Distribution and fluidizing action of soluble and aggregated amyloid beta-peptide in rat synaptic plasma membranes.

The effects of soluble and aggregated amyloid beta-peptide (Abeta) on cortical synaptic plasma membrane (SPM) structure were examined using small angle x-ray diffraction and fluorescence spectroscopy approaches. Electron density profiles generated from the x-ray diffraction data demonstrated that soluble and aggregated Abeta1-40 peptides associated with distinct regions of the SPM. The width of the SPM samples, including surface hydration, was 84 A at 10 degrees C. Following addition of soluble Abeta1-40, there was a broad increase in electron density in the SPM hydrocarbon core +/-0-15 A from the membrane center, and a reduction in hydrocarbon core width by 6 A. By contrast, aggregated Abeta1-40 contributed electron density to the phospholipid headgroup/hydrated surface of the SPM +/-24-37 A from the membrane center, concomitant with an increase in molecular volume in the hydrocarbon core. The SPM interactions observed for Abeta1-40 were reproduced in a brain lipid membrane system. In contrast to Abeta1-40, aggregated Abeta1-42 intercalated into the lipid bilayer hydrocarbon core +/-0-12 A from the membrane center. Fluorescence experiments showed that both soluble and aggregated Abeta1-40 significantly increased SPM bulk and protein annular fluidity. Physico-chemical interactions of Abeta with the neuronal membrane may contribute to mechanisms of neurotoxicity, independent of specific receptor binding.     

Proline transport activity in Escherichia coli membrane vesicles of different buoyant densities.

Cytoplasmic membrane vesicles prepared by lysis of Escherichia coli W 3110 spheroplasts in a French press at 0 degrees C are heterogeneous with respect to density due to membrane protein aggregation as a result of lateral phase separation of membrane phospholipids and to the presence of more or less outer membrane. These different vesicle classes can be separated on isopycnic density gradients. Assays for various membrane-associated functions show that the membranes differ not only with respect to density and structure but also with respect to function. The proline transport system (as detected by uptake experiments with the artificial electron donor ascorbate-phenazine methosulfate) shows maximal activities in membrane fractions that have considerably higher densities than the normal cytoplasmic membrane. This is always the case, whether vesicles are isolated from membranes that exhibit a temperature-induced protein aggregation or not. A correlation between high proline transport activity and the presence of vesicles with double membranes (consisting of outer and inner membrane) has been established. The possibility that the outer membrane protects the transport system in the cytoplasmic membrane during the isolation of vesicles is discussed.     

Effect of Ca2+ binding on the profile structure of the sarcoplasmic reticulum membrane using time-resolved x-ray diffraction.

A number of studies have indicated that Ca(2+)-ATPase, the integral membrane protein of the sarcoplasmic reticulum (SR) membrane, undergoes some structural change upon Ca2+ binding to its high affinity binding sites (i.e., upon conversion of the E1 to the CaxE1 form of the enzyme). We have used x-ray diffraction to study the changes in the electron density profile of the SR membrane upon high-affinity Ca2+ binding to the enzyme in the absence of enzyme phosphorylation. The photolabile Ca2+ chelator DM-nitrophen was used to rapidly release Ca2+ into the extravesicular spaces throughout an oriented SR membrane multilayer and thereby synchronously in the vicinity of the high affinity binding sites of each enzyme molecule in the multilayer. A critical control was developed to exclude possible artifacts arising from heating and non-Ca2+ photolysis products in the membrane multilayer specimens upon photolysis of the DM-nitrophen. Upon photolysis, changes in the membrane electron density profile arising from high-affinity Ca2+ binding to the enzyme are found to be localized to three different regions within the profile. These changes can be attributed to the added electron density of the Ca2+ bound at three discrete sites centered at 5, approximately 30, and approximately 67 A in the membrane profile, but they also require decreased electron density within the cylindrically averaged profile structure of the Ca(2+)-ATPase immediately adjacent (< 15 A) to these sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the sarcoplasmic reticulum membrane profile induced by enzyme phosphorylation to E1 approximately P at 16 A resolution via time-resolved x-ray diffraction.

Time-resolved x-ray diffraction studies of the isolated sarcoplasmic reticulum (SR) membrane have provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is transiently trapped exclusively in the first phosphorylated intermediate state, E1 approximately P, in absence of detectable enzyme turnover vs. that before ATP-initiated phosphorylation of the enzyme. These diffraction studies, which utilized the flash-photolysis of caged ATP, were performed at temperatures between 0 and -2 degrees C and with a time-resolution of 2-5 s. Analogous time-resolved x-ray diffraction studies of the SR membrane at 7-8 degrees C with a time resolution of 0.2-0.5 s have previously provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is only predominately in the first phosphorylated intermediate state under conditions of enzyme turnover vs. that before enzyme phosphorylation. The two difference profiles, compared at the same low resolution (approximately 40 A), are qualitatively similar but nevertheless contain some distinctly different features and have therefore been analyzed via a step-function model analysis. This analysis was based on the refined step-function models for the two different electron density profiles obtained independently from x-ray diffraction studies at higher resolution (16-17 A) of the SR membrane before enzyme phosphorylation at 7.5 and -2 degrees C. The step-function model analysis indicated that the low resolution difference profiles derived from both time-resolved x-ray diffraction experiments arise from a net movement of Ca2+ ATPase protein mass from the outer monolayer to the inner monolayer of the SR membrane lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proline transport activity in Escherichia coli membrane vesicles of different buoyant densities

Cytoplasmic membrane vesicles prepared by lysis of Escherichia coli W 3110 spheroplasts in a French press at 0° C are heterogeneous with respect to density due to membrane protein aggregation as a result of lateral phase separation of membrane phospholipids and to the presence of more or less outer membrane. These different vesicle classes can be separated on isopycnic density gradients. Assays for various membrane-associated functions show that the membranes differ not only with respect to density and structure but also with respect to function.The proline transport system (as detected by uptake experiments with the artificial electron donor ascorbate-phenazine methosulfate) shows maximal activities in membrane fractions that have considerably higher densities than the normal cytoplasmic membrane. This is always the case, whether vesicles are isolated from membranes that exhibit a temperature-induced protein aggregation or not. A correlation between high proline transport activity and the presence of vesicles with double membranes (consisting of outer and inner membrane) has been established. The possibility that the outer membrane protects the transport system in the cytoplasmic membrane during the isolation of vesicles is discussed.     

Myelin Organization in the Nodal,Paranodal, and Juxtaparanodal Regions Revealed by Scanning X-Ray Microdiffraction

X-ray diffraction has provided extensive information about the arrangement of lipids and proteins in multilamellar myelin. This information has been limited to the abundant inter-nodal regions of the sheath because these regions dominate the scattering when x-ray beams of 100 µm diameter or more are used. Here, we used a 1 µm beam, raster-scanned across a single nerve fiber, to obtain detailed information about the molecular architecture in the nodal, paranodal, and juxtaparanodal regions. Orientation of the lamellar membrane stacks and membrane periodicity varied spatially. In the juxtaparanode-internode, 198–202 Å-period membrane arrays oriented normal to the nerve fiber axis predominated, whereas in the paranode-node, 205–208 Å-period arrays oriented along the fiber direction predominated. In parts of the sheath distal to the node, multiple sets of lamellar reflections were observed at angles to one another, suggesting that the myelin multilayers are deformed at the Schmidt-Lanterman incisures. The calculated electron density of myelin in the different regions exhibited membrane bilayer profiles with varied electron densities at the polar head groups, likely due to different amounts of major myelin proteins (P0 glycoprotein and myelin basic protein). Scattering from the center of the nerve fibers, where the x-rays are incident en face (perpendicular) to the membrane planes, provided information about the lateral distribution of protein. By underscoring the heterogeneity of membrane packing, microdiffraction analysis suggests a powerful new strategy for understanding the underlying molecular foundation of a broad spectrum of myelinopathies dependent on local specializations of myelin structure in both the PNS and CNS.     

Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins.

A considerable interest exists currently in designing innovative strategies to produce two-dimensional crystals of membrane proteins that are amenable to structural analysis by electron crystallography. We have developed a protocol for crystallizing membrane protein that is derived from the classical lipid-layer two-dimensional crystallization at the air/water interface used so far for soluble proteins. Lipid derivatized with a Ni(2+)-chelating head group provided a general approach to crystallizing histidine-tagged transmembrane proteins. The processes of protein binding and two-dimensional crystallization were analyzed by electron microscopy, using two prototypic membrane proteins: FhuA, a high-affinity receptor from the outer membrane of Escherichia coli, and the F(0)F(1)-ATP synthase from thermophilic Bacillus PS3. Conditions were found to avoid solubilization of the lipid layer by the detergent present with the purified membrane proteins and thus to allow binding of micellar proteins to the functionalized lipid head groups. After detergent removal using polystyrene beads, membrane sheets of several hundreds of square micrometers were reconstituted at the interface. High protein density in these membrane sheets allowed further formation of planar two-dimensional crystals. We believe that this strategy represents a new promising alternative to conventional dialysis methods for membrane protein 2D crystallization, with the additional advantage of necessitating little purified protein.     

Location of high-affinity metal binding sites in the profile structure of the Ca+2-ATPase in the sarcoplasmic reticulum by resonance x-ray diffraction.

Resonance x-ray diffraction measurements on the lamellar diffraction from oriented multilayers of isolated sarcoplasmic reticulum (SR) membranes containing a small concentration of lanthanide (III) ions (lanthanide/protein molar ratio approximately 4) have allowed us to calculate both the electron density profile of the SR membrane and the separate electron density profile of the resonant lanthanide atoms bound to the membrane to a relatively low spatial resolution of approximately 40 A. Analysis of the membrane electron density profile and modeling of the separate low resolution lanthanide atom profile, using step-function electron density models based on the assumption that metal binding sites in the membrane profile are discrete and localized, resulted in the identification of a minimum of three such binding sites in the membrane profile. Two of these sites are low-affinity, low-occupancy sites identified with the two phospholipid polar headgroup regions of the lipid bilayer within the membrane profile. Up to 20% of the total lanthanide (III) ions bind to these low-affinity sites. The third site has relatively high affinity for lanthanide ion binding; its Ka is roughly an order of magnitude larger than that for the lower affinity polar headgroup sites. Approximately 80% of the total lanthanide ions present in the sample are bound to this high-affinity site, which is located in the "stalk" portion of the "headpiece" within the profile structure of the Ca+2 ATPase protein, approximately 12 A outside of the phospholipid polar headgroups on the extravesicular side of the membrane profile. Based on the nature of our results and on previous reports in the literature concerning the ability of lanthanide (III) ions to function as Ca+2 analogues for the Ca+2 ATPase we suggest that we have located a high-affinity metal binding site in the membrane profile which is involved in the active transport of Ca+2 ions across the SR membrane by the Ca+2 ATPase.     

Structure of human serum lipoproteins in solution. I. Theory and techniques of an x-ray scattering approach using solvents of variable density.

A theoretical framework is given for the interpretation of the small-angle X-ray scattering experiments of solutions of macromolecules with a non-uniform electron density distribution, using solvents of variable density. This provides an introduction to an experimental study of human serum lipoproteins, described in the accompanying paper (Tardieu et al., 1976). Four theoretical cases are considered, specified by the following conditions. (A): the sample is a perfect solution of identical particles. (B): the electron density inside the particles is independent of the density of the solvent. (C): the electron density inside the particles takes only two values. (D): the particles display a quasi-spherical symmetry. If conditions (A) and (B) are satisfied several parameters can be determined: the volume, radius of gyration and surface area of the shape function; the second moment and the square average of the electron density contrast at buoyancy. Moreover the intensity curves can all be put on an absolute scale. If conditions (A), (B) and (C) are satisfied it is possible to determine the density and volume of the low and high density regions, the area of the interface between the two regions and the distribution of chords corresponding to the shape function and to each of the two regions (a chord is a straight segment joining two points at the interface). If conditions (A), (B) and (D) are satisfied it is possible to determine the spherical average of the electron density distribution at buoyancy and of the shape function. Moreover, if condition (C) is satisfied it is also possible to determine the fraction of each spherical shell which is occupied by the low and the high density regions. The validity of the different conditions is discussed, and criteria are suggested to test some of them.     

The distribution of anionic sites on the surfaces of mitochondrial membranes. Visual probing with polycationic ferritin

Polycationic ferritin, a multivalent ligand, was used as a visual probe to determine the distribution and density of anionic sites on the surfaces of rat liver mitochondrial membranes. Both the distribution of bound polycationic ferritin and the topography of the outer surface of the inner mitochondrial membrane were studied in depth by utilizing thin sections and critical-point dried, whole mount preparations for transmission electron microscopy and by scanning electron microscopy. Based on its relative affinity for polycationic ferritin, the surface of the inner membrane contains discrete regions of high density and low density anionic sites. Whereas the surface of the cristal membrane contains a low density of anionic sites, the surface of the inner boundary membrane contains patches of high density anionic sites. The high density anionic sites on the inner boundary membrane were found to persist as stable patches and did not dissociate or randomize freely when the membrane was converted osmotically to a spherical configuration. The observations suggest that the inner mitochondrial membrane is composed of two major regions of anionic macromolecular distinction. It is well-known that an intermembrane space exists between the two membranes of the intact mitochondrion; however, a number of contact sites occur between the two membranes. We determined that the outer membrane, partially disrupted by treatment with digitonin, remains attached to the inner membrane at these contact sites as inverted vesicles. Such attached vesicles show that the inner surface of the outer membrane contains anionic sites, but of decreased density, surrounding the contact sites. Thus, the intermembrane space in the intact mitochondrion may be maintained by electronegative surfaces of the two mitochondrial membranes. The distribution of anionic sites on the outer surface of the outer membrane is random. The nature and function of fixed anionic surface charges and membrane contact sites are discussed with regard to recent reports relating to calcium transport, protein assembly into mitochondrial membranes, and membrane fluidity.     

Liquid diffraction analysis of sarcoplasmic reticulum. II. Solvent electron contrast variation.

Intensities of x-ray scattering from rabbit muscle sarcoplasmic reticulum membrane have been measured over the range of s = 0.05-0.25, at solvent densities varying between p0 = 0.335 and 0.389 electrons/A3. Analysis of the results shows agreement with the elements of structure deduced earlier by a different technique, based on the variation of the lipid-protein concentration ratio. In addition, the present work extends the analysis to allow isolation of the lipid contribution to the total scattering, from which a profile of the lipid electron density normal to the membrane face is evaluated. The scattering arising from electron correlations within the plane of the bilayer has also been identified.     

X-Ray Diffraction of Myelin Membrane: II. Determination of the Phase Angles of the Frog Sciatic Nerve by Heavy Atom Labeling and Calculation of the Electron Density Distribution of the Membrane

The phase signs of the five main X-ray reflections from normal frog sciatic nerve have been determined as all positive using a technique of labeling with very small amounts of heavy metal. The changes in intensity of the individual reflections were studied as a function of uptake of metal label by the membrane. The possible localization of the metal label was decided from computer-analogue studies and from Patterson calculations. These phases are different from those determined by previous workers using techniques of trial of the best set of phases, or a step model, to give the best fit of the combined intensity data of normal and swollen myelin membranes. The electron density map has been calculated using eight reflections and their experimentally determined phases. The map shows an inner low electron density region which is different from that shown by earlier calculations. The center of the low electron density region shows a small region of increased electron density. However, without fixing absolute electron density levels in the map, it is not yet possible to allocate regions of low electron density to pure lipid or lipoprotein. The map shows the two sides of the membranes to be different in molecular structure without significant water spaces between the membranes.     

Structural studies on reconstituted reaction center-phosphatidylcholine membranes.

Reaction center protein, isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26 mutant, was incorporated into phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. Cytochrome c, added to preformed reaction center-phosphatidylcholine vesicles, rapidly reduced up to 90% of the laser-generated (BChl)2+ of the reaction center (with kinetics of electron transfer similar to those in the chromatophore membrane) which suggests that the portion of the reaction center which accommodates functional cytochrome c binding sites is exposed predominantly on the exterior of the vesicles. Unit cell electron density profiles were derived from lamellar X-ray diffraction from oriented reaction center-phosphatidylcholine membrane multilayers at varying lipid/protein ratios. The analysis of these profiles showed that the reaction center protein incorporates into the phosphatidylcholine membrane with unique sidedness and that the profile of the reaction center protein itself is asymmetric and spans the membrane.     

Characterization of a low density cytoplasmic membrane subfraction isolated from Escherichia coli

We have used freeze fracture electron microscopy to study the distribution of membrane proteins in the cytoplasmic membrane of Escherichia coli W 3110. While these proteins were distributed randomly at the growth temperature (37 °C), there was extensive protein lipid segregation when the temperature was lowered, resulting in bare patches containing no visible particles (protein), and areas of tightly packed or aggregated particles. To understand the segregation process, we have separated the bare patches from the particle rich membrane areas. Lysis of spheroplasts at 0 °C leads to cytoplasmic membrane fragments with different amounts of membrane particles per unit area; such fragments have been separated on isopycnic sucrose gradients. The bare patches occurred as low density membranes which were completely devoid of particles. They were compared to normal density cytoplasmic membranes with respect to fatty acid composition, protein distribution as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their content of several cytoplasmic membrane marker enzymes.The phospholipid to protein ratio of low density membranes was five times greater than that of normal membranes; unsaturated fatty acids were more abundant in the low density membranes. Most proteins had disappeared from the low density membranes. One protein, which had an apparent molecular weight of 26000 on sodium dodecyl sulfate gels appeared to be concentrated in the low density membranes; it accounted for about 50% of the total protein found in this membrane fraction.Of the cytoplasmic membrane markers tested, NADH oxidase and succinate dehydrogenase were excluded, while d-lactate dehydrogenase remained, and even appeared to be concentrated in the low density membranes.These results indicate that while most membrane proteins are associated with the fluid portion of the bilayer, some proteins evidently associate preferentially with phospholipids in the gel or frozen state.     

Membrane movements and fluidity during rotational motility of a termite flagellate. A freeze-fracture study

Freeze-fracture electron microscopy was used to examine the structure of a region of plasma membrane that undergoes continual, unidirectional shear. Membrane shear arises from the continual clockwise rotation of one part (head) of a termite flagellate relative to the rest of the cell. Freeze-fracture replicas show that the lipid bilayer is continuous across the shear zone. Thus, the relative movements of adjacent membrane regions are visible evidence of membrane fluidity. The distribution and density of intramembrane particles within the membrane of the shear zone is not different from that in other regions of the cell membrane. Also, an additional membrane shear zone arises when body membrane becomes closely applied to the rotating axostyle as cells change shape in vitro. This suggests that the entire membrane is potentially as fluid as the membrane between head and body but that this fluidity is only expressed at certain locations for geometrical and/or mechanical reasons. Membrane movements may be explained solely by cell shape and proximity to rotating structures, although specific membrane-cytoskeletal connections cannot be ruled out. The membrane of this cell may thus be viewed as a fluid which adheres to the underlying cytoplasm/cytoskeleton and passively follows its movements.     

Interactions of normal and mutant vesicular stomatitis virus matrix proteins with the plasma membrane and nucleocapsids.

We demonstrated recently that a fraction of the matrix (M) protein of vesicular stomatitis virus (VSV) binds tightly to cellular membranes in vivo when expressed in the absence of other VSV proteins. This membrane-associated M protein was functional in binding purified VSV nucleocapsids in vitro. Here we show that the membrane-associated M protein is largely associated with a membrane fraction having the density of plasma membranes, indicating membrane specificity in the binding. In addition, we analyzed truncated forms of M protein to identify regions responsible for membrane association and nucleocapsid binding. Truncated M protein lacking the amino-terminal basic domain still associated with cellular membranes, although not as tightly as wild-type M protein, and could not bind nucleocapsids. In contrast, deletion of the carboxy-terminal 14 amino acids did not disrupt stable membrane association or nucleocapsid interaction. These results suggest that the amino terminus of M protein either interacts directly with membranes and nucleocapsids or stabilizes a conformation that is required for M protein to mediate both of these interactions.     

Cytoplasmic surface structure in postsynaptic membranes from electric tissue visualized by tannic-acid-mediated negative contrasting

In this study, acetylcholine receptor-rich postsynaptic membranes from electric tissues of the electric rays Narcine brasiliensis and Torpedo californica are negatively contrasted for thin-section electron microscopy through the use of tannic acid. Both outer (extracellular) and inner (cytoplasmic) membrane surfaces are negatively contrasted, and can be studied together in transverse sections. The hydrophobic portion of the membrane appears as a thin (approximately 2 nm), strongly contrasted band. This band is the only image given by membrane regions which are devoid of acetylcholine receptor. In regions of high receptor density, however, both surfaces of the membrane are seen to bear or be associated with material which extends approximately 6.5 nm beyond the center of the bilayer. The material on the outer surface can be identified with the well-known extracellular portion of the receptor molecule. A major portion of the inner surface image is eliminated by extraction of the membranes at pH 11 to remove peripheral membrane proteins, principally the 43,000 Mr (43K) protein. The images thus suggest a cytoplasmic localization of the 43K protein, with its distribution being coextensive with that of the receptor. They also suggest that the 43K protein extends farther from the cytoplasmic surface than does the receptor.     

X-ray diffraction of myelin membrane. I. Optimal conditions for obtaining unmodified small angle diffraction data from frog sciatic nerve

The X-ray diffraction pattern of myelin of frog sciatic nerve has been investigated, using a Kratky small angle slit camera to obtain the electron density distribution across the membrane. All major reflections observed were related to a fundamental repeat distance of 171 ± 2.8 A. There was no further increase in the number of reflections on varying the experimental conditions (varying pH, applying tension, immersion in various isotonic buffer solutions, etc.) or by varying the camera slit arrangement. The degree of disorder within the myelin sheath was examined by comparing the crystallite size to the half-width of the diffraction peak at half-height. The limiting of the diffraction spectra to five major reflections was determined not to be caused by disorder. It is concluded that the observed X-ray diffraction pattern is a consequence of the particular electron density distribution of the membrane. Therefore, the membrane cannot contain sharply distinct step-function regions of electron density, but approaches a modified cosine distribution.     

Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli.

Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance. Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form. Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed. In E. coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small. This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier. When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm. In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes. Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion. Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E. coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin. These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm. A quantitative model of tetracycline fluxes in E. coli cells of various types is presented.     

ULTRASTRUCTURE OF THE MATURE UNGERMINATED RICE (ORYZA SATIVA) CARYOPSIS. THE STARCHY ENDOSPERM

The structure of the starchy endosperm of rice (Oryza sativa) was studied by using light and transmission electron microscopy coupled with proteolytic enzyme digestions. The starchy endosperm was divided into two regions, the subaleurone and central, based on the number and types of protein bodies observed. The subaleurone region contained three different types of membrane bounded protein bodies—large spherical, small spherical, and crystalline protein bodies. The small spherical protein bodies were most numerous and the large spherical ones were least numerous. The crystalline protein bodies displayed crystal lattice fringes and were a composite of smaller angular components. The central region lacked both the small spherical and crystalline protein bodies. The large spherical protein bodies of this region were located in pockets of densely stained proteinaceous material. In contrast to the relatively well preserved cytoplasm of the subaleurone region, the central endosperm zone consistently was poorly preserved.