Metabolism of Cytokinin

Abstract

Two forms (F-I and F-II) of 5'-nucleotidases (5'-ribonucleotide phos-phohydrolase, EC 3.1.3.5) which catalyze the dephosphorylatlon of N-(A2-lsopentenyl)adenosine S'-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Ticwum aesiwm) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5'-nucleotidases dephosphorylate the ribonu-cleotides at an optimum pH of7. The Km values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millmolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside S'-phosphates, AMP being the preferred substrate. N6-(A2-isopentenyl)Adenosine 5'-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 32-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5'-nucleotidases may play an important role in regulating levels of "active cytokinin" in plant cells. The occurrence of cytokinin nrbonucleotide and cytokinin ri-bonucleoside in plant cells is well documented (2, 4, 5, 10, 11). The cytokinin ribonucleotide can be formed from cytokinin base (3, 11, 18, 20), the ribonucleoside (3, 7, 17, 18) or turnover of cytokinin-containing tRNA (13, 22). Alternatively, the ribonu-cleotide i6Ado-5'-P2 can be synthesized by a de novo pathway from the simple metabolites AMP and A2-isopentenylpyrophos-phate (6, 24). In the de novo biosynthetic pathway using a crude enzyme preparation, cytokinin ribonucleoside was also formed. Thus, 5'-nucleotidases which are capable of dephosphorylating the ribonucleotide may be contained in the crude enzyme preparation. The 5'-nucleotidase has been isolated from animal cell membrane (8, 9, 14) and cytosol3 (19). Although the precise physiological function of this enzyme system in plant and animal cells is still unclear, the 5'-nucleotidase may play a role in the regulation of cytokinin metabolism. We describe here the partial purification of 5'-nucleotidase from wheat germ cytosol, the properties of the enzyme, and kinetics of 1 This work was supported by National Science Foundation Research Grant PCM 79 03832 (to C.-M. C). 2Abbreviations: i6Ado-5'-P, N6-(A2-isopentenyl)adenosine 5'-mono-phosphate; Ade, adenine; Ado, adenosine; i6Ado, N6-(A2-iso-pentenyl)adenosine. 3 The term "cytosol" is used only in distinction to "membrane bound." The extraction procedures do not rigorously preclude the possibility of contamination by nucleus enzymes. the dephosphorylation of cytokinin ribonucleotide by this enzyme system. MATERIALS AND METHODS CHEMICALS AND ENZYMES Ado, AMP, i6Ado, concanavalin A (grade IV), carbowax (mol wt, 6,000), 5'-nucleotidase (Crotalus adamteus venom), and wheat (Triticum aestivum) germ were from Sigma; i6Ado-5'-P was from P-L Biochemical Co., and [8-'4C]Ado (59 mCi/mmol) was from Amersham-Searle Corp. ANALYTICAL TECHNIQUES Protein concentration was determined according to the method of Bradford (1). A Cary model 14 spectrophotometer was used to quantify purine nucleosides and nucleotides. Dephosphorylated nucleosides were separated from the corresponding nucleotides by paper electrophoresis (Camag TLE Cell, Whatman No. 3MM paper) with 0.5 M Tris-citrate buffer (pH 3.5) at 20 C and by paper chromatography (Whatman No. 3MM) in a descending fashion using the following solvent systems (v/v): A, 95% ethanol: 100 mm (NH4)3B03 (pH 9.0) (1:9); B, ethylacetate:l-propanol:H20 (4:1:2); C, l-propanol:concentrated NH40H:H20 (60:20:20). Radioactivity was measured in a Nuclear-Chicago Unilux II scintillation system. EXTRACTION AND FRACTIONATION OF ENZYMES Wheat germ (45 g) frozen with liquid N2 was homogenized in a Waring Blendor in 7 volumes/weight of buffer A [50 mm Tris-HCl buffer (pH 7), 10 mm 2-mercaptoethanol, and 12 mM MgCl2J containing polyvinylpolypyrrolidine (4.5 g). The homogenate was filtered through double layers of cheesecloth. The filtrate was centrifuged for 15 min at 15,000g and the resulting supernatant was centrifuged again for 20 min at 20,000g. The supernatant is referred to as extract. The following steps were employed to further purify the extract: Step 1. Ammonium Sulfate Fractionation. Solid ammonium sulfate was added to the extract to 20%o saturation. After 30 min, the precipitate was removed by centrifugation at 20,000g for 20 min and the supernatant was brought to 60%1o by ammonium sulfate. The precipitate, collected by centrifugation at 25,000g for 20 min, was redissolved in 20 ml buffer A. The protein solution (136 mg protein) was dialyzed against 2 liters buffer A for 16 h and reduced to 10 ml by carbowax. Step 2: DEAE-cellulose Chromatography. The concentrated ,protein solution was applied onto a DEAE-cellulose (Whatman DE-23) column (2.5 x 27 cm) equilibrated with buffer A. The column was eluted with 1.4 bed volumes buffer A followed by linear gradient of NaCl (0 to 0.5 M; total volume, 180 ml) in the 494