Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.     

Occurrence of Lectin in the Silkgland of the Silkworm, Bombyx mori

Monoclonal antibodies were prepared against the 350 kDa lectin purified from larval hemolymph of the silkworm, Bombyx mori . The antibodies inhibited the hemagglutinating activity (HA activity) and bound specifically to the hemolymph 350 kDa lectin on Western blotting analysis. Immunohistological observations revealed the occurrence of lectin in the cuticular intima of the anterior silk gland, but not the middle or posterior silk glands of fifth instar larvae of Bombyx mori . Extracts from the anterior silk glands showed HA activity and exhibited the same biochemical characteristics as those of the 350 kDa lectin in the hemolymph. These results suggested that lectin-like molecules in epithelial tissues may be important in histolysis during molting and metamorphosis.     

Regulation of glutamine uptake in the cyanobiont Nostoc ANTH

Abstract Glutamine uptake in the cyanobiont Nostoc ANTH was energy-dependent and repressed in ammonia-grown cells. l -Methionine- dl -sulphoximine (MSX), a glutamate analogue and an inhibitor of glutamine synthetase (GS), did not affect glutamine uptake whereas azaserine, an inhibitor of glutamate synthase (GOGAT) did, suggesting that GS activity is not necessarily involved in the glutamine uptake system and that increased intracellular glutamine level regulates its own uptake. Repression of glutamine uptake by ammonia did not require de novo protein synthesis but required GS activity, suggesting that ammonia itself was not the repressor signal. The derepression of the glutamine uptake system did not require GS activity but required de novo protein synthesis.     

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent K _m values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent K _m values were 0.1 mM for -ketoglutarate and 0.22 mM for glutamine.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase     

Changes in the activity of dopa quinone imine conversion factor during the development of Bombyx mori

The activity of DOPA quinone imine conversion factor (QICF) in tissues at different developmental stages of the silkworm, Bombyx mori, was determined. QICF activity was detected in all developmental stages from egg to pupa although the activities, other than in fifth instar larvae, were quite low. Activity in whole larvae peaked one day before the onset of larval-pupal development and declined to a low level shortly before ecdysis. In whole pupae, maximal QICF activity was obtained 1 h after pupation. The activity in larval cuticles was elevated on the last day of the fourth instar and again between days 4 and 8 of the fifth instar, decreasing to very low levels before pupal ecdysis. QICF was detectable in pupal cuticles with most of the pupal activity found in homogenates of mid and hind guts. A major part of the total larval QICF activity was found in hemolymph. Activity in hemolymph varied in a different manner from that in cuticles, with markedly raised levels immediately before pupal ecdysis when the cuticular activity had declined. It is postulated that QICF in cuticles plays some role in wound healing and/or sclerotizatio,, while QICF in hemolymph participates in melanization in the humoral immune system.     

Effect of glutamine on enzymes of nitrogen metabolism in Bacillus subtilis.

An earlier study of the regulation of glutamate synthase (GOGAT) in Bacillus subtilis (Deshpande et al., Bichem. Biophys. Res. Commun. 95:55--60, 1980) revealed an inverse relationship between the specific activity of this essential ammonia-assimilatory enzyme and the intracellular pool of glutamine: GOGAT activity decreased when the internal glutamine concentration reached or exceeded 2.5 mM. This finding prompted the present investigation of the intracellular events linking glutamine formation to the regulation of GOGAT. A growing culture of B. subtilis was shifted from glutamate plus NH+4 medium (high GOGAT activity) to glutamate medium (low GOGAT activity). At various times after the shift, the intracellular concentrations of aspartate, glutamate, glutamine, alanine, and NH+4 and the activities of GOGAT and glutamine synthetase (GS) were measured. After 30 min, the only significant pool level change was an eightfold increase in glutamine, which paralleled a 2- to 3-fold increase in GS activity. Approximately 15 min after the glutamine pool reached its peak, GOGAT activity began to decrease and eventually declined 2.5-fold. In contrast, when B. subtilis was shifted from glutamate medium to glutamate plus NH+4 medium, there was a 1- to 2-h lag before the glutamine pool and GS activity approached a steady state. As a result, GOGAT activity was low until the concentration of glutamine dropped below 2.5 mM. We propose that glutamine is an important regulatory element in the control of GOGAT activity and that one form of GOGAT regulation involves enzyme inactivation. In addition, these results indicate that glutamine is neither a corepressor nor a feedback inhibitor of GS.     

Isolation of auxotrophic mutants in support of ammonia assimilation via glutamine synthetase in Methanobacterium ivanovii

Various enzymes involved in the initial metabolic pathway for ammonia assimilation by Methanobacterium ivanovii were examined. M. ivanovii showed significant activity of glutamine synthetase (GS). Glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) were present, wheras, glutamate dehydrogenase (GDH) was not detected. When M. ivanovii was grown with different levels of NH ₊ ⁴ (i.e. 2, 20 or 200 mM), GS, GOGAT and ADH activities varied in response to NH ₊ ⁴ concentration. ADH was not detected at 2 mM level, but its activity increased with increased levels of NH ₊ ⁴ in the medium. Both GS and GOGAT activities increased with decreasing concentrations of NH ₊ ⁴ and were maximum when ammonia was limiting, suggesting that at low NH ₊ ⁴ levels, GS and GOGAT are responsible for ammonia assimilation and at higher NH ₊ ⁴ levels, ADH might play a role. Metabolic mutants of M. ivanovii that were auxotrophic for glutamine were obtained and analyzed for GS activity. Results indicate two categories of mutants: i) GS-deficient auxotrophic mutants and ii) GS-impaired auxotrophic mutants.Abbreviations GS Glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase     

Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography–tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49 %) were predicted to function as binding proteins and 46 % were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.     

The regulation of ammonia assimilating enzymes in Lemma minor

Summary Lemna minor has the potential to assimilate ammonia via either the glutamine or glutamate pathways. A 3-4 fold variation in the level of ferredoxindependent glutamate synthase may occur, when plants are grown on different nitrogen sources, but these changes show no simple relationship to changes in the endogenous pool of glutamate. High activities of glutamate synthase and glutamine synthetase at low ammonia availability suggests that these two enzymes function in the assimilation of low ammonia concentrations. Increasing ammonia availability leads to a reduction in level of glutamate synthase and glutamine synthetase and an increase in the level of glutamate dehydrogenase. Glutamine synthetase and glutamate dehydrogenase are subject to concurrent regulation, with glutamine rather than ammonia, exerting negative control on glutamine synthetase and positive control on glutamate dehydrogenase. The changes in the ratio of these two enzymes in response to the internal pool of glutamine could regulate the direction of the flow of ammonia into amino acids via the two alternative routes of assimilation.Abbreviations GS Glutamine synthetase - GDH Glutamate dehydrogenase - GOGAT Glutamate synthase     

Studies on ammonium-assimilating enzymes of Platymonas striata Butcher (Prasinophyceae)

Platymonas striata Butcher displays significant levels of glutamate synthase (GS) (EC 2.6.1.53) and glutamine synthetase (GOGAT) (EC 6.3.1.2.), but very low levels of glutamate dehydrogenase (GDH) (EC 1.4.1.4). This suggests that the GS/GOGAT pathway is important for nitrogen assimilation. The in vitro rates of enzyme activity can however only account for about 10% of the in vivo rates of nitrogen assimilation. Nitrogen-starvation reduced GS activity to undetectable levels. On nitrate or ammonium ion refeeding the cellular GS activity was rapidly restored, and reached levels of 56% and 91% greater than the unstarved values 24h after refeeding nitrate or ammonium respectively.Abbreviations NAR nitrate reductase - NIR nitrate reductase     

The involvement of glutamine synthetase/glutamate synthase in ammonia assimilation by Aspergillus nidulans

Wild-type Aspergillus nidulans grew equally well on NH4Cl, KNO3 or glutamine as the only nitrogen source. NADP+-dependent glutamate dehydrogenase (EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) activities varied with the type and concentration of nitrogen source supplied. Glutamate synthase (GOGAT) activity (EC 1.4.7.1) was detected but it was almost unaffected by the type and concentration of nitrogen source supplied. Ion exchange chromatography showed that the GOGAT activity was due to a distinct enzyme. Azaserine, an inhibitor of the GOGAT reaction, reduced the glutamate pool by 60%, indicating that GOGAT is involved in ammonia assimilation by metabolizing the glutamine formed by GS.     

The pathway of ammonia assimilation in the silkworm,Bombyx mori

Ammonia can easily be assimilated into amino acids and used for silk-protein synthesis in the silkworm, Bombyx mori. To determine the metabolic pathway of ammonia assimilation, silkworm larvae were injected with methionine sulfoximine (MS), a specific inhibitor of glutamine synthetase (GS). Activity of GS in the fat body 2h after treatment with 400&mgr;g MS decreased to less than 10% of the control activity, whereas MS had no effect on the activity of glutamate dehydrogenase (GDH), another enzyme which could possibly be responsible for ammonia assimilation. Glutamine concentration in the hemolymph rapidly decreased after MS treatment, while the ammonia level in the hemolymph sharply increased. Glutamine concentration in the hemolymph 4h after injection decreased with increasing doses of MS, whereas ammonia concentration increased in proportion to the MS dose. MS strongly blocked the incorporation of (15)N label into silk-protein in larvae injected with (15)N ammonia acetate, while it slightly inhibited the incorporation of (15)N-amide glutamine into silk-protein. These results suggest that ammonia is mainly assimilated into glutamine via the action of GS and then converted into other amino acids for silk-protein synthesis and that GDH does not play a major role in ammonia assimilation in B. mori.     

A new chitinase-related gene, BmChiR1, is induced in the Bombyx mori anterior silk gland at molt and metamorphosis by ecdysteroid.

A novel ecdysteroid-inducible gene was isolated from the anterior silk gland of the silkworm by mRNA differential display and named Bombyx mori chitinase-related gene 1 (BmChiR1). cDNA for BmChiR1 is 3.7 kbp encoding 1080 amino acids. Its predicted protein sequence consists of two tandem-repeated sequences, both showing high similarities to arthropod chitinases but lacking the active site glutamate essential for catalytic activity, suggesting that BmChiR1 protein has no chitinolytic activity. BmChiR1 mRNA was expressed simultaneously with chitinase mRNA in the anterior silk gland at the ends of the penultimate and last larval instar. Injection of 20-hydroxyecdysone (20E) into feeding last instar larvae induced accumulation of BmChiR1 mRNA. Topical application of a juvenile hormone analog, fenoxycarb, just after the 20E injection, suppressed this induction. BmChiR1 expression is therefore upregulated by ecdysteroid and downregulated by juvenile hormone.     

南瓜种子萌发及子叶发育时谷氨酰胺合成酶和其它氨同化酶的变化

在发育的新生组织中 ,来自种子胚乳储存蛋白的降解和氨基酸分解代谢产生的氨由谷氨酰胺合成酶 ( Glutamine synthetase,GS)重新同化 ,生成的谷氨酰胺 ( Gln)被转运到正在生长着的部分。GS是高等植物氮素代谢的关键酶 [1] ,这个酶能同化不同来源的氨。 GS有多种同工酶 ,存在于植物的各种组织和器官中。它们是由一小的同源但分离的核基因家族编码的 [2 3 ] ,这些不同的 GS在植物氮素同化中起着非重叠的作用 [4] ,它们的表达受到环境、发育进程以及组织或细胞类型等许多因素的影响。在大多数已研究过的植物叶片中存在两种 GS,即胞液型GS(…     

The pathways of ammonium assimilation in Rhizobium meliloti

Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase. The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12. All the strains had active glutamine synthetase and NADP-linked glutamate synthase. Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12. Three glutamate synthase deficient mutants were isolated from strain 2011. They were unable to use 1 mM ammonium as a sole nitrogen source. However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase. It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase     

Glutamate formation by a new In vitro enzyme system consisting of purified glutamine synthetase and glutamate synthase

After the addition of ammonia to the culture medium, the concentration of glutamine in B. flavum cells increased in 20 s with a decrease in glutamate. In the subsequent 30 s, the glutamine concentration deceased again with an increase in glutamate. An enzyme system, which consisted of purified glutamine synthetase (GS) and glutamate synthase (GOGAT) with ATP- and NADPH-regenerating systems, was made up to study the functions of the GS/GOGAT pathway: concentrations of the substrates and of the enzymes were decided on according to the intracellular conditions. Changes in the concentrations of amino acids caused by the addition of ammonia to the system were very similar to those of intracellular glutamate and glutamine when ammonia was added to the bacterial culture. The time required for the complete formation of glutamate from 0.5 mM ammonia was about 4-times shorter in the GS/GOGAT system than in the system using purified glutamate dehydrogenase (GDH) and the NADPH-regenerating system. The glutamate synthase reaction in the GS/GOGAT system was inhibited by some amino acids much more markedly than in the standard assay mixture consisting of glutamine, α-ketoglutarate and NADPH. These results gave further evidence elucidating the operation of the GS/GOGAT pathway in ammonia assimilation, and suggested that a reconstructed enzyme system is useful for studying physiological mechanisms.     

Antisense reduction of serine hydroxymethyltransferase results in diurnal displacement of NH4+ assimilation in leaves of Solanum tuberosum

Serine hydroxymethyltransferase (SHMT) is part of the mitochondrial enzyme complex catalysing the photorespiratory production of serine, ammonium and CO(2) from glycine. Potato plants (Solanum tuberosum cv. Solara) with antisensed SHMT were generated to investigate whether photorespiratory intermediates accumulated during light lead to nocturnal activation of the nitrogen-assimilating enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT). The transformant lines contained 70-90% less SHMT protein, and exhibited a corresponding decrease in mitochondrial SHMT activity. SHMT antisense plants displayed lower photosynthetic capacity and accumulated glycine in light. Glycine was converted to serine in the second half of the light period, while serine, ammonium and glutamine showed an inverse diurnal rhythm and reached highest values in darkness. GS/GOGAT protein levels and activities in the transgenics also reached maximum levels in darkness. The diurnal displacement of NH(4)(+) assimilation was accompanied by a change in the subunit composition of GS(2), but not GS(1). It is concluded that internal accumulation of post-photorespiratory ammonium is leading to nocturnal activation of GS/GOGAT, and that the time shift in ammonia assimilation can constitute part of a strategy to survive photorespiratory impairment.     

Growth of Bacillus fastidiosus on glycerol and the enzymes of ammonia assimilation

Bacillus fastidiosus was able to grow on glycerol as a carbon source when allantoin or urate was used as nitrogen source. The primary assimilatory enzyme for glycerol was glycerol kinase; glycerol dehydrogenase could not be detected. The glycerol kinase activity was increased 30-fold in allantoin/glycerol-grown cells as compared to alantoin-grown cells. Under both growth conditions high levels of glutamate dehydrogenase were found. Glutamine synthetase and glutamate synthase activities could not be demonstrated, while low levels of alanine dehydrogenase were present. It is concluded that B. fastidiosus assimilates ammonia by the NADP-dependent glutamate dehydrogenase.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase     

Ammonia assimilation and nitrogen fixation in Rhizobium meliloti

Summary The enzymes involved in ammonia assimilation by Rhizobium meliloti 4l and their role in the regulation of nitrogen metabolism were studied. Glutamine synthetase (GS) and glutamate synthase (GOGAT) were present at relatively high levels in cells grown in media containing either low or high concentrations of ammonia. NADP-linked glutamate dehydrogenase could not be detected.GOGAT and GS mutants were isolated and characterised. A mutant lacking GOGAT activity did not grow even on high concentrations of ammonia, it was a glutamate auxotroph and was effective in symbiotic nitrogen fixation. The GS and assimilatory nitrate reductase activities of this mutant were not repressible by ammonia but still repressible by casamino acids. A mutant with low GS activity required glutamine for optimal growth. It was ineffective and its nitrate reductase was not inducible.These findings indicate that ammonia is assimilated via the GS/GOGAT pathway in free-living R. meliloti and bacterial GOGAT is not important in symbiosis. Furthermore, GS is suggested to be a controlling element in the nitrogen metabolism of R. meliloti.