Transport of the large-neutral amino acids by the gamma-glutamyl cycle: a proposal.

The γ-glutamyl cycle has been proposed by Meister (1973) as one possible mechanism for the mediation of amino acid transport. The high energy requirement of the pathway, the very low specificity of γ-glutamyl transpeptidase and the inability to account for trans membrane stimulation of amino acid entry are but three criticisms of this hypothesis. It is proposed that the various objections can be overcome by postulating that the soluble form of γ-glutamyl transpeptidase transfers the γ-glutamyl moiety from gluthathione to glutamine (in the case of brain) and that the membrane sequestered form of this enzyme catalyzes the exchange of the γ-glutamyl group between γ-glutamyl glutamine and an entering neutral amino acid. The released glutamine leaves the cell. The γ-glutamyl amino acid then passes into the cytoplasm where it is acted upon by either γ-glutamyl cyclotransferase or the soluble γ-glutamyl transpeptidase which transfers the γ-glutamyl group to another molecule of glutamine. It is postulated that access to the membrane-bound enzyme is dependent on the relative lipophilia of the entering large-neutral amino acids. The available data support this mechanism. By regeneration of γ-glutamyl glutamine, a low expenditure of energy is required for the transport process. Specificity of transpeptidation is attained by the constraints of access to the membrane bound enzyme site.     

gamma-Glutamyl transpeptidase from WI-38 fibroblasts: purification and active site modification studies

γ-Glutamyl transpeptidase has been purified to homogeneity from WI-38 human fetal lung fibroblasts, following extraction with Triton X-100 in the absence of added proteases. The specific activity of the purified enzyme is 16 units/mg protein at the optimum of pH 8.0. Although this activity value is low, the WI-38 enzyme is very similar to previously described γ-glutamyl transpeptidases in its molecular properties. The native molecule (apparent molecular weight of 82,000) is composed of one light and one heavy subunit (apparent molecular weights of 20,000 and 62,000, respectively). Papain digestion reduces the native molecular weight to an apparent value of 73,000 by proteolysis of the heavy chain. The known active site modifying agent and glutamine analog 6-diazo-5-oxo-l-nor-leucine, completely inactivates the enzyme, coincident with its stoichiometric incorporation into the light subunit. This inactivation is accelerated by maleate and prevented by S-methylglutathione. The WI-38 γ-glutamyl transpeptidase is also inactivated by the fluorescent alkylating agent, 5-iodoacetamidofluorescein. Selective reaction of this reagent with an active site residue is suggested by prevention of the inactivation by S-methylglutathione, the stoichiometric incorporation of the fluorescein moiety, and the loss of one methionine residue per molecule of protein accompanying inactivation.     

gamma-Glutamyl transpeptidase of human seminal fluid.

γ-Glutamyl transpeptidase, which is present in high levels in human seminal fluid plasma, was purified about 870-fold from this source. The enzyme is present in seminal fluid plasma in particulate form. Purification by a procedure involving treatment with bromelain gave a protein (apparent molecular weight, about 70,000), which exhibited catalytic properties characteristic of γ-glutamyl transpeptidase preparations isolated from rat kidney and other mammalian tissues. The physiological significance of seminal fluid γ-glutamyl transpeptidase and its potential clinical value are considered.     

Structure of the Hydrolyzed Product (F-2) Released from γ-Polyglutamic Acid by γ-Glutamyl Hydrolase YwtD of Bacillus subtilis

The structure of the hydrolyzed product (F-2) with a molecular mass of about 2 kDa released from γ-polyglutamic acid by the γ-glutamyl hydrolase YwtD of Bacillus subtilis was analyzed. The results showed that F-2 is an optically heterogeneous polymer consisting of D- and L-glutamic acid in an 80:20 ratio with D-glutamic acid on both the N- and C-terminal sides, suggesting that YwtD is an enzyme that cleaves the γ-glutamyl bond between D- and D-glutamic acid recognizing adjacent L-glutamic acid toward the N-terminal region.     

Diversity of γ- glutamyl peptides and oligosaccharides,the “kokumi” taste enhancers,in seeds from soybean mini core collections

Soybeans (Glycine max (L,) Merr,) contain γ-glutamyl peptides and oligosaccharides, and these components play an important role in imparting the “kokumi” taste to foods. To gain insight into the genetic diversities and molecular mechanisms of accumulation of γ-glutamyl peptides and oligosaccharides in soybean, we measured the contents of these components using the Japan and World mini core collections. Similar to other previously reported traits, wide variations were detected among the accessions in the core collections with respect to the content of γ-glutamyl peptides and oligosaccharides. We found a positive relationship between the content of γ-glutamyl tyrosine and γ-glutamyl phenylalanine and between the content of raffinose and stachyose. Furthermore, there were unique accessions that included high levels of γ-glutamyl peptides and oligosaccharides. These accessions may be helpful in understanding the accumulation mechanism of γ-glutamyl peptides and oligosaccharides and to increase the “kokumi” taste components in soybean by performing a genetic analysis.     

Effects of diet on female fecundity and larval development in the carrion beetle Necrophila japonica

The carrion beetle subfamily Silphinae (Coleoptera: Silphidae) contains dominant macroinvertebrates of soil ecosystems in temperate zones. However, their feeding habits, which determine the role of each species in the ecosystem, have not been sufficiently studied. Moreover, although a diet shift from necrophagy on vertebrate carcasses to predatory feeding on invertebrates is known to occur in this subfamily, the processes and mechanisms of this shift have also been inadequately addressed. We examined female fecundity and larval development on various diets in a Silphinae species, Necrophila (Eusilpha) japonica (Motschulsky). The experimental diets included a meat diet and various invertebrate diets, which reflect the ‘ancestral’ feeding habit in Silphinae, necrophagy, and the ‘derivative’ feeding habit, predatory feeding. Female fecundity was significantly higher on the meat diet (minced beef) than on an insect larvae diet (mealworms and dipteran larvae) but did not significantly differ from that on an earthworm diet. Larval developmental performance was significantly higher on the earthworm diet than on the meat and insect larvae diets. Our results for larval development were consistent with those of previous stable isotope analyses of the same species, in which isotopic values of larval samples agree with those of hypothetical consumers that utilize earthworms. The consistency of results among different methods indicates that N. japonica larvae are most likely earthworm feeders. In contrast, our results for the female fecundity experiment differed from those of previous stable isotope analyses, in which vertebrate carcasses unlikely serve as the staple diet of adults in the field; thus, the feeding habits of N. japonica adults remain unresolved. Our observations that females and larvae performed best on the meat and earthworm diets, respectively, may indicate that, in Silphinae, the diet shift from necrophagy to predatory habits occurs earlier in larvae than in adults.     

Properties of γ-Glutamyltransferase from Lentinus edodes

An enzyme preparation catalyzing p-nitroaniline release from γ-glutamyl-p-nitroanilide was obtained in a 200-fold purified state from fruit bodies of an edible mushroom, Lentinus edodes. Analysis of the final preparation by differential centrifugation revealed that the enzyme was still bound with subcellular particles. The enzyme catalyzed both the hydrolysis and transfer of the γ-glutamyl moiety from γ-glutamyl-p-nitroanilide, but exhibited essentially no activity of glutaminase, glutamine aminotransferase, glutamine synthetase or γ-glutamyl cyclotransferase. With γ-glutamyl-p-nitroanilide the activity was maximal at about pH 7.6. The enzyme activity increased with an increasing concentration of Tris-HCl buffer, but not with phosphate buffer which was inhibitory. An apparent Michaelis constant of 4 mm was obtained in 0.5 m Tris-HCl buffer at pH 7.6. S-Alkylcysteine sulfoxide served as the best glutamyl acceptor. A serine-borate mixture, pCMB, Cu²⁺, Hg²⁺ and Zn²⁺ were potent inhibitors. All the experimental results, including the insoluble nature of the enzyme, allowed us to classify the Lentinus enzyme in the family of γ-glutamyl transferase.     

Purification and Properties of an Exocellular γ-Glutamyl Arylamidase from Bacillus sp. Strain No. 12

An exocellular γ-glutamyl arylamide-hydrolyzing enzyme was produced by a Bacillus sp. in L-glutamate-containing medium. This enzyme was a tetrameric simple protein composed of two heavy subunits (Mr 56,000) and two light subunits (Mr 46,000). It hydrolyzed γ-amido, acyl and aryl bonds in L- and D-glutamyl compounds, and the activity on L-glutamic acid γ-p-nitroanilide was inhibited by the addition of glutamate and γ-glutamyl compounds but not by α-glutamyl compounds. The activity was stimulated by various dipeptides but not by free amino acids, L-Alanine, glycine, L-serine and L-cysteine inhibited the enzyme competitively. Addition of hy-droxylamine had no effect on the activity.     

Cross reaction of antibodies against liver gap junction protein (26K) with lens fiber junction protein (MIP) suggests structural homology between these tissue specific gene products

Detergent solubilization of γ-glutamyl transpeptidase was deduced from measuring sedimentation equilibrium behaviour in an air-driven ultra-centrifuge. Sequential fractionation of the tube contents and analysis of the radial concentration dependence permits direct determination of molecular weight. Studies using D₂O and H₂O indicate that the Triton X-100 solubilized enzyme has a molecular weight of approximately 175,000 and contains 55% bound detergent. The glycoprotein nature of the enzyme is demonstrated by its interaction with concanavalin A.     

Gamma-glutamyl transpeptidase of rat seminal vesicles; effect of orchidectomy and hormone administration on the transpeptidase in relation to its possible role in secretory activity.

γ-Glutamyl transpeptidase was prepared from rat seminal vesicles by two methods and was found to be similar to rat kidney γ-glutamyl transpeptidase with respect to substrate specificity, stimulation of “glutaminase” activity by maleate, and apparent molecular weight. Histochemical studies demonstrated that γ-glutamyl transpeptidase is concentrated in the secretory epithelium of the seminal vesicle. Like the epithelium itself, the enzyme responds to the presence or absence of testosterone. The content and specific activities of γ-glutamyl transpeptidase and γ-glutamyl cyclotransferase in rat seminal vesicles are low in orchidectomized animals, an effect which is reversed by administration of testosterone but accentuated by estradiol administration. These enzymes may be involved in the secretory functions of the seminal vesicles.     

Isolation of anthglutin, an inhibitor of gamma-glutamyl transpeptidase from Penicillum oxalicum.

Anthglutin, a new inhibitor of γ-glutamyl transpeptidase, has been isolated from the cultured medium of Penicillium oxalicum and its structure established as l-γ-l-glutamyl-2-(2-carboxyphenyl)hydrazine. The isolation of anthglutin was achieved by ion-exchange chromatography. Anthglutin inhibited γ-glutamyl transpeptidase specifically and the kinetic analysis of the inhibition showed that anthglutin inhibited the enzyme competitively with regard to the glutamyl donor, γ-glutamyl-p-nitroanilide, and noncompetitively with regard to the glutamyl acceptor, glycylglycine. K₁ values were 5.7 μm for the hog kidney enzyme, 18.3 μm for the human kidney enzyme, 13.6 μm for the human liver soluble enzyme, and 10.2 μm for the bound enzyme. After oral administration of [¹⁴C]methionine and anthglutin to rats, no effect of anthglutin was observed on the absorption of methionine in the intestine.     

Temperature: Diet Interactions Affect Survival through Foraging Behavior in a Bromeliad‐Dwelling Predator

Temperature, food quantity and quality play important roles in insect growth and survival, influencing population dynamics as well as interactions with other community members. However, the interaction between temperature and diet and its ecological consequences have been poorly documented. Toxorhynchites are well‐known biocontrol agents for container‐inhabiting mosquito larvae. We found that Toxorhynchites haemorrhoidalis larvae (Diptera: Culicidae) inhabiting water‐filled rosettes of tank bromeliads catch and eat prey of both aquatic (mosquito larvae) and terrestrial origin (ants), using distinct predatory methods. They carried out frontal attacks on ants, but ambushed mosquito larvae. In choice tests, T. haemorrhoidalis favored terrestrial prey. Temperature had a significant effect on predator development and survival through its interaction with diet, but did not alter the preference for ants. T. haemorrhoidalis larvae emerged quickly when fed only mosquito larvae, whereas all individuals died before pupation when fed only ants. We conclude that behavioral factors (i.e., attraction to ants that disturb the surface of the water) overtake physiological factors (i.e., the adverse outcome of elevated temperature and an ant‐based diet) in determining a predator's response to temperature:diet interactions. Finally, because T. haemorrhoidalis larvae preferentially feed on terrestrial insects in tank bromeliads, mosquito larvae may indirectly benefit from predation release.     

Relationships among catalpol sequestration,metabolism and nutritional efficiencies of the catalpa sphinx,Ceratomia catalpae (Lepidoptera: Sphingidae)

Sequestration of defensive compounds acquired from the environment can be costly to herbivorous insects. The catalpa sphinx, Ceratomia catalpae Boisduval (Lepidoptera: Sphingidae) sequesters the iridoid glycoside catalpol, which it acquires from Catalpa spp. (Lamiales: Bignoniaceae) host plants, at concentrations above those in host plant leaves. Previous research has shown that C. catalpae larvae are avoided by predatory arthropods; however, catalpol sequestration is negatively associated with the larval immune response, suggesting a cost to sequestration. By measuring the mass of C. catalpae larvae, their frass, and their diet, as well as respiration rate and catalpol sequestration, this study aimed to determine whether sequestration is also associated with reduced growth and nutritional efficiency. I found that respiration rate was negatively correlated with the amount of catalpol sequestered and positively correlated with the concentration, or proportion dry mass of catalpol sequestered, and that the amount of catalpol sequestered was positively correlated with three of four measures of nutritional efficiency. Larger larvae that were closer to pupation sequestered very low proportions dry mass of catalpol and had a reduced respiratory rate per unit mass. These results suggest that sequestration has some costs for expenditure of energy, but is positively correlated with several growth and nutrition‐related processes. Instead, the immunological cost of sequestration measured in previous studies could be due to direct effects on immune system components. Further research on the potential metabolic costs of chemical defense in other specialist species is recommended.     

Purification and Characterization of Poly(γ-glutamic acid) Hydrolase from a Filamentous Fungus,Myrothecium sp. TM-4222

Poly(γ-glutamic acid) (PGA) hydrolase was purified from the culture filtrate of a filamentous fungus, Myrothecium sp. TM-4222 and its general properties, especially the mode of hydrolytic action on the γ-glutamyl bond of PGA, were investigated. The purified preparation demonstrated a homogeneous band on an acidic slab gel of pH 4.3 with polyacrylamide gel electrophoresis. The enzyme showed its maximum activity at 37°C and at pH 5.0, being stable up to 40°C. The molecular mass was estimated to be 68 kDa by gel filtration. The hydrolytic action of the enzyme was specific for PGA, but not for other γ-glutamyl peptides or amides. The enzyme converted 38% of the original PGA with an average molecular mass of 500 kDa to smaller peptides, and then depolymerized these fragments to a mixture of γ-oligopeptides which consisted of only L-glutamic acid. L-Glutamic acid monomer was negligible in the reaction mixture. The remaining 62% of PGA was resistant to the enzyme action, in which D-glutamic acid was mainly detected. This study demonstrated a novel endo-type specificity of hydrolysis on PGA by the enzyme.     

Occurrence and some properties of a novel γ-glutamyltransferase responsible for the syntheis of γ-L-glutamul-D-alanine in pea seedlings

Occurrence of a novel γ-glutamyltransferase responsible for the formation of γ-L-glutamyl-D-alanine was demonstrated in pea seedlings, and the enzyme was purified 600-fold. The enzyme preparation catalyzed the transfer of the γ-glutamyl moiety of L-glutamine and other γ-glutamyl compounds to D-amino acids. In the formation of γ-L-glutamyl peptides of D-amino acids, L-glutamine served as the most effective γ-glutamyl donor and D-alanine acted as a highly-specific acceptor. The maximum activity of the γ-glutamyl transfer reaction between L-glutamine and D-alanine was observed at pH 9.5 and the apparent K_m values for these amino acids were estimated to be 2.0 and 2.9mM, respectively. This unique γ-glutamyltransferase activity was always accompanied by the catalytic activities of the known γ-glutamyltransferases during the purification procedure.     

gamma-glutamyl transpeptidase and related enzyme activities inthe reproductive system of the male rat.

The γ-glutamyl transpeptidase activity of the epididymis is much higher than that of the several other organs of the reproductive system of the male rat. The epididymal caput has much more activity than the epididymal cauda. Relatively low activity was found in spermatozoa. The enzyme is present in the epididymal fluid in a particulate form suggesting that it originates from membranes of epididymal epithelial cells. The epididymal caput exhibits high γ-glutamylcysteine synthetase activity indicating an active γ-glutamyl ycle in this tissue, which plays an important role in transport phenomena.     

gamma-Glutamyl transpeptidase: relation to immunoglobulin A and free secretory component.

The experiments reported show that bovine γ-glutamyl transpeptidase can be separated from free secretory component. An ion-exchange Chromatographic procedure was developed to analyze the incubation mixtures of the enzyme with glutathione or S-(2-acetamido)-glutathione and glycylglycine. Using this system or the γ-glutamyl p-nitroanilide assay, no significant transpeptidase activity could be detected in the free secretory component-containing fractions of DEAE-cellulose chromatography. Gel filtration on Biogel A-5M showed that the bovine whey transpeptidase chromatographed in the void volume suggesting an aggregate of a minimum molecular weight of about 5 × 10⁶. The transpeptidase could be separated from all immunoglobulins in bovine whey and human colostrum by a combination of agarose gel filtration and immunoadsorption. Concentrated samples of human and sheep saliva showed normal amounts of secretory component, but no detectable γ-glutamyl transpeptidase activity. These experiments show that (1) the transpeptidase and secretory component are two different proteins, and (2) the transpeptidase is present in bovine and human milk as a high molecular weight aggregate which does not include any of the immunoglobulins.     

γ-Glutamyl transpeptidase architecture: Effect of extra sequence deletion on autoprocessing,structure and stability of the protein from Bacillus licheniformis

γ-Glutamyl transpeptidases (γ-GTs, EC 2.3.2.2) are a class of ubiquitous enzymes which initiate the cleavage of extracellular glutathione (γ-Glu-Cys-Gly, GSH) into its constituent glutamate, cysteine, and glycine and catalyze the transfer of its γ-glutamyl group to water (hydrolysis), amino acids or small peptides (transpeptidation). These proteins utilize a conserved Thr residue to process their chains into a large and a small subunit that then form the catalytically competent enzyme. Multiple sequence alignments have shown that some bacterial γ-GTs, including that from Bacillus licheniformis (BlGT), possess an extra sequence at the C-terminal tail of the large subunit, whose role is unknown. Here, autoprocessing, structure, catalytic activity and stability against both temperature and the chemical denaturant guanidinium hydrochloride of six BlGT extra-sequence deletion mutants have been characterized by SDS-PAGE, circular dichroism, intrinsic fluorescence and homology modeling. Data suggest that the extra sequence has a crucial role in enzyme activation and structural stability. Our results assist in the development of a structure-based interpretation of the autoprocessing reaction of γ-GTs and are helpful to unveil the molecular bases of their structural stability.     

Polymorphic growth in larvae of Maculinea butterflies, as an example of biennialism in myrmecophilous insects

The presence of annual and biennial individuals within the same population has been recently demonstrated in the myrmecophilous butterflies Maculinea rebeli and Maculinea alcon, which present a cuckoo strategy inside Myrmica nests, and Maculinea arion which is a predatory species. Here, we present field and laboratory data on polymorphic larval growth in two other predatory species of Maculinea: M. teleius and M. nausithous. Body mass distributions of pre-pupation larvae were bimodal in both species. These results point to the existence of larvae that develop in 1 or 2 years. We also showed that the probability of pupation depended on larval body mass. In the case of M. teleius, the critical body mass at which larvae have a 50% probability of pupation is about 80 mg. We suggest that polymorphism in Maculinea may have evolved as an adaptation to life in ant nests, a habitat which protects them from predators and provides food. However, the quality of this resource is highly variable and unpredictable. According to the bet-hedging hypothesis, if the habitat is unpredictable, females should have an advantage by producing more variable offspring. In the case of Maculinea butterflies, this may involve maintaining larvae that develop in 1 or 2 years.     

gamma-Glutamyl transpeptidase in Hydra littoralis.

$\text{Hydra}$$\text{littoralis}$ exhibits high γ-glutamyl transpeptidase activity, i.e., about 12% of the activity (determined with glutathione) of rat kidney. Histochemical studies show that the enzyme is located mainly in the gastric and sub-hypostome regions; the enzyme is also present in the tentacles and basal disc. These results and the presence of other enzymes of the γ-glutamyl cycle suggest that the cycle plays a role in the metabolism of glutathione in hydras and that γ-glutamyl transpeptidase may function in their digestive and absorptive processes and possibly also in the behavioral response to glutathione.