Isolation of a ribonucleus from grain sorghum (Sorghum vulgare pers) and initial studies on its physico-chemical properties and mode of action

Abstract

The present thesis is concerned with the detection, isolation and characterization of a ribonuclease from a cereal, namely grain sorghum (Sorghum vulgare Pers.). Grain sorghum is a very important cereal forming the staple diet of large populations throughout the world. Information regarding the proteins of sorghum is rather limited, and we have initiated an investigation in this area. Ribonucleases play an important part in the metabolism of ribonucleic acids in all biological systems. Investigations on the isolation, structure, specificity and mode of action of these enzymes have in recent years added considerably to our knowledge of their key role in life processes. Although considerable information is available about some of the animal and microbial ribonucleases, information is rather limited in the case of higher plant ribonucleases. There are many reports about their presence in higher plants; however, there are only few instances where they have been obtained in any degree of purity from higher plant sources for studies of their structure and function. A systematic investigation was carried out to determine the distribution of ribonuclease activity in several strains of grain sorghum. All the strains exhibited ribonuclease activity with small differences among them. The strain M 35 1, one of the higher activity strains, was chosen for further studies on ribonuclease from grain sorghum. Ribonuclease activity in germinating grain sorghum was determined in order to assess the changes, if any, in the levels of the enzyme during development. The specific activity of the enzyme was found to increase from the 2nd day of germination onwards until at least the 8th day. A large part of the activity was located in the endosperm (seed coat intact) portion of the seedlings. The specific activity of ribonuclease in the endosperm portion of 8 day old seedlings was four fold higher than in resting seeds. The increase in specific activity of the enzyme in the axis (both root and shoot included), on the other hand, was very small and even on the 8th day it was only about the same as that present in resting seeds. Thus, the endosperm part of 6-7 day old seedlings appeared to be suitable material for further studies on ribonuclease from grain sorghum. Studies were undertaken to purify the enzyme from this source. The purification of plant proteins is often complicated by factors not usually present in the study of animal proteins. Plant extracts normally contain a low concentration of proteins and a high concentration of secondary metabolite products. Quinones, especially, are known to react with amino acid groups in enzymes, resulting in their modification and inactivation. In attempts to purify ribonuclease from endosperm of germinating grain sorghum, various methods were tried to select a procedure that would be consistent and reproducible. Attempts to purify the enzyme from buffer extracts of fresh endosperm or acetone powders thereof were unsuccessful, due to large losses of activity during further purification and poor quality protein preparations, for example intense coloration of the protein samples. Acid extracts of fresh endosperm were found to have ribonuclease activity and were suitable for purification of the enzyme. Accordingly, the enzyme was extracted with cold dilute sulfuric acid containing ascorbic acid and polyvinylpolypyrrolidone (PVP), an insoluble polymer known to remove phenolic compounds from plant tissue. The acid extract was fractionated with ammonium sulfate, and the active 40-70% ammonium sulfate fraction was obtained. This fraction was further purified by column chromatography on CM cellulose (pH 6), Sephadex G 50 (pH 6) and Sephadex G 75 (pH 7.5). The ribonuclease preparation obtained had high specific activity and migrated as a single component on acrylamide gel electrophoresis. The preparation was free from related nucleolytic activities such as phosphomonoesterase, phosphodiesterase and deoxyribonuclease activities. It was also free from proteolytic activity. Studies on the physicochemical properties, specificity and mode of action of the enzyme were carried out with this preparation. Sorghum ribonuclease did not irreversibly lose significant activity when incubated with 5.5 M urea. Treatment with 8 M urea or 6.5 M guanidine hydrochloride, however, irreversibly denatured the enzyme, resulting in loss of 80% of its initial activity. Sorghum ribonuclease was found to be stable over a wide pH range, pH 2-11. Above pH 11, the enzyme lost its activity sharply, with almost complete loss at pH 12. In its high pH stability the enzyme resembled pancreatic RNase. Sorghum ribonuclease was also highly stable to heat in the pH range 2-7.5. In contrast to pancreatic RNase, sorghum ribonuclease was inactivated by treatment with trypsin and chymotrypsin at room temperature. The enzyme was more sensitive to trypsin than to chymotrypsin. The UV absorption spectrum of sorghum ribonuclease was typical of a protein, with a maximum at 278 nm and a minimum at 256 nm. A shoulder around 290 nm indicated the presence of tryptophan residues. The protein showed no absorbance in the visible region. The molecular weight of sorghum ribonuclease was found to be about 20,000 daltons. The enzyme retained this molecular weight even after treatment with sodium dodecyl sulfate and mercaptoethanol, indicating that it consisted of a single polypeptide chain. Amino acid analysis showed that the enzyme contained 177 residues. It contained all the amino acids normally present in proteins. It was rich in acidic amino acids, leucine, glycine, alanine, valine, serine, tyrosine and tryptophan. There was one methionine residue and three histidine residues. No detectable carbohydrate was present; the protein appeared to be composed entirely of amino acids. Its amino acid composition differed markedly from those of RNase A and RNase T , and resembled more closely that of corn RNase I. The N terminal amino acid of sorghum ribonuclease was identified by the dansylation procedure as glycine. The pH activity profile of sorghum ribonuclease resembled that of pancreatic RNase, with maximal activity at pH 7-8. Sorghum ribonuclease retained ~8% of maximal activity at pH 3 and pH 10. RNase A, in contrast, had no activity at pH 3 and about 28% activity at pH 10. To obtain preliminary information on functional groups essential for activity, the effects of specific chemical reagents were examined. Sulfhydryl reagents (p chloromercuribenzoate and N ethylmaleimide) had no effect, indicating the absence of essential sulfhydryl groups. Reducing agents such as mercaptoethanol and cysteine hydrochloride also had no effect. Sorghum ribonuclease was inactivated by iodoacetate at pH 7.5. At pH 2.6 the reagent had no effect, and at pH 5.0 the activity loss was much lower, suggesting involvement of histidine and/or lysine residues at the active site. Sorghum ribonuclease did not require any metal ions for activity; many divalent metal ions were strongly inhibitory. The specificity and mode of action were investigated using synthetic and natural substrates. The enzyme was shown to be an endoribonuclease that cleaves polynucleotide chains at multiple points. 2 ,3 cyclic nucleotides were formed as intermediates and were subsequently hydrolysed to 3 phosphates exclusively. Of the four homopolyribonucleotides (poly A, poly U, poly C and poly G), poly A and poly U were readily cleaved; poly C was cleaved slowly; poly G was completely resistant. However, sorghum ribonuclease cleaved the dinucleoside monophosphate guanylyl 3 5 cytidine (GpC), and it cleaved phosphodiester bonds adjacent to any of the four bases in yeast RNA, demonstrating base non specificity. The failure to act on poly G was likely due to the stable higher order structure of that polymer. In conclusion, this new, highly purified ribonuclease from grain sorghum-exhibiting properties both similar to and distinct from known RNases-appears to be well suited for comparative studies of the structure and function of ribonucleases from animal, microbial and higher plant sources.