Effect of drugs inhibiting spermidine biosynthesis and metabolism on the in vitro development of Plasmodium falciparum

Treatment of Plasmodium falciparum with the potent inhibitor dicyclohexylamine completely arrests in vitro cell proliferation of the chloroquine-susceptible P. falciparum strain NF54 and the R strain, which shows less sensivity to chloroquine. The average inhibitory concentration (IC50) values determined for both strains revealed different inhibition profiles. The IC50 value for the chloroquine-sensitive NF54 strain was 97 microM and 501 microM for the R strain. Monitoring polyamine pools after treatment with dicyclohexylamine leads to a significant decrease in the intracellular spermidine content, which was nearly reversed by supplementation with spermidine. Since spermidine is an important precursor for the biosynthesis of hypusine and homospermidine in eukaryotes, we studied the developmental effect on both P. falciparum strains of 1,7-diaminoheptane as an inhibitor of deoxyhypusine synthase (EC 1.1.1.249) in mammalian cells, and agmatine as a moderate inhibitor of homospermidine synthase (EC 2.5.1.44). Inhibition profiles with 1,7-diaminoheptane resulted in an IC50 value of 466 microM for the NF54 strain and 319 microM for the R strain. Spermidine pools changed significantly. Inhibition with agmatine caused a strong decrease in parasitemia for the chloroquine-susceptible NF54 strain, with a determined IC50 value of 431 microM and an IC50 value of 340 microM for the less chloroquine-susceptible R strain. Spermidine was not detectable after inhibition. The uncommon triamine homospermidine occurred in both P. falciparum strains. To our knowledge this is the first evidence of homospermidine in P. falciparum. The use of specific inhibitors of spermidine metabolism might be a novel strategy for the design of new antimalarials, and suggests the occurrence of both enzymes in the parasite.     

Effect of putrescine on the respiration of Trypanosoma brucei brucei

When bloodstream forms of Trypanosoma brucei brucei were exposed to exogenous putrescine for 24 h during in vitro culture, the rate of O2 consumption increased significantly in a concentration-related and time-dependent manner. Trypanosomes cultured with 100 microM DL-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, were depleted of intracellular putrescine, and the rate of O2 consumption decreased by more than 50%. This effect could be abrogated if 100 microM putrescine was also present. A similar pattern was observed in trypanosomes harvested from rats after 36 h of DFMO treatment. If such trypanosomes were placed in culture for 2 h with 100 microM putrescine, the rate of O2 consumption returned to that of controls. When an intraperitoneal injection of putrescine was given to infected rats 18 h after commencement of DFMO treatment, rates of O2 consumption in the trypanosomes were found to return to control values. The addition of putrescine, spermidine or Mg2+ did not affect rate of O2 consumption in enriched mitochondrial preparations. However, when putrescine was present throughout the preparation of mitochondrial fractions, there was an increase of 23% in O2 uptake, which was 23% higher than in the controls. Putrescine may modulate trypanosomal respiration by stabilizing mitochondrial membranes.     

Polyamine N-acetyltransferase from Fasciola hepatica.

A cytosolic polyamine N-acetyltransferase which catalyses polyamine and diamine acetylation has been partially purified from the liver fluke Fasciola hepatica. The enzyme has an apparent Mr of 50,000 and unlike the corresponding mammalian liver counterpart is capable of putrescine acetylation. Among the substrates tested, spermidine had the highest reaction rate but putrescine had a lower Km value. The Km values for spermidine, spermine, norspermidine, putrescine, cadaverine and 1,3-diaminopropane were 20 microM, 1.30 mM, 20 microM, 7 microM, 10 microM and 50 microM, respectively. Acetylated polyamines were also substrates for the trematode acetylase, but histones were inactive. The partially purified enzyme had no deacetylase activity. The Km for acetyl-CoA was 4.4 microM. Coenzyme A was strongly inhibitory with a Ki value of 5.3 microM. Bis(benzyl)polyamine analogue MDL 27695 was a potent competitive inhibitor of the enzyme with a Ki of 22 microM. Inhibition by 1,4-dimethyl-putrescine was non-competitive and had a Ki value of 15 microM. The trematode acetylase is highly dependent on sulfhydryl groups for its activity. As had been reported in nematodes, polyamine acetylation could represent a process by which trematodes convert excess polyamines to forms suitable for transport and excretion. On the other hand, this could be the regulatory step of a functional interconversion pathway in these parasites.     

In vivo effects of alpha-DL-difluoromethylornithine on the metabolism and morphology of Trypanosoma brucei brucei

The EATRO 110 isolate of Trypanosoma brucei brucei was grown in rats for 60 h and the animals treated with the ornithine decarboxylase inhibitor alpha-DL-difluoromethylornithine 12 h or 36 h prior to sacrifice. Control untreated animals died 72-80 h after infection. Treated parasites were shorter and broader than the predominantly long slender forms found in untreated controls and many had two or more nuclei and kinetoplasts. Trypanosomes were purified from blood and examined for disruption of polyamine metabolism. ODC activity decreased by more than 99% after 12 h treatment and putrescine and spermidine levels also decreased dramatically. Spermine, not normally present in control cells, increased to detectable, low levels (less than 1 nmol mg-1 protein) after 36 h treatment. alpha-DL-Difluoromethylornithine-treated cells were unable to synthesize putrescine from [3H]ornithine but were able to convert [3H]putrescine + methionine to spermidine. 12-h treated parasites responded to polyamine depletion by assimilating radiolabeled polyamines in vitro at 2- to 4-times the rate of untreated cells. The metabolism of S-adenosylmethionine was also altered in treated parasites: decarboxylated S-adenosylmethionine increased more than 1000-fold over untreated cells while S-adenosylmethionine decarboxylase activity, associated with the formation of spermidine and spermine in other eukaryotes, paradoxically declined in treated cells. Synthesis of macromolecules was perturbed in treated parasites: rates of DNA and RNA synthesis declined 50-100%, while protein synthesis increased up to 4-fold in 36-h treated cells. alpha-DL-Difluoromethylornithine treatment progressively limits the parasites' ability to synthesize nucleic acids and blocks cytokinesis while inducing morphological changes resembling long slender leads to short stumpy transformation.     

Molecular characterisation of Trypanosoma brucei alkyl dihydroxyacetone-phosphate synthase

Alkyl dihydroxyacetone-phosphate synthase is the second enzyme of the ether-lipid biosynthetic pathway which is responsible for the introduction of the ether linkage between a fatty alcohol and a glycerol present in a subclass of phospholipids, the plasmalogens and possibly in glycolipid membrane anchors. In this study the gene coding for alkyl dihydroxyacetone-phosphate synthase was isolated from Trypanosoma brucei. Southern blot analysis of total genomic DNA suggested the presence of a single copy gene. The analysis, together with sequencing of different cDNA clones showed that the two alleles of the gene differ in only one nucleotide. The gene encodes a protein of 612 amino acids with a calculated molecular mass of 68,891, not counting the initiator methionine. It carries a type-1 peroxisomal targeting signal (a C-terminal tripeptide--AHL) and a calculated overall positive charge of +10. The gene was expressed in a bacterial system and the corresponding protein carrying a His-tag was purified. The recombinant alkyl dihydroxyacetone-phosphate synthase and the enzyme isolated directly from the glycosomes of bloodstream-form trypanosomes have comparable kinetics. The Km for hexadecanol was 42 microM, while approximately 100 microM of palmitoyl dihydroxyacetone phosphate (DHAP) was necessary for optimal activity. Sodium chloride inhibited both the His-tagged protein and the enzyme isolated from the glycosomes of bloodstream-form and insect stage T. brucei.     

The anti-trypanosomal agent lonidamine inhibits Trypanosoma brucei hexokinase 1

Glycolysis is essential to the parasitic protozoan Trypanosoma brucei. The first step in this metabolic pathway is mediated by hexokinase, an enzyme that transfers the gamma-phosphate of ATP to a hexose. The T. brucei genome (TREU927/4 GUTat10.1) encodes two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. Our previous efforts have revealed that TbHK2 is an important regulator of TbHK1 in procyclic form parasites. Here, we have found through RNAi that TbHK1 is essential to the bloodstream form parasite. Silencing the gene for 4 days reduces cellular hexokinase approximately 60% and leads to parasite death. Additionally, we have found that the recombinant enzyme is inhibited by lonidamine (IC(50)=850 microM), an anti-cancer drug that targets tumor hexokinases. This agent also inhibits HK activity from whole parasite lysate (IC(50)=965 microM). Last, lonidamine is toxic to cultured bloodstream form parasites (LD(50)=50 microM) and procyclic form parasites (LD(50)=180 microM). Interestingly, overexpression of TbHK1 protects PF parasites from lonidamine. These studies provide genetic evidence that TbHK1 is a valid therapeutic target while identifying a potential molecular target of the anti-trypanosomal agent lonidamine.     

Regulation of polyamine content in cultured fibroblasts

The content of putrescine and of the polyamines (spermidine and spermine) and the activities of their biosynthetic enzymes were measured in 3T3 mouse fibroblasts and SV40-transformed mouse fibroblasts over the entire period from subculturing in fresh medium until confluence. The transformed cells had a substantially higher content of putrescine and spermidine than the 3T3 cells and higher activities of all of the biosynthetic enzymes. However, the ratio of spermine synthase to spermidine synthase was higher in the 3T3 cells, which correlated with their higher spermine-to-spermidine ratio. All of the biosynthetic enzymes increased in activity during cell growth. Ornithine decarboxylase increased 20-fold with a maximum at 24-36 h after culturing whereas S-adenosylmethionine decarboxylase increased 3-fold at the same time. Spermidine synthase increased 10- to 16-fold during the growth period whereas spermine synthase increased 2- to 3-fold. The relative enzyme activities and the changes in total polyamine content suggested that 1) the activity of S-adenosylmethionine decarboxylase limited the production of the polyamines and 2) the relative amounts of spermidine and spermine synthase determined the predominant polyamine that the available decarboxylated S-adenosylmethionine is used to synthesize. When 3T3 cells become quiescent at confluence, there was a substantial fall in the intracellular spermidine level because of a greatly increased excretion of spermidine into the medium. Spermine content also fell because there was an increased conversion of spermine into spermidine, which was then excreted. The specific excretion of spermidine did not occur with the transformed SV-3T3 cells.     

Characterization of genes coding for two major metacyclic surface antigens in Trypanosoma brucei

Procyclic Trypanosoma brucei brucei strain 366D is susceptible to DL-alpha-difluoromethylornithine (DFMO) with an in vitro ED50 value of 225 microM. A mutant of the procyclic strain resistant to 20 mM of DFMO was isolated by serial in vitro passages of the organisms in increasing concentrations of the drug. Drug resistance remains unchanged after at least ten serial passages in the absence of DFMO. The mutant contains the same level of ornithine decarboxylase activity as the wild-type procyclic, and the mutant enzyme exhibits a similar susceptibility toward DFMO as the wild type. Neither the rate of decarboxylation of ornithine, nor the membrane potential in the mutant cell is changed. The only observed change in the mutant is its significantly decreased uptake of DFMO which reaches a saturating level of 18 microM inside the cells; a concentration seven times below the Ki value of DFMO on T. brucei ornithine decarboxylase (130 microM). Apparently, the failure of DFMO uptake in the mutant strain has provided the basis of drug resistance. The results also raise the question on whether the uptake of DFMO by T. brucei is by passive diffusion or by transporter(s) mediation. DFMO does not compete with the uptake of ornithine, arginine or putrescine, and the reverse holds also true. However, the mutant strain cultivated under DFMO for several generations has a greatly enhanced uptake of ornithine and a moderately heightened uptake of putrescine. Both are reduced to the normal level upon further propagations of the mutant strain in the absence of DFMO.     

Studies on the glycosomal orotate phosphoribosyl transferase of Trypanosoma cruzi

The orotate phosphoribosyltransferase of the epimastigote form of Trypanosoma cruzi was studied in its particulate state in preparations derived from glycosomes. Maximum activity was observed at pH 9. There was little activity in the absence of Mg2+; optimum [Mg2+] was related to [5'-phosphoribosyl-alpha-1-pyrophosphate]; Mn2+ could partially substitute for it. Kinetic analyses ruled out a substituted mechanism and suggested instead that the mechanism may be sequential. The apparent Km orotate was 2 microM; that for 5'-phosphoribosyl-alpha-1-pyrophosphate was 8 microM. The enzyme could not use uracil as substrate and was apparently not regulated by naturally-occurring nucleotides. It was, however, sensitive to inhibition by a wide range of pyrimidine analogues, the most active of which was 5-fluoroorotate. These inhibitors were as effective against the enzyme activity of intact glycosomes as broken preparations. This observation, when considered with an apparent lack of latency, suggests that the enzyme is located on the outside of the glycosome. The product of its reaction, orotidine 5'-phosphate, did not exchange readily with exogenous orotidine 5'-phosphate, suggesting that it is channeled directly to orotidine 5'-phosphate decarboxylase, the next enzyme in the pathway.     

Active transport of 2-deoxy-D-glucose in Trypanosoma brucei procyclic forms

The characteristics of glucose transport by procyclic forms of Trypanosoma brucei were examined in a rapid transport assay using the glucose analogue 2-deoxyglucose. In contrast to bloodforms where the Km for 2-deoxyglucose transport is about 1 mM, procyclic forms have a Km of about 38 microM. Procyclic forms show temperature-dependent, saturable import, and import of 2-deoxyglucose is competitive with glucose and mannose. Unlike the bloodforms which employ facilitated diffusion, the procyclic forms actively transport glucose. Use of inhibitors and ionophores suggests that a protonmotive force is required for glucose transport in procyclic forms. Unlike the human erythrocyte glucose transporter, the glucose transporter of the T. brucei procyclic form is relatively insensitive to inhibition by cytocholasin B.     

Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity

The promastigote form of Leishmania donovani is sensitive to growth inhibition by DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, with an EC50 value of approximately 30 microM. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania, DFMO-10, which was capable of proliferating in 10 mM DFMO. DFMO-10 cells possessed an EC50 value for DFMO greater than 4 mM, and were cross-resistant to alpha-methylornithine, alpha-monofluoromethyl-3,4-dehydroornithine methyl ester, and delta-methyl-acetylenic putrescine, three other inhibitors of ODC activity. DI700 and DFMO-10 cells accumulated and/or transported [3H]DFMO and a spectrum of basic, neutral, and acidic amino acids at comparative rates. However, the DFMO-resistant Leishmania, if suspended in culture medium in the absence of DFMO for several days, expressed up to 15-fold greater levels of ODC activity than did wild-type cells. The overexpressed ODC in mutant cells appeared kinetically normal, since the ODC activities from DI700 and DFMO-10 cells possessed similar apparent Km values for ornithine and were equally sensitive to inactivation by DFMO. Incubation of extracts of DFMO-10 cells, but not of wild-type parental cells, with [3H]DFMO for 1 h resulted in the labeling of a polypeptide, presumably ODC, which migrated with a molecular weight of 76,000 +/- 4000 on SDS-gel electrophoretograms. As a consequence of the elevated ODC activities, the levels of putrescine in mutant cells released from DFMO exposure were also elevated by about 15-fold over those of wild-type cells, although spermidine levels in DI700 and DFMO-10 cells were similar. In the absence of prolonged selective pressure, the resistance to DFMO, the ODC activity, and the putrescine levels of DFMO-10 cells all returned to those of wild type cells, indicating that the mutant phenotype of DFMO-selected L. donovani was unstable.     

Triazinyl derivatives that are potent inhibitors of glucose transport in Trypanosoma brucei

Glucose transport in Trypanosoma brucei is facilitated by a transporter that is kinetically markedly different from its mammalian homologue. In this regard, the trypanosomal transporter may be selectively targeted. We investigated the potential of a series of triazinyl derivatives as inhibitors of glucose transport in T. brucei. A graded response of glucose transporter inhibition by these compounds was observed, with Cibacron blue 3GA, CiB, being the most potent. This inhibited transport by up to 90% in a concentration-dependent manner, with an IC50 of about 19.4 microM. A Dixon plot of different concentrations of this triazine and the rate of transport suggested that inhibition may be simple and competitive. The inhibition constant Ki was 14.8 microM. Although cytochalasin B has been widely reported to inhibit glucose uptake by mammalian and other eukaryotic glucose transporters, it had no effect at all on the trypanosome transporter at concentrations equivalent to those of the triazines. This may suggest structural differences between the trypanosome and mammalian glucose transporters and also suggests that the triazine moiety may serve as a template for the design potent trypanocides targeted at the glucose transporter.     

Trypanothione biosynthesis in Leishmania major

Trypanothione plays a crucial role in regulation of intracellular thiol redox balance and in defence against chemical and oxidant stress. Crithidia fasciculata requires two enzymes for the formation of trypanothione, namely glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and a glutathionylspermidine-dependent trypanothione synthetase (TryS; EC 6.3.1.9), whereas Trypanosoma cruzi and Trypanosoma brucei use a broad-specificity trypanothione synthetase to make trypanothione from glutathione (GSH) and spermidine. Here, we report the identification of two genes in Leishmania major with similarity to previously identified GSPS and TRYS. GSPS is an apparent pseudogene containing two frame shift mutations and two stop codons, whereas TRYS is in a single open-reading frame. The enzyme encoded by TRYS was expressed and found to catalyse formation of trypanothione with GSH and either spermidine or glutathionylspermidine. When GSH is varied as substrate the enzyme displays substrate inhibition (apparent Km=89 microM, Ki(s)=1mM, k(cat)=2s-1). At a fixed GSH concentration, the enzyme obeys simple hyperbolic kinetics with the other substrates with apparent Km values for spermidine, glutathionylspermidine and MgATP of 940, 40 and 63 microM, respectively. Immunofluorescence and sub-cellular fractionation studies indicate that TryS localises to the cytosol of L. major promastigotes. Phylogenetic analysis of the GspS and TryS amino acid sequences suggest that in the trypanosomatids, TryS has evolved to replace the GspS/TryS complex in C. fasciculata. It also appears that the L. major still harbours a redundant GSPS pseudogene that may be currently in the process of being lost from its genome.     

The incorporation of radiolabeled polyamines and methionine into turnip yellow mosaic virus in protoplasts from infected plants

Turnip yellow mosaic virus contains large amounts of nonexchangeable spermidine and induces an accumulation of spermidine in infected Chinese cabbage. By 7 days after inoculation, a majority of protoplasts isolated from newly emerging leaves stain with fluorescent antibody to the virus. These protoplasts contain 1-2 X 10(6) virions per cell and continue to produce virus in culture for at least 48 hr. [14C]Spermidine (10 microM) was taken up by these cells in amounts comparable to the original endogenous pool within 24 hr. However, after an initial rise, the spermidine content of the cell returned to its original level, implying considerable regulation of the endogenous pool(s). Putrescine and spermine were major products of the metabolism of exogenous spermidine. Radioactivity from exogenous [14C]spermidine was also readily incorporated into the ribonucleoprotein component(s) of the virus, where it appeared as both spermidine and spermine. The specific radioactivities of the viral polyamines were approximately twice those of spermidine and spermine extracted from the whole cell. Radioactivity from [2-14C]methionine was readily incorporated into the protein, spermidine, and spermine of the virus. Again, the specific activities of these amines were substantially higher in the virus than in the whole cell. Thus, newly formed virus contained predominantly newly synthesized spermidine and spermine. However, inhibition of spermidine synthesis by dicyclohexylamine led to incorporation of preexisting spermidine and increased amounts of spermine into newly formed virus.     

Guanosine triphosphate cyclohydrolase in Plasmodium falciparum and other Plasmodium species

GTP cyclohydrolase (EC 3.5.4.16), the first enzyme in the pteridine pathway leading to the de novo formation of folic acid, has been identified and isolated from the human malaria parasite, Plasmodium falciparum. The enzyme was purified 200-fold by high performance size-exclusion chromatography on a TSK-G-3000 SW protein column. The molecular weight was estimated at 300 000. Optimal enzyme activity was observed at pH 8.0 and 42 degrees C. The Km for GTP was 54.6 microM. Products of the enzyme reaction were identified as the carbon-8 of GTP and D-erythro-dihydroneopterin triphosphate. ATP was a competitive inhibitor (Ki = 600 microM) of the enzyme. Activity of the enzyme was Mg2+-independent, whereas Mn2+, Cu2+ and Hg2+ (5 mM) were inhibitory. GTP cyclohydrolase activity was also identified in a murine parasite, Plasmodium berghei, and a simian parasite, Plasmodium knowlesi. Activity of the enzyme in P. knowlesi, an intrinsically synchronous quotidian parasite, was found to be dependent on the stage of parasite development.     

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.     

In vivo effects of difluoromethylornithine on trypanothione and polyamine levels in bloodstream forms of Trypanosoma brucei

The effect of D,L-alpha-difluoromethylornithine (DFMO) on thiol and polyamine levels in Trypanosoma brucei was investigated by isolating trypanosomes from infected rats treated with DFMO for 12-48 h. Concentrations of thiols, polyamines and other amino-compounds were measured by an automated high-performance liquid chromatography method. The levels of DFMO in rat plasma (0.02-1.34 mM) is similar to that found in the parasites (0.27-0.99 mM), concentrations which exceed the Ki of DFMO for T. brucei ornithine decarboxylase. Treatment with DFMO increases intracellular levels of ornithine, S-adenosylmethionine and decarboxylated S-adenosylmethionine and decreases putrescine and spermidine. Putrescine is undetectable after 12 h treatment with DFMO and after 48 h spermidine is decreased by 76%. By 48 h, the spermidine-glutathione conjugates glutathionylspermidine and dihydrotrypanothione (bis(glutathionyl)spermidine) are also decreased by 41 and 66%, respectively. In contrast, levels of glutathione show a slight increase. These changes in metabolite levels are consistent with the biosynthetic pathway proposed for Crithidia fasciculata, where trypanothione is synthesized from spermidine and glutathione via the intermediates N1- and N8-glutathionyl-spermidine. Trypanothione is thought to have two important roles in trypanosomatid metabolism: the maintenance of intracellular thiols in the correct redox state and in the removal of hydrogen peroxide and other hydroperoxides. Thus, it is proposed that depletion of this metabolite may be an important contributory factor to the selective toxic effect of DFMO, particularly in its synergistic effect with other trypanocidal drugs.     

Purification and characterisation of a trypsin-like serine oligopeptidase from Trypanosoma congolense.

Trypanosoma brucei contain a serine oligopeptidase (OP-Tb) that is released into (and remains active in) the blood of trypanosome-infected animals. Here a similar enzyme from Trypanosoma congolense is described. This oligopeptidase, called OP-Tc, was purified using three-phase partitioning, and ion-exchange and affinity chromatography. OP-Tc is inhibited by alkylating agents, by serine peptidase-specific inhibitors including 3,4-dichloroisocoumarin, 4-(2-aminoethyl)benzenesulfonylfluoride and diispropylfluoro-phosphate and by other peptidase inhibitors including leupeptin, antipain and peptidyl chloromethyl ketones. Reducing agents such as dithiothreitol enhanced activity as did heparin, spermine and spermidine. The enzyme has trypsin-like specificity since it cleaved fluorogenic peptides that have basic amino acid residues (Arg or Lys) in the P1 position. Potential substrates without a basic residue in P1 were not hydrolysed. Although OP-Tc has weak arginine aminopeptidase activity, the enzyme clearly preferred substrates that had amino acids in the P2 and P3 positions. Overall, OP-Tc appears to be less efficient than OP-Tb because it usually displayed lower k(cat)/Km values for the substrates tested. However, like OP-Tb, the best substrate for OP-Tc was Cbz-Arg-Arg-AMC (Km = 0.72 microM, k(cat) = 96 s(-1)). OP-Tc preference for amino acids in the P2 position was (Gly,Lys,Arg) > Phe > Leu > Pro. The results also suggest that the P3-binding site has hydrophobic characteristics. OP-Tc may not be a naturally immunodominant molecule because neither IgG nor IgM anti- OP-Tc antibodies were detected in the blood of experimentally infected cattle.     

Putrescine N-acetyltransferase in Onchocerca volvulus and Ascaris suum, an enzyme which is involved in polyamine degradation and release of N-acetylputrescine

A novel type of N-acetyltransferase, clearly different from the nuclear and cytosolic polyamine N-acetyltransferases of mammals, was recently found in the intestinal nematode Ascaris suum. The occurrence of this putrescine N-acetylating enzyme has also been noted in the filarial parasite Onchocerca volvulus. The enzyme was partially purified from adults of O. volvulus and A. suum by chromatography on DEAE-cellulose and cadaverine-Sepharose. Substrate specificities of the filarial enzyme resemble those of the N-acetyltransferase from A. suum, with respect to its preference for putrescine and other diamines above polyamines and histones. Additionally, both nematode enzymes acetylated histamine, whereas dopamine and serotonin were not accepted as substrates. The activities of the N-acetyltransferase from O. volvulus and A. suum were potently inhibited by the drug berenil; the type of inhibition was competitive with respect to putrescine. The inhibition constants for berenil were determined as 4.2 and 1.2 microM for the enzymes of O. volvulus and A. suum, the Km values for putrescine were found to be 330 microM and 250 microM, respectively. Putrescine N-acetyltransferase is discussed as a regulatory step in the degradation of excessive polyamines via polyamine oxidase to putrescine. At this branching point, putrescine is retained in the cell for de novo synthesis of spermidine and spermine, catabolized via diamine oxidase or acetylated to a suitable transport product for excretion.