM dithiothreitol. The buffer and dithiothreito1 were flushed with nitrogen for 50 min before addition of enzyme, and preincubation was then carried out under nitrogen in a sealed tube. As shown in Fig. 1, the same extent of inhibition by 0.25 mM dithiothreitol was observed whether preincubations of the enzyme were carried out in air or nitrogen. These experiments tend to rule out a mechanism of inhibition which is dependent on hydrogen peroxide formation or an oxidative mechanism dependent on air and lend further support to the metal-chelation inactivation mechanism. Incubation of the enzyme with lower concentrations of chelating agents resulted in a time-dependent inhibition of activity which could be easily monitored with different substrates. The time-dependent inhibition by EDTA, o-phenanthroline, 8-hydroxyquinoline, and QSA at pH 8.5 and 3o”C, as determined by assaying residual activity with nitrophenylpdT, is shown in Fig. 2. It is apparent that, in addition to inhibition being time dependent, the rate of inhibition is also dependent on the concentration of chelating agent. Similar inhibition studies were carried out by measuring residual activity with the substrates UDP-glucose and GDPmannose. With the same concentration of a given inhibitor, the rate of inhibition was similar whether nitrophenyl-pdT or a sugar nucleotide was used as substrate (data not shown). These results are consistent with our previous conclusion (1) that a single enzyme is catalyzing hydrolysis of the different substrates. No inhibition was noted upon incubation of the enzyme for up to 90 min with 2 mM mphenanthroline when assayed with nitrophenyl-pdT or UDP-glucose, whereas 1.0 mM o-phenanthroline resulted in 50% inhibition in approximately 5 min when assayed with either substrate. Prevention and Reversal of EDTA tion by Other Metals

Inhibi-

Experiments patterned after those of Gracy and Noltmann for phosphomannose

30

90

60 TIME

120

(min)

FIG. 2. Time-dependent inhibition of nucleotide pyrophosphatase by metal-binding agents. Enzyme (0.1 mg of protein/ml) was incubated at 30°C in 0.1 M Tris-HCl buffer, pH 8.5, with the various metalbinding agents. Aliquots were removed at various times and were assayed for enzyme activity with 1.0 mM nitrophenyl-pdT in 0.1 M Tris-HCl buffer, pH 8.5. (x-x) 2.0 mM m-phenanthroline; (O0) 0.5 mM o-phenanthroline; (0-O) 1.0 mM ophenanthroline; (A-A) 0.5 mM QSA; (0-O) 0.125 mM EDTA; (m-m) 0.25 mM EDTA; (AA) 2.0 mM Shydroxyquinoline. Enzyme activity is expressed as a percentage of activity in the control sample incubated with no metal-binding agent.

isomerase (8) were carried out to determine if the inhibition of nucleotide pyrophosphatase by EDTA was prevented by preincubation of the chelating agent with stoichiometric quantities of other metals before addition of enzyme. The data in Table III demonstrate that such a prevention of inhibition by EDTA does occur, and that the extent of prevention parallels, in general, the stability constants of the metal-EDTA complexes. The only exception to this parallelism was Ni2+, which has a higher stability constant for EDTA than does Zn2+ or Co2+, but which afforded slightly less protection against EDTA inactivation of the enzyme than did Zn2+ or co2+. Attempts to reactivate with metal ions the enzyme which had been completely inhibited by EDTA were only partially successful. For these experiments, the en-

ZINC TABLE

IN YEAST

NUCLEOTIDE

253

PYROPHOSPHATASE

III

ABILITY OF METALS TO PREVENT EDTA INHIBITION OF NUCLEOTIDE PYROPHOSPHATASE” Log stability Metal ion Activity (%) constant None Zn2+ co’+ NiS+ Fez+ Mn2+ Ca2+ Mgz+

0 87 84 82 76 46 17 8

16.5 16.3 18.6 14.3 14.0 10.6 8.7

” Enzyme (1 pg) was incubated for 4.5 h at 30°C pH 8.5, 1 mM in 0.02 ml containing 0.2 M Tris-HCl, EDTA, and 1 mM metal ion as indicated. The EDTA and metal ion were mixed before addition of enzyme. Residual activity was determined with 1 mM nitrophenyl-pdT and is expressed as a percentage of activity recovered from enzyme incubated with no EDTA or metal ion added. The stability constants are taken from Chaberek and Martell (5).

zyme was incubated for 2 h with 0.25 mM EDTA, at which time no activity on nitrophenyl-pdT was observed. Aliquots of the EDTA-enzyme solution were then diluted loo-fold and were assayed for activity on nitrophenyl-pdT after addition of metal ions as shown in Fig. 3. Fifty percent recovery of activity was obtained with Zn2+, but only at relatively high final concentrations (>10W3 M). Addition of either Co*+ or Fez+ to a final concentration of lob3 M resulted in 40% recovery of activity, and Mn2+ at a 5 x 10e2 M concentration gave 30% recovery. Metal Content of Nucleotide tase

Pyrophospha-

Analysis by atomic absorption spectrometry of calcium, magnesium, copper, and zinc was carried out on several of the fractions obtained during purification of the enzyme. As shown in Table IV, the only metal which was enriched during purification was zinc, and the slower rate of increase in zinc content as compared to specific activity indicates that other zinccontaining proteins were being removed. Calcium, copper, and magnesium were not enriched, and these three metals plus cobalt and iron (data not shown) were undetectable in the purified enzyme. The con-

-8

-7

-6

-5 LOG

-4

-3

-2

Mu=’

FIG. 3. Metal ion activation of EDTA-inhibited nucleotide pyrophosphatase. Enzyme (10 pg in 0.1 ml) was incubated for 2 h at 30°C in 100 mM TrisHCl, pH 8.5, containing 0.25 mM EDTA. Aliquots (0.01 ml) were then withdrawn and added to a reaction mixture (1.0 ml) containing 1 mM nitrophenyl-pdT and 0.2 M Tris-HCl, pH 8.5. Metal ions were then added, and initial velocities were recorded. Without addition of metal ions, no reaction was observed for a IO-min period. Activity is expressed as a percentage of that observed for enzyme which was incubated for 2 h without EDTA and which was assayed in the absence of added metal ions. Metal ion concentrations on the abscissa are the free metal ion concentrations, calculated from the amounts of EDTA and metal ions added and the stability constants given in Table III.

centration of zinc increased during the purification shown in Table IV to 702 pg/ g of protein. The highest level of zinc obtained on other samples of purified enzyme was 932 Fg/g of protein. Using a molecular weight of 65,000 for the enzyme (l), the zinc content is 0.70 g atom/m01 of enzyme, as calculated from the data in Table III, or 0.93 g atom/m01 of enzyme, as calculated from another sample. This most likely means that the yeast nucleotide pyrophosphatase, which is a single polypeptide, contains 1 g atom of zinclmol. The zinc content of the enzyme was also determined by measuring the absorption of the Zn(QSA), complex (12) at 370 nm. During titration of the enzyme with QSA, enzyme activity was also determined. It was observed that the absorption at 370 nm did increase upon titration with QSA, and that enzyme activity was progressively lost. Using an extinction coefficient of 1.1 x lo4 M-’ for Zn(QSA), (121, 1 A,,,,/ 0.63 mg for the enzyme, and a molecular weight for the enzyme of 65,000 (11, the moles of Zn(QSAj3 formed per mole of

254

TWU, HAROZ, AND BRETTHAUER TABLE IV METAL CONTENT OF NUCLEOTIDE PYROPHOBPHATASEDURING PURIFICATION

Fraction

Specific activity _^“. limits X lU~/mgJ 1.82

Extract Heat pH 4.5 Hydroxylapatite Sephadex G-200 ’ Not determined. b Undetectable.

2.02 2.00

19.0 640

0 100 0

= 80 5 :: 60 I= IE 40

0

K-7

Metal content (pg/g of protein) Calcium

Copper

Magnesium

33 9

13 5

ND” 155

Ub U U

U U U

66

105

U U

Zinc

Zinc (g atoms/m01 of enzyme)

70

0.07

79 213

0.08 0.11 0.21

702

0.70

0.75 mol of Zn(QSA),/mol of enzyme. These data are in good agreement with the atomic absorption analysis of a single zinc atom per mole of enzyme.

0

DISCUSSION

This investigation was carried out to substantiate our previous suggestion that “9 0 the yeast nucleotide pyrophosphatase is a 0 metal-containing protein (metalloen, A, 1 I zyme). Several different types of experi0.2 0.4 0.6 0.0 1.0 ments were carried out to ascertain that MOLES Zfl(QSA)3/MOLE ENZYME the requirements generally set forth for FIG. 4. Titration of nucleotide pyrophosphatase identification of a metalloenzyme (13) be with QSA. A Cary Model 15 spectrophotometer with met. Thus, the enzyme is inhibited by l-cm light-path cells was utilized. The sample cell several metal-binding agents including contained enzyme (1.01 mglml) in 0.02 M Tris-HCl, thiols, EDTA, o-phenanthroline, &hypH 7.2, and 0.05 M NaCl. The reference cell condroxyquinoline, and QSA. Inhibition by tained buffer and NaCl, but no enzyme. Aliquots of these compounds is both time and concena 10 mM solution of QSA were added (to a final tration dependent. The inhibition of the concentration of 0.48 mM) to both cells, and the enzyme by EDTA can be partially preabsorptions at 370 nm were recorded after stabilizing at maximal values. After recording the 340~nm vented by complexing of the EDTA with metal ions before addition of enzyme, the absorption, aliquots were removed from both cells for determination 1 h later of residual enzyme extent of activity then paralleling the staactivity with 0.5 mM nitrophenyl-pdT as substrate. bility constants of the EDTA-metal cheThe enzyme assay mixtures contained the same lates. That the enzyme is not irreversibly concentrations of QSA as did the sample cell utilized denatured by EDTA is indicated by at for determination of absorptions at 370 nm. Without least partial recovery of activity when exthis precaution, some reversal of inhibition due to cess metal ions are added to the completely dilution apparently occurs at the lower QSA conceninactivated enzyme. trations, as the line of Fig. 4 becomes a convex The direct demonstration by atomic abcurve. sorption spectroscopy of zinc in the purienzyme were calculated for each point in fied protein confirms that the previous the titration curve. These ratios were plot- inhibition by metal-binding agents was ted against residual enzyme activities due to chelation of an essential metal and (Fig. 4). A direct correlation between the not to other secondary effects. We have amounts of ZnCQSA), formed and the loss previously demonstrated (1) the apparent of enzyme activity was observed, and the homogeneity of the nucleotide pyrophosend point of the titration revealed (by phatase and thus believe that zinc is an extrapolation) the total loss of enzyme integral part of the enzyme as isolated, activity upon formation of approximately rather than a mere contaminant. During 2 0” 20 t

ZINC

IN YEAST

NUCLEOTIDE

purification of the enzyme, other metals (calcium, copper, magnesium, cobalt, and iron) were removed, whereas zinc was continuously enriched to a final value of 700900 pglg of protein. For an enzyme of 65,000 molecular weight, this amount of zinc approaches 1 g atom/m01 of enzyme. Similar results were obtained by titration of the enzyme with QSA and measuring the absorption of the (QSA),Zn complex. Although this method resulted in detection of approximately 1 mol of (QSA),Zn/mol of enzyme, the result may be questioned because of the uncertainty that the zinc was removed from the enzyme and, if not, whether the molar extinction coefficient utilized for the QSA *Zn complex is correct. Attempts to isolate an apoenzyme free of metal have so far been unsuccessful. Likewise, attempts to reactivate inhibited enzyme after removal of excess metal-binding agent by gel filtration or dialysis have been unsuccessful, although some reversal of inhibition could be demonstrated by either addition of excess metal ions to the mixture of enzyme and metal-binding agent (Fig. 3) or by dilution of the metal-binding agent concentration (legend to Fig. 4). Our results do not allow a conclusion to be made as to whether inhibition by the chelating agents is due to removal of the metal ion from the protein or to combination with the metal ion without removal. It is interesting that zinc is typically found as the metal ion of yeast metalloenzymes, others being alcohol dehydrogenase (14), fructose-1,6-diphosphate aldolase (15), n-lactate cytochrome reductase (16), pyruvate carboxylase (17), and phosphomannose isomerase (8). Our conclusions in this report would thus add another enzyme, nucleotide pyrophosphatase, to this list. Several nucleotide pyrophosphatases of mammalian tissues have also been shown to be inhibited by metal chelating agents (18-21). In particular, the reactivation by zinc ions of the inhibited enzyme activity in several tissues of rat (19) correlates well with the results presented in this communication for the yeast enzyme. As nucleotide pyrophosphatase, at least in rat tissues, is found as a component of

PYROPHOSPHATASE

membranes (18, 21, 24) which are also used in in vitro studies of glycosyltransferase reactions (22, 23, 25-28) the hydrolysis of sugar nucleotide substrates by this enzyme is often a serious complication and can lead to erroneous results and conclusions. The inhibition of many nucleotide pyrophosphatases by 5’-nucleotides (18, 20, 21, 27, 28; Bretthauer, unpublished) and by metal chelating agents (1, 18-21; this report) suggests ways of at least reducing the hydrolytic activity in membrane preparations where sugar nucleotides must be used as substrates. As examples, Lau (29) has reported that Mn*+dependent galactosyltransferase activities in rat tissue extracts can be more readily determined after inhibition of the nucleotide pyrophosphatase with EDTA, and Geren and Ebner (27) have demonstrated the enhanced galactosyltransferase activity of rat kidney extracts obtained in the presence of either 5’-AMP or folic acid. REFERENCES

4.

5.

6. 7. 8. 9. 10. 11. 12. 13. 14.

HAROZ, R. K., Twu, J. S., AND BRETTHAUER, R. K. (19’72) J. Biol. Chem. 247, 1452-1457. GOODWIN, T. W., AND MORTON, R. A. (1946) Biochem. J. 40, 628-630. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28, 350-356. BJERRUM, J., SCHWARZENBACH, G., AND SILLEN, L. G. (1957) Stability Constants, Part 1, The Chemical Society, London. CHABEREK, S., AND MARTELL, A. E. (1959) Organic Sequestering Agents, John Wiley, New York. LENZ, G. R., AND MARTELL, A. E. (1964) Biochemistry 3, 745-750. COOMBS, T. L., FELBER, J. P., AND VALLEE, B. L. (1962) Biochemistry 1, 889-905. GRACY, R. W., AND NOLTMANN, E. A. (1968) J. Biol. Chem. 243, 4109-4116. ZIELKE, C. L., AND SUELTER, C. H. (1971) J. Biol. Chem. 246, 2179-2186. HAYMAN, S., AND PATTERSON, E. K. (1971) J. Biol. Chem. 246, 660-669. TROTTA, P. P., PINKUS, L. M., AND MEISTER, A. (1974) J. Biol. Chem. 249, 1915-1921. SIMPSON, T. R., AND VALLEE, B. L. (1969)Znorg. Chem. 8, 1185-1186. VALLEE, B. L. (1955) Aduan. Protein Chem. 10, 317-384. VALLEE, B. L., AND HOCH, F. L. (1955) Proc. Nat. Acad. Sci. USA 41, 327-338.

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15. KOBES, R. D., SIMPSON, R. T., VALLEE, B. L., AND RUTTER, W. J. (1969) Biochemistry 8,

585-588. 16. CREMONA, T., AND SINGER, T. P. (1964) J. Biol. Chem. 239, 1466-1473. 17. SCRUTTON, M. C., YOUNG, M. R., AND UTTER, M. F. (1970) J. Biol. Chem. 245, 6220-6227. 18. SCHLISELFELD, L. H., VAN EYS, J., AND TOUSTER, 0. (1965) J. Biol. Chem. 240, 811-818. 19. CORDER, C. N., AND LOWRY, 0. H. (1969) Biochim. Biophys. A& 191, 579-587. 20. KRISHNAN, N., AND APPAJI RAO, N. (1972)Arch. Biochem. Biophys. 149, 336-348. 21. DECKER, K., AND BISCHOFF, E. (1972) FEBS Lett. 21, 95-98. 22. JENTOFT, N., CHENG, P.-W., AND CARLSON, D.

M. (1976) in The Enzymes of Biological Mem-

branes; Vol. 2: Biosynthesis of Cell Components (Martonosi, A., ed.), pp. 343-383, Plenum Press, New York. 23. SELA, B., LIS, H., AND SACHS, L. (1972) J. Biol. Chem. 247, 7585-7590. 24. TOUSTER, O., ARONSON, N. N., JR., DULANEY, J. T., AND HENDRICKSON, H. (1970) J. Cell Biol. 47, 604-618. 25. CARLSON, D. M., DAVID, J., AND RUTTER, W. J. (1973) Arch. Biochem. Biophys. 157, 605-612. 26. KIRSCHBAUM, B. B., AND BOSMANN, H. B. (1973) Biochem. Biophys. Res. Commun. 50,510-516. 27. GEREN, L. M., AND EBNER, K. E. (1973) Biothem. Biophys. Res. Commun. 59, 14-21. 28. MOOKERJEA, S., AND YUNG, J. W. M. (1975) Arch. Biochem. Biophys. 166, 223-236. 29. LAU, J. T. Y. (1977)Fed. Proc. 36, 744.