ABSTRACUChlorsulfuron, an inhibitor of acetolactate synthase (EC 4.13.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemma minor at concentrations of 10' molar and above, but had no inhibitory effects on growth at 10' molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10'8 molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as a-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of a-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10 molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 104 times greater than that required to inhibit growth. Tracer studies with "N demonstrate that chlorsulfuron inhibits the incorporation of '"N into valine, leucine, and isoleucine. The a-amino-nbutyrate accumulated in the presence of chlorsulfuron and ["NjR.+ was heavily labeled with "N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isolencine branch of the pathway, and transamination of 2-oxobutyrate to a-aminon-butyrate by a constitutive transaminase utilizing either glutamate or alanine as a-amino-N donors.
Plants from several families (Chenopodiaceae, Gramineae, Compositae) accumulate betaine (glycine betaine) in response to salt or water stress via the pathway: choline -) betainal (betaine aldehyde) -* betaine. Betaine
When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with I'5NIH.4, prior to supplying the inhibitor. Analyses of the 'IN abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [I5NIH4', are '4N enriched and are not apparently derived from '5N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in rivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.It is now well established that GS2 occupies a central position in plant N metabolism (12, 13). The GS-GOGAT cycle is thought to be responsible for the assimilation of most, if not all, of the ammonia derived from nitrate reduction and photorespiration (2-4, 10, 18, 19, 21, 23, 25). Studies with the potent inhibitor of GS, MSO, appear to rule out any major contribution of GDH to ammonia assimilation (1,3,8,19,21,23). However, recent investigations with isolated plant mitochondria suggest that a small fraction of the ammonia generated from glycine decarboxylation can be directly reassimilated into glutamic acid via a mitochondrial GDH (26). The quantitative significance of this latter pathway in vivo still remains obscure. '
The free sterols and phospholipids of the demospongeAplysina fistularis were isolated and analyzed. The free sterols consisted mainly of the unusual 26-methylated sterols aplysterol (53%) and 24(28)-dehydroaplysterol (7%) together with 7 commonly occurring sterods. The major phospholipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine and diphosphatidylglycerol. The major fatty acyl components of the phospholipids consisted of 85% C14-C20 acids, including the unprecedented 2,6,10-trimethyl-5-tetradecenoic acid and 11-methyloctadecanoic acid. The remaining 15% were C27-C30 demospongic acids, including 2 novel acids tentatively assigned the structures 5,9,23-octacosatrienoic acid and 5,9,23-nonacosatrienoic acid, and 3 novel acids proven to be 5,9,21-octacosatrienoic acid, Z,Z-20-methyl-5,9-hexacosadienoic acid and Z,Z-22-methyl-5,9-octacosadienoic acid. The biosyntheses of the novel demospongic acids are proposed to occur by chain elongation of monoenoic or branched precursors followed by desaturation. The large quantities of typically bacterial phospholipids and fatty acids found implied the presence of bacteria in the sponge, in agreement with microscopic studies. Analysis of the phospholipid-bound fatty acids in a sponge cell-enriched fraction indicated that the demospongic acids, including the 2 branched structures, were the major acids of the sponge cells. The presence inA. fistularis of demospongic acids containing membrane disordering groups-methyl branches or double bonds-on the ω7 carbon is proposed to be due to the need by the sponge for membranes possessing fluidity near the middle of the phospholipid bilayer. It is also proposed that the C26 methyl group of aplysterol causes disordering of the phospholipid bilayer in the same region, and thus also evolved in response to this need.
Two cardioacceleratory peptides from the corpora cardiaca of Periplaneta americana have been purified by gel filtration and reversed-phase liquid chromatography. Based on analysis of the intact factors and their chymotryptic fragments, we have assigned the primary structure of these octapeptides as pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, designated periplanetin CC-1, and pGlu-Leu-Thr-Phe-Thr-ProAsn-Trp-NH2, designated periplanetin CC-2. They represent new members of a family of invertebrate peptides that includes locust adipokinetic hormone and crustacean red-pigment concentrating hormone. Both peptides show adipokinetic activity in grasshoppers and hyperglycemic activity in cockroaches.One of these peptides (CC-2) has provocative sequence homology with the N112-terminal portion of glucagon.The insect corpora cardiaca (CC) are major neurohemal organs that are analogous to the vertebrate hypothalamohypophyseal system. Not only do the CC store and release products synthesized in the brain, but also they contain intrinsic glandular cells producing a variety of bioactive factors affecting developmental, metabolic, and myotropic processes (1). Many of these factors appear to be peptides; only one, adipokinetic hormone (AKH) from Locusta migratoria, has been identified (2).Corpora cardiaca of the cockroach Periplaneta americana have proven a rich and accessible source of bioactive factors. Previous studies have demonstrated that CC homogenates and partially purified fractions affect the cockroach heartbeat (3-6) and elevate the concentration of hemolymph trehalose (the main sugar in blood of most insects) (7-9). These factors appear to be peptides (3, 7), but it is difficult to assess how many are actually distinct substances or whether some of them have multiple activities (8,(10)(11)(12) In this paper we report the isolation and sequence determination of two structurally related octapeptides from the CC of P. americana that have cardioacceleratory and hyperglycemic activity in the host insect.MATERIALS AND METHODS Insects. Cockroaches (P. americana) were raised at 28°C and 50% relative humidity under a 16-hr light/8-hr dark photo regime and were fed on dry dog food. Corpora cardiaca with corpora allata attached were dissected from 0-to 6-wk-old cockroaches and collected in saline (5 mM CaCl2/1 mM MgCl2/5 mM KCI/140 mM NaCl/4 mM NaHCO3/5 mM trehalose/20 mM Hepes, pH 7.0) at 0°C prior to freezing (-20°C). A total of -4000 cockroach CC were used.Heart Bioassay. Aliquots of all fractions from purifications were assayed for bioactivity by using a semiisolated heart preparation (6). Heart rate was monitored with an impedance converter (UFI model 2991) connected to a frequency integrator and recorder. A heart was selected on the basis of frequency (-60 beats per min) and regularity and then was bathed in saline to stabilize (30 min). Test samples to be assayed were applied to the heart in a volume of 50 ,u.Carbohydrate and Lipid Bioassays. Hemolymph carbohydrate levels in P. americana and hemolymph lipid levels in th...
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