Proceedings of the NATIONAL ACADEMY OF SCIENCES Volume 40 . Number 9 . September 15, 1954 GLUCOSAMINE KINASE OF SCHISTOSOMA MANSONI* BY ERNEST BUEDINGt HOWARD RUPPENDERf AND JOAN MACKINNON DEPARTMENT OF PHARMACOLOGY, WESTERN RESERVE UNIVERSITY SCHOOL OF MEDICINE, CLEVELAND, OHIO Communicated by Harland G. Wood, May 28, 1964 The parasitic trematode Schistosoma marsoni is dependent for survival on ana- erobic glycolysis which occurs at an extremely rapid rate.1 As in mammalian- tis- sues, formation of lactic acid by homogenates and extracts of the worms proceeds via the Embden-Meyerhof scheme.2 Opportunities for inhibiting glycolysis of schistosomes without injury to the host would be available if the-glycolytic enzymes catalyzing the same reactions in the parasite and in mammalian tissues were not identical. The existence of such differences has been observed recently. For example, the functional integrity of phosphohexose isomerase of Schistosoma man- soni is inhibited by an antibody which does not affect the activity of the isomerase of rabbit muscle.3 The lactic dehydrogenases of schistosomes and of rabbit muscle can be distinguished from each other not only by their behavior toward specific antibodies4 but also by their affinity to one of their substrates (pyruvate) and by the effect of pH on their activities.5 Furthermore, comparison of the properties of the hexokinases of the parasite with those of the host have revealed differences in the kinetics as well as in substrate specificities.' 6 Sols and Crane7 have demon- strated that purified brain hexokinase catalyzes the phosphorylation of glucose, of fructose, of mannose, of glucosamine, and of other hexoses by adenosine triphos- phate (ATP). The same is true for crystallized yeast hexokinase.8' 9 On the other hand, Schistosoma mansoni contains three distinct hexokinases,3' 6 each one of which reacts specifically with only one hexose, either glucose, fructose, or mannose. None of these three kinases reacts with glucosamine.2 In the present communication it is reported that the worms contain a fourth hexokinase which catalyzes the Phos- phorylation of glucosamine by ATP. On the basis of these differences in the nature of the hexokinases of schistosomes from those of the host, the effect of glucosamine on the parasite was tested, and it was observed that this hexose is active against Schistosoma mansoni in vitro and in vivo. Glucosamine kinase and glucokinase activities were measured by incubating the enzyme preparation of the worms with the hexose and with ATP for a period of 10 minutes at 370 C. The final molar concentrations of the constituents in the reaction mixture were as follows: glucosamine (or glucose), 1.5 X 10-3; ATP, 7 X 10-3; MgCl2, 1 X 10-2; potassium glycylglycine buffer (pH 7.1 for the deter- mination of glucosamine kinase and 7.5 for that of glucokinase activity), 5 X 10-2. 773 Downloaded by guest on September 23, 2021 1-74. BIOCHEMISTRY: BUEDING ET AL. PROC. N. A. S. ATP was added to the experimental samples before, and to the control samples immediately after, incubation. Proteins and phosphate esters were precipitated with barium hydroxide and zinc sulfate,'0 and the concentration of the nonphos- phorylated hexose was determined in the filtrate."' 12 Hexose utilization was calculated from the difference between the free hexose concentration (reaction mixture incubated without ATP) and the experimental sample. Glucosamine-6- phosphate was prepared according to Brown.9 Glucosamine kinase was purified as follows: adult male schistosomes were homogenized in 0.01 M glycylglycine buffer (pH 7.5; 1 ml. of buffer was used per 140 worms), and the protein concentration of the homogenate was adjusted to 5.0 mg/ml by dilution with the buffer. 0.04 ml. of alumina gel CQ (23 mg. of dry weight per milliliter of gel) were added per milli- liter of homogenate. The mixture was stirred and then centrifuged. Addition of 0.04 ml. of the same gel per milliliter of supernatant and subsequent elution of the residue with glycylglycine buffer (0.06 M; pH 7.5) yielded a fraction containing glucokinase activity. All operations were carried out at temperatures ranging between 00 and 4° C. Incubation of schistosome homogenates with glucosamine, ATP, and MgCl2 resulted in the disappearance of the free amino sugar. This suggested that the TABLE 1 GLUCOSAMINE KINASE AND GLUCOKINASE ACTIVITIES OF A HOMOGENATE OF Schistosoma mansoni GLUCOSAMINE KINASE GLUCOKINA8E Per Cent Per Cent ADDITION Activity* Inhib. Activity* Inhib. None 0.21 .. 0.65 0.004 M glucosamine-6-phos- phate 0.12 43 0.28 57 0.0016 M glucosamine-6-phos- phate 0.18 14 0.54 17 0.01 M N-acetylglucosamine 0 05 76 0.003 M N-acetylglucosamine 0.13 38 * Micromoles of free hexo8e phosphorylated per milligram of protein. enzyme preparation of the worms catalyzed the phosphorylation of glucosamine. Evidence for the formation of a phosphate ester of glucosamine under these conditions was obtained by the isolation of a water-soluble, alcohol-insoluble barium salt from the deproteinized reaction mixture. After the removal of the barium, analysis of this material for acid-stable organic phosphate and for glucosamine"2 as well as fractionation by ion-exchange chromatography" revealed that the com- pound had characteristics identical with those of glucosamine-6-phosphate.9 13 These data indicated that the compound was not identical with glucosamine-l- phosphate. Because of the limited amount of available organisms, it was not possible to obtain this compound in sufficient quantities to determine whether phos- phorylation had occurred in positions 6, 3, 4, or 5. Glucose did not inhibit the phosphorylation of glucosamine, nor did glucosamine affect the activity of glucokinase. Therefore, neither of these two hexoses inter- fered with the phosphorylation of the other by schistosome homogenates. It has been reported that N-acetylglucosamine inhibits the phosphorylation of glucosamine by brain hexokinase.14 Similarly, N-acetylglucosamine reduced the activity of the worm enzyme which catalyzes the phosphorylation of glucosamine (Table 1). Crane and Sols have demonstrated that glucose-6-phosphate inhibits the activity Downloaded by guest on September 23, 2021 VOL. 40Y 19D54 BIOCHEMISTRY: BUEDING ET AL. 775 of brain hexokinase.7' 15 While this ester did not affect the phosphorylation of fruc- tose, it did inhibit the phosphorylation of glucose and of mannose by homogenates and by extracts of schistosomes.6 Glucosamine-6-phosphate inhibited glucosamine kinase and glucokinase activity of schistosomes (Table 1); however, higher concen- trations were required to produce this effect than in the case of glucose-6-phosphate. On fractionation of homogenates of male schistosomes by the use of alumina gel C,, an enzyme extract was obtained which contains glucosamine kinase, free of glucokinase activity (Table 2). It has been found previously that purified gluco- kinase, fructokinase, and mannokinase of schistosomes do not react with glucos- amine.3 Therefore, in Schistosoma mansoni phosphorylations of glucosamine and of glucose are catalyzed by two different enzymes. TABLE 2 GLUCOSAMINE KINASE ACTIVITY OF A FRACTION OF A MALE SCHISTOSOME HOMOGENATE PURIFIED BY USE OF ALUMINA CTy Experiment Glucosamine Kinase Glucokinase No. Activity* Activity* 1 7.6 0 2 5.4 0 3 9.2 0 * Micromoles of free hexose phosphorylated per milligram of protein. The occurrence in the worms of a glucosamine kinase whose activity is not affected by glucose raised the question whether a phosphate ester of glucosamine or a metabolic product thereof interferes with chemical reactions essential for the survival of the worms. Such a possibility was suggested by the inhibitory effect of glucosamine-6-phosphate on the glucokinase of the parasite as well as by the fact that glucosamine reduced the rate of glyoolysis of intact schistosomes.3 This problem was studied by determining the effect of glucosamine on the parasites in vitro and in vivo. In a chemically defined medium the schistosomes survive for a period of 5-6 days. 16 Addition of glucosamine to this medium resulted in a signifi- cant reduction of their survival (Table 3). The action of glucosamine was not affected by the concentration of glucose in the medium. Accordingly, there was no evidence for a competition between glucose and glucosamine. Reduction of the survival of the parasite by glucosamine was observed also when horse serum was used as the medium (Table 3). TABLE 3 EFFECT OF GLUCOSAMINE ON SURVIVAL OF Schistosoma manmoni IN VITRO Glucosamine Average Survival Reduction of Survival Medium (Mg/Ml) (Days) (Per Cent) 6.2 Synthetic{ 3. 1.1 82 ]1.2 2.3 63 t0.4 3.9 37 23.0 Serum 3 0 6.2 73 1.2 9.8 58 O0.4 15.9 31 Oral administration of glucosamine to mice infected with Schistosoma mansoni resulted in a change in the distribution of the worms in the host. The adult schistosomes are located in the mesenteric veins, in the portal vein, and in the liver Downloaded by guest on September 23, 2021 776 BIOCHEMISTRY: BUEDING ET AL. PROC. N. A. S. sinuses. When the worms are damaged by chemotherapeutic agents, such as organic antimonials, they lose their attachment in the mesenteric veins and migrate toward the liver. Such an effect was observed after oral or intraperitoneal administration of glucosamine; under these conditions a significant reduction in the number of worms in the mesenteric veins occurred which was associated with a corresponding increase of worms in the liver (Table 4). The differences in the distribution of the TABLE 4 EFFECT OF ORAL ADMINISTRATION OF GLUCOSAMINE TO MICE INFECTED WITH Schistosoma mansoni ON DISTRIBUTION OF WORMS IN PORTALHEPATO-VENOUS SYSTEM OF HOST* TOTAL No. .-PERCENTAGE OF WORMS IN-- EXPERIMENT No. OF OF WORMS Mesenteric Portal No. GROUP MICE (Av.) Veins Vein Liver fControl 7 28.7 54 40 6 1 \Experimental 13 28.7 30 41 29 2 fControl 11 25.9 47 41 12 Experimental 10 24.7 32 38 30 * Glucosamine was administered twice daily (10 per cent solution; pH 6.0) at a single dose of 50 mg.