The second cysteine protease inhibitor, EhICP2, has a different localization in trophozoites of Entamoeba histolytica than EhICP1.

The genome of Entamoeba histolytica contains two genes encoding inhibitors of cysteine proteases of the chagasin family. In contrast to that of EhICP1, the derived primary structure of the second inhibitor, EhICP2, possesses a typical N-terminal signal sequence. Processed EhICP2 is as weakly related to amoebiasin-1 (27% identity) as to chagasin (identity 30%), indicating a different evolutionary origin of both amebic genes.

Characterization of a proteasome alpha-chain from Giardia lamblia.

To begin to characterize the components of the 20S proteasome of Giardia lamblia, we have cloned a genomic sequence encoding an alpha-chain (type alpha3/C9, predicted size 244 amino acid residues). Southern analysis indicated that a single gene codes for this protein, and a Northern blot exhibited a single signal at 850 nt. An antiserum against a C-terminal fragment of the alpha-chain expressed in Escherichia coli reacted with a single protein band of Mr 27,000 that was present at constant levels in trophozoites and encysting cells.

neo-inositol polyphosphates in the amoeba Entamoeba histolytica.

We have reexamined the structure of inositol phosphates present in trophozoites of the parasitic amoeba Entamoeba histolytica and show here that, rather than being myo-inositol derivatives (Martin, J.-B., Bakker-Grunwald, T.

Functional identification of alpha 1-giardin as an annexin of Giardia lamblia.

A protein with a relative molecular mass of 31 kDa was specifically extracted by EGTA from a detergent-insoluble fraction of Giardia lamblia. N-terminal sequencing showed this protein to be identical to alpha 1-giardin, a component of the ventral disc which, based on its predicted amino acid sequence, has been classified as annexin XIX. Purified alpha 1-giardin associated with multilamellar phosphatidyl serine-containing vesicles in a Ca(2+)-dependent manner, confirming that it is a functional annexin.

Characterization of ubiquitin genes and -transcripts and demonstration of a ubiquitin-conjugating system in Entamoeba histolytica.

We have investigated the genomic organization of Entamoeba histolytica ubiquitin and looked for the occurrence of a ubiquitin-conjugating system in this organism. Southern blots indicated the presence of > or = 5 ubiquitin-coding regions. One of these, EhUBI1, was cloned and sequenced and found to correspond to a monoubiquitin gene; as shown by a polymerase chain reaction, E.

Evidence for the existence of both proteasomes and a novel high molecular weight peptidase in Entamoeba histolytica.

To screen for high molecular weight proteases in Entamoeba histolytica, we subjected a soluble amebal extract to density gradient centrifugation and tested the fractions for activity against the chymotryptic peptide substrate, Suc-leucyl-leucyl-valyl-tyrosyl-4-methylcoumaryl-7-amide. Two peaks of activity, of approximately 11 and 20 S, were clearly separated. Based on SDS-electrophoretic pattern and immunoblot analysis, we ascribe the 20 S activity to proteasomes.

Characterization of glycogen and amino acid pool of Entamoeba histolytica by 13C-NMR spectroscopy.

Trophozoite extracts of axenic Entamoeba histolytica were investigated by natural-abundance 13C-NMR spectroscopy. The extracts were found to contain a high level of glycogen (30 mM glucose equivalents), which had a compact structure as suggested by alpha (1-->6) branch points every 5-6 glucose residues. As other major metabolites, we identified putrescine (9.

Evidence for the existence of a single ubiquitin gene in Giardia lamblia.

All eukaryotes investigated so far contain multiple copies of ubiquitin genes, most of which are arranged in fusions coding for either polyubiquitin or ubiquitin-ribosomal protein constructs; the former are normally under the control of a heat shock promoter. Giardia lamblia, an intestinal parasite, is the most primitive eukaryote known to date. We have investigated the arrangement and expression of ubiquitin genes in this organism by Southern and Northern blotting.

31P-NMR analysis of Entamoeba histolytica. Occurrence of high amounts of two inositol phosphates.

Perchloric-acid extracts of axenic Entamoeba histolytica were investigated by 31P-NMR spectroscopy. All major 31P resonances observed were assigned to specific compounds. The cells contained inorganic phosphate (1039 nmol/g wet cells), pyrophosphate (16 nmol/g wet cells), nucleoside diphosphates (91 nmol/g wet cells), nucleoside triphosphates (275 nmol/g wet cells), NAD(P) (60 nmol/g wet cells), phosphocholine (184 nmol/g wet cells), phosphoethanolamine (214 nmol/g wet cells), cytidine 5'-diphosphocholine (41 nmol/g wet cells) and cytidine 5'-diphosphoethanolamine (55 nmol/g wet cells).

Entamoeba histolytica as a model for the primitive eukaryotic cell.

Entamoeba histolytica is a structurally simple eukaryote lacking mitochondria, peroxisomes and a well-developed Golgi apparatus, also in its biochemistry, it deviates substantially from the more complex eukoryotes. These features have alternatively been interpreted as archaic, ie. the ancestor of Entamoeba branched off before the primitive eukaryotic cell obtained proto-mitochondria, or as regressive, ie.

Ion transport in parasitic protozoa.

Many parasitic protozoa go through complex life cycles in the course of which they adapt to widely different environments; ion transport processes are expected to play a role both in pathogenicity and in adaptation. So far, studies on ion transport have been virtually limited to Leishmania, Plasmodium and Entamoeba. The distribution of ion pumps in the former two organisms generally appears to conform to the picture established for other protozoa, i.

Ubiquitin of Entamoeba histolytica deviates in six amino acid residues from the consensus of all other known ubiquitins.

The amino acid sequence of ubiquitin from Entamoeba histolytica, as deduced from a cDNA nucleotide sequence, deviated at six positions from the consensus of all other known ubiquitins (ranging from Trypanosoma cruzi to Homo sapiens). The corresponding residues were scattered over the primary sequence, but came close together on the surface of the folded protein structure. We conclude that (i) E.

Sulfate metabolism in Entamoeba histolytica.

Sulfate fluxes and sulfate metabolites in Entamoeba histolytica were characterized employing [35S]sulfate as a marker. Sulfate was taken up both across the plasma membrane and by pinocytosis; in growth medium (sulfate concentration, 1.1 mM) total uptake was 1.

Subcellular distribution of amebapain, the major cysteine proteinase of Entamoeba histolytica.

Amebapain, the major cysteine proteinase of E. histolytica, appears to be important both for digestion and as a cytotoxic factor. By immunocytology we established that amebapain was bound to matrix-like structures both on the cell surface and within subcellular vesicles; the amebapain co-localized with pinocytized iron oxide, suggesting that the enzyme recycles between plasma membrane and pinocytic vesicles.

Evidence for a vacuolar-type proton ATPase in Entamoeba histolytica.

Entamoeba histolytica is a primitive eukaryote that lacks mitochondria. Golgi and a well-developed endoplasmic reticulum. Close to half of the cell volume is occupied by pinocytic vesicles, which are in continuous turnover with the plasma membrane and perform functions that in higher eukaryotic cells are taken over by lysosomes.

Magnetic separation of pinocytic vesicles of defined age from Entamoeba histolytica.

We describe a rapid and simple method to isolate pinocytic vesicles of defined age (residing time within the cell) from Entamoeba histolytica. Amoebas are allowed to pinocytize for greater than 5 min a suspension of superparamagnetic iron oxide particles, washed, and resuspended for predetermined periods (up to 150 min) in iron oxide-free medium. Subsequently, the cells are homogenized and iron oxide-containing vesicles are separated magnetically.

Pore-forming protein from Entamoeba histolytica forms voltage- and pH-controlled multi-state channels with properties similar to those of the barrel-stave aggregates.

Pore-forming protein from Entamoeba histolytica forms cation-selective channels in planar bilayers. With increasing potentials, the open-state probability of these channels decreases, and channel aggregates collapse (Young, J.D.

Pathogenic and non-pathogenic Entamoeba: pore formation and hemolytic activity.

Pore-forming activity in planar lipid bilayers and liposomes of extracts from differentially pathogenic Entamoeba and the capacity of trophozoites and subcellular fractions to lyse human red blood cells (hrbc) were investigated. In all amebas studied, the two activities paralleled each other. They were high in E.

Effects of amiloride on Na+ content and pinocytosis in Entamoeba histolytica.

Amiloride, a blocker of Na+ leak and Na+-H+ exchange in animal cells, caused cells of Entamoeba histolytica to release Na+ (up to 40% of their original Na+ content within 90 min, at an amiloride concentration of 3 mM); K+ content was not affected. By comparing the unidirectional uptake of 22Na+ with that of the fluid-phase marker 125I-labeled poly(vinylpyrrolidone) we established that the amiloride-induced Na+ loss was due to inhibition of pinocytic Na+ uptake rather than to blockage of an amiloride-sensitive transport system in the plasma membrane. Amiloride penetrated the cells, and both its intracellular concentration and its effect on pinocytosis increased with pH.

Effects of ATP and cyclic AMP on the (Na+ + K+ + 2Cl-)-cotransport system in turkey erythrocytes.

As turkey erythrocytes were progressively depleted of ATP by preincubation with dinitrophenol, the (Na+ + K+ + 2Cl-)-cotransport system (assayed by the bumetanide-sensitive fraction of 86Rb+ influx) became less responsive to activation. The dependence upon intracellular ATP concentration was significantly steeper for transport activated by hypertonic shock (halfmaximal activity at 0.7 mM ATP) than for that activated by either epinephrine or cyclic AMP (halfmaximal activity at 1.

Effects of cytochalasin B on Na+ content and cell volume of Entamoeba histolytica.

Cells of Entamoeba histolytica accumulated K+ and extruded Na+ compared to the concentrations of those ions present in the growth medium. Pinocytic activity, measured by the uptake of horseradish peroxidase of 125I-polyvinylpyrrolidone, was high (up to 0.3 ml/ml cells per h).