| dc.description.abstract | SYNOPSIS OF THE THESIS ENTITLED
“POLYAMINE METABOLISM IN HIGHER PLANTS: ENZYMATIC ASPECTS OF PUTRESCINE BIOSYNTHESIS IN CUCUMIS SATIVUS”
Submitted by G. L. Prasad for the award of the Ph.D. degree of the Indian Institute of Science, Bangalore, India
As part of an ongoing programme in this laboratory to unravel the molecular mechanisms involved in polyamine biosynthesis in higher plants, this thesis deals with the enzymatic aspects of putrescine production and its regulation in Cucumis sativus (cucumber), used here as a model system.
Earlier work from this laboratory had shown that cucumber cotyledons grown in long-term organ culture could be used to examine amine-hormone interrelationships and to correlate changes in levels of arginine decarboxylase (ADC)-the first and rate limiting enzyme-with changes in putrescine levels in response to phytohormones and altered environmental conditions such as potassium and acid treatments.
To minimize the generalized pleiotropic growth responses associated with long term cultures treated with growth regulators or other test compounds, a short term culture system was preferred in this study to assess the in vivo influence of amines and amino acids known to modulate ornithine decarboxylase (ODC) activity in animal systems (Chapter I).
Upon culturing cucumber cotyledons with 10 mM agmatine for 12 h, a 50% inhibition of ADC activity was observed. Prolonging the culture period or increasing agmatine concentration did not further suppress ADC. The inhibition was gradually relieved after 12 h exposure to the guanido amine.
Among various di- and polyamines tested for potential inhibitory effects on ADC, putrescine proved most potent (60% inhibition), while N carbamylputrescine (NCP) was the least effective. Among amino acids, L homoarginine, the higher homologue of arginine (and a competitive inhibitor of Lathyrus sativus ADC), dramatically enhanced ADC activity (3-4 fold), with maximal stimulation at 12 h of culture. This stimulatory effect, like agmatine inhibition, was transient.
The possible involvement of non dialysable macromolecular effectors in the modulation of ADC by agmatine or homoarginine was ruled out. De novo protein synthesis was essential for homoarginine stimulated ADC activity, whereas agmatine inhibition of basal activity did not require new protein synthesis in a 12 h culture.
Altered ADC activity also appeared to arise from changes in substrate affinity. The enzyme exhibited Km values of 0.8 mM (control), 0.3 mM (homoarginine treated) and 1.5 mM (agmatine treated) cotyledons. Unlike mammalian ODC, the rate of ADC degradation (t½ 5 h) did not change significantly with altered enzyme activity.
Despite being a potent ADC stimulator, homoarginine failed to induce ADC in K treated cotyledons, reaffirming the known overriding control of potassium on amine metabolism.
Previous studies showed that ADC purified from different higher plants exhibited different molecular organizations-for example:
1. Lathyrus sativus ADC: homo hexamer, native Mr 220,000
2. Oat seedling ADC: two molecular species of Mr 195,000 and 118,000
Hence, it was of interest to explore the molecular features of ADC from another higher plant-Cucumis sativus. It was also necessary to compare in vivo and in vitro regulation of this enzyme to obtain a cohesive picture of its modulation. These studies are described in Chapter II.
A simple and reproducible three step purification protocol involving DEAE cellulose chromatography and Sephadex G 150 gel filtration was devised for ADC purification from cucumber seedlings. The final preparation was homogeneous by PAGE (pH 8.3) and analytical ultracentrifugation.
However, on SDS PAGE under reducing conditions, the enzyme resolved into 48 kDa, 44 kDa and 15 kDa polypeptides, whereas under non reducing conditions it migrated as a single species of Mr 150 kDa. Two dimensional tryptic peptide mapping of radio iodinated 48 kDa and 44 kDa bands established that the 44 kDa (and 15 kDa) species originated by proteolytic degradation of the 48 kDa polypeptide.
On storage at 4°C for ~2 weeks, the 48 kDa species was degraded by associated/intrinsic proteases to smaller peptides while the 44 kDa species remained intact.
The purified ADC also displayed ornithine decarboxylase (ODC) activity. Interestingly, ODC activity could not be assayed in crude extracts, likely due to a lost inhibitor during purification. Various ligands (agmatine, Pi, difluoromethylornithine, putrescine) influenced ADC and ODC activities differently, indicating two distinct but interacting catalytic domains. This is the first demonstration that arginine and ornithine are decarboxylated by the same enzyme protein.
The agmatine putrescine conversion pathway had not been thoroughly examined in higher plants until recent work in this laboratory identified a multifunctional enzyme-putrescine synthase-in Lathyrus sativus seedlings. This enzyme possesses the following activities:
1. Agmatine iminohydrolase (AIHase)
2. Putrescine transcarbamylase (PTCase)
3. Ornithine transcarbamylase (OTCase)
4. Carbamate kinase (CKase)
Preliminary evidence for an “agmatine cycle” in other plants, including cucumber, placed putrescine synthase at the centre of putrescine biosynthesis.
Chapter III details purification and characterization of putrescine synthase from cucumber seedlings using putrescine CH-Sepharose affinity chromatography. The purified enzyme contained all the component activities listed above and catalyzed the complete agmatine putrescine conversion via OTCase or CKase.
On PAGE (pH 8.3), the enzyme resolved into two entities (EA and EM), with EM more abundant. On SDS PAGE under reducing conditions, both forms resolved into 48 kDa and 44 kDa polypeptides, along with smaller degradation products; the unreduced enzyme migrated as a single ~150 kDa band. Two dimensional electrophoresis established that EA and EM consist of identical polypeptides.
Purifying the enzyme from seedlings at different developmental stages showed that the 48 kDa and 44 kDa species are in vivo degradation products of a 66 kDa precursor. Isolation of RNA from six day old seedlings and its translation in a cell free rabbit reticulocyte lysate system confirmed that putrescine synthase is synthesized as a 66 kDa primary translation product. In contrast, in a homologous cell free system (S extract of cucumber cotyledons), both the 66 kDa precursor and a 48 kDa species were detected.
EA exhibited higher activity per unit protein for all component and overall reactions relative to EM or the putrescine Sepharose eluate. Both ADC and putrescine synthase showed similar chromatographic and electrophoretic behavior and were immunologically indistinguishable.
Using [¹ C]-arginine, putrescine-rather than agmatine-was the sole product of the ADC reaction in purified preparations, indicating tightly regulated AIHase activity. Thus, while ADC is the rate limiting step, the committed step in putrescine biosynthesis appears to be agmatine N carbamylputrescine conversion.
These observations clearly demonstrate that arginine putrescine and ornithine putrescine conversions in cucumber seedlings are mediated by a single versatile multifunctional enzyme. | |
