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We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase-sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.

Using x-ray microanalysis technique the distribution of potassium, phosphorus and sulphur was analysed in Acanthamoeba castellanii cells. Distribution of potassium was nonuniform; the high level of the element was observed in the cortex region of these cells. Distribution of phosphorus was shown to be similar to that of potassium, whereas sulphur was rather uniformly distributed.

Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. The phosphate content is less than 0.2 mol/mol. There is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. Unique two-dimensional maps of tryptic and chymotryptic peptides and complete absence of antibody cross-reactivity show that Acanthamoeba actophorin, profilin, capping protein, and actin are separate gene products with minimal homology. Actophorin has features of both an actin monomer-binding protein and an actin filament-severing protein. Actophorin reduces the extent of actin polymerization at steady state in a concentration-dependent fashion and forms a complex with pyrene-labeled actin that has spectral properties of unpolymerized actin. During ultracentrifugation a complex of actophorin and actin sediments more rapidly than either actin monomers or actophorin. Although actophorin inhibits elongation at both ends of actin filaments, it accelerates the late stage of spontaneous polymerization like mechanical shearing and theoretical predictions of polymer fragmentation. Low concentrations of actophorin decrease the length and the low shear viscosity of actin filaments. High concentrations cause preformed filaments to shorten rapidly. Ca2+ is not required for any of these effects. Muscle and amoeba actin are equally sensitive to actophorin.

Polypeptides of whole-cell extracts of Naegleria fowleri flagellates and growing amebae were resolved by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms of the [35S]methionine-labeled polypeptides of amebae and flagellates were analyzed by two dimensional densitometry to determine whether there were correlations between intracellular concentration of a protein and subunit size or charge. The majority of the polypeptides of amebae and flagellates had molecular sizes in the range of 20 to 60 kilodaltons. The radioactivity per polypeptide species in the size range of 20 to 60 kilodaltons was greater in amebae than in flagellates. The greatest number of polypeptides detected in amebae and flagellates was in the isoelectric focusing range of pH 6 to 7. The radioactivity per polypeptide species in the isoelectric focusing gradient below 6.3 was greater in amebae than in flagellates. Polypeptides in the size range of 20 to 60 kilodaltons had a median isoelectric point below pI 6.3, whereas those larger than 60 kilodaltons had a median pI value above 6.3. These data indicated that molecular size and charge were not entirely independent variables and that the size and charge of a polypeptide might have an important influence in determining its intracellular concentration in both amebae and flagellates. Autoradiograms were also compared so that changes in intracellular protein complement and concentrations occurring during differentiation could be recognized. The relative amounts of a limited number of polypeptides increased markedly, and others decreased markedly, during enflagellation.

Crystalline sheets of Acanthamoeba actin induced by the trivalent lanthanide gadolinium exist in three different polymorphic forms, which show different striation patterns and surface topographies. We have called these different forms "rectangular" and "square" sheets, and "cylinders" and have shown that each of the three forms is constructed from common "basic" lattices associated in different ways. We have used image processing of electron micrographs to obtain a model for the actin molecule in projection to a resolution of 1.5 nm. The overall dimensions observed in these images are 5.6 x 3.3 x 4.5 nm, and the molecule itself appears distinctly bilobed with the two lobes separated by a cleft. actin monomers in the sheets are arranged with P2 symmetry and are therefore packed in a manner different from that of the molecules in actin filaments. Because approximately 35% of the surface area of the actin molecule is exposed on the surface of these sheets, the sheets should be useful to study the stoichiometric binding of actin-binding proteins to the actin molecule.

Previous studies have established that 'informational molecules' present in cytoplasmic fractions of A. discoides may be transferred by microinjection into A. proteus. Clones derived from injected cells showed various changers, including lowered sensitivity to growth in streptomycin and neomycin, in which respects they resembled A. discoides. These changes in response to antibiotics were transferred independently and were permanent, the information being replicated over many generations. The most 'active' material in terms of the number of clones showing character changes was found following injection of 16S ribonucleoprotein obtained after sucrose density gradient centrifugation of the mcirosomal fraction. Polyacrylamide gel electrophoresis of the 16S material showed 3 small peaks of RNA. In order to obtain adequate amounts of material, these peaks of RNA were identified in electrophoresis profiles of RNA extracted from the whole microsomal fraction, and RNA eluted from these latter gels was injected into A. proteus. Although the number of surviving clones was low, all were examined for their response to growth in either streptomycin, neomycin, erythromycin or chloroquine. After injection of RNA eluted from the 3 small peaks of RNA (slices 26-33), 8 out of 10 and 9 out of 10 clones showed lowered sensitivity to growth in streptomycin and neomycin respectively, and resembled the donor A. discoides. No changes in responses to antibiotics were obtained from clones derived from cells injected with RNA eluted from another region of the gel, or after ribonuclease treatment of the RNA from slices 26-33. The relative molecular weights of these 'informational' RNA molecules were found to be between 9 and 13 X 10(4) Daltons.

The cytology of the testaceous ameba, Lesquereusia spiralis (EHRENBERG) PENARD, has been described using a broad spectrum of cytochemical methods. Most cytological characteristics lie within the range of variability reported for free living non-shelled amebas. The shell of L. spiralis is complex, being composed of siliceous platelets positioned in an organic matrix. This interplatelet matrix is composed of sulfomucin complexed with basic protein. The matrix contains a complex of pore-like structures. The structure and possible origins and function of this matrix complex are discussed.

Acanthamoebe profilin has a native molecular weight of 11,700 as measured by sedimentation equilibrium ultracentrifugation and an extinction coefficient at 280 nm of 1.4 X 10(4) M-1cm-1. Rabbit antibodies against Acanthamoeba profilin react only with the 11,700 Mr polypeptide among all other ameba polypeptides separated by electrophoresis. These antibodies react with a 11,700 Mr polypeptide in Physarum but not with any proteins of Dictyostelium or Naeglaria. Antibody-binding assays indicate that approximately 2% of the ameba protein is profilin and that the concentration of profilin is approximately 100 mumol/liter cells. During ion exchange chromatography of soluble extracts of Acanthamoeba on DEAE-cellulose, the immunoreactive profilin splits into two fractions: an unbound fraction previously identified by Reichstein and Korn (1979, J. Biol. Chem., 254:6174-6179) and a tightly bound fraction. Purified profilin from the two fractions is identical by all criteria tested. The tightly bound fraction is likely to be attached indirectly to the DEAE, perhaps by association with actin. By fluorescent antibody staining, profilin is distributed uniformly throughout the cytoplasmic matrix of Acanthamoeba. In 50 mM KCl, high concentrations of Acanthamoeba profilin inhibit the elongation rate of muscle actin filaments measured directly by electron microscopy, but the effect is minimal in KCl with 2 MgCl2. By using the fluorescence change of pyrene-labeled Acanthamoeba actin to assay for polymerization, we confirmed our earlier observation (Tseng, P. C.-H., and T. D. Pollard, 1982, J. Cell Biol. 94:213-218) that Acanthamoeba profilin inhibits nucleation much more strongly than elongation under physiological conditions.

The repeat unit coding for the precursor to 18S, 5.8S, and 26S ribosomal ribonucleic acids (rRNAs) has been cloned from the free-living soil amoeba Acanthamoeba castellanii. The cloned deoxyribonucleic acid (DNA) was mapped with 11 restriction endonucleases and by R-loop mapping. The entire repeat unit is 12 kbp (kilobase pairs) in length and contains sites for EcoRI, SmaI, BglII, SstI, Bam-HI, PstI, KpnI, HindIII, and XbaI but not for XhoI or SalI. All of the repeat units in the nuclear DNA appear to be identical, and no introns were detected. However, the regions which code for the two RNAs which comprise the 26S RNA are separated by a gap of approximately 200 base pairs. Unlike some other lower eukaryotes, the 5S RNA gene is not linked to this repeat unit. A fragment of the repeat unit which contains the initiation sequence of the putative precursor has been subcloned into pBR322 for use in vitro transcription studies.

The DNA content of five species of Acanthamoeba was determined by flow microfluorometry. Acanthamoeba castellanii (AC-30), acanthamoeba polyphaga (APG and P-23), acanthamoeba rhysodes, acanthamoeba culbertsoni (A-1), and acanthamoeba royreba were grown in a casitone based medium 24-48 HR. The trophozoites were harvested, and evaluated for DNA-bound fluorescence. All species tested has DNA values between 2.0-5.0 pg/cell. These results placed DNA/cell values of Acanthamoeba slightly lower than DNA/cell values of other eucaryotic cells and much lower than Amoeba proteus values. These results indicate that FMF may be a useful adjunct in distinguishing Acanthamoeba cells from either eucaryotic cells or some other amoeba. However, differences in DNA/cell between species of Acanthamoeba are small and would not be useful in identification of species.

The Naegleria ameba cytopathogenic material (NACM) obtained from free-living and/or pathogenic amebae has been partially isolated and characterized. The evidence indicates that the material is a protein or has a protein component, and is in the estimated molecular weight range of 50,000 daltons. The partially isolated NACM, like its counterpart in the amebic lysates, is capable of sustaining itself in tissue cultures at a temperature which otherwise would inactivate it, and in serial passages through multiple dilutions. No distinctions between the NACMs from amebae of different Naegleria species or strains have been found in the biological response to the various treatments studied.

A polar lipid accounting for 12.5% of the total lipid nitrogen has been isolated from the protozoan Acanthamoeba castellanii. On the basis of thin-layer chromatography and mass spectral analysis, the lipid has been identified as diacylglyceryltrimethylhomoserine (DGTS). Fast atom bombardment (FAB) mass spectra of DGTS are reported for the first time and are compared to the FAB mass spectra of phosphatidylcholines and the electron ionization (EI) and field desorption (FD) mass spectra of DGTS. Gas-liquid chromatographic-mass spectrometric (GLC-MS) analysis of the acyl chain composition of this lipid has shown that 87.5% consists of cis-9-octadecenoic acid. Plasma membrane isolated from this organism has shown that labeled DGTS appears in the plasma membrane but is not enriched in this fraction. DGTS has been isolated previously only from a limited number of green plants and one species of fungus. Identification of this lipid in Acanthamoeba indicates that this lipid is distributed among a diverse group of lower eucaryotes.

Disc electrophoresis was employed in an attempt to determine its usefulness as a means of differentiating and classifying some free-living species of amoebae. The Rp values and the percentage of similarity of the protein bands were used as taxonomic criteria. Some species of the genera Hartmanella and Naegleria were examined. The protein patterns of all species studied were highly reproducible. Four strains of Hartmannella castellanii showed an absolute identity to the original H. castellanii strain. One strain isolated by Neff and H. rhysodes were similar to all the other strains although their protein patten were not completely identical. On the other hand, H. culbertsoni, H. astronyxis and H. lammellipodia were different from all the other examined species of Hartmannella. Pathogenic N. fowleri-strains were identical to each other and were different in their protein patterns from non-pathogenic N. gruberi strain. The similarities and differences of protein bands of strains of ameobae and their correlations with other attributes were discussed. The results have shown that the disc electrophoresis appears useful in differentiating between species of Hartmannella and Naegleria.

A technique is described for isolating nuceoli from Acanthamoeba castellanii. Nuclei isolated by a modification of the technique of F. J. Chlapowski and R. N. Band (1971) are sonicated in a surcrose-Tris-MgSO4-KC1-Triton X-100 buffer and centrifuged on a linear sucrose gradient extending from 1.3 M to 1.5 M with a 2.6 M cushion, at 41000 rpm for 90 min. The only apparent contaminants in the nucleolar preparation are outer cyst walls. A procedure is described for the isolation of chemically pure outer cyst walls, and a comparison of the proteins with the nucleolar preparation reveals that outer cyst walls represent negligible contaminants. The ultrastructure of these isolated nucleoli examined with transmission electron microscopy is found to be identical with that of nucleoli from whole cells, fixed in an identical manner. The 50 nucleolar proteins separated by SDS gel electrophoresis have been examined throughout the growth cycle of Acanthamoeba and into the strat of induced encystment, at which time 10 protein bands disappear, 11 bands are observed to decrease, and 8 are seen to increase in concentration. Phenol-soluble proteins are extracted from the nucleolus which correspond to 29 of the 50 nucleolar proteins, with 17 of these proteins corresponding to nucleolar proteins that change at the onset of encystment. Thes nucleolar proteins are also compared with those of rat liver nucleoli by gel electrophoresis, resulting in the observation that extremely few protein homologies exist between the two. Numerous quantitative and qualitative changes in the gel pattern of phenol-soluble nuclear proteins during early and late log phase growth and the onset of stationary phase were also observed.

Amoebae of Dictyostelium discoideum were attached to a surface coated with polylysine, and the upper portion of the cells was sheared off with a stream of buffer. Scanning and transmission electron microscopy showed that the cytoplasmic surface of the exposed membrane was covered with fibers consisting of actin-containing filaments. The actin was identified by its solubility properties and its ability to interact with muscle myosin.

We describe a case of primary amebic meningoencephalitis due to Naegleria fowleri. This is the first reported case, to our knowledge, in Missouri. Emphasis is placed on the morphologic identification of degenerating amebas in a cytospin preparation of cerebrospinal fluid using Wright's stain during the rapidly progressive meningoencephalitic stage.

Fifteen aminoacyl-transfer ribonucleic acids (tRNA's) from vegetative cells (trophozoites) and mature cysts of Acanthamoeba castellanii were compared by reversed-phase 5 chromatography. Little or no differences were detected in reversed-phase 5 chromatography elution profiles of alanyl-, arginyl-, isoleucyl-, phenylalanyl-, prolyl-, seryl-, threonyl-, tryptophanyl- and valyl-tRNA's. Significant differences in the relative proportions of isoaccepting species of leucyl-, lysyl-, methionyl-, aspartyl-, histidyl-, and tyrosyl-tRNA's were observed. Based upon the criterion of cyanogen bromide reactivity with the modified nucleoside queuosine, the content of queuosine in aspartyl-tRNA of A, castellanii is significantly greater in mature cysts than in trophozoites. The similarity of change in reversed-phase 5 chromatography elution profiles of aspartyl-, histidyl-, and tyrosyl-tRNA suggests that a common mechanism is responsible for alterations in the chromatographic patterns.

A protein has been purified from Acanthamoeba which, like cytochalasin B, caps the end of actin filaments normally favoured for monomer addition and inhibits the interactions of actin filaments. In addition, this 'capping' protein nucleates the polymerization of actin monomers and blocks the annealing of actin filament fragments.

We report further characterization of the physical and immunologic properties, mechanism of action, and intracellular localization of Acanthamoeba castellanii capping protein, an actin regulatory protein discovered by Isenberg (Isenberg, G., U. Aebi, and T. D. Pollard, 1980, Nature (Lond.) 288:455-459). The native molecular weight calculated from measurements of Stokes' radius (3.8 nm by gel filtration chromatography) and sedimentation coefficient (4.8 S by sucrose gradient velocity sedimentation) was 74,000 daltons. The subunit molecular weights were 31,000 and 28,000 daltons, so the native molecule is a heterodimer. The two subunits did not immunologically cross-react with each other or with any other proteins from Acanthamoeba or several other organisms. In studies of the mechanism of action, Isenberg (see above reference) found that capping protein blocked polymerization from the barbed end of actin filaments and sedimented with actin filaments. We confirmed that capping protein binds to actin filaments with a gel filtration assay. Capping protein decreased the length distribution and high shear viscosity of actin filaments. Capping protein did not bundle or cross-link actin filaments. Low concentrations of capping protein increased the critical concentration for muscle and ameba actin polymerization from 0.1 to 0.6 microM in Mg++ and EGTA. Increasing amounts of capping protein did not increase the critical concentration further. In Ca++ capping protein did not change the critical concentration for muscle actin, but did increase the critical concentration for ameba actin. Ca++ had no effect on the ability of capping protein to decrease the low or high shear viscosity of actin filaments. By indirect fluorescent antibody staining, capping protein was localized to the cell cortex, an area rich in actin filaments. During subcellular fractionation of homogenates, about 1/3 of cellular capping protein banded with a crude membrane fraction. The other 2/3 of cellular capping protein was soluble, with a Stokes' radius equal to that of the purified protein. The molar ratio of capping protein to actin in the cell was 1:150.

The cellular DNAs of Acanthamoeba castellanii have been characterized by their behaviour in CsC1 density gradients, by their thermal denaturation and by their renaturation kinetics. Whole-cell DNA exhibits, on CsC1 density gradients, a major peak with a density of 1.717 g/cm3 (major component) and a minor peak with a density of 1.692 g/cm3 (minor component). The major component is nuclear and the minor component is of cytoplasmic origin. The latter contains mitochondrial DNA as well as an extramitochondrial DNA fraction. Reiterated sequences make up approximately 14% of the total and are mainly cytoplasmic. They are characterized by three families of nucleotide sequences. The mitochondrial DNA exhibits a complex renaturation pattern. The fast renaturing component has a calculated complexity of 4.107 daltons. The slower renaturing component has a kinetic complexity tentatively estimated as 1.1010 daltons. The melting profile of mitochondrial DNA suggests heterogeneity in base composition.

A microbial agent was isolated previously from a case of Viluy encephalomyelitis and named the 'KPN agent' after the initials of the patient. Here a detailed characterization of nucleic acids extracted from the purified KPN agent is presented. The agent contains both DNA and RNA, and has its own tRNAs and some other low-Mr RNAs, including 5S RNA. These findings, and the isolation of eukaryotic-type ribosomes, suggest that the KPN agent is not a virus, as believed before, but a more complex micro-organism, with protein-synthesizing capacity. The nucleotide sequence of the 5S RNA in the ribosomes of the KPN agent is identical with the sequence of 5S RNA of Acanthamoeba castellanii. The novel protozoan nature of the KPN agent is discussed in relation to other unusual properties of this micro-organism. Some implications of these results for the aetiology of Viluy encephalomyelitis are also discussed.

Actin from amebae of Naegleria gruberi has been purified to homogeneity. The purified actin shares many attributes with numerous other actins that have been characterized, including molecular weight, strong binding to DEAE-cellulose, binding to DNase I, reversible polymerization to F-actin, binding of rabbit myosin subfragment 1 to give distinctive arrowheads , formation of Mg paracrystals, and activation of myosin Mg2+-ATPase. In two respects the attributes of Naegleria actin are unusual. Isoelectric focusing resolves three distinct isoforms of the actin, which raises questions about the function of multiple isoforms in a unicellular eukaryote. The amino acid composition closely resembles other actins except that Naegleria actin lacks N tau-methylhistidine. This result indicates that N tau-methylhistidine is not a prerequisite for actin-actin or actin-myosin interactions.

Fully polymerization-competent fluorescein-labeled actin from skeletal muscle was microinjected into both normal moving and experimentally treated Amoeba proteus. Its intracellular distribution was followed by integral image intensification of the fluorescence on a television screen and compared with controls injected with rhodamine-labeled serum albumin. The labeled actin was incorporated into the endogenous actin pool and exhibited a characteristic redistribution depending on the cellular morphology. Increased amounts of labeled actin could be detected within a thin layer separating the hyalo- and granuloplasm or running immediately beneath the plasma membrane when hyaloplasmic regions were absent. The topography of the fluorescent layer demonstrated in living cells is in agreement with the cortical microfilament layer described ultrastructurally recently in corresponding cells. The combined results emphasize the important role of the cortical filament layer in both morphogenetic processes (e.g., hyalo-granuloplasm separation or changes in cell shape) and motive force generation for cytoplasmic streaming and amoeboid movement.

The amoeboflagellate transformation in Physarum polycephalum involves a series of dramatic changes in cell shape and motile behavior. This report describes the morphological and behavioral changes through which a synchronously transforming population of cells passes, stressing that, although there are a series of distinguishable stages, cells at all stages display striking plasticity. Our previous studies showed that amoeboflagellates transiently display a flattened motile extension--the ridge--that projects from a specific location on the cell surface and contains a laminar core densely packed with a series of crisscrossing arrays of actin microfilaments. Details are presented here concerning the movements of the ridge as well as the dynamics of ridge formation and disassembly in relation to other morphogenetic events of the transformation. The ridge forms at about the same time as transforming cells begin to elongate, propagates undulations parallel to the long axis of the cell as the transformation proceeds, and disassembles late in the transformation. Staining of fixed cells with the fluorescent probe rhodaminephalloidin shows that the actin of amoeboid cells is strikingly redistributed as the transformation proceeds. Amoeboflagellates contain most of the stainable actin in the ridge and in a ventral-posterior spot that may be a site of cell-substratum adhesion. These results provide additional insights into the possible functions of the ridge and the roles of actin during the amoeboflagellate transformation.

Actin was extracted from isolated nuclei of Acanthamoeba castellanii and purified to homogeneity under nondenaturing conditions by diethylaminoethylcellulose and Sephadex G-100 chromatography. The pure protein has the same molecular weight as cytoplasmic Acanthamoeba actin and a very similar amino acid composition. Isoelectrofocusing shows that nuclear actin is slightly more acidic than the major cytoplasmic species, and comparative analysis of peptides from tryptic and cyanogen bromide digests shows that both actins are very similar but not chemically identical. In an assay that is specific for most actins, the inhibition of DNase I through the formation of a 1:1 G-actin-DNase I complex, the nuclear and cytoplasmic actins are equally effective. By use of a similar procedure for the purification of both actins, it is estimated that the amount of nuclear actin is about 1.5% of the amount of cytoplasmic actin, a major protein of the amoeba. It is concluded that a minor isoelectric species of actin associates selectively with the nuclei of A. castellanii.

1. The degree of homology between ribosomal RNA isolated from microplasmodia and amoebae of the slime mould Physarum polycephalum has been determined by competive hybridisation of the RNA from the two sources to homologous DNA in solution. The extent of competition was measured both by hybridisation to saturation and by following the kinetics of hybrid formation. In each case competition was found to be 100%, indicating that the ribosomal RNAs from the two, quite different vegetative forms of the organism exhibit a high degree of homology and are probably transcribed from the same genes. 2. The relationship between the amount of nuclear DNA that codes for ribosomal RNA (rDNA) and ploidy has been investigated in three strains of P. polycephalum which exhibit a 1:2:5 variation in the amount of DNA per nucleus. Ribosomal RNA saturation values were determined by hybridisation to DNA isolated from prophase nuclei of plasmodia. The proportion of rDNA was found to be constant at 0.16--0.18% of the total genome in the three strains.

Lipophosphonoglycan, a major component of the plasma membrane of Acanthamoeba castellanii, has now been shown to contain 8% inositol and 13% C25- and C24-phytosphingosines in addition to the previously identified content of neutral sugars (26%), amino sugars (3%), aminophosphonates (10%), acidhydrolyzable phosphate (3%), and long chain fatty acids (14%). The fatty acids and phytosphingosines are in ceramide groups. Lipophosphonoglycan can be separated by dodecyl sulfate-polyacrylamide electrophoresis into two major components that are similar in composition except for different oligosaccharide groups. A tentative structural model incorporating these features is proposed in which each of the two components of lipophosphonoglycan is conceived as an oligomeric inositol-containing glycosphingolipid.

By amino acid sequence studies, only one form of cytoplasmic actin was detected in Acanthamoeba castellanii. Its amino acid sequence is very similar to the sequences of Dictyostelium and Physarum actins, from which Acanthamoeba actin differs in only nine and seven residues, respectively, including the deletion of the first residue. Acanthamoeba actin is unique in containing a blocked NH2-terminal neutral amino acid (glycine), while all other actins sequenced thus far have a blocked acidic amino acid (aspartic or glutamic) at the NH2 terminus. Acanthamoeba actin is also unique in that it contains an N epsilon-trimethyllysine residue at position 326. Like other actins, Acanthamoeba actin contains an NT-methylhistidine residue at position 73. The protein sequence is in complete agreement with the sequence derived from the nucleotide sequence of an expressed actin gene.

By transplanting nuclei between labeled and unlabeled cells, we determined the localization of the major proteins of amebas and described certain features of their intracellular distributon. We identified approximately 130 cellular proteins by fluorography of one-dimensional polyacrylamide electrophoretic gels and found that slightly less than half of them (designated NP, for nuclear proteins) are almost exclusively nuclear. About 95 percent of the other proteins (designated CP for cytoplamsic proteins) are roughly equally concentrated in nucleus and cytoplasm, but-because the cytoplasm is 50 times larger than the nucleus-about 98 percent of each of the latter is in the cytoplasm. Of the CP, roughly 5 percent are not detectable in the nucleus. Assuming that these are restricted to the cytoplasm only because, for example, they are in structures too large to enter the nucleus and labeled CP readily exit a nucleus introduced into unlabeled cytoplasm, we conclude that the nuclear envelope does not limit the movement of any nonstructural cellular protein in either direction between the two compartments. Some NP are not found in the cytoplasm (although ostensibly synthesized there) presumably because of preferential binding within the nucleus. Almost one half of the protein mass in nuclei in vivo is CP and apparently only proteins of that group are lost from nuclei when cells are lysed. Thus, while an extracellular environment allows CP to exit isolated nuclei, the nuclear binding affinities for NP are retained. Further examination of NP distribution shows that many NP species are, in fact, detectable in the cytoplasm (although at only about 1/300 the nuclear concentration), apparently because the nuclear affinity is relatively low. These proteins are electrophoretically distinguishable from the high-affinity NP not found in the cytoplasm. New experiments show that an earlier suggestion that the nuclear transplantation operation causes an artifactual release of NP to the cytoplasm is largely incorrect. Moreover, we show that cytoplasmic "contamination" of nuclear preparations is not a factor in classifying proteins by these nuclear transplantation experiments. We speculate the no mechanism has evolved to confine most CP to the cytoplasm (where they presumably function exclusively) because the cytoplasm's large volume ensures that CP will be abundant there. Extending Bonner's idea of "quasi-functional nuclear binding sites" for NP, we suggest that a subset of NP usually have a low affinity for available intranuclear sites because their main function(s) occurs at other intranuclear sites to which they bind tightly only when particular metabolic conditions demand. The other NP (those completely absent from cytoplasm) presumable always are bound with high affinity at their primary functional sites.

1. The G + C content of the DNAs of Amoeba proteus and Amoeba indica was determined by buoyant density gradient centrifugation. 2. Generally, different clones of the same strains did not differ in the G + C content of their main DNA or their satellite DNA. 3. The G + C content of the main DNA of the TD and XD strains of A. proteus were the same (25.3 and 29.3% respectively) as were the G + C contents of the satellite DNA (54.7 and 61.8% respectively). 4. The main and satellite DNAs are reversed for the F strain of A. proteus and the I strain of A. indica; the G + C contents of the main DNA being 57.4 and 59.8% respectively and those of the satellite DNA being 23.5 and 28.4% respectively. 5. The heterologous hybrids produced by nuclear transplantation between the A strain of A. proteus and the I strain of A. indica have the same G + C content in their main and satellite DNAs as do their cytoplasmic parents. 6. The data suggest that the occurrence of phenotypic variations does not necessarily mean an alteration in the G + C content of either main or satellite DNA. 7. There is also evidence that the G + C content of these DNAs in under cytoplasmic control.

For purposes of studying the degree of polymerization of actin in nuclei, nuclei from 35S-labeled amoebas (Amoeba proteus) were transplanted into unlabeled cells, which were immediately lysed and extracted under conditions considered to stabilize preexisting fibrous actin. The enucleated 35S-donor cells were similarly treated for analysis of cytoplasmic actin. The extraction conditions permitted separation of soluble (unpolymerized or G) actin from pelletable (polymerized or F) actin, and the radioactivity of each was determined after the actin was separated from other proteins by polyacrylamide gel electrophoresis. We found that about 2/3 of the actin within the nucleus is pelletable, whereas only about 1/3 of the cytoplasmic actin is pelletable. We speculate that polymerized actin in the nucleus is involved in the condensation of chromatin.

A hemolytic activity associated with postnuclear supernatant fractions of Naegleria fowleri has been partially characterized in an attempt to isolate cytolytic molecules that may participate in naeglerial cytopathogenicity. Hemolysis by naeglerial postnuclear supernatant fractions was sensitive to heat and trypsin hydrolysis, and was inhibited by divalent cations. The majority of the hemolytic activity was nonlatent and associated with a particle fraction sedimenting at 48,000 X g (maximum) for 1 h. This particle-associated hemolytic activity appears to be membrane associated, as high salt concentration, chelating agents, and pH extremes were ineffective in solubilizing the hemolytic activity, whereas treatment with 0.15% Zwittergent 3-12, a dipolar ionic detergent, results in 98% release of the sedimentable hemolysin. The sigmoidal nature of the progress curve of postnuclear supernatant hemolysis, as well as synergistic interactions between fractions of amoebal whole cell extracts, suggests that the hemolytic activity has a multicomponent nature, with at least two and possibly three components participating in the hemolytic event. The significance of these findings in the context of naeglerial cytopathogenicity is discussed.

I have purified a high molecular weight actin filament gelation protein (GP-260) from Acanthamoeba castellanii, and found by immunological cross-reactivity that it is related to vertebrate spectrins, but not to two other high molecular weight actin-binding proteins, filamin or the microtubule-associated protein, MAP-2. GP-260 was purified by chromatography on DEAE-cellulose, selective precipitation with actin and myosin-II, chromatography on hydroxylapatite in 0.6 M Kl, and selective precipitation at low ionic strength. The yield was 1-2 micrograms/g cells. GP-260 had the same electrophoretic mobility in SDS as the 260,000-mol-wt alpha-chain of spectrin from pig erythrocytes and brain. Electron micrographs of GP-260 shadowed on mica showed slender rod-shaped particles 80-110 nm long. GP-260 raised the low shear apparent viscosity of solutions of Acanthamoeba actin filaments and, at 100 micrograms/ml, formed a gel with a 8 microM actin. Purified antibodies to GP-260 reacted with both 260,000- and 240,000-mol-wt polypeptides in samples of whole ameba proteins separated by gel electrophoresis in SDS, but only the 260,000-mol-wt polypeptide was extracted from the cell with 0.34 M sucrose and purified in this study. These antibodies to GP-260 also reacted with purified spectrin from pig brain and erythrocytes, and antibodies to human erythrocyte spectrin bound to GP-260 and the 240,000-mol-wt polypeptide present in the whole ameba. The antibodies to GP-260 did not bind to chicken gizzard filamin or pig brain MAP-2, but they did react with high molecular weight polypeptides from man, a marsupial, a fish, a clam, a myxomycete, and two other amebas. Fluorescent antibody staining with purified antibodies to GP-260 showed that it is concentrated near the plasma membrane in the ameba.

Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741-1751, 1987). In this study, indirect immunofluorescence microscopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggregating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrane and that the actin-binding activity of ponticulin may be tightly controlled. Indirect immunofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified against D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.