N-acetylglucosaminyltransferase from Ascaris suum.

The occurrence of N-acetylglucosaminyltransferase, the initial step in the synthesis of the carbohydrate moiety of N-linked glycoproteins, is demonstrated in the microsomal fraction of the nematode Ascaris suum. Phosphatidylglycerol stimulated enzyme activity three- to six-fold without affecting the Km values of either substrates, uridinediphospho-N-acetylglucosamine or dolichylphosphate. The Km values were determined to be about 12 microM and 100 micrograms ml-1, respectively. The enzyme activity was strongly inhibited by tunicamycin acting as a competitive inhibitor with respect to the substrate uridinediphospho-N-glucosamine.

Biosynthesis of dolichyl diphosphate N-acetylglucosamine intermediates in Ascaridia galli microsomes.

1. N-acetylglucosamine-1-phosphate transferase was demonstrated in the microsomal fraction of Ascaridia galli. 2. The transferase reaction depends on exogenous dolichyl phosphate as lipid acceptor and was found to be inhibited by tunicamycin. 3. The enzyme activity was optimal in the presence of sodium deoxycholate as detergent and Mg cations after 10 min of incubation. 4. The product of the transferase reaction--dolichyl diphosphate N-acetylglucosamine was converted into lipid-disaccharide-dolichyl diphosphate N,N'-diacetylchitobiose. 5. The maximum level of the conversion was achieved at 5 mM concentration of unlabelled UDP-N-acetylglucosamine, while this conversion was negligible at lower UDP-N-acetylglucosamine concentrations (0.1 and 0.5 mM).

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.

Dolichol kinase in Ascaris suum and Onchocerca volvulus.

Dolichol kinase was demonstrated in the microsomal fraction of Ascaris suum and Onchocerca volvulus. The enzyme from nematodes exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. Enzyme activity was optimal at pH 7.4, in the temperature range between 30 degrees and 37 degrees C, and in the presence of 0.5% Triton X-100. In addition, the enzyme was found to depend on divalent cations for activity; Mg2+ being more effective than Mn2+ and Ca2+. The dolichol kinase from both nematodes was shown to be independent of Ca2+-calmodulin for activity. The apparent Km values for dolichol were determined to be 7.5 and 9.0 micrograms ml-1 for the enzyme from A. suum and O. volvulus, respectively. Those for CTP were estimated to be 0.85 and 0.75 mM, respectively.

Effect of amoscanate derivative CGP 8065 on aminoacyl-tRNA synthetases in Ascaris suum.

Evidence is provided for the occurrence of a multienzyme complex consisting of several aminoacyl-tRNA synthetases besides 'free enzymes' in Ascaris suum. The molecular mass of this complex was calculated to be about 10(6) daltons, compared to about 150 000 daltons for the seryl-tRNA synthetase. Leucyl-, isoleucyl-, arginyl- and lysyl-tRNA synthestases were found in the high molecular weight fraction. The Michaelis constants of these aminoacyl-tRNA synthetases were found to be in the range of 4 to 10 microM for amino acids and of 0.1 to 1.0 mM for ATP. Leucyl- and isoleucyl-tRNA synthetase interact with the amoscanate-derivative CGP 8065. The inhibition constants were determined to be 34 microM and 8 microM, respectively. The type of inhibition was found to be competitive with respect to ATP. It is proposed that the interference of CGP 8065 with the charging of tRNA might be another target for the chemotherapeutic attack of this amoscanate derivative.

Poly(ADP-ribosylation) in Ascaris suum.

Poly(ADP-ribosylation) was demonstrated in the intestinal parasite Ascaris suum, especially in the reproductive tissues. The activity of the ADP-ribosyltransferase was found to depend on divalent cations and to be stimulated by deoxyribonuclease I about 5-fold. The reaction rate was optimal at a temperature of 30 degrees C and at pH about 8.4. The apparent Km value for NAD was estimated to be 0.2mM. The enzyme activity was effectively inhibited by nicotinamide (Ki = 65 microM) benzamide (6 microM), 3-aminobenzamide (10 microM), theophylline (35 microM) and thymidine (50 microM). The type of inhibition by these compounds was found to be competitive with respect to NAD.

Cyclic AMP-dependent and independent protein kinases in Ascaridia galli.

The occurrence of multiple protein kinases, distinguished with respect to molecular weight and preference for acceptor proteins, was demonstrated in Ascaridia galli. The molecular weights of the cyclic AMP-dependent protein kinase and of phosvitin kinase I and II - both independent of cyclic AMP-were determined to be 160000, greater than 200000 and 40000, respectively. The cyclic AMP-dependent protein kinase preferred histones and kemptide as acceptor substrates; stimulation of enzyme activity was up to 4-fold by cyclic AMP. The activities of phosvitin kinase I and II were found to be effectively inhibited by suramin. The inhibition constants were calculated to be 2 microM and 5 microM, respectively. In addition, stibophen turned out to be a potent inhibitor of phosvitin kinase I; the inhibition constant was determined to be 10 microM.