The enzymatic malonylation of 1-aminocyclopropane-1-carboxylic acid in homogenates of mung-bean hypocotyls

Homogenates of hypocotyls of light-grown mung-bean (Vigna radiata (L.) Wilczek) seedlings catalyzed the formation of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) from the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-coenzyme A. Apparent K_m values for ACC and malonyl-CoA were found to be 0.17 mM and 0.25 mM, respectively. Free coenzyme A was an uncompetitive inhibitor with respect to malonyl-CoA (apparent K_i=0.3 mM). Only malonyl-CoA served as an effective acyl donor in the reaction. The d-enantiomers of unpolar amino acids inhibited the malonylation of ACC. Inhibition by d-phenylalanine was competitive with respect to ACC (apparent K_i=1.2 mM). d-Phenylalanine and d-alanine were malonylated by the preparation, and their malonylation was inhibited by ACC. When hypocotyl segments were administered ACC in the presence of certain unpolar d-amino acids, the malonylation of ACC was inhibited while the production of ethylene was enhanced. Thus, a close-relationship appears to exist between the malonylation of ACC and d-amino acids. The cis- as well as the trans-diastereoisomers of 2-methyl- or 2-ethyl-substituted ACC were potent inhibitors of the malonyltransferase. Treatment of hypocotyl segments with indole-3-acetic acid or CdCl₂ greatly increased their content of ACC and MACC, as well as their release of ethylene, but had little, or no, effect on their extractable ACC-malonylating activity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Germacranolides from Melampodium americanum and Melampodium pilosum

Chemical analysis of Melampodium americanum yielded, besides the known melampodinin A, four new melampolides, melampodinin B, melampodinin C, 9-des     

Inhibition of anthocyanin formation in seedlings and flowers by the enantiomers of α-aminooxy-β-phenylpropionic acid and their N-benzyloxycarbonyl derivatives

Both enantiomers of -aminooxy--phenylpropionic acid (AOPP), potent inhibitors of L-phenylalanine ammonia-lyase, and their N-benzyloxycarbonyl (N-BOC) derivatives inhibit anthocyanin formation in developing flowers of Ipomoea tricolor Cav. and Catharanthus roseus Don. as well as in seedlings of Brassica oleracea var. caulo-rapa DC (kohlrabi) and B. oleracea var. capitata L. (red cabbage) with little interference with their normal development. Kohlrabi seedlings tolerate up to 0.3 mM L-AOPP and N-BOC-L-AOPP without a reduction of fresh weight or chlorophyll content, while anthocyanin is reduced to less than 20%.Abbreviations AOPP -aminooxy--phenylpropionic acid - N-BOC N-benzyloxycarbonyl - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Purification of chorismate synthase from a cell culture of the higher plant Corydalis sempervirens Pers

Chorismate synthase (EC 4.6.1.4) was purified from a cell suspension culture of Corydalis sempervirens almost 1000-fold to near homogeneity. The subunit Mr estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 41,900. The Mr of the native enzyme was estimated to be 80,100 by gel filtration, suggesting a dimeric structure. Antisera directed against the 41.9-kDa protein also reacted with the native enzyme. Further confirmation of the identity of the purified protein was obtained by sequence comparison of a tryptic peptide with known sequences of the Escherichia coli and Neurospora crassa chorismate synthases.     

Substitution of Gly-96 to Ala in the 5-enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae results in a greatly reduced affinity for the herbicide glyphosate

The aroA gene of Klebsiella pneumoniae encoding the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, which is the target of the herbicide glyphosate, was cloned and sequenced from both the wild-type and the glyphosate-resistant mutant K. pneumoniae K1, which possesses a glyphosate-insensitive EPSP synthase. Both genes were expressed in Escherichia coli and were capable of complementing an auxotrophic aroA mutation. The transformed cells showed increased tolerance to glyphosate due to the overproduction of either the mutant or the wild type EPSP synthase. Nucleotide sequence analysis of the K. pneumoniae aroA gene indicated a protein-coding region of 427 amino acids with a derived Mr for the EPSP synthase of 45,976. Comparison of the two aroA alleles showed a single base change resulting in a substitution of Gly-96 to Ala in the deduced amino acid sequence. By comparison with other known EPSP synthase sequences the mutation was shown to be located in a highly conserved region, indicating that this region is essential for the binding of the herbicide glyphosate.     

On the turnover of polyamines spermidine and spermine in mouse brain and other organs

The apparent biological half-lives of spermidine and spermine in mouse brain and other organs were determined by measurement of the specific radioactivities of these compounds over long periods of time. The endogenous polyamine pools were labeled by repeated intraperitoneal injections of [1,4-¹⁴C]putrescine·2HCl, [2-¹⁴C]d,l-methionine, [2-³H]l-methionine, andS-adenosyl-[2-³H]l-methionine. Repeated injections were given to ensure labeling of both fast and slow polyamine pools. It was shown that the two parts of the polyamine molecules which derive from ornithine and methionine have significantly different life spans, especially in the brain. Actual turnover rates of polyamines could not be determined because of the active interconversion between spermine and spermidine, and between spermidine and putrescine. The observed reutilization of putrescine originating from spermidine degradation for spermidine biosynthesis, and the analogous reutilization of spermidine in spermine biosynthesis is discussed with respect to its physiological significance and its relationship to cellular organization.     

Regulatory interrelations between GABA and polyamines. I. Brain GABA levels and polyamine metabolism

Elevation of brain GABA levels by GABA-T inhibition is accompanied by a decrease ofS-adenosylmethionine decarboxylase activity. This is followed by an increase of ornithine decarboxylase activity and a severalfold increase of brain putrescine levels. Spermidine and spermine levels are not significantly affected under these conditions. These unexpected findings support a regulatory interaction between GABA and polyamine metabolism.     

The estimation of phenylalanine ammonia-lyase (PAL)-activity in intact cells of higher plant tissue

From cell cultures of Haplopappus gracilis, an enzyme, catalyzing the glucosylation of cyanidin at the 3 position using uridine diphosphate-D-glucose (UDPG) as glucosyl-donor, has been isolated and purified 50-fold. The enzyme was not specific for cyanidin alone, but also glucosylated other anthocyanidins and flavonols in position 3. However, apigenin, luteolin, naringenin and dihydroquercetin were not glucosylated. The reaction has an optimum pH of approximately 8, and the apparent K _m values for UDPG and cyanidin were 0.5 and 0.33 mM respectively. The enzyme reaction is strongly inhibited by cyanidin (above 0.25 mM).     

A spectrophotometric assay for meso-diaminopimelate decarboxylase and L-alpha-amino-epsilon-caprolactam hydrolase

A spectrophotometric assay for the activities of mesodiaminopimelate decarboxylase and L-alpha-amino-epsilon-caprolactam hydrolase is described. With the commercially available enzyme saccharopine dehydrogenase lysine formed either by decarboxylation of meso-diaminopimelate or by hydrolysis of L-alpha-amino-epsilon-caprolactam is converted to saccharopine with the concomitant oxidation of NADH, which is monitored by the decrease in absorbance at 340 nm. For meso-diaminopimelate decarboxylase this assay can be performed either as an endpoint determination, when working with crude extracts, or as a continuous spectrophotometric assay of partially purified enzyme preparations. The activity of L-alpha-amino-epsilon-caprolactam hydrolase can only be assayed by the endpoint method because of the great differences in the pH optima of the hydrolase and the saccharopine dehydrogenase.