James T. Madison

Structure of a Ribonucleic Acid

The complete nucleotide sequence of an alanine transfer RNA, isolated from yeast, has been determined. This is the first nucleic acid for which the structure is known.

Nucleotide sequence of a yeast tyrosine transfer RNA.

The nucleotide sequence of a tyrosine transfer ribonucleic acid is described and compared to the known sequence of an alanine transfer RNA. It is possible to construct very similar base-paired models for the two molecules in spite of only limited similarities in sequences. The evidence indicates that the sequence containing guanosine, pseudouridine, and adenosine in the middle of the polynucleotide chain is the anticodon.

Threonine synthetase from higher plants: stimulation by S-adenosylmethionine and inhibition by cysteine.

Abstract S-Adenosylmethionine greatly stimulates the formation of threonine from O-phosphohomoserine by an enzyme from sugar beet leaves. The stimulation due to S-adenosylmethionine is inhibited by cysteine. Cysteine and O-phosphohomoserine are incorporated into cystathionine by another enzyme. The results suggest that the conversion of O-phosphohomoserine to either threonine or cystathionine is regulated by the relative amounts of cysteine and S-adenosylmethionine present.

Effect of methionine on growth and protein composition of cultured soybean cotyledons

Abstract lmmature soybean cotyledons were cultured in vitro on a ‘complete’ medium with and without supplementation with methionine. The supplement increased dry wt by 23 %. The growth increase indicated that under these conditions the cotyledons could not synthesize methionine rapidly enough to supply the methionine required for maximum protein synthesis. This indication was supported by finding that aminoacylation of methionyl-transfer RNA was increased 18 % by methionine supplementation. Supplemental methionine also increased the methionine content of the protein fraction by more than 20 %, decreased the arginine content by 11 % and significantly affected several other amino acids. These latter results indicate that the amino acid composition of seed protein can be influenced by the supply of amino acids.

Soybeans and radish leaves contain only one of the sulfonium diastereoisomers of S-adenosylmethionine

Abstract Using a liquid chromatography method that separates the two sulfonium diastereoisomers of adenosylmethionine, we have found that immature soybeans, soybean callus culture, radish leaves, yeast and rat liver contain only the ( S )-sulfonium form of S -adenosylmethionine. Our findings contradict the suggestion by Stolowitz and Minch that 10–20% of naturally-occurring adenosylmethionine may have the ( R )-configuration at the sulfonium pole. Absence of the ( R )-sulfonium isomer of adenosylmethionine in biological materials indicates that the ( R )-sulfonium form of adenosylmethionine present in commercial adenosylmethionine samples is an artifact of the isolation procedure. Our method of measuring the isomers of adenosylmethionine enabled us to readily determine the rate of racemization and hydrolysis of adenosylmethionine. Our rate constants for racemization ( K r ) and hydrolysis ( K h ) were 2.4 × 10 −6 sec −1 and 12.3 × 10- −6 sec −1 , respectively; values which are noticeably different from those of Wu and co-workers which were obtained with a more complicated method ( K r = 8 × 10 −1 sec −1 ; K h = 6 × 10 −6 sec −1 ). We believe the absence of the ( R )-isomer in vivo is best explained by stabilization of the ( S )-isomer as suggested by Wu et al . Although the tissues we have analysed contained the ( S )-sulfonium form of adenosylmethionine exclusively, when ethionine-resistant soybean cell lines were given ethionine, they accumulated both sulfonium diastereoisomers of adenosylethionine.

Characterization of soybean tissue culture cell lines resistant to methionine analogs.

Several hundred soybean [Glycine max (L.) Merr.] cell lines resistant to ethionine were isolated either with or without chemical mutagenesis. of these, 26 were found to contain 2 to 22 times higher than normal levels of uncombined methionine. These 26 cell lines also contained higher than normal levels of S-adenosylmethionine and S-methylmethionine, but the levels of free lysine, threonine, cysteine, valine, tyrosine and phenylalanine were not elevated. Isoleucine levels were only slightly elevated. These results suggest that the regulation of methionine synthesis in vivo is more likely to be later in the pathway (after homoserine phosphate) than early in the pathway.

Homoserine kinase and threonine synthase in methionine-overproducing soybean tissue cultures.

To gain understanding of the regulation of methionine level in plants, we assayed homoserine kinase and threonine synthase in extracts of wild type and several methionine-overproducing soybean [Glycine max (L.) Merr.] callus lines. The specific activity of homoserine kinase was depressed by 45–73%, and that of threonine synthase by 26–43% in the high methionine lines. Cysteine inhibited threonine synthase in wild type and variant lines. Threonine synthase in two variant lines showed significantly less inhibition by cysteine and in one line was inhibited by threonine. Depressed threonine synthase activity may increase the availability of homoserine phosphate to the competing methionine biosynthetic pathway.

Biochemical and physiological studies of soybean β-amylase.

Abstract Soybean ( Glycine max L. Merr.) seed β-amylase has been identified among the ethanol-soluble proteins, purified and several cDNA clones isolated [Ren et al. (1993) Phytochemistry 33, 535]. Both soybean and barley ( Hordeum vulgare L.) β-amylases were soluble in ethanol solutions up to 60%. The solubility of the enzyme decreased with increasing ethanol concentration and dropped sharply between 50 and 60% ethanol. The results showed that β-amylase is an enzyme which can tolerate extensive ethanol treatment at room temperature without losing activity. In soybean seeds, β-amylase appeared to be located neither in protein bodies nor in a membrane fraction. The β-amylase accumulated in the developing seed and disappeared from the germinating cotyledons at a rate similar to that of the total seed protein. Soybean β-amylase was resistant to digestion by some proteases, such as trypsin, chymotrypsin and a protease from Staphylococcus aureus V8. Very little of the total amylase activity in soybean leaves and roots was ethanol soluble. In eight other legume seeds tested, amylase activity was very low compared to the activity present in soybean seeds.

Methionine analogs inhibit production of β-subunit of soybean 7S protein

Abstract We have previously reported that exogenous methionine inhibits production of the β-subunit of the 7 S storage protein in cultured soybean cotyledons, and that this inhibition involves lack of functional m RNA for the β-subunit. Analogs of methionine were used to study this inhibition. Cycloleucine, norleucine, norvaline and S -ethylcysteine treatments prevented accumulation of the β-subunit. The effects of cycloleucine and norleucine on β-subunit synthesis might have been indirect, since these compounds inhibited growth and caused a 2- to 3-fold increase in free methionine concentration. Norvaline did not affect free methionine concentration, but it did inhibit growth. Treatment with a combination of S -ethylcysteine and aminoethoxyvinylglycine prevented appearance of the β-subunit without inhibiting growth or raising the S -adenosylmethionine concentration. Thus, accumulation of S -adenosylmethionine does not appear to mediate the effect of exogenous methionine on β-subunit production. Treatment with S -ethylcysteine raised free methionine concentration only 34%, so S -ethylcysteine was probably acting directly to inhibit β-subunit production. Measurements of free methionine concentrations in seeds of different sizes, taken from intact plants, suggested that the relatively late appearance of the β-subunit in normal soybean seed development may be due to the presence of high levels of free methionine in very young seeds.

Determination of aconitate isomerase in plants.

We have developed a method for measuring aconitate isomerase (EC 5.3.3.7) in plants which depends on the release of tritium from labeled trans-aconitate. The released tritium is separated from labeled aconitate by passage through a column of strong anion-exchange resin. This method is more sensitive, simpler, and more specific than previous methods, especially with crude extracts. The validity of the method was demonstrated by a comparison of the quantity of tritium released with the amount of cis-aconitate isomerized and this comparison demonstrated an isotope effect. Aconitase did not interfere. The method was tested by measuring aconitate isomerase in crude extracts of several higher plant species and tissues and these analyses showed that wheat and corn have more aconitate isomerase than the other species tested.