| dc.description.abstract | A survey of literature indicates that although the occurrence of B-vitamins in higher plants is well known and also that several plant enzymes require the presence of these for their action, very little work has been carried out regarding the forms in which these vitamins occur in plants and their metabolism. The abundance of riboflavin, in particular, is very well known in a number of cereals and pulses and much of the work reported in literature relates to the nutritional aspects.
The present investigations were undertaken to characterize the naturally occurring forms of the flavin in plants and their quantitative relation during germination and distribution in various parts of the seedling. By the application of paper chromatography the flavins from a few plant sources were separated from each other and identified by various criteria such as spectrophotometry, chromatography, specific enzyme assay and degradation, as the well-known nucleotide forms of riboflavin, viz., flavin mononucleotide and flavin adenine dinucleotide. For the routine assay of these nucleotide forms of riboflavin in seeds and seedlings, the fluorimetric method of Bessey et al. was slightly modified to suit the estimations in plant material.
Eight pulse seeds were examined for their flavin nucleotide content. The major flavin in all cases was FAD with varying amounts of FMN and no free riboflavin. On germination for six days there was significant increase in both FMN and FAD contents. Maximum synthesis occurred when the seeds were grown in dark. The studies on inter-organal distribution of the flavin nucleotides in the seedlings of Phaseolus radiatus (green gram) revealed that the major flavin in the plumule was FMN and the radicle was FAD. After 5 days of germination, the plumule and radicle contained 30 and 6% of total flavins, respectively. Of the total FAD 87% was present in the radicle and 13% in the plumule, while 37% of the total FMN was present in the radicle and 47% in the plumule.
These results have been interpreted as follows: (1) the growing parts are the sites of synthesis of the flavins, (2) the predominance of FMN in the plumule is probably due to the requirement of FMN in photosynthesis when the plumule forms the green shoot, (3) the predominance of FAD in the radicle is probably due to the high metabolic activity of this part during the early stages of development of the seedling and (4) the increased synthesis of flavins when the seeds are grown in dark may be so because in the absence of photosynthesis the only source of energy is through oxidative phosphorylation and flavin being an important constituent of the chain it is synthesised more in order to satisfy the demand (Chapter I).
Having established the naturally occurring forms of riboflavin as FMN and FAD, the next step is to study the mode of formation and degradation of these. By an earlier investigation, Prof. K.V. Giri and his colleagues in this laboratory had shown that FMN is synthesised by a typical kinase reaction. The enzyme catalysing the reaction,
Riboflavin + ATP ? FMN + ADP
was partially purified from resting seeds of green gram (P. radiatus) and characterised by them. Following this study, the biosynthesis of FAD was undertaken in the present investigations.
The enzymatic synthesis of flavin adenine dinucleotide was found to proceed according to the following equation:
FMN + ATP ? FAD + P-P
pH 7.4, 37°C
The partial purification of the enzyme and the description of the system in plants has been reported. The enzyme from green gram seeds was solubilised by mcerhonate and was purified 85-fold by ethanol fractionation, adsorption and elution gel. The reaction was a condensation between FMN and AMP, the latter being donated by the ATP molecule. The optimum conditions were pH 7.4 and 37°C. The pathway for the biosynthesis of FAD differed from the system reported by Trufanov wherein riboflavin directly reacted with ATP to yield FAD and FMN. The system described by us is similar to the system reported in yeasts and animal tissues. The enzyme synthesising FAD is of common occurrence in plants (Chapter II).
During the studies on the enzymes synthesising FMN and FAD, it was observed that the crude extracts possessed high hydrolytic activity against these nucleotides and hence an inhibitor was to be used in order to demonstrate the synthetic reactions. The characterisation of the enzymes hydrolysing FMN and FAD were studied.
The enzyme catalysing the hydrolysis of FMN was purified from extracts of green gram seeds by an effective combination of classical methods of fractionation and chromatography on CM-cellulose. The method described in text yielded a preparation about 200-fold purified. During the process of purification, a gross portion of the non-specific acid phosphatase, inorganic pyrophosphatase, ATPase, 5? and 3?-nucleotidases were removed. Although it retained an appreciable ?-glycerophosphatase activity, in view of its higher affinity (1.25 × 10?? M) for FMN and a greater rate of reaction with FMN as substrate, suggested its identity as a specific FMN-hydrolase. However, the FMN-hydrolase was entirely different from the FMN-synthesising enzyme "flavokinase". The FMN-hydrolase functioned optimally at a pH 5.2 and a temperature 49°C (Chapter III).
The enzyme was capable of transferring the cleaved phosphoryl moiety to suitable acceptors such as simple alcohols, thiamine, pyridoxal, pyridoxamine and nucleosides, yielding the corresponding phosphate derivatives. It is interesting to note that the phosphorylation of pyridoxal and thiamine, which normally demands the participation of energy-rich pyrophosphate bonds (P~P) like those of ATP, are synthesised by a simple hydrolytic enzyme involving a transfer of the cleaved moiety to the available acceptor. The identity of pyridoxal phosphate and thiamine monophosphate was established by absorption spectra and co-chromatography with authentic samples. The enzyme also showed a certain degree of specificity towards the acceptors of phosphate moiety. While pyridoxal, pyridoxamine, were phosphorylated, pyridoxine, in spite of the availability of two -CH?OH groups, was not phosphorylated. Similarly, in the case of thiamine, thiamine disulphide was not phosphorylated. Amongst the purine and pyrimidine nucleosides, the pyrimidine nucleosides were preferred.
Thus the enzyme functioned as a synthetase as well and participated in the synthesis of coenzymatic forms of related vitamins (Chapter IV).
The enzyme hydrolysing FAD was purified partially (85-fold) from extracts of green gram (P. radiatus) seedlings by classical methods such as fractionation with ammonium sulphate, ethanol and adsorption on alumina. The hydrolysis of FAD was optimal at pH 7.2 and 37°C. The products of hydrolysis were FMN and AMP. The enzyme was inhibited by cyanide and fluoride. It was unlikely to ageing in the cold (0–4°C). Thus it differed in characteristics from the well-known nucleotide pyrophosphatase purified extensively and well characterised by Kornberg and Pricer. Therefore, it is suggested that this enzyme may be one of the species of the nucleotide pyrophosphatase class of enzymes. | |
