Hydrophobic and ionic effects upon the electrophoretic mobilities of the subunits of coupling factor 1 from mitochondria

A sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system was used to investigate certain properties of the subunits of the beef heart mitochondrial ATPase, (native F1, nF1). By examining the affects of urea concentration and acrylamide concentration upon the electrophoretic mobilities of the polypeptides comprising the nF1 enzyme, we have obtained conditions under which all five subunits are simultaneously resolved when the discontinuous buffer system of Laemmli is used (U. K. Laemmli (1970) Nature (London) 277, 680-685). The determination of the apparent molecular weights by analysis of Ferguson plots (K. A. Ferguson (1964) Metabolism 13, 985-1002) revealed that the addition of urea to the SDS gels resulted in a decrease in the apparent molecular weight of the beta subunit. A dramatic increase in the apparent molecular weight of the delta subunit was also brought about by the presence of urea in the SDS gels. In addition, the apparent molecular weight of both the alpha and the beta subunits was dependent upon the acrylamide concentration used, indicating that these subunits contain either areas highly resistant to denaturation by the combined action of urea and SDS, or covalent modifications leading to anomalous electrophoretic mobility. The results of experiments in which urea analogs were used indicate that the interactions of urea with the beta subunit involve the formation of hydrogen bonds between urea and regions of this subunit. On the other hand, the interactions of urea with the delta subunit are primarily of a hydrophobic nature, suggesting that these interactions could involve domains of the delta subunit required for binding of the coupling factor to the mitochondrial membrane.     

The effect of cross-links on the mobility of proteins in dodecyl sulphate–polyacrylamide gels

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate-polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate-polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.     

Tissue-specific forms of actin in the developing chick

Actin has been identified in nonmuscle and muscle tissues as a highly conserved homogeneous protein. We have identified and characterized actin from embryonic and adult chick brain and muscle, and have compared these actins by SDS and urea/SDS gradient polyacrylamide gel electrophoresis. In the presence of SDS alone, embryonic or adult brain and muscle actin co-migrate as homogeneous polypeptides. Electrophoresis of both actins in the presence of urea and SDS, however, reveals that brain and muscle actins migrate with distinctly different mobilities. Actin from embryonic thigh muscle at different stages of development migrates as two separate components. In early muscle development, only the “brain” type actin is present. As muscle development progresses the “muscle” type actin becomes relatively more abundant, so that by day 20 of embryonic development, “muscle” actin becomes predominant. These results may be interpreted as due to differences in the primary structure of actin.     

Evidence for the presence of tropomyosin in the cytoskeletons of ADP- and thrombin-stimulated blood platelets

Stimulation of porcine platelets with ADP or thrombin and subsequent analyses of their cytoskeletons by SDS-polyacrylamide gel electrophoresis have shown the presence of a 30.5-kDa polypeptide in the cytoskeletons of activated as well as aggregated platelets. This polypeptide comigrates with pure porcine platelet tropomyosin in SDS gels, their mobilities being similarly and markedly decreased in the presence of 6 M urea. One-dimensional peptide mapping after limited proteolysis by Staphylococcus aureus protease gives the same pattern for pure tropomyosin and the 30.5-kDa polypeptide. This latter may thus be identified as the porcine platelet tropomyosin subunit, the role of which may not be solely structural.     

Specific colorimetric detection of o-diphenols and 3,4-dihydroxyphenylalanine-containing peptides

Interpretation of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis results for polypeptides which contain both collagenous and noncollagenous regions may be somewhat ambiguous since collagenous chains obey a different molecular weight vs mobility relationship than reduced globular proteins. In a recent study [Freytag, J. W., Noelken, M. E., and Hudson, B. G., 1979, Biochemistry18, 4761–4768], however, it was found that the α chains of calf skin collagen obeyed the same size-mobility relationship as reduced globular proteins when the number of residues was used as a measure of size. We extended that study over a broad size range and found the same result for 581 to 2104 residue polypeptides when 5% gels were used, and for 217 to 1052 residue polypeptides with 9% gels. On the other hand, SDS complexes of collagenous chains having fewer than 300 residues migrated considerably more slowly through 12.5% gels than their counter-parts from globular proteins. Also, SDS complexes of α_s1-, β-, and γ₂-casein which have 8.5, 16.7, and 20 mol% proline, respectively, had mobilities between those of SDS complexes of collagenous polypeptides and their reduced globular protein counterparts with the same number of residues. Our results indicate that SDS-polyacrylamide electrophoresis can be used to determine accurately the number of residues of collagenous polypeptides in the 217 to 2104 residue size range if appropriate gel concentrations are used. However, this conclusion does not apply to high-proline polypeptides in general.     

Polypeptides of the oxygen-evolving photosystem II complex. Immunological detection and biogenesis

Oxygen-evolving photosystem II complex was isolated from spinach chloroplasts. The individual polypeptides of the complex were isolated from sodium dodecyl sulfate (SDS)-polyacrylamide gels and antibodies were raised in rabbits against these polypeptides. After washing of the isolation complex by 0.8 M Tris to release the extrinsic proteins, a distinct diffused protein band was revealed at the position of 33 kDa in SDS gels containing 4 M urea. When this band was electroeluted from the gel and subsequently electrophoresed on SDS gels, three distinct protein bands became apparent. Antibodies raised against each one of these polypeptides cross-reacted with the other two polypeptides to varying degrees but not with the other subunits of the complex. The three polypeptides were denoted as "34," "33," and "32" kDa and the 33 being the herbicide-binding protein. Using the antibodies, the relative amounts of the photosystem II polypeptides were followed during greening of etiolated spinach seedlings. While all three extrinsic polypeptides were present in etiolated leaves at relatively high amounts, the other polypeptides could not be detected prior to an approximate 6-h illumination period. Further illumination induced the appearance of all of the rest of the subunits in a relatively similar rate. The oxygen evolution activity was developed parallel to the increase in the amounts of these polypeptides. Therefore, the assembly of the active photosystem II during greening is a two-step process in contrast with the photosystem I reaction center, which is assembled step by step, and the rest of the chloroplast protein complexes, which are assembled by a concerted mechanism.     

Urea-induced binding of histone 1 to nucleosomes lacking linker DNA.

The binding of H1 (and H5) to nucleosome core particles was demonstrated by separating mononucleosomes according to their DNA size on acrylamide gels containing high molarity urea. The presence of urea causes a redistribution of H1 so that it associates with some particles of all linker lengths, including no linker. When the urea is removed the H1 remains associated with particles of all DNA sizes if the different size classes are not mixed with each other. Therefore, urea can effect the transfer of H1 from particles with linker to particles with no linker. When nucleosomes of uniform DNA fragment length, some containing and some lacking H1, are re-electrophoresed under native conditions, they migrate as two widely separated bands. The mobilities of these variants do not depend on linker length and are identical to the mobilities of native H1-containing and H1-lacking particles. When the same collection of particles is electrophoresed in the presence of high molarity urea they migrate with a uniform mobility. These results suggest that H1-containing nucleosomes are conformationally different from H1-lacking particles, but that this difference is eliminated when histone-histone interactions are disrupted by urea.     

Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification

Acrylamide concentration, urea content, and the trailing ion used for sodium dodecyl sulfate (SDS)-gels modify electrophoretic protein mobilities in a protein-dependent way. Varying these parameters we coupled two SDS-gels to a two-dimensional (2-D) electrophoresis system. Protein spots in 2-D gels are dispersed around a diagonal. Hydrophobic proteins are well separated from water-soluble proteins which is the essential advantage of the novel technique. Mass spectrometric identification of previously unaccessible hydrophobic proteins is now possible.     

Striated flagellar roots: isolation and partial characterization of a calcium-modulated contractile organelle

We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1% SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 Mr. The 20,000 Mr protein band represents approximately 63% of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 Mr flagellar root protein into two components of nearly identical Mr, but of differing isoelectric points (i.e., pl's of 4.9 and 4.8), which we have designated 20,000-Mr-alpha and 20,000-Mr-beta, respectively. Densitometric scans of two-dimensional gels of cell extracts indicate that the 20,000-Mr-alpha and -beta polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000-Mr-beta component is phosphorylated and the 20,000-Mr-alpha component contains no detectable label. These results suggest that the 20,000-Mr-alpha component is converted to the more acidic 20,000-Mr-beta form by phosphorylation. Both the 20,000-Mr-alpha and -beta flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-Mr protein binds to both the 20,000-Mr-alpha and 20,000-Mr-beta forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-IgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 Mr calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.     

Human factor VIII: purification from commercial factor VIII concentrate, characterization, identification and radiolabeling

Human factor VIII was purified from commercial factor VIII concentrate with a 12% yield. The specific coagulant activity of purified factor VIII was 8,000 units/mg. In the presence of SDS the purified factor VIII consisted of a variety of polypeptides on polyacrylamide gels, ranging between Mr 80,000 and Mr 208,000. In the absence of SDS the purified factor VIII showed an apparent molecular weight of 270,000 upon Sephadex G200 gel-filtration. The purified factor VIII could be activated by thrombin, which resulted in the disappearance of Mr 108,000-208,000 polypeptides in favor of an Mr 92,000 polypeptide. Treatment with factor Xa also activated factor VIII, whereas treatment with activated protein C resulted in the inactivation of coagulant activity. Coagulant-active 125I-factor VIII was prepared using a lactoperoxidase radioiodination procedure. This 125I-factor had the same characteristics as unlabeled factor VIII. All polypeptides could be precipitated with monoclonal antibodies directed against factor VIII. With 125I-factor VIII a pIapp of 5.7 was found in the presence of urea.     

Rhythmic chloroplast migration in the green alga Ulva: dissection of movement mechanism by differential inhibitor effects

Rhodopseudomonas viridis thylakoid membrane polypeptides were characterised by SDS gels, 2 D gels and surface-specific iodination. Four polypeptides with apparent molecular weights of 38 000, 33 000, 27 000, and 24 000 (reaction centre) and three low molecular weight polypeptides 11 000, 8000 and 6000 (probably light harvesting polypeptides) were identified. Antibodies were produced against the polypeptides eluted from SDS gels and tested for specificity by an immunoblotting assay. The antibodies were bound to the membranes and viewed by electron microscopy using a modification of the ferritin labelling technique. It is suggested that antigenic determinants for the 38 000, 33 000, and 27 000 reaction centre polypeptides and the 11 000 and 8000 low molecular weight polypeptides are present on the cytoplasmic membrane surface. The 33 000, 27 000, 11 000 and 6000 polypeptides appear to have surface-located residues which can be iodinated. The photosynthetic membrane of Rps. viridis appears to be a highly asymmetrical membrane.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH2: cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands. 2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c1, band 5 and Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c1 and identified with the so-called oxidation factor, and band 7 a polypeptide peptide associated with cytochrome b. 3. The validity of molecular weight estimate for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively. 4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide band.     

Gel electrophoresis in studies of protein conformation and folding

Electrophoresis through polyacrylamide gels is a useful method for distinguishing conformational states of proteins and analyzing the thermodynamic and kinetic properties of transitions between conformations. Although the relationship between protein conformation and electrophoretic mobility is quite complex, relative mobilities provide qualitative estimates of compactness. Conformational states which interconvert slowly on the time scale of the electrophoretic separation can often be resolved, and the rates of interconversion can be estimated. If the transitions are more rapid, then the electrophoretic mobility represents the equilibrium distribution of conformations. Protein unfolding transitions induced by urea are readily studied using slab gels containing a gradient of urea concentration perpendicular to the direction of electrophoresis. Protein applied across the top of such a gel migrates in the presence of continuously varying urea concentrations, and a profile of the unfolding transition is generated directly. Transitions induced by other agents could be studied using analogous gradient gels. Electrophoretic methods are especially suited for studying small quantities of protein, and complex mixtures, since the different components can be separated during the electrophoresis.     

Anomalies in the electrophoretic resolution of Na+/K(+)-ATPase catalytic subunit isoforms reveal unusual protein--detergent interactions

Three different isozymes of the Na+/K(+)-ATPase have slightly different different electrophoretic mobilities in sodium dodecyl sulfate (SDS). Certain procedures (reduction and alkylation, heating, and the use of sodium tetradecyl sulfate) have been reported either to improve the electrophoretic separation of isoforms or to reveal the presence of new isoforms. The variables affecting gel electrophoretic mobility were investigated here. Reduction and alkylation decreased the mobility of all three isozymes, and slightly improved the separation of alpha 1 from alpha 2 and alpha 3 without causing a qualitative change in the alpha isoforms detected. Heating the enzyme in SDS caused splitting into two bands. Both bands were intact polypeptides but migrated differently in 5% and 15% polyacrylamide, disclosing an anomalous conformation in detergent. The use of sodium tetradecyl or decyl sulfate instead of dodecyl sulfate altered the relative mobilities of the isozymes, revealing differences in detergent affinity, but no new isoforms were found. In conclusion, Na+/K(+)-ATPase alpha-subunit mobility reflects complex detergent-protein interaction that can be affected by experimental conditions. The existence of more than one band on gels may reflect different conformations in detergent, but should not be accepted alone as evidence for subunit structural heterogeneity.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH₂:cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands.

2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c₁, band 5 the Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c₁ and identified with the so-called oxidation factor, and band 7 a polypeptide associated with cytochrome b.

3. The validity of molecular weight estimates for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively.

4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide bands.     

The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons

This study of the slow component of axonal transport was aimed at two problems: the specific identification of polypeptides transported into the axon from the cell body, and the identification of structural polypeptides of the axoplasm. The axonal transport paradigm was used to obtain radioactively labeled axonal polypeptides in the rat ventral motor neuron and the cat spinal ganglion sensory neuron. Comparison of the slow component polypeptides from these two sources using sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis revealed that they are identical. In both cases five polypeptides account for more than 75% of the total radioactivity present in the slow component. Two of these polypeptides have been tentatively identified as tubulin, the microtubule protein, on the basis of their molecular weights. The three remaining polypeptides with molecular weights of 212,000, 160,000, and 68,000 daltons are constitutive, and as such appear to be associated with a single structure which has been tentatively identified as the 10-nm neurofilament. The 212,000-dalton polypeptide was found to comigrate in SDS gels with the heavy chain of chick muscle myosin. The demonstration on SDS gels that the slow component is composed of a small number of polypeptides which have identical molecular weights in neurons from different mammalian species suggests that these polypeptides comprise fundamental structures of vertebrate neurons.     

Electrophoretic mobility is a reporter of hairpin structure in single-stranded DNA oligomers

The electrophoretic mobilities of 24 single-stranded DNA oligomers, each containing 26 nucleotide residues, have been measured in polyacrylamide gels and in free solution. The mobilities observed at 20 degrees C differed by approximately 20% in polyacrylamide gels and by approximately 10% in free solution, even though the oligomers contained the same number of bases. Increasing the temperature or adding urea to the solution equalized the mobilities of the oligomers, suggesting that the variable mobilities observed at 20 degrees C are due to the formation of stable secondary structures, most likely hairpins. Thermal melting profiles were measured for eight oligomers in 40 mM Tris acetate buffer. The observed melting temperatures of most oligomers correlated roughly with the mobilities observed at 20 degrees C; however, one oligomer was much more stable than the others. The melting temperatures of four of the oligomers were close to the values predicted by DINAMelt [Markham, N. R., and Zuker, M. (2005) Nucleic Acids Res. 33, W577-W581]; melting temperatures of the other oligomers differed significantly from the predicted values. Thermal melting profiles were also measured for two oligomers as a function of the Tris acetate buffer concentration. The salt concentration dependence of the melting temperatures suggests that 0.15 Tris+ ion per phosphate is released upon denaturation. Because the apparent number of Tris+ ions released is greater than that observed by others for the release of Na+ ions from similar hairpins, the results suggest that DNA hairpins (and, presumably, duplexes) bind more Tris+ ions than Na+ ions in solution.     

Two-dimensional electrophoresis of soybean root plasma membrane proteins solubilized by SDS and other detergents

Proteins solubilized from enriched soybean root plasma membrane with sodium dodecyl sulphate (SDS) and selected non-denaturing detergents (octyl-β-d-glucopyranoside, Zwittergent 312, Zwittergent 314, Zonyl FSK, and Nonidet P-40) were electrophoresed in two-dimensions by standard procedures. The basic electrophoretogram ‘fingerprint’ was similar for all detergents tested. However, differences in the total number of polypeptides resolved and the presence or absence of certain polypeptides on specific two-dimensional gels indicated some selectivity. Of all detergents tested, SDS solubilized the most polypeptides (ca 95) and provided the best resolution. The other detergents solubilized 50–80 polypeptides with varying resolution. Of those tested, octyl-β-d-glucopyranoside consistently provided the best balance between the number of polypeptides resolved (ca 70) and the level of resolution. The results suggest that selected detergents may prove useful in plant plasma membrane studies which require non-denaturing conditions.     

The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii

The sites of synthesis of the major thylakoid membrane polypeptides have been studied in the green alga Chlamydomonas reinhardtii by pulse labeling of cells with [14C]acetate in the presence of inhibitors specific for chloroplast and cytoplasmic protein synthesis. The labeled membrane polypeptides were separated by an improved method of sodium dodecyl sulfate (SDS) gradient gel electrophoresis, and autoradiographs were made of the dried gels. The results demonstrate that of the 33 polypeptides resolved in the gels, at least nine are made on chloroplast ribosomes. Two of these (polypeptides 2 and 6) are associated with the reaction centers of photosystems I and II. Another polypeptide (polypeptide 5) appears from genetic data to be coded by chloroplast DNA. Experiments with a mutant whose chloroplast ribosomes are resistant to spectinomycyn (spr-u-1-6-2) show that polypeptides whose synthesis takes place on chloroplast ribosomes are made in the presence of spectinomycin in the mutant although their synthesis is blocked by this antibiotic in wild type cells.     

Properties and topography of the major integral plasma membrane protein of a unicellular organism

The cellular distribution, membrane orientation, and biochemical properties of the two major NaOH-insoluble (integral) plasma membrane proteins of Euglena are detailed. We present evidence which suggests that these two polypeptides (Mr 68 and 39 kD) are dimer and monomer of the same protein: (a) Antibodies directed against either the 68- or the 39-kD polypeptide bind to both 68- and 39-kD bands in Western blots. (b) Trypsin digests of the 68- and 39-kD polypeptides yield similar peptide fragments. (c) The 68- and 39-kD polypeptides interconvert during successive electrophoresis runs in the presence of SDS and beta- mercaptoethanol. (d) The 39-kD band is the only major integral membrane protein evident after isoelectric focusing in acrylamide gels. The apparent shift from 68 to 39 kD in focusing gels has been duplicated in denaturing SDS gels by adding ampholyte solutions directly to the protein samples. The membrane orientation of the 39-kD protein and its 68-kD dimer has been assessed by radioiodination in situ using intact cells or purified plasma membranes. Putative monomers and dimers are labeled only when the cytoplasmic side of the membrane is exposed. These results together with trypsin digestion data suggest that the 39- kD protein and its dimer have an asymmetric membrane orientation with a substantial cytoplasmic domain but with no detectable extracellular region. Immunolabeling of sectioned cells indicates that the plasma membrane is the only cellular membrane with significant amounts of 39- kD protein. No major 68- or 39-kD polypeptide bands are evident in SDS acrylamide gels or immunoblots of electrophoresed whole flagella or preparations enriched in flagellar membrane vesicles, nor is there a detectable shift in any flagellar polypeptide in the presence of ampholyte solutions. These findings are considered with respect to the well-known internal crystalline organization of the euglenoid plasma membrane and to the potential for these proteins to serve as anchors for membrane skeletal proteins.