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393

Partial Purification of Adenosine 3': 5'-Cyclic Monophosphate Phosphodiesterase from Phaseolus vulgaris L. :Associated Activator and Inhibitors E. G. BROWN, T. AL-NAJAFI and R. P. NEWTON Department 01Biochemistry, University College of Swansea, Singleton Park, Swansea SA2 8PP, U.K.

The occurrence of cyclic AMP in tissues of higher plants has now been demonstrated by a number of different procedures (see, e.g., Brown &Newton, 1973; Brewin &Northcote, 1973a; Giannattasio et al., 1974~).The associated formative enzyme, adenylate cyclase, has also been detected in higher plants (Wellburn et al., 1974; Newton, 1974; Giannattasio & Macchia, 1973), as has the corresponding catabolic enzyme, cyclic AMP phosphodiesterase (GianndttaSiO & Macchia, 1973; Amrhein, 1974; Newton, 1974). The present communication concerns the partial purification and properties of the cyclic AMP phosphodiesterase activity of Phaseolus vulgaris L. Initially, cyclic AMP phosphodiesterase activity was extracted from 200g of fresh tissue by homogenizing at W C in 335m1 of 5Om~-Tris-HCIbuffer, pH 7.4. The homogenate was centrifuged at l00000g for 1 h and the pellet resuspended in 35ml of 50mMTris-HCI buffer, pH8.2. After dialysis against the same buffer, the phosphodiesterase activity of the non-diffusible fraction was assessed by an adaptation of the method of Butcher & Sutherland (1962). In addition to a portion of theextract, incubation mixtures contained cyclic AMP, MgCI2and (NH4)2S04,all at 2 m ~and , l00pg of 3'-nucleotidasc and 5'-nucleotidase. After 60min at 31"C, released PI was determined by the method of Fiske & SubbaRow (1925). Each assay incorporated a no-substrate control. This procedure showed mature dry seeds to possess cyclic AMP phosphodiesterase activity equivalent to 10.4nmol of PI released/min per g. Activity was also found in the shoot, root and cotyledon of seedlings. On a dry-weight basis, that within the root and shoot decreased with age, from 99.4nmol of PI released/min per g in 2-day-old seedlings to 34.2nmol of PI released/min per g in 12-day-old seedlings. Further purification of the crude phosphodiesterase activity was effected by first centrifuging the homogenate (300g of fresh tissue in 500ml of 50m~-Tris-HCl buffer, pH7.4) at lOooOg for 10min. The supernatant was decanted and dialysed as before. The diffusible fraction (B) was retained and the non-diffusible fraction (A) subjected to fractional precipitation with (NH4)2S04.Protein fractions were obtained at 0-37% saturation, 3 7 4 8 % saturation and 48-100% saturation. The 0-37%-sat~rated-(NH,)~SO~ fraction (C) and the 48-l00%-saturated-(NH4),SO4 fraction (D) were retained, and the 37-48 % - ~ a t u r a t e d - ( N H ~ ) ~fraction S O ~ was again dialysed against Tris-HCI buffer, pH7.4, before chromatography on a column of Stphadex G-200. Two peaks ( I and 11) exhibiting cyclic AMP phosphodiesterase activity were obtained. An inactive protein fraction (E) was eluted before peak I, another (F) was collected between peaks I and 11, and a third inactive fraction ( G )was collected immediately after peak I1 had been eluted. A summary of the partial purification procedure is given in Table I . Increases in activity in excess of 100% of the original activity were shown to be due to removal, during dialysis, of a diffusible inhibitor, probably phosphate, as indicated by the work of Giannattasio et al. (19746). The active material in peaks I and 11 had apparent molecular weights of 3 . 3 7 ~lo5 and 8 . 1 2 lo3 ~ respectively. Both activities required addition of Mgz+ and (NH4)2S04 for full effect; omission of either resulted in a loss of about 20% of the activity. The specificities of the phosphodiesterase activity in the two peaks were assessed relative to cyclic AMP as substrate. Cyclic G M P and cyclic UMP were about 90% as effective as substrates, whereas cyclic IMP, cyclic XMP, cyclic CMP and cyclic dTMP were about SO%aseffective. Dibutyryl cyclic AMPand cyclicdAMPgavevaluesofabout 50%. The results with peak I were essentially similar to those obtained with peak 11. Although fractions B, C, D, E, F and G exhibited no cyclic AMP phosphodiesterase

Vol. 3

Crude homogenate Dialysed homogenate lOOOOg supernatant 37-48 %-satn.-(NH,),SO, ppt. Peak I Peak TI

Preparation

375 382 368 40 81 80

0.81 1.2 1.4 8.8 3.8 2.2

40.5

19 12 13.5 1.4 0.4

Enzyme activity (uni ts/ml)

Protein content (mg/ml)

58 159

2.72 5.5

1

101

0.11 0.65

100

% of original activity 151 169 115

0.02 0.06

Specific activity (units/mg of protein)

For details of the assay method see the text; activities were measured at pH6.5 in 0.1M-cacodylate buffer. Incubations were at 31°C for 60min. One enzymic unit is defined as that amount of enzyme releasing 1 nmol of P,/min.

Table 1. Partial purification of cyclic AMP phosphodiesterase from Phaseolus vulgaris

r

‘d

h

s

5

z 8

P

w

,395

555th MEETING, ABERYSTWYTH

Table 2. Modification of Phaseolus vulgaris cyclic A M P phosphodiesterase activity by errdogenous protein fractions Activity was determined a t 31°C in cacodylate buffer, pH6.5. Change in activity of phosphodiesterase (% of control) Fraction added

Peak I

Peak I1

B C D E F G

-1.2 -77 0

-6.3 -87 0 0 +118 -12.9

-8.1 $1 34 -12.4

activity themklves, fractions C and F had a strong modifying effect on that of peaks I and 11; fraction F was strongly stimulatory whereas fraction C was strongly inhibitory (Table 2). The presence of a cyclic A M P phosphodiesterase inhibitor that does not diffuse during dialysis has been indicated by Brewin & Northcote (19736),but there is n o previous report of the presence of an activator in plant tissues. Such activators have, however, been found in animal tissues (see, e.g., Uzunov & Weiss, 1972). Isoelectric-focusing studies of peaks 1and 11 revealed the presence, in each, of several bands of protein. Only two of these bands in peak I exhibited cyclic A M P phosphodiesterase activity and they had the same pl values as two corresponding bands in peak 11. Preincubation of material of either peak 1 or 11 with the activator (fraction F ) caused all the phosphodiestcrase activity to appear in a single band. The authors gratefully acknowledge a supporting grant from the Science Research Council. Amrhein, N. (1974) Z. PflanzenPhysiol. 72, 249-261 Brewin, N. J. & Northcote, D. M. (1973~)J. Exp. Bot. 24, 881-888 Brewin, N. J. & Northcote, D. M. (19736) Biochim. Biophys. Acra 320, 104-122 Brown, E. G. & Newton, R. P. (1973) Phytochemistry 12, 2683-2686 Butcher, R. W. & Sutherland, E. W. (1962) J. Biol. Cheni. 237, 1244-1250 Fiske, C. M. & SubbaRow, Y . (1925) J. Biol. Chem. 66, 375-381 Giannattasio, M. & Macchia, V. (1973) Plant Sci. Lett. 1,259-264 Giannattasio, M., Mandato, E. & Macchia, V. (1974~)Biochem. Biophys. Res. Commun. 57, 365-372

Giannattasio, M., Sica, G. & Macchia, V. (19746) Phytochernistry 13, 2729-2733 Newton, R. P. (1974) Biochem. SOC.Trans. 2, 385-387 Uzunov, P. & Weiss, B. (1972) Biochim. Biophys. Acra 284, 220-226 Wellburn, A. R., Ashby, J. P. & Wellburn, F. A. M. (1974) Biochim. Biophys. Acra320,363-371

Rhythmic Oscillations in Carbohydrate Metabolism during Growth of Sycamore (Acer pseudopfutunus L.) Cells in Continuous (Chemostat) Culture MICHAEL W. FOWLER and A N N E CLIFTON Department of Biochemistry, University of Shefield, Shefield S10 2TN, U.K. Rhythmic oscillations in metabolic activity are well known in bacterial systems (Chance et a/., 1973). Similar phenomena have also been noted recently in plant tissues (Dietzer VOl. 3

Partial purification of adenosine 3':5'-clclic monophosphate phosphodiesterase from Phaseolus vulgaris l.: associated activator and inhibitors.

555th MEETING, ABERYSTWYTH 393 Partial Purification of Adenosine 3': 5'-Cyclic Monophosphate Phosphodiesterase from Phaseolus vulgaris L. :Associate...
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