Synthesis and degradation of 1-aminocyclopropane-1-carboxylic acid by Penicillium citrinum.
1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 220.127.116.11] and ACC deaminase [EC 18.104.22.168], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M(r) 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the Km for S-adenosyl-L-methionine of 1.74 mM and kcat of 0.56 s-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M(r) 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a Km for ACC of 4.8 mM and kcat of 3.52 s-1. The presence of 7 mM Cu2+ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification. (+info)
Expression of 1-aminocyclopropane-1-carboxylate oxidase during leaf ontogeny in white clover.
We examined the expression of three distinct 1-aminocyclopropane-1-carboxylic acid oxidase genes during leaf ontogeny in white clover (Trifolium repens). Significant production of ethylene occurs at the apex, in newly initiated leaves, and in senescent leaf tissue. We used a combination of reverse transcriptase-polymerase chain reaction and 3'-rapid amplification of cDNA ends to identify three distinct DNA sequences designated TRACO1, TRACO2, and TRACO3, each with homology to 1-aminocyclopropane-1-carboxylic acid oxidase. Southern analysis confirmed that these sequences represent three distinct genes. Northern analysis revealed that TRACO1 is expressed specifically in the apex and TRACO2 is expressed in the apex and in developing and mature green leaves, with maximum expression in developing leaf tissue. The third gene, TRACO3, is expressed in senescent leaf tissue. Antibodies were raised to each gene product expressed in Escherichia coli, and western analysis showed that the TRACO1 antibody recognizes a protein of approximately 205 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed preferentially in apical tissue. The TRACO2 antibody recognizes a protein of approximately 36.4 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed in the apex and in developing and mature green leaves, with maximum expression in mature green tissue. No protein recognition by the TRACO3 antibody could be detected in senescent tissue or at any other stage of leaf development. (+info)
Glucose regulation of glutaminolysis and its role in insulin secretion.
Leucine or the nonmetabolized leucine analog +/- 2-amino-2-norbornane-carboxylic acid (BCH) (both at 10 mmol/l) induced biphasic insulin secretion in the presence of 2 mmol/l glutamine (Q2) in cultured mouse islets pretreated for 40 min without glucose but with Q2 present. The beta-cell response consisted of an initial peak of 20- to 25-fold above basal and a less marked secondary phase. However, BCH produced only a delayed response, while leucine was totally ineffective when islets were pretreated with 25 mmol/l glucose plus Q2. With Q2, 10 mmol/l BCH or leucine caused a nearly threefold increase, a twofold increase, or had no effect on cytosolic Ca2+ levels in islets pretreated for 40 min with 0, 5, or 15 mmol/l glucose, respectively. Thus, pretreatment of islets with high glucose inhibited BCH- and leucine-induced cytosolic Ca2+ changes and insulin release. Glucose decreased glutamine oxidation in cultured rat islets when BCH was present at 10 mmol/l, but not in its absence, with a lowest effective level of approximately 0.1 mmol/l, a maximum of 18-30 mmol/l, and an inhibitory concentration, 50%, of approximately 3 mmol/l. The data are consistent with the hypothesis that glucose inhibits glutaminolysis in pancreatic beta-cells in a concentration-dependent manner and hence blocks leucine-stimulated insulin secretion. We postulate that in the basal interprandial state, glutaminolysis of beta-cells is partly turned on because glutamate dehydrogenase (GDH) is activated by a decreased P-potential due to partial fuel depletion and sensitization to endogenous activators such as leucine. Additionally, it may contribute significantly to basal insulin release, which is known to be responsible for about half of the insulin released daily. The data explain "leucine-hypersensitivity" of beta-cells during hypoglycemia and contribute to the elucidation of the GDH-linked syndrome of hyperinsulinism associated with elevated serum ammonia levels. Thus, understanding the precise regulation and role of beta-cell glutaminolysis is probably central to our concept of normal blood glucose control. (+info)
Putative partial agonist 1-aminocyclopropanecarboxylic acid acts concurrently as a glycine-site agonist and a glutamate-site antagonist at N-methyl-D-aspartate receptors.
1-Aminocyclopropanecarboxylic acid (ACPC) has been shown to protect against neuronal cell death after ischemic insult in vivo. Such results can be correlated with in vitro assays in which ACPC protected neurons against glutamate-induced neurotoxicity by reducing the activity of N-methyl-D-aspartate (NMDA) channel activation. Electrophysiological studies have determined that ACPC inhibits NMDA receptor activity by acting as a glycine-binding site partial agonist. In this study, rapid drug perfusion combined with whole-cell voltage-clamp was used to elicit and measure the effects of ACPC on NMDA receptor-mediated responses from cultured hippocampal neurons and cerebellar granule cells. The ACPC steady-state dose-response curve had both stimulatory and inhibitory phases. Half-maximal activation by ACPC as a glycine-site agonist was 0.7 to 0.9 microM. Half-maximal inhibition by ACPC was dependent on NMDA concentration. Peak responses to a >100 microM ACPC pulse in the presence of 1 microM glutamate were similar to those of glycine but decayed to a steady-state amplitude below that of glycine. The removal of ACPC initially caused an increase in inward current followed by a subsequent decrease to baseline levels. This suggests that relief of low-affinity antagonism occurs before high-affinity agonist dissociation. Simulations of ACPC action by a two glutamate-binding site/two glycine-binding site model for NMDA channel activation in conjunction with the concurrent role of ACPC as a glycine-site full agonist and glutamate-site competitive antagonist were able to successfully approximate experimental results. (+info)
Regulation of L-methionine and L-lysine uptake in chicken jejunal brush-border membrane by dietary methionine.
In the chicken intestine, L-methionine is transported by systems that are specific for neutral amino acids (L- and B-like) and by systems that can also transport cationic amino acids (y(+)m and b(0,+)-like). These four uptake pathways have been investigated in brush-border membrane vesicles from the jejunum of chickens fed a diet enriched with 0.4% L-methionine. Methionine supplementation from the 1st to the 6th wk of age has no effect on body weight or on the efficiency of food utilization. The kinetic analysis of L-methionine influx across the transport systems specific for neutral amino acids shows, for system L, no dietary effect on the Michaelis constant (Km) and a 30% reduction in maximal velocity (Vmax); for system B it shows a decrease in Km (30%) and in Vmax (51%). Transport systems shared by cationic and neutral amino acids show no dietary effect on b(0,+) activity and a significant reduction in y(+)m Vmax, similar for L-methionine and L-lysine, both in the absence and in the presence of Na+ (L-methionine, 30 and 26% reduction; L-lysine, 19 and 28% reduction, respectively). The downregulation induced by L-methionine supplementation may be an adaptive response to reduce the risk of intoxication by dietary excess of L-methionine. These results support the view that the toxicity of the supplemented substrate can be an important factor in the regulation of amino acid transport by dietary content. (+info)
Hypaphorine from the ectomycorrhizal fungus Pisolithus tinctorius counteracts activities of indole-3-acetic acid and ethylene but not synthetic auxins in eucalypt seedlings.
Very little is known about the molecules regulating the interaction between plants and ectomycorrhizal fungi during root colonization. The role of fungal auxin in ectomycorrhiza has repeatedly been suggested and questioned, suggesting that, if fungal auxin controls some steps of colonized root development, its activity might be tightly controlled in time and in space by plant and/or fungal regulatory mechanisms. We demonstrate that fungal hypaphorine, the betaine of tryptophan, counteracts the activity of indole-3-acetic acid (IAA) on eucalypt tap root elongation but does not affect the activity of the IAA analogs 2,4-D ((2,4-dichlorophenoxy)acetic acid) or NAA (1-naphthaleneacetic acid). These data suggest that IAA and hypaphorine interact during the very early steps of the IAA perception or signal transduction pathway. Furthermore, while seedling treatment with 1-amincocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, results in formation of a hypocotyl apical hook, hypaphorine application as well as root colonization by Pisolithus tinctorius, a hypaphorine-accumulating ectomycorrhizal fungus, stimulated hook opening. Hypaphorine counteraction with ACC is likely a consequence of hypaphorine interaction with IAA. In most plant-microbe interactions studied, the interactions result in increased auxin synthesis or auxin accumulation in plant tissues. The P. tinctorius / eucalypt interaction is intriguing because in this interaction the microbe down-regulates the auxin activity in the host plant. Hypaphorine might be the first specific IAA antagonist identified. (+info)
Expression of AtPRP3, a proline-rich structural cell wall protein from Arabidopsis, is regulated by cell-type-specific developmental pathways involved in root hair formation.
The tightly regulated expression patterns of structural cell wall proteins in several plant species indicate that they play a crucial role in determining the extracellular matrix structure for specific cell types. We demonstrate that AtPRP3, a proline-rich cell wall protein in Arabidopsis, is expressed in root-hair-bearing epidermal cells at the root/shoot junction and within the root differentiation zone of light-grown seedlings. Several lines of evidence support a direct relationship between AtPRP3 expression and root hair development. AtPRP3/beta-glucuronidase (GUS) expression increased in roots of transgenic seedlings treated with either 1-aminocyclopropane-1-carboxylic acid (ACC) or alpha-naphthaleneacetic acid (alpha-NAA), compounds known to promote root hair formation. In the presence of 1-alpha-(2-aminoethoxyvinyl)glycine (AVG), an inhibitor of ethylene biosynthesis, AtPRP3/GUS expression was strongly reduced, but could be rescued by co-addition of ACC or alpha-NAA to the growth medium. In addition, AtPRP3/GUS activity was enhanced in ttg and gl2 mutant backgrounds that exhibit ectopic root hairs, but was reduced in rhd6 and 35S-R root-hair-less mutant seedlings. These results indicate that AtPRP3 is regulated by developmental pathways involved in root hair formation, and are consistent with AtPRP3's contributing to cell wall structure in Arabidopsis root hairs. (+info)
Salicylates of intact Salix myrsinifolia plantlets do not undergo rapid metabolic turnover.
Salicylates, the main phenolic glucosides of northern willow (Salix spp.), play an important role in plant-herbivore interactions. Salicylates are labile metabolites that are thought to undergo metabolic turnover. Salicylates are synthesized from phenylalanine (Phe) via the shikimate pathway. 2-Aminoindan-2-phosphonic acid (AIP), a strong inhibitor of Phe ammonia-lyase (EC 22.214.171.124), was used to block the biosynthesis of salicylates. The aim of this study was to investigate long-term turnover of salicylates in intact micropropagated plantlets of Salix myrsinifolia Salisb. The biosynthesis of salicylates was inhibited efficiently but not completely by 30 microM 2-aminoindan-2-phosphonic acid. Inhibitor treatment, aside from leading to a high accumulation of Phe, also led to an increase in tyrosine and tryptophan, indicating that 2-aminoindan-2-phosphonic acid may also inhibit enzymes other than Phe ammonia-lyase. Salicylates were shown to be unexpectedly stable metabolites that did not undergo marked metabolic turnover in intact plants; in leaves no significant turnover occurred, and in the stems the five salicylates studied were turned over slowly, with half-lives of 11 to 25 d. The total amount of salicylate in mature shoots decreased only 0.6% per day. (+info)