Detection, purification and partial characterization of beta-amylase from trophozoites of Entamoeba invadens.

Trophozoites of Entamoeba invadens were able to ingest glucopolysaccharides and metabolize them. An activity capable of degrading these substrates was purified to apparent homogeneity. The enzyme was identified as beta-amylase (alpha-1,4-D-glucan maltohydrolase EC 3.2.1.2): It was active against glycogen, amylose and amylopectin in a ratio of 100:198:133 and was also able to attack linear alpha-1,4-glucooligosaccharides with more than three glucose moieties. All degradation experiments yielded maltose as reaction product, and no free glucose could be detected. While amylose was completely degraded, amylolysis of glycogen and amylopectin yielded limit dextrins besides maltose. The enzyme was completely inactive against cyclohexaamylose, cycloheptaamylose and pullulan, indicating its lack of endo-glucosidase specificity. Hydrolysis of 4-nitrophenyl-maltoheptaoside resulted in the successive removal of maltose units from the non-reducing end yielding 4-nitrophenyl-maltopentaoside, -trioside and -glucoside. No 4-nitrophenyl-glycosides with even numbered glucose moieties were formed from this substrate. The enzyme exhibited a relative molecular mass of M(r) = 45,000 +/- 5% by gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis and the N-terminal sequence 1 VEVNVMLPL 9. Optimal hydrolysis was observed at pH 5.5 and a temperature of 42 degrees C. On the basis of inhibition by mercury ions and p-chloro-mercurybenzoate and abrogation of this effect by thiol reagents beta-amylase was identified as sulfhydryl-enzyme.

Purification and substrate specificity of two cysteine proteinases of Giardia lamblia.

The proteinase activity present in homogenates of trophozoites of Giardia lamblia, active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of Mr = 95,000 and 35,000 +/- 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (Mr = 95,000) and proteinase II (Mr = 35,000) were active against the beta-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1-6, 8-18, and 20-30 of the insulin beta-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.

Detection of glycogen-debranching system in trophozoites of Entamoeba histolytica.

Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4-alpha-glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl-alpha-glucoside yielding successively 4-nitrophenyl-alpha-maltoside and 4-nitrophenyl-alpha-maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of Mr = 180,000 +/- 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.

Breakdown of glucopolysaccharides in Entamoeba histolytica by phosphorylase.

Homogenates of trophozoites of Entamoeba histolytica released glucose 1-phosphate from amylopectin, glycogen, and amylose in a ratio of 100:78:74 at glucopolysaccharide concentrations of 0.1%. By use of self-generating Percoll gradients this activity was shown to be particulate and associated with glycogen. The phosphorylase was extracted from the 40,000 g pellet in aqueous medium and purified to homogeneity by gel filtration on Fractogel TSK HW-55(F) followed by chromatography on Blue Sepharose CL-6B. The purified enzyme was active not only against the glucopolysaccharides but also on dextrins with more than 3 glucose moieties, which were primarily formed by the action of amoebic amylases. At substrate concentrations of 1 mM nonreducing ends of each glucan, the phosphorolysis rate of the branched polysaccharides was about 1.75 x 10(4) times higher than those of the maltodextrins. By means of HPLC the sequential degradation of 4-nitrophenyl-maltoheptaoside (G(7)-pNP) was studied. Native phosphorylase exhibited a relative molecular mass of M(r) = 200,000 by gel filtration and gel electrophoresis. The SDS electrophoresis, under reducing conditions, indicated that the native enzyme was a dimer. Optimal degradation of the polysaccharides and dextrins was achieved at pH values of 7.5 and 7.0 respectively.

Specificity of a cysteine proteinase of Entamoeba histolytica against various unblocked synthetic peptides.

The activity of highly purified cysteine proteinase of Entamoeba histolytica against different peptides of the sequence X-Gly-Phe-Phe was compared. The synthetic peptide Arg-Gly-Phe-Phe of the insulin B-chain was readily hydrolyzed yielding Arg-Gly and Phe-Phe as split products. Lys-Gly-Phe-Phe and Tyr-Gly-Phe-Phe were cleaved at rates of 20 and 4%, respectively. Val-Gly-Phe-Phe, Gly-Gly-Phe-Phe, Glu-Gly-Phe-Phe, and Ser-Gly-Phe-Phe were hydrolyzed at rates far below 1%. Gly-Arg-Phe-Phe, Gly-Phe-Phe, and Gly-Phe were completely resistant to the enzyme. Another good substrate was found in Arg-Gly-Leu-Hyp, which represents a model compound of a scissile site in collagen type I. Furthermore, peptide Arg-Arg-Phe-Phe was attacked by the enzyme releasing Arg-Arg and Phe-Phe. Compared with Arg-Gly-Phe-Phe at substrate concentrations of 2 mM the rates of hydrolysis of Arg-Arg-Phe-Phe and Arg-Gly-Leu-Hyp were 37 and 127%. The enzyme exhibited dipeptidyl peptidase activity against the nonapeptide Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala releasing Arg-Gly.

Secretory hydrolases of Entamoeba histolytica.

Cells of Entamoeba histolytica grown over a period of four days contained NADP+-dependent alcohol dehydrogenase exclusively inside the cells. No activity of this enzyme could be found in the growth medium after harvesting the cells. Under the same conditions, acid phosphatase, beta-N-acetylglucosaminidase, esterase, alpha-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium. The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively. The comparison of the elution diagrams revealed that NADP+-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells. These activities, as well as beta-N-acetylglucosaminidase and alpha-glucosidase, were released into the extracellular environment to a different degree. The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum. Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate.

Specificity of a cysteine proteinase of Entamoeba histolytica towards the alpha 1-CB2 peptide of bovine collagen type I.

The cysteine proteinase of Entamoeba histolytica was shown to hydrolyze the cyanogen bromide peptide alpha 1-CB2 of calf skin collagen type I yielding two split products. Amino acid analyses of the formed peptides and estimation of the amino-terminal residues revealed that the alpha 1-CB2 peptide was exclusively cleaved between Gly10 and Leu11 within the sequence -Arg9-Gly10-Leu11-yielding two peptides with 7 and 29 amino acids, respectively. Under identical conditions the ratio of hydrolysis of the Gly10-Leu11 bond in the alpha 1-CB2 peptide to the Gly23-Phe24 bond within the internal sequence -Arg22-Gly23-Phe24- of bovine insulin B-chain was 100:65. The enzyme was found to split both benzyloxycarbonyl-Arg-Arg-methoxy-2-naphthylamide and benzyloxycarbonyl-Arg-Gly-2-naphthylamide. The ratio of hydrolysis of these substances was 100:11.6. Benzyloxycarbonyl-Gly-Arg-2-naphthylamide was a very poor substrate for the enzyme.

Studies on the substrate specificity of alpha- and beta-amylase of Entamoeba histolytica.

From the supernatant fraction of cell homogenates of Entamoeba histolytica alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) and beta-amylase (1,4-alpha-D-glucan maltohydrolase, EC 3.2.1.2) were separated and purified by gel filtration and isoelectric focusing, followed by DEAE- and CM-chromatography, respectively. Both enzymes catalyzed the degradation of amylose, amylopectin and glycogen. Hydrolysis of polysaccharides by alpha-amylase yielded as reaction products maltose, maltotriose, maltopentaose and maltohexaose, but no free glucose. beta-Amylase produced as main degradation product of glucopolysaccharides maltose and to a minor extend maltotriose, but no glucose. alpha- and beta-amylase were able to hydrolyze 4-nitrophenyl-alpha-D-glucooligosaccharides (Gn-pNP) containing more than two glucose units per molecule. Under identical conditions G6-pNP was cleaved at highest velocity by alpha-amylase, while beta-amylase exhibited the highest activity with G4-pNP as substrate. alpha-Amylase hydrolyzed G4-pNP, G5-pNP and G6-pNP yielding as main reaction product G2-pNP, but also the formation of G1-pNP and G3-pNP from G4-pNP, of G1-pNP, G3-pNP and G4-pNP from G5-pNP, of G1-pNP, G3-pNP, G4-pNP and G5-pNP from G6-pNP was observed. alpha-Amylase as endo-glucohydrolase attacked all glycosidic bonds in G6-pNP, G5-pNP and G4-pNP, while beta-amylase successively removed maltose units from the non-reducing ends of the glycosides.

Cysteine proteinase of Entamoeba histolytica. I. Partial purification and action on different enzymes.

A method for a 50-60-fold purification of a cysteine proteinase from trophozoites of Entamoeba histolytica using 35-80% ammonium sulphate fractionation, gel chromatography on Sephadex G-75, and preparative isoelectric focusing is described. The enzyme was examined for its proteolytic potencies towards native enzyme substrates. The amebic proteinase directly inactivates aldolase and glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle as well as glucose-6-phosphate dehydrogenase from yeast. The inactivation of citrate synthase from porcine heart proceeds rather slowly, whereas malate dehydrogenase from porcine heart is not affected by the amebic proteinase under the condition used. With the exception of aldolase all inactivated enzyme substrates have been cleaved by limited proteolyses yielding major cleavage products. The inactivation of aldolase probably functions by the release of a small segment from a terminus being essential for aldolase activity.

Cysteine proteinase of Entamoeba histolytica. II. Identification of the major split position in bovine insulin B-chain.

The substrate specificity of a cysteine proteinase of Entamoeba histolytica was tested using the oxidized B-chain of bovine insulin. 28 proteolytic peptides were formed after different intervals of incubation and could be separated by high-performance liquid chromatography. Two dominant peptides appearing after 10 min of incubation were isolated and analyzed for amino acid composition. The results obtained indicate that these two peptides were formed by cleavage of the peptide bond between the amino acids 23 and 24. Thus as major split position the Gly-Phe bond in the insulin B-chain could be identified.

A weakly acidic protease has a powerful proteolytic activity in Entamoeba histolytica.

A thiol dependent protease from homogenates of the parasite Entamoeba histolytica has been identified and partially purified by means of ammonium sulphate fractionation, gel filtration and isoelectric focusing. The protease, having a molecular mass of 21 +/- 2 kDa as judged by gel chromatography, represents a highly potent proteolytic capacity. The protease shows maximal activity against azocasein around the slightly acidic pH of 4.4 at 37 degrees C, but is also active at pH 3.4 and 8.5. At optimal pH, the turnover increases with increasing temperature up to 85 degrees C. The enzyme possesses a thiol group essential for activity, which is inhibited by the thiol blocking reagent p-chloromercuribenzoate. Solutions containing low concentrations of free Hg2+ cause conservation of protease activity. The native protein exhibits an isoelectric point of 4.9. This protease resembles the thiol endopeptidases of mammalian lysosomes, and appears to be a major proteolytic enzyme in Entamoeba histolytica.

The occurrence of beta-amylase in Entamoeba histolytica.

A beta-amylase like activity isolated from a cell homogenate of Entamoeba histolytica, and separated from other glucohydrolytic activities by gel filtration and isoelectric focusing, hydrolyzed amylose, amylopectin and glycogen, yielding maltose as reaction product. From the non-reducing ends of the 4-nitrophenyl-alpha-D-glycosides of maltotriose, maltotetraose, maltopentaose and maltohexaose the enzyme removed maltose units. The pH optimum for hydrolyzing the poly- and oligosaccharides was pH 6.5, and the temperature optimum was 29 degrees C. Estimation of relative molecular mass by gel filtration on Sephadex G 75 gave values around Mr = 32 000. The isoelectric point was found to be 5.7.

Degradation of biogenic oligosaccharides by beta-N-acetyl-glucosaminidase secreted by Entamoeba histolytica.

beta-N-Acetylglucosaminidase secreted by Entamoeba histolytica was extracted from the growth medium by affinity chromatography on CH-Sepharose 4 B coupled to p-aminophenyl-1-thio-beta-2-acetamido-2-deoxyglucopyranoside. The enzyme was further purified by isoelectric focusing, by sequential chromatography on DEAE-cellulose and Sephadex G-150, and by preparative disc gel electrophoresis. Chitobiose (betaGlcNAc1-4GlcNAc) derived from chitin as well as the oligosaccharides betaGlcNAc1-4 betaGlcUA1-3GlcNAc, betaGlcNAc1-4 betaGlcUA1-3 betaGlcAc1-4GlcUA, and betaGlcNAc1-4 betaGlc-UA1-3 betaGlcNAc1-4 betaGlcUA1-3 betaGlcNAc1-4GlcUA derived from hyaluronic acid were tested as potential physiological substrates. All these oligosaccharides are susceptible to action of beta-N-acetylglucosaminidase from E. histolytica. Under identical conditions chitobiose is cleaved 38-48 times faster than hyhyauronate oligosaccharides. No release of N-acetylglucosamine was observed when glycopeptides from ovalbumin were used as substrate. The pH optimum of hydrolase activity was 4.5 when chitobiose was used as substrate. Optimal hydrolysis of aluronate oligosaccharides was observed at pH 3.0 for trisaccharide and pH 2.0 for tetra- and hexasaccharide, respectively. Estimation of molecular weight by means of gel filtration gave values of 75 000. The isoelectric point was 5.02 beta-N-Acetylglucosaminidase from E. histolytica does not act on macromolecular chitin and hyaluronic acid.

Evidence for different glycohydrolase and glycosyltransferase activities of beta-N-acetylglucosaminidases A and B.

1) Two forms of beta-N-acetylglucosaminidase--known as form A and form B - were purified from bovine spleen homogenates and efficaciously separated by preparative disc electrophoresis on polyacrylamide gel. Studies on the enzymatic specificity revealed that the two forms have different glycoside hydrolase and glycosyl transferase activities towards substrates of natural origin. 2) With the trisaccharide GlcNAc-GlcUA-GlcNAc from hyaluronate as substrate, form A released free N-acetylglucosamine at a rate 35-40 times higher than form B. The B form, however, transferred N-acetyl-[6-3H]glucosamine from phenyl-beta-N-acetyl-D[6-3H]glucosaminide to the tetrasaccharides GlcUA-GalNAc-4-sulfate-GlcUA--GalNAc-4-sulfate or GlcUA-GlcNAc-GlcUA-GlcNAc isolated from chondroitin 4-sulfate or hyaluronate at rates 5-10 times higher than beta-N-acetyl-glucosaminidase A, the corresponding 3H-pentasaccharides being isolated as reaction products. 3) The pH optimum of the glycoside hydrolase activity is 4.5, while optimum glycosyl transfer proceeds at pH 6.5. Under condition optimum for glycoside transferase, hydrolytic activity is still observed with each form, but the B form exhibits about equal glycoside hydrolase and glycoside transferase activity, whereas the A form has a predominant glycoside hydrolase action.