Indialt J. PlaRt Physiol., Vol. XXVII, No.2 pp 209-213 (April 1984)
SHORT COMMUNICATION
PARTIAL PURIFICATION AND CHARACTERIZATION OF' STARCH PHOSPHORYLASE FROM CABBAGE (Elephantopus seabar) LEAVES
ANiL KUMAR
Department of Biochemistry, Lucknow University. Lucknow-226 007, India. (Revised: April 3, 1984)
SUMMARY
Starch phosphorylase was partially purified from cabbage leaves using ammonium sulfate fractionation and DEAB-cellulose chromatogra phy. The enzyme showed optimum activity at pH 6.0 and 40°C. It showed absolute specificity for ,Iucose-I-phosphate and utilized starch or amylose as primer with equal efficiency. This was followed by amylopectin and glycogen. However. Schardinger dextrin and cellulose failed to act as primer. L-tyrosine. non-competitively, inhibited the enzyme activity.
Starch phosphory lase (EC 2.4.1.1; -L 4-g1ucan: Orthophosphate glucosyl transferase) has been extensively studied and purified from tubers Lee, 1960; Ariki & Fukui, 1975), and seeds (Bliss & Nayler, 1946; Matheson & Richardson, 1976», but only preliminary studies are reported from leaves (Khanna et al., 1971), probably due to the presence of inhibitory endogenous phenolics. Recently, starch phosphorylase has been homogeneously purified from spinach leaves (Preiss et 01., 1980; Earlier, the purification and characterization of this enzyme from banana (Kumar & Sanwal, 1981, 1982) and tapioca (Kumar
& Sanwal, 1982a) leaves were reported. The present study deals with the the partial purification and characterization of starch phosphorylase from cabbage leaves.
210 ANIL E:.UMA:R
to 100 mland centrifuged at 1600 x g for 30 min in an IEC. PR-2 Refrigerated Centrifuge.
Starch phosphorylase assay in the direction of polysaccharide synthesis was carried out according to the method of Green and Stumpf (1942) with some modifications. The enzyme assay system consisted of 0.2 ml tris-maleate buffer, pH 6.0 (0.1 M).O.l ml freshly prepared soluble starch (3%),0.1 ml sodium fluoride (0.2 M) and 0.5 ml enzyme preparation and water. The reaction was started by the addition of 0.1 ml glucose-I-phosphate (0.05M) and incub ated for 30 min at 30°C. The reaction was stopped by the addition of 1.0 ml 10% trichloroacetic acid. The precipitate formed was removed by contrifug ation in the cold and inorganic phosphate in the clear supernatant was deter mined according to the method of Fiske and Subbarow (1925). A control which received glucose-I-phosphate after deproteinization was run simultaneously. One unit of the enzyme activity was defined as the amount of the enzyme that causes the liberation of one micromole of inorganic phosphate in 30 min under the experimental conditions. Protein was determined according to the procedure of Lowry et al. (1951) as modified by Khanna et aT. (1969) using bovine serum albumin as standard protein.
Unless otherwise stated, the following procedure was carried out at 4-6"C for enzyme purification.
STAR.CH PHOSPHOR.YLASE FROM CABBAGE LEAVES
211
The effect of the composition of isolating media on the activity of starch phosphorylase is shown in Table I. The enzyme activity was present on using pH 7.5 buffer. There was no enzyme activity with pH 6.0 buffer. The presence of Triton-X-IOO in the isolation medium had no effect on the enzyme activity.
Table 1: Effect of the composition of the isolating medium on the activity of starch phosphorylase
Medium used Enzyme activity Protein Spocific activity
(units) (mg) (units/l11I protein)
Medium a1 0 S8 0
Medium b 0 S7 0
Mediumc 0 58 0
Mediumd 29 62 0.47
Medium e 30 64 0.47
Twenty gram tissue was used for the preparation of enzyme nomoaenate.
1 The composition of various media are described in the text.
30-90% Ammonium sulfate fraction showed 1.9 fold enzyme enrichment with 51
%
recovery Table 2. On DEAE-cellulose chromatography, the enzyme was eluted at 0.25-0.30 M NaCI, resulting in overall 7 fold purification with 29% recovery.The optimum pH and temperature both for crude and partially purified enzyme were 6.0 and 40°C, respectively. The ions such as MgH, MnH, CaH,
cr
and SO,-" in 5 mM concentration did not affect the activity but heavy metals such as Ag"'-, Hg~, Fe+2 and CuH in same concentration, inhibited the activity almost completely. The enzyme utilized starch and amylose (3g/0 with equal Table II : Partial purification of starch phosphorylase from cabbage leavesFraction Enzyme activity Protein Sq. act. Fold Recovery (units) (mg) (units/mg
enrich-protein) mont
Initial extract 119.3 262.2 0.45 100
30-90% Ammonium
Sulfate fraction 61.3 68.1
0.'1
1.9 SIDEAE-ceHulose
chromatography 34.7· 11.0 3.1S 7.0 29
~~
. . . .,_ . . . ..r ' .
\
212 ANIL KUMAR
efficiency, but amylopection and glycogen at same concentration were only 70 and 45% active compared to starch. Schardinger dextrin and cellulose failed to act as primer. Glucose-I-phosphate showed absolute specificity. Glucose-6 phosphate, fractose-6-phosphate, fructose L 6-bisphosphate or ribose-5-phosphate could not replace glucose-I-phosphate in the reaction. Aromatic compound, L tyrosin inhibited the enzyme activity non-competitively with a Kivalue of 5
±
1.0 mM as calculated by Dixon plot. The aromatic compounds inhibition of rabbit muscle glycogen phosphorylase (Soman & Philip, 1975), banana fruit starch phosphorylase (Singh & Sanwal, 1973), and banana leaf starch phosphory lase (Kumar Sanwal, 1982) are well known. It may be concluded therefore, a-glucan phosphorylase has a separate binding site for aromatic compounds which regulates the enzme activity in vivo as was shown earlier (Soman & Philip. 1975; Singh & Sanwal, 1974; Kumar & Sanwal, 1982).
ACKNOWLEDGEMENTS
The author thanks Professor G.G. Sanwal for encouragement, enthusiastic discussions and constructive suggestions. The work was supported by grants from the University Grants Commission, New Delhi under the Special Assistance Programme to this Department and United Nations Development Programme.
REFERENCES
Ariki, M. & Fukui, T. (1975). (X-glucan phosphorylase from sweet potato : isolation and properties of the partially degraded enzyme. Biochem. Biophys. Acta., 386: 301-308.
Bliss, L. & Naylor, N.M. (1946). The phosphorylase of waxy maize Cereal Chem., 23: 177-186. Fiske, C.H. & Subbarow, Y. (1925). The colorimetric determination of Phosphorus
J. Bioi. Chem., 66 : 375-400.
Green, D.E. & Stumpf, P.K. (1942). Starch phosphorylase from potato. J. Bioi Chem., 142 : 355-366.
Khanna, S.K., Mattoo, R.L. Vi shwanathan , P.N. Tiwari, C.P. & and Sanwal. G.O. (1969). Colorimetric determination of protein and orthophosphate. Indian J. Biochem .• 6: 21-25.
Khanna S.K., Sanwal, 0.0. & Krishnan, P.S. (1971). Olucan phosphorylase in tbe leaves of Dendrophthoe /alcata: purification and characterization of enzyme. Phytochemis try, 10 : 551-56.
Kumar, A. & Sanwal. 0.0. (1981). Immobilized starch phosphorylase from mature banana (Musa paradisjaca) leaves. Indian J. Biochem. Biophys .• 18 : 114-119.
STARCH PHOSPHORYLASE FROM CABBAGE LEAVES 213
Kumar, A. & Sanwal G.G. (1982&). Starch phosphorylase from tapioca leavea. Absence of pyridoxal phosphate. Arch. Biochem. Biophys., 117 : 341-350.
Lee, Y.P. (1960). Potato phosphorylase 1. Purification. physico-chemical properties and catalytic properties. Biochem. Biophys. Acta., 43 : 18-24.
Lowry. O.H., Rosebrough, N.J. Farr, A.L. & Randall, R.L. (1951). Protein measurement with folin phenol reagent. J. 101. Chem., 193 : 265·275.
Matheson, N.K. & Richardson, R.H. (1976). Starch phosphorylase enzymes in developing and germinating pea seeds. Phyochemistry, 15 : 887-892.
Preiss, J. Okita, T.W. & Greenberg, E. (1980). Characterization of spinach leaf phosphory lase. Plant Physiol., 66 : 864-869.
Sin,h, S. & SanwaJ, G.G. (1973). An allosteric «-glucan phosphorylase (rom banana fruits. Biochem. Biophys. Acta., 30.9: 280-288.
Soman, G. & Philip, G. (I97S). The nature of the biDding site for aromatic compounds in glycogen phosphorylase b. Biochem.I., 147 : 369·371.
