Much research has been carried out over the years examining cell wall glucans from Saccharomyces cerevisiae and this Study further examines aspects of the binding of (1-->4)-alpha-D-glucan in the yeast cell wall, using a number of isolation techniques as well as monoclonal antibodies able to recognize a mixed (1-->4)-alpha-D-glucan/(1-->6)-beta-D-glucan. Extraction of purified glucan, from S. cerevisiae cell wall, with 0.1N HCl, at 80 degrees C for 6 h., released into the solution (1-->4)-alpha-D-glucan and (1-->6)-beta-D-glucan as the major polysaccharides, along with an insoluble pellet highly enriched in (1-->3)-beta-D-glucan. The released (1-->4)-alpha-D-glucan was composed of a high molecular size >100 kDa fraction (7.2% w/w) and a medium 5-50 kDa polysaccharide (10.2% w/w), with the (1-->4)-alpha-D-glucan covalently bound to the (1-->6)-beta-D-glucan. The average molar ratio of the alpha:beta glucan was 47: 53 in this mixed polysaccharide. The structure of this polysaccharide was different from the structure of plant starch or animal glycogen as monoclonal antibodies specific to yeast (1-->4)-alpha-D-glucan/(1-->6)-beta-D-glucan did not recognize the plant starch or animal glycogen standards.

The intestinal epithelial cell and specifically the cytoskeleton of the brush border are thought to be controlled by micromolar levels of free calcium. Calcium-binding proteins of this system include intestinal calcium binding protein (CaBP), calmodulin (CaM), villin, and a 36,000-mol-wt protein substrate of tyrosine kinases. To assess the sequence of events as the intracellular Ca++ level rises, we determined the amount of CaM and CaBP in the intestinal epithelium by western blotting and tested the Ca++ binding of CaM and CaBP by equilibrium dialysis. The Ca++-dependent actin severing activity of villin was analyzed in the presence of physiological CaM levels and increasing calcium concentrations. In addition, we analyzed the Ca++ levels required for interaction between CaM and the microvillus 110,000-mol-wt protein as well as fodrin and the interaction between a polypeptide of 36,000 mol wt (P-36) and actin. The results suggest that CaBP serves as the predominant Ca++ buffer in the cell, but CaM can effectively buffer ionic calcium in the microvillus and thus protect actin from the severing activity of villin. CaM binds to its cytoskeletal receptors, 110,000-mol-wt protein and fodrin differently, governed by the free Ca++ and pH. The interaction between P-36 and actin, however, appears to require an unphysiologically high calcium concentration (10(-4) to 10(-3) M) to be meaningful. The results provide a coherent picture of the different Ca++ regulated events occurring when the free calcium rises into the micromolar level in this unique system. This study would suggest that as the Ca++ rises in the intestinal epithelial cell an ordered sequence of Ca++ saturation of intracellular receptors occurs with the order from the lowest to highest Ca++ requirements being CaBP less than CaM less than villin less than P-36.

Spectrin from erythrocytes and two other tissues (brain and intestine) were isolated from two distant species, pig and chicken; some structural and functional properties were compared. A quantitative antibody inhibition assay was used to determine that antibodies to mammalian red cell spectrin cross-react very poorly, if at all, with their non-erythroid (brain) counterpart and similarly antibodies to pig brain spectrin (fodrin) cross-react very weakly with erythroid spectrin. By contrast, antibodies which were directed against the 240000-Mr subunit of avian fodrin were completely inhibited with avian spectrin and vice versa. To analyze the structural relatedness of these molecules further we compared the chymotryptic iodinated peptide maps generated from each individual subunit. Consistent with the antibody results, we find little (less than 10%) homology between peptides derived from mammalian fodrin and spectrin, but complete homology (100%) of the peptides derived from the 240000-Mr subunits of chicken fodrin, spectrin and another related molecule from intestine, TW260/240. Whereas the peptide maps of fodrin (brain spectrin) revealed striking similarity between divergent species, suggesting a high degree of structural conservation, the peptide maps of erythrocyte spectrin was highly variable between species, indicating that it has diverged considerably in mammalian evolution. In addition we have compared a functional activity of mammalian spectrins, the ability to bind calmodulin, using two different assays. Both results show that, whereas fodrin-calmodulin interaction can be readily demonstrated, the binding to mammalian erythroid spectrin is negligible. This suggests that the high-affinity calmodulin site present on fodrin has been lost from spectrin in mammalian evolution.

A protein of 110,000 MW connects actin filaments to the plasma membrane in microvilli of intestinal epithelial cells. In the present study four independent lines of evidence suggest that the 110K protein is directly bound to the lipid bilayer. The solubilization of the 110K protein requires detergents and removal of detergent after solubilization results in aggregation. The 110K protein partitions into the detergent phase in Triton X-114 solutions. It is selectively incorporated into liposomes. It is specifically labeled with the hydrophobic probe 14C-phenylisothiocyanate. In addition we present a purification scheme for the 110K protein in milligram amounts. This represents the simplest system of membrane to filament attachment, in which an integral membrane protein is also a cytoskeletal protein.

Friend erythroleukemic cells can be used as a model of erythroid cell differentiation with the synthesis of the erythrocyte-specific products hemoglobin and spectrin stimulated by agents such as DMSO. In the present study we investigated the expression of both erythroid spectrin and non-erythroid fodrin in uninduced and DMSO-treated Friend cells. We report that both spectrin and fodrin co-exist at low levels in uninduced Friend cells and both are induced by treatment with DMSO. After longer times both spectrin and fodrin appear to undergo rearrangements into submembranous ‘patches’ and ‘caps’. Although both molecules co-localize in most of these cells, they can be independently immunoprecipitated, suggesting that significant amounts of hybrid molecules are not formed.

Previous studies have shown that molecules related to erythrocyte spectrin are present in the cortical cytoplasm of nonerythroid cells. We report here the localization by immunoelectron microscopy of one such molecule, TW-260/240, in the brush border of intestinal epithelial cells. Using highly specific antibodies against TW-260 and TW-240 as well as antibodies against fodrin, another spectrinlike molecule, we have found that the TW-260/240 molecules are displayed between rootlets at all levels of the terminal web. Occasionally, extended structures appear labeled suggestive of the fine filaments known to cross-link actin bundles. These results are in line with previous in vitro studies showing that TW-260/240 binds to, and cross-links, actin filaments. The results are discussed in terms of a model in which rootlets are immobilized in the terminal web in a matrix of TW-260/240.

Fodrin is an axonally transported F-actin crosslinking rod-shaped multidomain protein having a length of 200 nm. It is thought to be related to erythroid spectrin (220,000 and 240,000 Mr) as it is built as a tetramer derived from a heterodimer comprised of two large subunits (235,000 and 240,000 Mr). A panel of 24 monoclonal antibodies has been used to probe the fodrin structure by direct microscopical decoration and identification of large tryptic fragments retaining antigenicity. The combined results unambiguously define the fodrin organization and show it to be strongly related to erythrocyte spectrin. They demonstrate the heterodimer organization, the head-to-head association of dimers, the proteaseresistant domain structure of each subunit and indicate that different termini of the polypeptides are localized at the free F-actin binding ends and the heterodimer association sites. They also show that within the same mammalian species even the highly related 240,000 Mr subunits of fodrin and spectrin are immunologically distinct, most likely reflecting different gene products.

Recently, molecules highly related to erythrocyte spectrin have been identified in nonerythroid cells. Here we summarize our current understanding of these molecules and suggest a model for their organization. Significant differences exist between this family of proteins isolated from mammalian cells and avian cells, and this may explain the variability in antibody preparations as well as differences in peptide maps of these subunits which have been reported. We have prepared antibodies specific for the variant subunits of the spectrinlike proteins fodrin, spectrin, and TW260/240 and analyzed the distribution of these variant subunits in different chicken cell types as well as their developmental distribution in the intestine. The results suggest that fodrin is the general member of this family of proteins and can even coexist with other spectrinlike proteins in the same cells.

The axonally transported high molecular weight protein fodrin, known to be present in the cortical cytoplasm of neurones and other cells, has been purified to homogeneity and several of its biochemical properties have been characterized. Fodrin is an F-actin-binding and cross-linking protein inducing actin gels. It is composed of two nonidentical polypeptide chains (Mr = 240,000 and 235,000) which form a tetrameric complex of a molecular weight close to 930,000. The similarity of fodrin with tetrameric erythrocyte spectrin is directly shown by rotary shadowed molecules both alone and in interaction with F-actin. The gelation and cross-linking activity of fodrin is influenced both by ionic strength and pH in a manner similar to other cross-linking factors. These results strengthen previous concepts concerning the existence of spectrin-related molecules in nonerythroid cells and point to a possible related function in the submembranous microfilament organization in nonmuscle cells.

A high molecular weight protein from the brush border of chicken intestinal epithelial cells has been purified. This protein (TW 260/240), a complex of two polypeptides with apparent molecular weights of 260,000 and 240,000, accounts for a significant amount of the terminal web organization. TW 260/240 is an F-actin-binding protein that also interacts with calmodulin. Rotary shadowing reveals long flexible rods of double-stranded morphology tightly connected at each end. TW 260/240 is quite distinct from smooth muscle filamin and macrophage actin-binding protein (APB), but, in spite of its higher contour length (265 nm), seems to be related to erythrocyte spectrin (194 nm for the tetramer). Immunofluorescence microscopy with antibodies against TW 260/240 indicates the existence of a submembranous organization distinctly different from that of stress fibers. We have compared TW 260/240 with fodrin, a brain protein known to occur in submembranous organization but not previously characterized in molecular terms. TW 260/240 and fodrin are clearly distinct molecules but are similar in many aspects. Ultrastructural, biochemical and immunological results indicate three distinct classes of rod-like high molecular weight actin-binding proteins, possibly reflected by the prototypes filamin (ABP), spectrin and TW 260/240 (fodrin). The latter group may be responsible for calmodulin control of submembranous microfilament structures in various nonmuscle cells.

Spectrin, fodrin, and TW-260/240 form a group of structurally and functionally similar but not identical high molecular weight actin-binding proteins from chicken erythrocytes, brain tissue, or intestinal epithelial brush borders. Immunological data and one-dimensional peptide maps of the separated subunits suggest that a common (Mr 240,000) and a variant (Mr 220,000, 235,000, or 260,000) subunit account for the three different heterodimers. These results are in line with the related but distinct morphology of the three proteins observed in micrographs of rotary-shadowed molecules and the finding that the common (Mr 240,000) subunit seems to account for the calcium-dependent calmodulin-binding activity displayed by the three proteins. The possible functions of spectrin-like molecules in nonerythroid cells are discussed.