Glucosaminephosphate synthase of human liver.

Although human liver contains glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19), its activity is rapidly lost during the course of extraction. The inactivation, however, is largely prevented if the extraction medium contains isopropanol at 1% concentration; using these "stabilized" extracts, the glucosaminephosphate synthase activity of human liver has been shown to be similar to the activity previously reported in rat liver. The enzyme precipitated from these extracts by (NH4)2SO4 is inhibited by UDP-N-acetylglucosamine, the concentration required to produce a half-maximal inhibition being 6 muM. These results seem to be sufficient to postulate that glucosaminephosphate synthase is important for UDP-N-acetylglucosamine synthesis in human liver. In contrast to the rat liver enzyme, the (NH4)2SO4-precipitated human liver enzyme is resistant to trypsin and undergoes no conversion reaction when incubated with glucose 6-phosphate.     

Regulation by testosterone of the glucosamine-6-phosphate synthase activity in the male reproductive organs of rat

Accessory reproductive organs of male rat were found to contain high activities of glucosamine-6-phosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19). Castration caused drastic reduction (75%) in the enzyme activity of ventral prostate. Testosterone propionate administration restored the enzyme activity while cortisol and estradiol-17β did not cause any effect. Cycloheximide blocked the stimulation caused by testosterone.     

Biosynthesis of biopterin in Ascaris lumbricoides suum

In in vivo experiments, radioactivity from [U-¹⁴C]GTP was incorporated into biopterin, and, in fact, all carbon atoms of biopterin synthesized in Ascaris lu lumbricoides suum originated from GTP.Biopterin was also biosynthesized in homogenates of tissue fluid and muscles of Ascaris lumbricoides suum.The enzyme which catalyzes sepiapterin synthesis from D-erythto-7,8-dihydroneopterin-3′-phosphate was found in A. lumbricoides suum extracts and extracted in the 0–30% (NH₄)₂SO₄ fraction from a 40 000 × g supernatant. The enzyme was purified by Sephadex G-200 column and DEAE-cellulose column chromatography. It is suggested that sepiapterin could be an intermediate compound in biopterin biosynthesis.     

Chitinsynthese bei dem Flußkrebs Orconectes limosus: Aktivität der Phosphoglucosamin-isomerase und Einbau von Glucose-U-14C in Chitin

Zusammenfassung Im Zusammenhang mit der Chitinsynthese wurde die Phosphoglucosamin-isomerase in den Organen des Flußkrebses Orconectes limosus untersucht. In Integumentgewebe, Kiemen und Enddarm lagen die Aktivitäten bei Tieren unmittelbar nach der Häutung hochsignifikant höher als bei Zwischenhäutungstieren.Der zeitliche Ablauf folgender Parameter wurde über einen Häutungszyklus hinweg bestimmt: Aktivität der Phosphoglucosamin-isomerase im Integumentgewebe, Einbaurate markierter Glucose in Chitin sowie das Chitingewicht. Der Kurvenverlauf zeigte unerwartete Phasendifferenzen. Die Phosphoglucosaminisomerase scheint ein die Chitinsynthese steuerndes Schlüsselenzym zu sein. Obgleich sich die Isomeraseaktivität zwischen den D₁- und D₂-Stadien verdoppelt, wird die Hämolymphglucose nicht zur Chitinsynthese herangezogen; ihr Einbau in Chitin erfolgt erst unmittelbar nach der Häutung.Die extrem hohen Einbauraten von Glucose in Chitin sprechen dafür, daß Kohlenhydrate als Energiequelle zumindest zu dieser Zeit keine große Rolle spielen; diese Aufgabe fällt wahrscheinlich den Lipiden zu.

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Chitin synthesis in the crayfish, Orconectes limosus: activity of glucosaminephosphate isomerase and incorporation of glucose-U-¹⁴C

Summary In connection with our studies of chitin synthesis the activity of glucosaminephosphate isomerase was determined in the organs of the crayfish, Orconectes limosus. The activities in integumentary tissues, gills and intestine were greater with a high significance for animals directly after molt than for intermolt animals.Within the molt cycle the time courses of the following parameters were determined: glucosaminephosphate isomerase activity in integumentary tissue, incorporation of glucose-U-¹⁴C into chitin and chitin weight. Unexpectedly these curves show significant phase-differences. Glucosaminephosphate isomerase seems to be a key enzyme regulating the chitin synthesis. But although the isomerase activity doubles between stages D₁ and D₂, hemolymph glucose is not used for chitin synthesis at this time. The incorporation of hemolymph glucose into chitin starts immediately after the molt.The extremely high rate of incorporation of glucose into chitin seems to demonstrate that carbohydrates are not very important as an energy source at least at that time. Most likely lipids are the energy source.

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Verwendete Abkürzungen AG-6-P N-Acetylglucosamin-6-phosphat - F-6-P D-Fructose-6-phosphat - G-6-P D-Glucosamin-6-phosphat - PGI Phosphoglucosamin-isomerase. Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Chitin synthesis in zygotes of Ascaris suum

In Ascaris suum chitin is formed in the zygote immediately after oocyte fertilization, and its synthesis is completed in the eggs from the distal half of the uterus. Incorporation of radiocarbon [14C] glucose into chitin of the eggshell was 40-fold higher than incorporation of [14C] glucosamine. The same rank order also holds for the incorporation of label from these isotopes into the glycogen of the ovaries. A large part of the radiolabel was incorporated first into oocyte glycogen and only after fertilization was it incorporated into eggshell chitin. Actinomycin D inhibited chitin synthesis in the eggs from the distal half of the uterus and it significantly reduced incorporation of radiocarbon from glucose into chitin.     

Glucosamine metabolism in Drosophila virilis salivary glands: Ontogenetic changes of enzyme activities and metabolite synthesis

In Drosophila virilis salivary glands the in vitro activities of enzymes involved in the glucosamine pathway were examined during the third larval instar and in the prepupa. While glutamine-fructose-6-phosphate aminotransferase (EC 5.3.1.19) becomes inactive at the time of puparium formation, glucosamine-6-phosphate isomerase (EC 5.3.1.10) and glucosamine-6-phosphate N-acetyltransferase (EC 2.3.1.3) show maximal activities in the prepupal gland. The activity of UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) may also decrease prior to puparium formation. Incubation of larval and prepupal glands in medium containing [³H]glucose + [¹⁴C]-uridine or [¹⁴C]glucosamine and subsequent separation of intermediates of the glucosamine pathway by chromatographic procedures reveal that the capacity of the glands to incorporate the isotopes into these intermediates decreases significantly at the time of puparium formation. The results suggest that in D. virilis salivary glands the formation of aminosugars is mainly controlled by the activities of the two enzymes glutamine-fructose-6-phosphate aminotransferase and UDP-N-acetylglucosamine pyrophosphorylase.     

Value of the specific distinction between Ascaris lumbricoïdes linné 1758 and Ascaris suum Goeze 1782

Ansel M. and Thibaut M. 1973. Value of the specific distinction between Ascaris lumbricoïdes Linné 1758 and Ascaris suum Goeze 1782. International Journal for Parasitology3: 317–319. Study by scanning electron microscopy of heads of Ascaris lumbricoides Linné 1758 and Ascaris suum Goeze 1782 showed typical distinctive characteristics between these two species. There are differences in the appearance of the rows of denticules and the shapes of the lips.     

Feedback inhibition of L-glutamine D-fructose 6-phosphate amidotransferase by uridine diphosphate N-acetylglucosamine in Neurospora crassa

The enzyme, l-glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) of Neurospora crassa, which catalyzes the formation of glucosamine 6-phosphate was shown to be subject to feedback inhibition by uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc). The conclusion is based on the following observations. UDP-GlcNAc, the direct precursor of chitin, did not accumulate in the cell even when its utilization for the synthesis of cell wall chitin was interrupted by the antibiotic polyoxin D, a competitive inhibitor of the chitin synthetase (EC 2.4.1.16). Furthermore, the cellular level of UDP-GlcNAc rose in a short period of time when the amidotransferase was bypassed in vivo by the addition of glucosamine to the growing medium of the fungus. The amidotransferase was purified from N. crassa approximately 85-fold. Kinetic studies showed that UDP-GlcNAc was a potent and specific inhibitor of the amidotransferase, and that it did not alter the Michaelis constant for either l-glutamine or d-fructose 6-phosphate, suggesting that the inhibitor binds at a site on the enzyme distinct from the active site.     

Further studies on the regulation of amino sugar metabolism in Bacillus subtilis

1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its K_m is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (K_m 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose.     

The incorporation of labelled amino sugars by Bacillus subtilis

1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its K_m is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (K_m 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose.     

Ascaris suum: aldolase I purification and immunologic study

Fructose-1,6-diphosphate d-glyceraldehyde-3-phosphate lyase (aldolase) (EC 4.1.2.13) from the body wall of Ascaris suum was purified 80-fold by a combination of salt precipitation and ion-exchange chromatography. A group of pigs was immunized with the purified aldolase preparation and was subsequently challenged with infective Ascaris larvae. The immunized animals showed clinical and histopathologic symptoms of acute sensitization reaction. Thrice as many larvae were found in the nonimmunized control pigs as compared to the immunized animals.     

Reversible inhibition of hatching of infective eggs of Ascaris suum (Nematoda)

Hurley L. C. and Sommerville R. I. 1982. Reversible inhibition of hatching of infective eggs of Ascaris suum (Nematoda). International Journal for Parasitology12: 463–465. Dilute solutions of an oxidising agent, iodine, reversibly inhibit hatching of infective eggs of Ascaris suum. The capacity to hatch is restored by exposure to reducing agent, hydrogen sulphide. These observations add to known similarities between hatching of infective eggs and exsheathment of infective larvae. It is proposed that the regulatory mechanisms for both processes are similar.     

Studies on sugar isomerases of Lactobacillus sp.: Whole cell immobilization of d-glucose isomerase

A new soil isolate of Lactobacillus sp. grown in Yamanaka medium under submerged conditions showed the presence of d-glucose, d-xylose and d-ribose isomerases in washed cell suspension and cell free extracts. d-Xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) and d-ribose isomerase (d-ribose ketol-isomerase, EC 5.3.1.20) activities reached a maximum in 48 h of growth and then declined. d-Glucose isomerase (d-glucose 6-phosphate isomerase, d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) activity was maximum after 72 h and remained constant for ～120 h of growth. d-Glucose isomerase activity increased with the increase in number of generations of culture and reached a maximum in 5–6 generations, whereas d-xylose and d-ribose isomerase activities decreased. The washed and starved whole cells could be heat treated and immobilized on the rough surface of glass rods or glass slides using acetone treatment. The heat treated immobilized cells showed only the presence of d-glucose isomerase activity and showed no d-xylose and d-ribose isomerase activities. d-Glucose isomerase activity of heat treated immobilized cells was inhibited less by sorbitol, mannitol, sodium arsenate, cysteine and calcium ions than the free d-glucose isomerase activity in fresh untreated washed whole cells and cell free extracts. EDTA inhibition had the same effect for both forms. Ca²⁺inhibition could be reversed by adding Mg²⁺ions.     

The occurrence of the second moult of Ascaris lumbricoides and Ascaris suum

Maung M. 1978. The occurrence of the second moult of Ascaris lumbricoides and Ascaris suum. International Journal for Parasitology 8: 371–378. Eggs of Ascaris lumbricoides and A. suum were cultured at 28°C and observed daily. Larvae were released by pressure, by artificial hatching with CO₂, and by natural hatching after infection of laboratory mice. The early stages of development in the egg were observed to comprise two moults, one occurring immediately after the other. Both moults were initiated within the egg, but the time of completion of the second moult varied considerably, and in some instances was not completed until the larvae reached the liver of experimentally infected animals.     

In vitro Induction of Entamoeba histolytica Cyst-like Structures from Trophozoites

Inhibition of encystment can be conceived as a potentially useful mechanism to block the transmission of Entamoeba histolytica under natural conditions. Unfortunately, amoeba encystment has not been achieved in vitro and drugs inhibiting the formation of cysts are not available. Luminal conditions inducing encystment in vivo are also unknown, but cellular stress such as exposure to reactive oxygen species from immune cells or intestinal microbiota could be involved. A role for certain divalent cations as cofactors of enzymes involved in excystment has also been described. In this study, we show that trophozoite cultures, treated with hydrogen peroxide in the presence of trace amounts of several cations, transform into small-sized spherical and refringent structures that exhibit resistance to different detergents. Ultrastructural analysis under scanning and transmission electron microscopy revealed multinucleated structures (some with four nuclei) with smooth, thick membranes and multiple vacuoles. Staining with calcofluor white, as well as an ELISA binding assay using wheat germ agglutinin, demonstrated the presence of polymers of N-acetylglucosamine (chitin), which is the primary component of the natural cyst walls. Over-expression of glucosamine 6-phosphate isomerase, likely to be the rate-limiting enzyme in the chitin synthesis pathway, was also confirmed by RT-PCR. These results suggest that E. histolytica trophozoites activated encystment pathways when exposed to our treatment.     

Stabilization of glucosaminephosphate synthase from rat liver by hexose 6-phosphates. Properties and interconversion of two molecular forms.

Glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19) prepared from rat liver by extraction in the presence of glucose 6-phosphate (Glc-6-P) followed by precipitation with (NH4)2SO4 is susceptible to digestion by trypsin. This enzyme, designated form A, can be converted to tryptic-insusceptible form B upon incubation with Glc-6-P or fructose 6-phosphate (Fru-6-P) at 37 degrees C. The two forms also differ in the degree of activation by dithiothreitol, the degree of inhibition by methyl-glyoxal and the behavior on DEAE-Sephadex and Sephadex G-200 column chromatography. During purification with DEAE-Sephadex followed by hydroxyapatite, form B is converted to form A if Fru-6-P is absent and form A to form B if Fru-6-P is present. The two forms are therefore intercovertible. Under the conditions of purification, form B is more stable than form A, since the purity and yield of the final product are greater with form B than with form A. These findings suggest that the two forms of glucosaminephosphate synthase differ conformationally and that the equilibrium position depends on the concentration of Fru-6-P. Glc-6-P is effective only when it gives rise to Fru-6-P by mediation of glucose-phosphate isomerase.     

Regulation of chitin synthesis during germ-tube formation in Candida albicans

The synthesis of chitin during germ-tube formation in Candida albicans may be regulated by the first and last steps in the chitin pathway: namely l-glutamine-d-fructose-6-phosphate aminotransferase and chitin synthase. Induction of germ-tube formation with either glucose and glutamine or serum was accompanied by a 4-fold increase in the specific activity of the aminotransferase. Chitin synthase in C. albicans is synthesized as a proenzyme. N-acetyl glucosamine increased the enzymic activity of the activated enzyme 3-fold and the enzyme exhibited positive co-operativity with the substrate, UDP-N-acetylglucosamine. Although chitin synthase was inhibited by polyoxin D (K _i =1.2M) this antibiotic did not affect germination. During germ-tube formation the total chitin synthase activity increased 1.4-fold and the expressed activity (in vivo activated proenzyme) increased 5-fold. These results could account for the reported 5-fold increase in chitin content observed during the yeast to mycelial transformation.Non-Standard Abbreviations GlcNac N-acetyl glucosamine - UDP-GlcNac UDP-N-acetyl glucosamine - PMSF phenylmethylsulphonylfluoride     

Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5′-diphospho-N-acetyl-d-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts d-fructose-6-phosphate (Fru-6P) and l-glutamine (Gln) into d-glucosamine-6-phosphate (GlcN-6P) and l-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme.     

Succinate dehydrogenase and fumarate reductase activity in Aspiculuris tetraptera and Ascaris suum and the effect of the anthelmintics cambendazole,thiabendazole, and levamisole

Comley John C. W. and Wright Spdenis J. 1981. Succinate dehydrogenase and fumarate reductase activity in Aspiculuris tetraptera and Ascaris suum and the effect of the anthelmintics cambendazole, thiabendazole, and levamisole. International Journal for Parasitology11: 79–84. Succinate dehydrogenase and fumarate reductase activities from a particulate fraction of A. tetraptera and a soluble extract of A. suum have been determined using spectrophotometric methods. Fumarate reductase activity in A. suum could only be detected anaerobically. Succinate dehydrogenase activity from A. suum was partially characterized and shown to exist in several multimolecular forms (isoenzymes). The in vitro effect of the anthelmintics cambendazole, thiabendazole and levamisole on succinate dehydrogenase and fumarate reductase activity from the above nematodes are described. Significant inhibition of fumarate reductase activity of both nematodes was only achieved using 5 mM levamisole and 1 mM thiabendazole. After in vivo anthelmintic treatment of A. tetraptera only thiabendazole significantly inhibited fumarate reductase. It is suggested that the succinate dehydro-ogenase-fumarate reductase complex in these nematodes is unlikely to be the primary site chemotherapeutic attack for any of the anthelmintics tested.