Biosynthesis of carcinine (beta-alanyl-histamine) in vivo

Carcinine was biosynthesized by Carcinus maenas from [14C]beta-alanine, [14C] histidine and [14C] histamine. Since carnosine (beta-alanyl-histidine) could not be detected in crab tissues, biosynthesis of carcinine could only be effected by direct coupling of beta-alanine and histamine resulting from histidine decarboxylation. Biosynthesis of carcinine was weak when [14C]beta-alanine and [14C] histidine were used as precursors. On the contrary when [14C] histamine was used, synthesis was important. Thus carcinine appears to be a product of histamine catabolism. After injecting [14C] histamine, radioactive carcinine was concentrated mainly in the heart and nervous system; nonmetabolized [14C] histamine was recovered mainly in the latter. The nervous system might therefore be the seat of carcinine biosynthesis and thus the site of action of histamine.     

Beta-alanylation, a means for neutralization of histamine in the central nervous system of Carcinus maenas

Histamine neutralization in Carcinus maenas is not ensured by oxidation, methylation, or acetylation. After injecting labelled histamine, radioactive carcinine (beta-alanylhistamine), biosynthesized in the central nervous system, rapidly accumulates in the heart. This synthesis is intense and proportional to the amount of histamine injected; on the contrary, it is very low after injecting labelled beta-alanine, whatever the amount injected. Ten days after injecting [14C]histamine, the amounts of radioactive carcinine stocked in the heart remain high. When incubated in the presence of labelled carcinine, various Carcinus tissues are unable to metabolize it. Thus it appears that carcinine would be the catabolite of histamine in Carcinus maenas and that beta-alanylation would be a novel pathway for histamine neutralization. Since carcinine synthetase activity is very high in the central nervous system, this enzyme might neutralize not only neuronal histamine, but also possibly exogenous histamine; thus it would constitute an element of the blood-brain barrier.     

Biosynthesis and metabolism of histamine in the central nervous system of Carcinus maenas

The central nervous system of Carcinus maenas synthesizes radioactive histamine when incubated in the presence of [14C] histidine and pyridoxal-5' phosphate. This biosynthesis increases linearly as a function of the amount of enzyme and the incubation time. It is not effected by heart, muscle or hepatopancreas extracts nor by haemolymph. Thus histamine appears to be synthesized mainly in the nervous system. The latter is also the seat of carcinine (beta-alanylhistamine) biosynthesis. Since carcinine seems to be a product of histamine neutralization, histamine metabolism should take place in its entirety in the nervous system. Thus histamine appears to be implicated in the neuronal activity of Carcinus. Different areas of the crustacean central nervous system: brain, eyestalks and thoracic ganglionic mass biosynthesize and metabolize histamine. Thus they all could contain sites of action for histamine. The nervous systems of two other Decapodes, Cancer and Astacus also effect histamine biosynthesis but don't metabolize it into carcinine.     

Carcinine (beta-alanyl-histamine): rapid synthesis and action on vertebrate blood pressure]

1.-- Synthesis of carcinine was performed by direct condensation of beta-alanine with histamine (free base), in the presence of N-N'-dicyclocarbodiimide. The amine radical of beta-alanine was protected by the t-butoxycarbonyl radical. This fast and simple method yielded a perfectly pure crystalline product. 2.-- Carcinine had no influence upon heartbeat frequency nor on respiratory movements in rats. 3. -- Carcinine had a vasodepressive action upon Vertebrates. It was active at half the concentration of carnosine and at about a thousand times the concentration of histamine. It appeared that histamine lost a great extent of its activity when linked with beta-alanine.     

Existence of carcinine, a histamine-related compound, in mammalian tissues

Carcinine (beta-alanylhistamine) was synthesized in vitro from histamine and beta-alanine. It was detected quantitatively using an HPLC method previously described for the quantification of the related compounds histamine, histidine, carnosine and 3-methylhistamine. Carcinine was identified in several tissue of the rat, guinea pig, mouse and human, and was then shown to be metabolically related in vivo to histamine, histidine, carnosine and 3-methylhistamine through radioisotopic labeling. The results demonstrate that carcinine may be concurrently quantitated using the same HPLC method as that used to measure histamine, histidine, carnosine and 3-methylhistamine. These findings suggest a role for carcinine in the carnosine-histidine-histamine metabolic pathway and in the mammalian physiologic response to stress.     

Metabolism of histamine in the CNS of Aplysia californica: cellular distribution of gamma-glutamylhistamine synthetase

1. The cellular distribution of the histamine-metabolizing enzyme, gamma-glutamylhistamine synthetase, was studied in the CNS of Aplysia californica. 2. Enzyme activity was assayed in single, re-identifiable neuronal cell bodies, clusters of nerve cells and neuropil and capsule tissue surrounding the ganglia. 3. The "histaminergic" C-2 cells and all other single nerve cell bodies contained measurable gamma-glutamylhistamine synthetase activity. 4. The cerebral E cluster, which houses the C-2 cells and several of its post-synaptic neurons, had an apparently higher specific enzyme activity than other neuronal clusters. 5. The finding of measurable enzyme activity in the histamine-rich C-2 cell bodies and in clusters of cells responsive to this imidazoleamine supports the hypothesis that one function for gamma-glutamylhistamine synthetase is in the disposal of neuronally released histamine. 6. The average specific activity of gamma-glutamylhistamine synthetase in single cells was 3.64 +/- 0.32 mumol g protein hr. This represented only 5% of the enzyme activity measured in the whole ganglion. 7. The bulk of synthetase activity was found to reside in the capsule tissue.     

An In Vitro Characterization of γ-Glutamylhistamine Synthetase: A Novel Enzyme Catalyzing Histamine Metabolism in the Central Nervous System of the Marine Mollusk, Aplysia californica

The properties of the histamine metabolizing enzyme, gamma-glutamylhistamine synthetase (gamma-GHA synthetase) were studied in Aplysia ganglia in vitro. This enzyme catalyzes the incorporation of histamine into peptide linkage with L-glutamate to form a peptidoamine, gamma-glutamylhistamine (gamma-GHA). gamma-GHA synthetase is a soluble enzyme with an apparent Km of 653 microM for histamine and 10.6 mM for L-glutamate. Synthesis of gamma-GHA is energy-dependent, having an absolute requirement for ATP. Magnesium ions and dithiothreitol are also essential for activity. Of a variety of gamma-glutamyl compounds and glutamate analogs tested, only L-glutamate was effectively incorporated into peptide linkage with histamine. Similarly, the enzyme has a higher affinity for histamine than for numerous imidazole analogs. In addition, 3,4-dihydroxyphenylethylamine (dopamine), 5-hydroxytryptamine, octopamine, and several other amines tested are effective inhibitors of gamma-GHA synthesis. Ganglia, nerve trunks, and the capsule surrounding the ganglion had the highest synthetase activity. The specific activity of the enzyme in muscle, heart, and hemolymph was less than 10% of that in ganglia. Differences in substrate specificity and effect of inhibitors distinguish gamma-GHA synthetase from gamma-glutamyl transpeptidase, glutamine synthetase, and carnosine synthetase.     

Effet de la farnesylacetone sur les syntheses proteiques de l'ovaire chez le crabe Carcinus maenas

Summary

We have analysed in vitro the effect of farnesylacetone, a substance produced by the androgenic gland of Crustacea, in a concentration of 20 ng/ml, on the protein synthesis in the ovary of Carcinus maenas. In winter, the farneslyacetone seems to be ineffective; the incorporation of labelled precursor per mass unity is then related to the weight of the sample. In summer, an activation of protein synthesis can be observed. These results do not depend on ovary maturation and concern all the proteins of the gonad.     

Activation of transglutaminase and production of protein-bound gamma-glutamylhistamine in stimulated mouse mast cells

The identification of transglutaminase in the growth-factor-dependent mouse mast cell line PT18 was accomplished through its characteristic catalytic properties (specificity, calcium dependency, and inhibition by iodoacetamide); and by both immunoprecipitation and Western blot analysis using affinity purified antibody. The enzymatic activity in these cells increased in association with the release of histamine from the cells induced by an IgE-dependent mechanism or by exposure to the ionophores A23187 or Br-x537A. The increase in transglutaminase activity was paralleled by a marked increase in the level of protein-bound gamma-glutamylhistamine, determined in radiolabeled form in mast cells that were either metabolically labeled with [3H]histidine or incubated with [3H]histamine before degranulation. The highest level of bound gamma-glutamylhistamine was found in the immunologically stimulated cells. Enzymatic activity and the gamma-glutamyl derivative were associated primarily with the cells, both before and after stimulation. Separation of gamma-glutamylhistamine in a proteolytic digest of these cells was carried out using a combination of ion exchange chromatography and high performance liquid chromatography. The gamma-glutamyl compound was identified and quantitated through the enzymatic production of histamine with the use of gamma-glutamylamine cyclotransferase, an enzyme specific for the disassembly of gamma-glutamylamines.     

Alteration of cytochrome C oxidase activity during spermatogenesis in Carcinus maenas

During spermatogenesis in the crab, Carcinus maenas, mitochondria in the developing spermatids degenerate. In close association with mitochondrial fragments in late spermatids, an elaborate lattice-like complex of fused membranes is formed and lies in a position adjacent to the acrosome. Mature sperm possess no mitochondria, but a whorl of membranes is present alongside the acrosome in the diffuse nucleus. To ascertain whether or not cytochrome c oxidase activity is lost as the mitochondria disintegrate, differentiating Carcinus maenas spermatids and mature sperm have been studied cytochemically. Cytochfome c oxidase activity was localised by means of the osmophilic compound 3,3'-diaminobenzidine (DAB). Cytochrome c oxidase activity is confined to the cristae of mitochondria in the testis. As spermatids mature most mitochondria are lost. A few mitochondrial fragments may be caught up within the lamellar complex. While they are recognisalbe as mitochondria they retain cytochrome c oxidase activity. The lamellar complex does not show this enzyme activity. These results therefore suggest that the mature sperm of Carcinus maenas do not contain the enzymes normally incorporated in the mitochondrial membrane, capable of oxidative phosphorylation.     

Angiotensin-converting enzyme-like activity in crab gills and its putative role in degradation of crustacean hyperglycemic hormone

Angiotensin-converting enzyme-like enzyme activity (ACELA) was found in Carcinus maenas using reverse phase high performance liquid chromatography (RP-HPLC) analysis of degradation kinetics of a synthetic substrate (Hippuryl-histidyl-leucine) and a specific inhibitor (captopril). Gills contained the highest ACELA, then brain, muscle, and testis, respectively, while no activity was detected in the following tissues: hepatopancreas, hindgut, hypodermis, heart, and hemolymph. ACELA present in gill membranes exhibited a K(m) of 0.23 mM and V(max) of 7.6 nmol with synthetic substrate. The enzyme activity was dependent on Cl- concentration and was markedly inhibited by captopril, lisinopril, and EDTA. Addition of Zn2+ to membranes previously treated with EDTA restored 89% activity, suggesting that C. maenas ACELA is a Zn2+ metalloenzyme. Gill membranes prepared from premolt crabs showed similar levels of ACELA to those of the intermolt animals. Administration of captopril in vivo lengthened the half life of circulating CHH, while in vitro incubation of gill membranes with captopril reduced CHH. These results suggest that C. maenas ACELA present in gills is likely to be involved in degradation of this neuropeptide.     

Beta-alanine synthesis in Escherichia coli.

The enzyme, aspartate 1-decarboxylase (L-aspartate 1-carboxy-lyase; EC 4.1.1.15), that catalyzes the reaction aspartate leads to beta-alanine + CO2 was found in extracts of Escherichia coli. panD mutants of E. coli are defective in beta-alanine biosynthesis and lack aspartate 1-decarboxylase. Therefore, the enzyme functions in the biosynthesis of the beta-alanine moiety of pantothenate. The genetic lesion in these mutants is closely linked to the other pantothenate (pan) loci of E. coli K-12.     

Biological methylations in the crab Carcinus maenas; in vitro inhibition by a fraction purified from androgen gland extracts]

Extracts from muscles, testis, seminal vesicles and ovaries of the Crab, Carcinus maenas, have been studied in vitro, in presence of [14C]-methyl S-adenosylmethionine, with an E. coli tRNA as methyl acceptor. The highest level of methylases is found in the testis. It has been reported previously that a purified fraction extracted from the androgenic glands of Carcinus maenas inhibits the vitellogenesis in ovaries. We now show that the same fraction inhibits tRNA methylation in an extract of testis as methylase; a 50% inhibition is obtained with about 10 mug of a purified fraction corresponding to 15 glands. With an enzymatic preparation from the ovaries, a 50% inhibition of the tRNA methylase is observed with the purified extract from 4 glands.     

Measurement of the Five Enzymes which Convert Chorismate to Tryptophan in Wheat Plants (Triticum aestivum cv. Kalyansona)

Conditions are described for measuring anthranilate synthetase, anthranilate-PRPP-phosphoribosyl transferase, N-5′-phosphoribosyl anthranilate isomerase, indole-3-glycerol phosphate synthetase and tryptophan synthetase in crude extracts from Triticum aestivum (wheat) plants. Only the last enzyme has been measured before in extracts from green plants. The extractable quantities of each enzyme in all plant parts at all stages of growth were sufficient to synthesize the amount of tryptophan present within the same tissue in 48 h. Anthranilate synthetase activity was the lowest of the five enzyme activities and was the only one inhibited by tryptophan in vitro, indicating that this enzyme may be the control point in tryptophan biosynthesis in wheat plants.     

Interspecific hybridization and mitochondrial introgression in invasive carcinus shore crabs

Interspecific hybridization plays an important role in facilitating adaptive evolutionary change. More specifically, recent studies have demonstrated that hybridization may dramatically influence the establishment, spread, and impact of invasive populations. In Japan, previous genetic evidence for the presence of two non-native congeners, the European green crab Carcinus maenas and the Mediterranean green crab C. aestuarii, has raised questions regarding the possibility of hybridization between these sister species. Here I present analysis based on both nuclear microsatellites and the mitochondrial cytochrome C oxidase subunit I (COI) gene which unambiguously argues for a hybrid origin of Japanese Carcinus. Despite the presence of mitochondrial lineages derived from both C. maenas and C. aestuarii, the Japanese population is panmictic at nuclear loci and has achieved cytonuclear equilibrium throughout the sampled range in Japan. Furthermore, analysis of admixture at nuclear loci indicates dramatic introgression of the C. maenas mitochondrial genome into a predominantly C. aestuarii nuclear background. These patterns, along with inferences drawn from the observational record, argue for a hybridization event pre-dating the arrival of Carcinus in Japan. The clarification of both invasion history and evolutionary history afforded by genetic analysis provides information that may be critically important to future studies aimed at assessing risks posed by invasive Carcinus populations to Japan and the surrounding region.     

Threonine Synthetase-Catalyzed Conversion of Phosphohomoserine to α-Ketobutyrate in Bacillus subtilis

An enzyme activity of Bacillus subtilis has been found that catalyzes the dephosphorylation and deamination of phosphohomoserine to alpha-ketobutyrate, resulting in a bypass of threonine in isoleucine biosynthesis. In crude extracts of a strain deficient in the biosynthetic isoleucine-inhibitable threonine dehydratase, phosphohomoserine was converted to alpha-ketobutyrate. Phosphohomoserine conversion to alpha-ketobutyrate was shown not to involve a threonine intermediate. Single mutational events affecting threonine synthetase also affected the phosphohomoserine-deaminating activity, suggesting that the deamination of phosphohomoserine was catalyzed by the threonine synthetase enzyme. It was demonstrated in vivo, in a strain deficient in the biosynthetic threonine dehydratase, that isoleucine was synthesized from homoserine without intermediate formation of threonine.     

Immunochemical evidence for glutamine-mediated degradation of glutamine synthetase in cultured Chinese hamster cells.

The specific activity of glutamine synthetase in cultured Chinese hamster cells is inversely related to the concentration of glutamine in the surrounding solution. Enzyme specific activity increases 8- to 10-fold when glutamine is removed from serum-free F12 growth media. The induction of glutamine synthetase activity occurs only after glutamine removal and not after the removal of other amino acids (methionine, leucine, or isoleucine). The analysis of the glutamine-mediated decrease in glutamine synthetase activity has been simplified by the finding that depression proceeds in nutrient-free buffered saline solution (141 mM NaCl, 5.4 mM KCl and 30 mM Tricine (pH 7.4). Under these conditions, 0.1 mM cyanide blocks glutamine-mediated depression. The cyanide inhibition is reversed by the addition of 1.0 mM glucose which suggests that ATP is required for depression. Glutamine-mediated depression is temperature-dependent, occurring between 25 and 45 degrees with an optimum rate at 37 degrees. Studies of the time course of induction and depression as a function of glutamine concentration suggest that glutamine regulates the rate at which the enzyme is either modified or degraded. We have employed an antibody prepared against homogeneous Chinese hamster liver glutamine synthetase to measure the amount of glutamine synthetase protein in extracts of cells containing induced or depressed levels of enzyme activity. A highly sensitive immunoprecipitation procedure enables quantitation of nanogram amounts of glutamine synthetase protein. Glutamine synthetase in cell extracts containing induced levels of enzyme activity possesses the same molecular specific activity (ratio of activity to antigenicity) as homogeneous Chinese hamster liver glutamine synthetase. The molecular specific activity of glutamine synthetase is almost the same in extracts of cells with depressed levels of enzyme obtained by growth for short (2 hours) and long (24 hours) times in the presence of glutamine. These data suggest that glutamine-mediated depression of glutamine synthetase results from degradation of enzyme molecules.     

Inhibition of carbonic anhydrase in the gills of two euryhaline crabs,Callinectes sapidus and Carcinus maenas,by heavy metals

Two subcellular fractions of gill tissue, cytoplasm and basolateral membranes, from two species of euryhaline decapod crustaceans, Callinectes sapidus and Carcinus maenas, acclimated to low salinity, were isolated via differential centrifugation. Carbonic anhydrase activity from both fractions was titrated against a variety of heavy metals in vitro. The metals Ag(+), Cd(2+), Cu(2+) and Zn(+) showed inhibitory action against the enzyme. Ki values for these metals against cytoplasmic CA from C. sapidus were in the range of 0.05-0.5 microM (for Ag(+), Cd(2+) and Cu(2+)) and 2-6 microM for Zn(+), some of the highest sensitivities reported for CA from an aquatic organism. The Ki values for these same metals were approximately 2-3 orders of magnitude higher for cytoplasmic CA from C. maenas, indicating that there are significant differences in heavy metal sensitivity in branchial CA from the two species, and that C. maenas possesses a metal-resistant CA isoform. It required concentrations of metals in the millimolar range, however, to inhibit CA activity from the membrane fraction of the gill of both species. There were no effects on either mortality or on hemolymph osmotic and ionic concentrations in C. maenas that were exposed to 10 microM Cd or Zn(+) at 32 per thousand salinity and subsequently transferred to 10 per thousand. The presence of a metal-resistant CA isoform in the gills of C. maenas suggests that this species would not be restricted from its normal estuarine environment by heavy metal pollution.     

Control of Tryptophan Biosynthesis in Plant Tissue Cultures: Lack of Repression of Anthranilate and Tryptophan Synthetases by Tryptophan

Tobacco (cv. Xanthi and cv. Wisconsin 38), rice, carrot, tomato, and soybean tissue cultures were grown in liquid media containing L-tryptophan. The addition of tryptophan increased the cellular tryptophan levels greatly (12–2500 fold), but did not lower appreciably the levels of two tryptophan biosynthetic enzymes, anthranilate synthetase and tryptophan synthetase. However, the addition of 50 μM tryptophan to the crude enzyme extract completely inhibited the anthranilate synthetase activity while 1 mM tryptophan inhibited the tryptophan synthetase activity by only 10–20°/o. This information indicates that tryptophan biosynthesis is controlled by the feedback inhibition of anthranilate synthetase by tryptophan and not by repression of enzyme synthesis. All of the species had significant enzyme levels. Anthranilate synthetase activity could not be detected in extracts from cells grown on tryptophan unless the extracts were first passed through two G-25 Sephadex columns with a short 30 °C warming step in between, a procedure shown to remove an inhibitor of the enzyme.