Metabolism of Cytokinin

Abstract

As part of the study of cytokinin metabolic pathways, an enzyme, adenosine phosphorylase (EC 2.4.2.-), which catalyzed the ribosylation of N6(A2-isopentenyl)adenine, N6-fUrfUryladenine, and adenine to form the corresponding nucleosides, was partially purifi from wheat (Triticum aestvum) germ. The pH optimum for the ribosylation of the cytokinins and adenine was from 6.5 to 7.8; for guanine and hypoxanthine it was from 7.0 to 8.5. At pH 7.2 (63 mfllimolar N-2-hydroxyethyl piperazine-N'-ethanesulfonic acid) and 37 C the Km for N -(A2-isopentenyl)adenine was 57.1 micromolar, NO-furfuryladenine, 46.5 micromolar, adenine, 32.2 micromolar, and the V,,= for N6-(A2-isopentenyl)adenWne, N-fUrfUryladenine, and adenine were 134.7, 137.1, and 193.1 nanomoles per millim protein per minute, respectively. The equilibrium constants of the phosphorolysis of N-(A2-isopentenyl)adenosine and adenosine by this enzyme indicated that the reaction strongly favored nucleoside formation. This enzyme was shown to be distinct from inosine-guanosine phosphorylase based on the differences in the Sephadex G-100 gel ffitration behaviors, pH optima, and the product and p-hydroxymercuribenzoate inhibitor studies. These results suggest that adenosine phosphorylase may play a significant role in the regulation of cytokinin metabolism.The metabolism of cytokinins has been studied in a variety of plant tissues (2-4, 7-9, 11, 13, 15) and some of the enzymes which regulate cytokinin metabolism have also been isolated (1, 6). One of the major metabolites formed from the cytokinin base is the corresponding ribonucleoside. Purine nucleoside phosphorylase (purine nucleoside, orthophosphate ribosyl transferase, EC 2.4.2.1) which catalyzes a reversible phosphorolysis of purine nucleosides is generally believed to be inactive toward Ado2 (14,16,19) and cytokinin nucleoside. One of the possible pathways by which the cytokinin base can be converted to the corresponding ribonucleoside is the phosphoribosylation of the cytokinin base to the ribonucleotide and then conversion to the corresponding ribonucleoside by 5'-nucleotidase (1). Senesi et al. (17) recently reported that in Bacillus subtilis adenosine phosphorylase (EC 2.4.2.-) is distinct from purine nucleoside phosphorylase and that this enzyme catalyzes the formation of Ado from Ade. Adenosine phosphorylase activity was also found in some species of mycoplasmas (10) and mammalian cells (5).This paper presents the first direct evidence that in plant cells the ribosylation of cytokinin base and Ade is catalyzed by adenosine phosphorylase, and that this enzyme is distinct from inosineguanosine phosphorylase. The equilibrium constant of the ribo- Chromatograms were cut into 1-cm sections and placed in vials containing scintillation fluid (4). Radioactivity was measured in a Nuclear-Chicago Unilux II scintillation system. Counting efficiency of paper chromatogram sections was 74% for 14C. A Cary model 14 spectrophotometer was used to measure the quantity of purine base and its corresponding riboside. P...

Metabolism of Cytokinin: Deribosylation of Cytokinin Ribonucleoside by Adenosine Nucleosidase from Wheat Germ Cells

“…Cytokinin bases and cytokinin ribonucleosides have been found in various plant cells, and these cytokinins are metabolized in the plant cell to form different metabolites (3,4,9,11,16,18). The relative amount of a specific metabolite formed may differ not only from one plant to another, but also in one particular plant or tissue under different physiological conditions.…”

“…Km values for the cytokinin nucleoside and esine are 238 and 1.43 micromolar, respectively, in 50 milHolar Tris-citrate buffer (pH 4.7) at 30 C. The presence of adenosine and other cytokinn nucleosides inlhbited the hydrolysis of N64A'.isopentenyl)adenosine but this reaction wa insensitive to guanosine, uridine, or 3'deoxy oine. It is hypothesized that an adequate level of "active cytokinin" in plant cells may be provided through the deribosylation of cytokinin riboside in concert with other cytokinin metabolic enzymes.Cytokinin bases and cytokinin ribonucleosides have been found in various plant cells, and these cytokinins are metabolized in the plant cell to form different metabolites (3,4,9,11,16,18). The relative amount of a specific metabolite formed may differ not only from one plant to another, but also in one particular plant or tissue under different physiological conditions.…”

Purine Catabolism in Plants

“…Also, the soluble fraction from pea seedlings produced inosine and hypoxanthine from substrate IMP (43), but these data did not explain whether inosine originating from IMP is converted to hypoxanthine hydrolytically or by phosphorolysis. In the extracts from germinating wheat embryos (38) and wheat germs (10), the activity of inosine phosphorylase ' This work is dedicated to Professor Jerzy Pawekkiewicz on the occasion of his sixtieth birthday.…”

Metabolism of Cytokinin

“…The occurrence of cytokinin nrbonucleotide and cytokinin ribonucleoside in plant cells is well documented (2,4,5,10,11).…”

“…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 ribonucleoside 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 ribonucleotide i6Ado-5'-P2 can be synthesized by a de novo pathway from the simple metabolites AMP and A2-isopentenylpyrophosphate (6, 24).…”