Biochemical Systematic$ and Ecology, Vol. 7, pp. 253 to 255, Pergamon Press Ltd. 1979. Printed in England,

Some Properties of Phosphodiesterase from Mycobacterium smegmatis

C. H. LEE* Department of Clinical Biochemistry, Faculty of Medicine, University of Otago, Medical School,

Dunedin, New Zealand

Key Word Index--Mycobacterium smegmatis; phosphodiesterase activity; nucleotides; nucleosides; deoxy- derivatives.

Abstract--The phosphodiesterase of Mycobacterium smegmatis was strongly inhibited by ATP and ADP. CTP, GTP, TTP, their corresponding deoxy derivatives and deoxy ATP were inhibitory to the enzyme while the mono- nucleotides AMP, CMP. GMP and TMP were slightly stimulatory. Adenosine at 2.0 mM stimulated enzyme activity by 50%.

Introduction The inhibitory effect of ATP on phosphodi- esterase activity may play a regulatory role in the cell [1] since it appears likely that the enzyme exists in vivo in a greatly inhibited state [2] and has been reported to be much in excess over adenylate cyclase in a number of tissues [2, 3]. Cyclic AMP has been purified from Mycobacterium smegmatis [4]; the adenylate cyclase has been partially character- ized in this non-pathogenic organism [5] and the role of cyclic AMP has been recently discussed [6]. This paper reports the inhibitory effect of purine and pyrimidine trinucleotides (and their deoxy derivatives) on phosphodi- esterase from this bacterium, and the stimu- latory effect of mononucleotides and adenosine on the enzyme's activity. The results suggest that in vivo phosphodiesterase activity in M. smegmatis may be regulated by ATP, ADP or other nucleotides and in turn controls intra- cellular cyclic AMP levels in this organism.

Results and discussion In the presence of excess Mg =+ (2.5 mM greater than the nucleotide), complete in- hibition of phosphodiesterase activity was observed in the presence of 15 mM ATP. Half-maximum inhibition was observed at 5 mM ATP in the presence of excess Mg =+ (Fig. 1). Inhibition by ADP was less marked, reaching maximum inhibition of 87% only at the highest nucleotide concentration tested (15 raM); the inhibition by ADP may be due to

• Present address: Depar tment o f Biochemistry and Microb io logy, Universi ty o f Agr icu l ture Malaysia, Serdang, Selangor, Malaysia.

(Received 16 December 1978)

~°° I .o p

"°r/// orl/o / , , ,

0 2.5 5.0 7.5 I0.0 t2.5 15.0

Nucleotide, mM

FIG. 1. EFFECT OF ATP AND ADP ON PHOSPHODIESTERAS ACTIVITY. A concentration of Mg 2+ in excess of the concentratio of ATP or ADP by 2.5 mM (unless otherwise indicated) wa maintained at each nucleotide concentration tested. 0 ~ - - 0 , ATP; • A , ATP (no Mg 2+ added); O ~ ' ~ O ADP.

its conversion to ATP by the presence of an adenylate kinase in the preparation used.

Although some workers found that a variety of nucleoside triphosphates were inhibitory to phosphodiesterase, it appears likely that their Mg=+-sequestering properties were at least partially responsible [1, 7]. However, other reports [8] showed that the inhibition by ATP was only partially reversible by the addition of excess Mg =+ or Mn =+ suggesting that the inhibition by ATP cannot be fully explained on the basis of metal chelation. However. bacterial phosphodiesterases might be metalloproteins as suggested by earlier experiments which showed that the phosphodiesterase of Brevi- bacterium liquefaciens and Escherichia coil were inhibited by reagents that chelated

253

254

TABLE 1. EFFECT OF NUCLEOTIDES AND RELATED COMPOUNDS ON PHOSPHODIESTERASE ACTIVITY

C. H. LEE

Compound (mM) %of control Compound (raM) %of control 5'GTP (0.5) 85 5"dTTP (0.5) 90

(2.0) 60 (2.0) 84 5'CTP (0.5) 87 5'AMP (0.5) 105

(2.0) 78 (2,0) 120 5'TTP (0.5) 94 5'G M P (0.5) 115

(2.0) 89 (2.0) 118 5'dATP (0.5) 84 5"CMP (0.5) 116

(2.0) 76 (2.0) 120 5"dGTP (0.5) 80 5"TMP (0.5) 102

(2.0) 65 (2.0) 105 5'dCTP (0.5) 89 Adenosine (0.5) 130

(2.0) 75 (2.0) 150 Enzyme activity in the absence of added nucleotides

compound tested by 2.5 mM was maintained; control readings.

was taken as 100%. A concentration of Mg 2+ in excess of the concentration of the tubes also contained 2.5 mM Mg 2+. Average values were obtained from triplicate

metals [9]. In the present studies, excess Mg =+ was added and the inhibit ion by ATP clearly appears to be more than a simple chelation of the metal on the enzyme. Speziali and Wijk [10] reported that ATP (0.2-2.0 mM) effectively inhibited Saccharomyces carlsber- gensis phosphodiesterase whi le in Serratia marcescens [11] ATP, ADP and other tri- nucleotides inhibited enzyme activity. Scott and Solomon [12] demonstrated that both ATP and ADP at relatively low concentrations (0.1 mM) inhibited phosphodiesterase activity by 88% in Neurospora crassa. Inhibit ion by nucleotides has also been reported in some animal tissues [2, 1 3].

The inhibitory effect of other tr inucleotides including GTP, CTP and -I-I-P on phosphodi- esterase activity is shown in Table 1. Enzymes from microbial sources are known to catalyse a direct reduction of r ibonucleotides to the corresponding deoxy derivative [14]. The deoxynucleot ides of ATP, GTP, CTP and TTP also inhibited phosphodiesterase activity in M. smegmatis.

However, the 5 ' -mononucleot ides including AMP, GMP, CMP and T M P were stimulatory and adenosine produced appreciable stimula- t ion (50%) of phosphodiesterase activity. Adenosine has been shown to stimulate the rat kidney phosphodiesterase [7].

Taken together, the concerted action of the stimulatory effect of mononucleot ides and adenosine, and the inhibitory effect of tri- nucleotides and their deoxy derivatives may possibly play a role in regulating intracellular cyclic A M P levels in M. smegmatis.

Exper imenta l Culture and harvest of microorganisms. Mycobactefium smegmatis (NCTC 523) was maintained without shaking at 37 ° in a medium containing (g/I.) L- asparagine, 4; citric acid, 2; glycerol, 30; magnesium

sulphate, 0.5; thiamine, 0.01; potassium dihydrogen phosphate, 0.5; ammonium ferric citrate, 0.05; DL- lactic acid solution (85%, w/w; boiled and neutralized), 10; and inorganic salt solution, 0.1%. The pH was adjusted to 7.2 with NaOH. The inorganic salt solution contained (g/l.): ammonium molybdate, 0.064; zinc sulphate, 2; and manganous chloride, 0.045. The bacteria were sub-cultured every 4th day. The bacteria were harvested by filtering through three layers of muslin, washing three times with water, and centri- fugation.

Preparation of the enzyme extract. Bacteria were suspended in 60 mM Tris-HCI buffer (pH 9.0) and sonicated in a 100 W ultrasonic disintegrator (MSE) for 20 min at 0 °. The disrupted cells were centrifuged at 40,000 xg for 40 rain at 4 °. The pellet was discarded and the supernatant kept at -80 °. The enzyme was stable for at least one month at this temperature and was free of adenylate cyclase [15].

Assay of phosphodiesterase. The assay mixture for phosphodiesterase contained 2.5 I~mol of 0.06 M Tris-HCI buffer (pH 9.0), Mg =+ (various concentra- tions), 0.005 IJmol purified cyclic AMP-[SH] (ca 40,000-50,000 cpm), nucleotide or nucleoside (various concentrations) and enzyme preparation (40.2 I~g protein) in a total volume of 40 tA, was incubated at 37 ° for 15 rain. Under these conditions less than 9% of the substrate was utilised and formation of 5'AMP was linear with time and enzyme concentration. No 5'- mononucleotidase activity was detectable under these assay conditions. The reaction was stopped with 5 t~1 of 50% (w/v) trichloroacetic acid. Tubes were centrifuged at 10,000 xg for 10 min to sediment the precipitate and 35 i~1 of the supernatant was spotted on Whatman No. 1 paper and developed in isopropanoI-NH =-H =O (7:1:2). The 5'AMP spots clearly separated from cyclic AMP were detected under UV light, cut out and counted in 10 ml toluene scintillation fluid.

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SOME PROPERTIES OF PHOSPHODIESTERASE FROM MYCOBACTERIUM SMEGMATIS 255

4. Lee, C. H. (1977) J. Bacteriol. 132, 1031. 5. Lee, C. H. (1978) J. Gen. Microbiol. 108, 325. 6. Lee, C. H. (1979) Arch. MicrobioL 120, 35. 7. Dousa, T. and Rychlik, I. (1970) Biochim. Biophys.

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15. Lee, C. H. (1973) Ph.D. Thesis, Univ. of Otago, New Zealand.