Planta (Berl.)131, 179-185 (1976)

P l a n t a 9 by Springer-Verlag 1976

Partial Purification and Properties of Adenosine Nucleosidase from Leaves of Spinach Beet (Beta vulgaris L.) Jonathan E. Poulton* and Vernon S. Butt Botany School, Oxford University, South Parks Road, Oxford OX1 3RA, U.K.

Summary. Adenosine nucleosidase (EC, which catalyses the irreversible hydrolysis of adenosine to adenine and ribose, has been isolated and purified about 40-fold from leaves of spinach beet (Beta vulgaris L.). The enzyme appeared to be specific for adenosine only among the naturally-occurring nucleosides, but comparable activity was also found with adenosine N-oxide. Adenosine hydrolysis, which had an optimum at pH 4.5, did not require phosphate ions nor was it stimulated by their presence. The Michaelis constant for this substrate was 11 gM. Whereas the rate of adenosine hydrolysis was unaffected by DL-homocysteine, L-methionine and ribose, it was sensitive to the presence of adenine, S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, A M P and deoxyadenosine. The role of this enzyme in plant metabolism is discussed.


Adenosine may be metabolized in living organisms by many reactions, whose relative importance may differ not only from one organism to another, but also in one particular organism or tissue under different physiological conditions. Whereas these reactions have been well studied in animal systems, information concerning adenosine metabolism and its control in plant tissues is lacking. Although the hydrolytic conversion of adenosine to adenine has been achieved using adenosine nucleosidase preparations from animal (Tarr, 1955), plant (Heppel and Helmoe, 1952; * Present address: Biologisches Institut II der Universitfit Freiburg, Sch/inzlestrage9-11, D-7800 Freiburg i.Br., Federal Republic of Germany Abbreviations: BSA=bovine serum albumin; SAH=S-adenosyl-L-homocysteine;SAM = S-adenosyl-L-methionine

Miller and Evans, 1955; Roberts, 1956; Mazelis and Creveling, 1963; Clark et al., 1972) and microbiological sources (Lampen and Wang, 1952; Takagi and Horecker, 1957), the metabolic role of these enzymes has not hitherto been defined. High adenosine nucleosidase activities were detected in extracts of spinach-beet (Beta vutgaris L.) leaves during an investigation of the metabolic interrelationship between the caffeic acid O-methyltransferase and the S-adenosyl-L-homocysteine hydrolase (EC in these leaves (Poulton and Butt, 1974, 1975, 1976). SAH hydrolase catalyses the reversible hydrolysis of SAH to adenosine and L-homocysteine. Since the equilibrium of this reaction lies heavily in favour of SAH synthesis, the hydrolysis of SAH can only be maintained when the products adenosine and homocysteine are removed from the system. The activity of the spinach-beet adenosine nucleosidase was shown to facilitate SAH hydrolysis in vitro, thereby reducing the extent of inhibition of caffeic acid methylation by SAH. This enzyme was therefore partially purified and its properties studied to determine whether they would be compatible with such a role in vivo.

Materials and Methods Chemicals

[8-14CJadenosine (52 mCi/mmole) was obtained from the Radiochemical Centre, Amersham, and diluted with unlabelled adenosine from Sigma (London) Chemical Co., S.W.6. The following compounds were also purchased from the latter source and used without further purification: inosine, uridine, cytidine,xanthosine, adenosine N-oxide, adenine N-oxide, thymine, hypoxanthine, xanthine, guanosine, D(-) ribose and adenosine-5-phosphate (disodium salt). The following compounds were obtained from British Drug Houses Ltd. : adenine, thymidine, uracil, cytosine, guanine and bovine serum albumin.


J.E. Poulton and V.S. Butt: Adenosine Nucleosidase from Leaves

Plant Materials

iii) Detection of ribose as product: The identification of ribose as the second product of the reaction was performed by thin-layer chromatography on Polygram CEL 300 UV254 sheets (R F 0.92) and on Polygram SIL N-HR/UV254 sheets (RF 0.83) using water for development. Ribose was detected on such sheets as an orangebrown zone by lightly spraying them with an aniline-oxalic acid mixture (composition: 0.21 g aniline/33 ml 0.05 M oxalic acid) followed by incubation for 3-5 min at 100~ C.

Leaves were harvested from spinach beet plants (Beta vulgaris L. ssp. vulgaris) which were grown outdoors. Only healthy adult leaves were used in these investigations.

Enzyme Assay Adenosine nucleosidase activity was assayed by measuring the rate of formation of [8-~4C]adenine from [8-14C]adenosine, which were separated by thin-layer chromatography. 0.1 gmol [8-14C]adeno sine (containing 222,000 dpm.), 5 gmol acetate buffer (pH 4.6) and 0.1 mg bovine serum albumin were incubated with up to 20 gl enzyme preparation in a total volume of 0.1ml at 30~ for 10 min. Control tubes, in which boiled enzyme preparation replaced the active extract, were included. The reaction was terminated by heating in boiling water for 2 min. The mixture was cooled and centrifuged, and an aliquot (I0 gl) applied to a Polygram CEL 300 UV254 sheet. The chromatogram was developed with water, after which adenosine and adenine could be seen in u.v. light. The zone containing adenine was cut out and counted in 2 ml of scintillation fluid, containing 0.5% (w/v) butyl phenyl biphenyl-oxadiazole in toluene, using a Tracerlab Corumatic/200 scintillation counter. The total amount of radioactivity added to the reaction mixture was determined by applying an aliquot (10 ~tl) of the heattreated reaction mixture to a piece (1 em x 1 cm) of the Polygram sheet and counting as above. One unit of adenosine nucleosidase activity is defined as the amount of enzyme required to catalyse the formation of one gmole of adenine/rain under the assay conditions described.

Chromatographic Identification of Reaction Products: i) Adenine was identified as the reaction product, when adenosine was supplied as substrate, by thin-layer chromatography using several solvent systems (Poulton and Butt, 1974, 1976). ii) Chromatography of other potential substrates and their respective products: Preliminary experiments showed that the majority of possible substrates could easily be separated from their respective products by thin-layer chromatography on Polygram CEL 300 UV254 sheets using water as solvent. The limit of detection for the reaction products on such thin-layer sheets lay between 0.5 1.0 nmol. The RF values observed are given in Table 1.

Table 1. Separation of nucleosides and their respective bases by thin-layer chromatography. The compounds below were applied to a Polygram CEL 300 UV254 sheet, which was later developed with distilled water. Each compound was easily visible in u.v. light after the sheets had been dried

Enzyme Purification The washed laminae of spinach-beet leaves (135 g) were macerated in an M.S.E. Ato-mix homogenizer with 200 ml ice-cold TrisKH2PO 4 buffer (10 mM with respect of phosphate), pH 7.3. The macerate was squeezed through two layers of muslin, and the resultant liquid was centrifuged at 30,000 g for 25 min. The supernatant liquid was brough to 30~ C, and its pH then adjusted to 4.4 over a period of 2.5 min by the addition of 1.0 M-acetate buffer, pH 4.0. After a further 4 min at 30~ C, the mixture was centrifuged at 30,000 g for 10 min. The supernatant liquid was cooled to 4 ~ C and then brought to 90% saturation by the addition of solid (NH4)2SO r over a period of 20 rain. The pH was maintained at 4.54.8 by addition of dilute KOH solution, and, after a further 25 min, the mixture was centrifuged at 10,000 g for 25 min. The precipitate was dissolved in a small volume of 10 mMacetate buffer, pH 4.5, and stored overnight at 4 ~ C. A brown precipitate which formed overnight was removed by centrifugation. Residual (NH4)2SO 4 in the supernatant liquid was removed by gel filtration through a column (33 cm x 2.5 cm) of Sephadex G-25, equilibrated in 1 mM-Tris-KHzPO4 buffer, pH 7.0. Elution was carried out with this buffer, and fractions containing protein were combined and applied to a column (2 cm x 1.25 cm) packed with DEAE-eellulose which had been equilibrated with 1 mM-TrisKH2PO4 buffer, pH 7.0. The column was washed with this buffer before elution was continued with a linear gradient up to 75 mMTris-KH2PO4 buffer, pH 7.0. Fractions possessing adenosine nucleosidase activity were stored at - 2 0 ~ and thawed when required.

Preparation of Buffer Solutions Tris-KHzPO4 buffer solutions were prepared by adding solid Tris (hydroxymethyl) aminomethane to the appropriate amount of KH2PO4 in solution to give the desired pH, before diluting to obtain the required final concentration of phosphate.

Protein Estimation The protein content of crude preparations was estimated by the Lowry method, as described by Leggett Bailey (1962), after precipitation from solution by 5% (w/v) trichloroacetic acid; crystalline bovine serum albumin, dessicated before use, was the standard. The protein content of column eluates was determined by the method of Warburg and Christian (1941).


RF value


Rv value

Adenosine Adenine

0.54 0.33

Xanthosine Xanthine

0.82 0.47

Inosine Hypoxanthine

0.76 0.58

Adenosine N-oxide Adenine N-oxide

0.75 0.60

Removal of Phosphate Ions from the Enzyme Preparation:

Uridine Uracil

0.85 0.74

guanosine guanine

0.57 0.33

Cyfidine Cytosine

0.80 0.70

thymidine thymine

0.80 0.73

After elution from the DEAE-cellulose column, the purified enzyme preparation contained low concentrations of phosphate ions (20-30 mM). These ions were removed by gel-filtration of an aliquot (2 ml) through a Sephadex G-25 column (12 x 1.5 cm), equilibrated and eluted with 10 mM Tris-HC1 buffer, pH 7.75.

J.E. Poulton and V.S. Butt: Adenosine Nucleosidase from Leaves


The Purification of Adenosine Nucleosidase from Spinach-beet Leaves Crude macerates, obtained by homogenizing spinachbeet leaves in ice-cold NaHCO3 solution, Tris-HC1 buffer or Tris-KHzPO4 buffer, catalysed the hydrolysis of adenosine to adenine at 30 ~ C at rates of 0.10.9 ~tmol/h/mg protein. On centrifugation at 30,000 g for 25 min, all of the adenosine nucleosidase activity was recovered in the supernatant liquid. The stability of this enzyme at acid pH allowed significant purification to be achieved merely by titration to pH 4.4 using acetate buffer. This preparation was then further purified by chromatography on DEAE-cellulose



0.5 t..

7 150-

~ 0.4




{ 03 100-



columns (Fig. 1) after its concentration by (NH4)2SO4 precipitation and gel filtration. Fractions eluting from the ion-exchange column were stored individually at - 2 0 ~ C, thawed when required, and used for the experiments described below. The overall scheme for the purification of a preparation of adenosine nucleosidase having a specific activity about 30 times greater than that of the crude homogenate is shown in Table 2.

Stability of the Enzyme The purified enzyme was stable for at least three months when stored at - 2 0 ~ C, but gradually lost activity on storage at 4 ~ C. It appeared however to be unstable in solutions of low protein concentration, and, for maximal enzymic activity, bovine serum albumin was added to the assay system. The optimum concentration of bovine serum albumin (1 mg/ml) was determined by titration and, unless otherwise specified, all experiments described below were performed with this addition.



50 ,e

-~ 0.2 N



;s 25 "6


8 - 10




Fraction number Fig. 1. Ion-exchange chromatography of adenosine nucleosidase on DEAE-cellulose. The fraction obtained by a (0 90)% (NH4)2SO4 precipitation was chromatographed on a column of DEAE-cellulose as described in the Methods section. 7 ml fractions were collected and assayed for protein content ( 9169 and adenosine nucleosidase activity(e--.). The Tris-KHzPO4 buffer concentration is shown by the broken line

pH Optimum The optimum pH for the hydrolysis of adenosine was determined in the absence of bovine serum albumin using several buffer systems (Fig. 2). The highest activity was observed at about pH 4.5, but more than 50% of this rate was observed between pH 3.0 and 7.75.

Time-course of Adenosine Hydrolysis The time-course of adenosine hydrolysis was determined in acetate buffer, pH 4.6, in the presence and

Table 2. Purification of adenosine nucleosidase from leaves of spinach beet Stage

Total volume (ml)

Crude Homogenate






Supernatant liquid after 30,000 g centrifugation






After titration to pH 4.4, and centrifugation






0-90% satd. (NH4)2SO4 precipitate Eluate from Sephadex G-25 Fractions from DEAE-cellulose chromatography (Fig. 1): Fraction 12 Fraction 13 Fraction 14

Protein content (mg/ml)

Nucleosidase activity (m-units/ml)

Nucleosidase specific activity (m-units/rag of protein)

Yield (% initial)











7.0 7.0 7.0

0.18 0.13 0.12

125 76 57

694 585 475

4 2.5 1.9


J.E. Poulton and V.S. Butt: Adenosine Nucleosidase from Leaves

suggesting that the purified enzyme was protected by this addition against thermal inactivation during prolonged incubation at 30 ~ C.

150 r

Volume Dependence

~100 i E %J

The extent of adenosine hydrolysis after incubation for 15 rain in acetate buffer, pH 4.6, was proportional to the added amount of preparation up to 5 gg of purified enzyme. This relationship was also observed in the absence of bovine serum albumin.

._> 50

N c"

Nature of the Reaction

LU .













Fig. 2. Effect of pH on the activity of purified adenosine nucleosidase. [8-~4C]adenosine was incubated for 15 min with the purified enzyme (10 p.1) using 50 mM concentrations of the following buffers: glycine-HCL ( o - - o ) , acetate (m--D), KH2PO~-K2HPO4 (~--zx), Tricine-NaOH ( e - - o ) . [8-~4C]adenine was estimated by liquid scintillation counting after thin-layer chromatography as described in the Methods section


.E ~400

The amount of adenine produced at each stage of the reaction was exactly accounted for by the quantity of adenosine utilized (Fig. 3). Since ribonucleosides can be cleaved either by phosphorolysis or by hydrolysis, residual phosphate ions were removed from the purified enzyme by gel-filtration as described in the Methods section. Only 35% of the applied enzyme activity was however recovered. This result probably only reflected the instability of the purified enzyme on dilution, since 88% of the applied enzyme activity was recovered when BSA (5 mg/ml) was added to the enzyme preparation before gel-filtration through the Sephadex G-25 column. The addition of phosphate ions (300 nmol) did not increase the activity of the resultant filtrate.

200 0.8!








Time (min) Fig. 3. The time-course of adenine production and adenosine hydrolysis. The hydrolysis of adenosine with the purified enzyme was carried out in the presence of bovine serum, using the following reaction mixture: 0.51 Ixmol adenosine (containing 1.11 x 106 dpm), 25 txmol acetate buffer pH 4.6, 0.5 mg BSA, 100 gl purified enzyme in a total volume of 0.5 ml. At intervals, aliquots (50 pl) were withdrawn, the reaction terminated, and [8-14C]adenine separated from [8-14C]adenosine as already described. Adenine produced (A--A), adenosine utilized ( o - - o ) . The production of adenine in a reaction mixture possessing no BSA was also followed ( e - - o )




i'o 2; absence of bovine serum albumin (Fig. 3). With this addition, the rate of reaction remained constant over the first 30 min and then progressively declined until the substrate was exhausted. In the absence of bovine serum albumin, the reaction did not go to completion,



1/[Ac:lenosine] Cram-t ) Fig. 4. Lineweaver-Burk plot of adenosine nucleosidase activity (v) against adenosine concentration (s). The rate of adenosine hydrolysis is expressed as nmol adenine produced/h/10 ~1 purified enzyme preparation

J.E. Poulton and V.S. Butt: Adenosine Nucleosidase from Leaves


Table 3. The effect of potential substrates on adenosine hydrolysis. The purified enzyme (10 gl) was incubated at 30~ for 15 min with 12nmol of [8-a4C]adenosine (containing 222,000dpm) and 5 gmol of acetate buffer, pH 4.6, in a total volume of 0.1 ml. When required, 0.4 gmol of the additions shown below were included. Control tubes, in which boiled enzyme preparations replaced the active extract, were also included. [8-14C]adenine was estimated as described in the Methods section

reducing sugars. These methods were not however used here, since, although both methods produced excellent linear calibration curves over the required range (0-0.5 gmol ribose), the development of the colours was found to be seriously affected by the presence of both adenosine and adenine. As difficulty was encountered with adenosine itself, the method was not extended to other substrates. Instead, a semiquantitative estimation, as described in the Methods section, was used to determine the substrate specificity of the purified enzyme. After 500 nmol of each of the substrates had been incubated with the enzyme in acetate buffer, pH 4.6, at 30~ C for 2 h, the reaction was terminated by heating for 2 min in boiling water. Precipitated protein was removed by centrifugation and an aliquot (10 gl) of the reaction mixture was chromatographed as described in Table 1. Upon viewing the sheets in u.v. light, adenine and adenine Noxide were detected from adenosine and adenosine N-oxide respectively. No reaction products were observed after incubating guanosine, xanthosine, inosine, uridine, cytidine or thymidine. The extent of [8-1~C]adenosine hydrolysis was determined under


Inhibition observed



(%) None Uridine Cytidine Inosine Thymidine

0 2.2 2.2 2.5 6.5

Inhibition observed

Purine riboside Xanthosine Adenosine N-oxide Guanosine

7.1 7.3 ll.1 11.8

Ribose was detected as the second reaction product by chromatography as described in the Methods section.

Miehaelis Constant for Adenosine The Michaelis constant for adenosine for the purified enzyme in acetate buffer (pH 4.6) was 11 gM, as determined from a Lineweaver-Burk plot (Fig. 4). The concentration range over which adenosine could be varied was limited at high concentrations, by its solubility and, at low concentrations, by the specific activity of the commercial [8-14C]adenosine available. Vmax for the purified enzyme was 49 gmol adenine/h/ mg protein at 30~ C.

Inhibition of Adenosine Hydrolysis by Potential Substrates Table 3 indicates that even in the presence of a 30-fold excess of each potential substrate, only small effects on adenosine hydrolysis were observed. Compounds with the greatest effect were all purine derivatives; these inhibited the reaction far more than equal concentrations of the pyrimidines, thymidine, cytidine or pridine. No products other than adenine were visible in u.v. light when the reaction mixtures were chromatographed on Polygram CEL 300 UV254 sheets using water as solvent.

Table 4. The effect of metabolites on adenosine hydrolysis. The purified enzyme (10 gl) was incubated for 20 rain at 30~ with 12 nmol of [18-14C]adenosine (containing 222,000 dpm) and 5 gmol of acetate buffer, pH 4.6, in a total volume of 0.1 ml. Where indicated, various metabolites were added. Control tubes, in which boiled enzyme extract replaced the active preparation, were included. [8-14C]adenine was estimated as described in the Methods section


Concentration (mM)

Relative activity (% control)


10 1

101 98


10 1

99 101


10 1

92 99



40 86


S-adenosyl-L-methionine Adenine


20 73

3.5 0.5

17 65


10 1

34 84



93 101


Substrate Specificity ADP

Many authors have followed the hydrolysis of adenosine by determining the production of ribose by the Nelson (1944) or Nelson-Somogyi (1945) methods for



10 1

94 95

10 1

48 82


J.E. Poulton and V.S. Butt: Adenosine Nucleosidase from Leaves

identical conditions; when 114 nmol adenosine was hydrolysed in 2 h, adenosine N-oxide was also hydrolysed significantly, but the hydrolysis of the other substrates must have been less than 2%. No further attempt was made to determine the rate of adenosine N-oxide hydrolysis, since this is unlikely to be a natural substrate for the enzyme.

weak or absent in crude homogenates from spinachbeet leaves (Poulton and Butt, 1975). During the purification of SAH hydrolase, the presence of adenosine nucleosidase activity was detected in these extracts. Although a similar enzyme had been extracted and purified from Brussel sprouts (Mazelis and Creveling, 1963) and from potato leaves (Clark et al., 1972), purification of this enzyme was undertaken and its properties studied especially in relation to its capacity to assist the hydrolysis of SAH in vivo by catalysing the removal of adenosine. A preparation having a specific activity 3040 times greater than that of the crude homogenate was obtained. In common with the above enzymes, it appeared to be specific for adenosine among the naturally-occurring purine and pyrimidine nucleosides, and likewise exhibited a low pH optimum. Closer examination of the reaction suggested that adenosine was being metabolized by a hydrolytic mechanism, since the reaction progressed to completion in the absence of phosphate ions. This view was strengthened by the absence of an effect when phosphate ions were added back to the system. A distinguishing feature of this enzyme however was its Michaelis constant for adenosine (11 ~tM), which was far smaller than that reported for the Brussel sprout enzyme, although both nucleosidases exhibited comparable specific activities. This difference may be due to interference with the Nelson method by adenosine and adenine. The rate of adenosine hydrolysis with the spinachbeet enzyme was unaffected by DL-homocysteine, Lmethionine, D-ribose, ADP or ATP. On the other hand, in common with the Brussel sprout enzyme which was inhibited 38% by 9 mM adenine, adenosine hydrolysis was significantly reduced by much lower concentrations of adenine (0.5-3.5 mM); the nature of this inhibition was not investigated. Furthermore, the enzyme was inhibited by deoxyadenosine, SAH, SAM and AMP; these effects are probably not of physiological significance however, since it is unlikely that these metabolites accumulate in such high concentrations in vivo. The role of adenosine nucleosidases in plant metabolism remains unclear. The crypticity of this enzyme under normal physiological conditions has been reported (Page, 1964). Uptake of adenosine via the petiole of potato leaves led to its rapid conversion to inosine and hypoxanthine; little adenine was detected in such leaf extracts. In contrast, injury by fungal invasion or by mechanical maceration was followed by the conversion of adenosine to adenine and ribose. It was suggested that this enzyme operates as part of a salvage pathway for the recovery of preformed purines which are then reutilized as precursors in the synthesis of ribonucleic-acid.

Effect of Metabolites on Adenosine Hydrolysis Among the metabolites investigated, DL-homocysteine, L-methionine and D-ribose had little effect upon the rate of adenosine hydrolysis with the purified enzyme (Table 4). The enzyme was however significantly inhibited by adenine itself, and by AMP and deoxyadenosine, which contain the adenine moiety in their structures. Moreover, SAM and SAH, each in 8-fold excess over adenosine, inhibited adenosine hydrolysis by 27% and 14% respectively, and, in 80-fold excess, by 80% and 60% respectively.


It is now well-documented that O-, S-, N- and Cmethyltransferases, which utilize SAM as methyl donor, are potently inhibited by low concentrations of the reaction product SAH (Chung and Law, 1964; Deguchi and Barchas, 1971). For example, SAH (Ki =4.4 gM) competitively inhibited the methylation of caffeic acid by SAM (Kin = 12.5 gM) in the presence of the purified caffeic acid O-methyltransferase from spinach-beet leaves (Poulton and Butt, 1975). It was proposed that the rate of this methylation reaction in vivo may be controlled by the ratio of the SAM and SAH concentrations. It would therefore be important that SAH is removed from the system in order for methylation to continue. The purification and properties of SAH hydrolase, an enzyme which catalyses the reversible hydrolysis of SAH to adenosine and homocysteine, have been reported (De la Haba and Cantoni, 1959, Poulton and Butt, 1976). It was shown that either or both of the products must be removed in vitro for SAH hydrolysis to be maintained. Investigations were therefore undertaken here to determine whether spinach-beet leaves possessed an enzymic system, whereby adenosine might be metabolized. Adenosine may be metabolized in living organisms by many reactions including phosphorylation, deamination and conversion to adenine by either hydrolytic or phosphorolytic mechanisms. Adenosine deaminase, which catalyses the deamination to inosine, is not widespread in the Plant Kingdom (Fiers and Vandendriessche, 1960) and this activity was very

J.E. Poulton and V.S. Butt: Adenosine Nucleosidase from Leaves


In our experiments (Poulton and Butt, 1974, 1975), adenosine nucleosidase facilitated the hydrolysis of SAH with crude spinach-beet leaf preparations even at alkaline pH and thereby relieved the inhibition of caffeic acid methylation by SAH. It seems more likely, however, that adenosine is conserved in vivo by its conversion to ADP through the successive action of adenosine kinase and adenylate kinase activities. In view of the high specific activity in crude leaf homogenates, acidic pH optimum and substrate specificity of the adenosine nucleosidase, it seems more possible that this enzyme may be spatially separated from the enzymes of SAH breakdown and may be involved in the release of adenine from adenosine during senescence. The acidic conditions which are likely to prevail as the leaf cells degenerate would provide a suitable environment for the action of this enzyme. This proposal requires a study of the changes in enzyme activity during the growth and senescence of the leaf. It is of interest in this connection that adenine accumulation within potato leaves resulted in severe dessication of the leaf tissue (Page, 1964).

adenosyl-L-homocysteine from adenosine and homocysteine. J. Biol. Chem. 234, 603~08 (1959) Deguchi, T., Barchas, J. : Inhibition of transmethylations of biogenic amines by S-adenosylhomocysteine. J. Biol. Chem. 246, 3175-3181 (1971) Fiefs, W., Vandendriessche, L.: Catabolism of nucleosides by barley-extracts. Arch. int. Physiol. Biochem. 68, 203-207 (1960) Heppel, L.A., Hilmoe, R.J. : Phosphorolysis and hydrolysis of purine ribosides by enzymes from yeast. J. Biol. Chem. 198, 683694 (1952) Lampen, J.O., Wang, T.P. : The mechanism of action of Lactobaeillus pentosus nucleosidase. J. Biol. Chem. 198, 385-395 (1952) Leggett Bailey, J. : In: Techniques in protein chemistry, pp. 293294. Amsterdam: Elsevier Publishing Co. 1962 Mazelis, M., Creveling, R.K. : An adenosine hydrolase from Brussel sprouts. J. Biol. Chem. 238, 3358-3361 (1963) Miller, G.W., Evans, H.J. : Nucleosidase from higher plants. Plant Physiol. 30, suppl. 37 (1955) Nelson, N.: A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153, 375-380 (1944) Page, O.T. : In vivo conversion of adenosine by potato leaves. Canad. J. Bot. 42, 951-952 (1964) Poulton, J.E. : The methylation of caffeic acid and related reactions. D. Phil. thesis, Oxford University, U.K. (1974) Poulton, J.E., Butt, V.S. : Purification and properties of S-adenosylL-methionine: caffeic acid O-methyltransferase from leaves of spinach beet (Beta vulgaris L.) Biochim. Biophys. Acta 403, 301-314 (1975) Poulton, J.E., Butt, V.S. : Purification and properties of S-adenosylL-homocysteine hydrolase from leaves of spinach beet. Arch. Biochem. Biophys. 172, 135-142 (1976) Roberts, D.W.A.: The wheat leaf phosphatases. II. Pathways of hydrolysis of some nucleotides at pH 5.5. J. Biol. Chem. 222, 259-270 (1956) Somogyi, M. : A new reagent for the determination of sugars. J. Biol. Chem. 160, 61~58 (1945) Takagi, Y., Horecker, B.J. : Purification and properties of a bacterial riboside hydrolase. J. Biol. Chem. 225, 77-87 (1957) Warburg, O., Christian, W. : Isolierung und Kristallisation des Gfirungsferments Enolase. Biochem. Z. 310, 384~421 (1941)

J.E. Poulton thanks the Science Research Council of Great Britain for a Research Studentship.

References Chung, A.E., Law, J.H. : Biosynthesis of cyclopropane compounds. VI. Product inhibition of cyclopropane fatty acid synthetase by S-adenosylhomocysteine and reversal of inhibition by a hydrolytic enzyme. Biochemistry 3, 1989-1993 (1964) Clark, M.C., Page, O.T., Fisher, M.G. : Purification and properties of N-ribosyladenine ribohydrolase from potato leaves. Phytochemistry 11, 3413-3419 (1972) De la Haba, G., Cantoni, G.L. : The enzymatic synthesis of S-

Received 16 April; accepted 26 April 1976

Partial purification and properties of adenosine nucleosidase from leaves of spinach beet (Beta vulgaris L.).

Adenosine nucleosidase (EC, which catalyses the irreversible hydrolysis of adenosine to adenine and ribose, has been isolated and purified ab...
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