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Egg whites were exposed to pulsed electric fields (PEFs) to investigate the protein-protein aggregation. No insoluble protein aggregate was found when egg whites were exposed to PEFs at 25, 30, and 35 kV/cm for 400 micros. However, atomic force microscopy showed that the sizes of the protein particles increased. Native polyacrylamide gel electrophoresis (PAGE) demonstrated the existence of aggregates under PEFs at 35 kV/cm for 400 micros. Sodium dodecyl sulfate (SDS)-PAGE in the presence and absence of 2-mercaptoethanol further indicated that sulfhydryl-disulfide interchange reactions occurred under PEFs. Differential scanning calorimetry scans showed 400 micros of PEF treatment at 35 kV/cm denatured 16.5% proteins. Insoluble egg white protein aggregates were induced by PEF (35 kV/cm, 800 micros) and heat (60 degrees C, 3.5 min) treatments. Disulfide bonds were the dominant binding forces in the formation of protein aggregates. However, the weakly noncovalent bonds play a much more important role in the protein aggregation forming in heat treatment (60 degrees C, 3.5 min) than that in PEF treatment (35 kV/cm, 800 micros).

Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 3.0-7.0 and 85 degrees C for 1 and 5 days, and the functional properties of the phosphorylated EWP (PP-EWP) were investigated. The phosphorylation was accelerated with a decrease of pH from 7.0 to 3.0 and for heating times from 1 to 5 days. The phosphorus content of EWP increased approximately 1.05% by dry-heating at pH 4.0 and 85 degrees C for 5 days in the presence of pyrophosphate, which was higher than that of casein. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The surface hydrophobicity of EWP increased by phosphorylation. The heat stability, emulsifying properties, and digestibility of EWP were improved by phosphorylation. The calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation. A firmer and transparent heat-induced gel of PP-EWP was obtained, and the water-holding capacity of heat-induced PP-EWP gel was higher that that of the control. These results suggest that phosphorylation by dry-heating in the presence of pyrophosphate is a useful method for improving the functional properties of EWP.

For centuries, the edible nests of Collocalia spp. ("Bird's Nests") have been used as a Chinese delicacy that had been claimed to be an effective health-giving tonic. However, clinical studies indicated that in Singapore, Bird's Nest is the most common cause of food-induced anaphylaxis in children, which could lead to potentially life-threatening allergenic reactions. The purpose of this study was to characterize the major allergens in Bird's Nest by using the combined technologies of two-dimensional gel electrophoresis (2-DE), immunochemistry, N-terminal protein sequencing, and mass spectrometry. Results from the immunostaining of the Western blots of the Bird's Nest 2-DE separated proteins with the sera from allergic patients indicated the presence of a major allergen of 66 kDa. Initial searches of the matrix assisted laser desorption/ionization--time of flight--mass spectrometry (MALDI-TOF-MS) tryptic peptide masses of the allergen in the SWISS-PROT and NCBI nonredundant databases revealed that this protein was novel. Based on the partial protein sequence information obtained from N-terminal microsequencing and nanoelectrospray-tandem MS, the 66 kDa immunoreactive allergen was found to be homologous to ovoinhibitor, a Kazal-type serine protease inhibitor, which is one of the dominant allergens found in chicken egg white.

The process of assembly of apolipoprotein (apo) B-containing lipoprotein particles occurs co-translationally after disulfide-dependent folding of the N-terminal domain of apoB but the mechanism is not understood. During a recent database search for protein sequences that contained similar amphipathic beta strands to apoB-100, four vitellogenins, the precursor form of lipovitellin, an egg yolk lipoprotein, from chicken, frog, lamprey, and C. elegans appeared on the list of candidate proteins. The X-ray crystal structure of lamprey lipovitellin is known to contain a "lipid pocket" lined by antiparallel amphipathic beta sheets. Here we report that the first 1000 residues of human apoB-100 (the alpha(1) domain plus the first 200 residues of the beta(1) domain) have sequence and amphipathic motif homologies to the lipid-binding pocket of lamprey lipovitellin. We also show that most of the alpha(1) domain of human apoB-100 has sequence and amphipathic motif homologies to human microsomal triglyceride transfer protein (MTP), a protein required for assembly of apoB-containing lipoproteins. Based upon these results, we suggest that an LV-like "proteolipid" intermediate containing a "lipid pocket" is formed by the N-terminal portion of apoB alone or, more likely, as a complex with MTP. This intermediate produces a lipid nidus required for assembly of apoB-containing lipoprotein particles; pocket expansion through the addition of amphipathic beta strands from the beta(1) domain of apoB results in the formation of a progressively larger high density lipoprotein (HDL)-like, then very low density lipoprotein (VLDL)-like, spheroidal lipoprotein particle.

A proteinase inhibitor, tentatively termed vitelloinhibitor, was purified from yolk of hen's ovarian follicles. It resembled egg-white ovoinhibitor not only in inhibitory spectrum (active for bovine trypsin and bovine chymotrypsin) but also in thermal stability, pH stability, antiserum reactivity and amino-acid composition. However, vitelloinhibitor had different molecular weight from that of ovoinhibitor. An alpha 2-proteinase inhibitor preparation, isolated from laying hen's serum in the present study, was found to exhibit two bands, and the larger one of the latter corresponded to vitelloinhibitor in molecular weight. The partial N-terminal amino-acid sequence of vitelloinhibitor was the same as those of the two components of serum inhibitor and all three agreed with that of ovoinhibitor. Vitelloinhibitor is likely to be an ovoinhibitor analog derived from a serum precursor, which might be the larger component of alpha 2-proteinase inhibitor.

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellenin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.

The crystalline lipovitellin/phosvitin complex has a molecular weight of 456 000 and contains nearly 100 molecules of bound phospholipid. Earlier work using electron microscopy and three-dimensional image reconstruction methods established the symmetrical dimeric nature of this lipoprotein, but the organization of the lipid was unknown. Under conditions where the lipoprotein is in solution, the high-resolution 31P nuclear magnetic resonance (NMR) spectra contain two well-resolved peaks which can be assigned to phosphoserine moieties in both lipovitellin and phosvitin and to the phospholipid microdomains. The spin-lattice relaxation times, T1, for the phosphoserines and the phospholipid head groups are distinctly different, with the serine phosphates having faster reorientation rates. 31P NMR spectra of crystalline lipoprotein contain a broad symmetric component with a chemical shielding anisotropy of about -50 ppm. By obtaining 31P MNR spectra from several modified forms of the lipoprotein as well as from the extracted lipids, it is shown that the chemical shielding anisotropy is characteristic of phospholipid in a bilayer arrangement. As a further check on 31P NMR results, samples of the lipovitellin/phosvitin complex containing deuterium-labeled 1,2-dielaidoyl-sn-glycero-3-phosphocholine were studied by 2H NMR methods. The resulting spectrum has characteristics similar to those obtained from model lipid systems in a lamellar state. The results of the 31P and 2H NMR experiments correlate with the low-resolution model of the crystalline lipovitellin complex obtained by diffraction studies. It is proposed that each subunit of lipovitellin contains a microdomain of phospholipid in a bilayer-like arrangement.

Ovomucoid and ovoinhibitor were isolated from chicken egg white. Enzyme-linked immunosorbent assay (ELISA) and affinity chromatography of ovoinhibitor-coupled Sepharose 4B showed that about 25% of rabbit anti-ovomucoid antibody reacted with ovoinhibitor. Ovoinhibitor required about 1000 times the concentration of ovomucoid to give 50% inhibition of anti-ovomucoid antibody binding to ovoinhibitor in the plate-binding ELISA. These results suggested that ovoinhibitor possesses antigenic determinants which are conformationally homologous with those of ovomucoid and cross-react with anti-ovomucoid antibodies.

The analysis of single crystals of the lipovitellin complex from lamprey has made it possible to compare electron density projections derived from X-ray diffraction and density modification methods with previously published electron micrographs. The close correlation between the images obtained by the two methods demonstrates that the fidelity of images obtained by electron microscopy is excellent despite the loss of specimen order. These correlations also attest to the ability of density modification methods, currently used in macromolecular crystallography, to estimate the phases of small angle X-ray reflections.

The polypeptide, lipid, and carbohydrate components of the lipovitellin complex from anostracan crustacean, Artemia salina, have been investigated. The lipovitellin isolated from the yolk granules is a carotenoid.lipoglycoprotein complex, containing 3.3% carbohydrate and 8.6% lipid. About half of the carbohydrate fraction is mannose and the rest consists of galactosamine, galactose, and glucosamine. The lipid fraction contained 67% phospholipid and 33% neutral lipid. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine are three major phospholipid components and their relative ratio changes by developmental stages. The neutral lipid fraction is composed of mainly triacylglycerol and cholesterol. Two polypeptide chains are identified as apoprotein components of the lipovitellin complex. Their molecular weights are estimated by sodium dodecyl sulfate gel electrophoresis to be 190,000 and 68,000, respectively. The third polypeptide with Mr = 85,000 is a very minor component and exists in nonstoichiometric levels in the lipovitellin complex. On the basis of an apparent molecular weight of the lipovitellin complex determined by gel filtration chromatography, the dimeric lipovitellin form is suggested, each monomer of which contains one set of Mr = 190,000 and one set of Mr = 68,000 apoproteins. Each dimeric lipovitellin complex contains approximately 70 associated lipid molecules, of which about 4 are attributed to canthaxantin, and 100 bound carbohydrate molecules.

The radioallergosorbent test (RAST) and RAST inhibition test were used to examine cross-allergenicity amongst the major hen's egg-white and egg-yolk proteins. Using ovalbumin as a reference allergen to compare cross-reactivity, it was apparent that the proteins conalbumin, ovomucoid and lysozyme substantially inhibited binding to ovalbumin discs of IgE in the sera of patients clinically hypersensitive to egg. The converse situation with conalbumin, ovomucoid and lysozyme on the discs and ovalbumin as the inhibitor also resulted in significantly decreased levels of IgE binding to the proteins on the discs. It was also demonstrated that cross-reactions occurred between ovalbumin and the yolk protein, apovitellenin I. Cross-reaction was also observed surprisingly when egg lysozyme was on the disc and the milk protein allergen alpha-lactalbumin was used as the inhibitor. The demonstration of cross-reaction between all of these proteins may signify that there are a number of common allergenic determinants on these egg proteins, thus providing a molecular basis for the phenomenon of cross-reactivity.

Lipid/protein and lipid/lipid interactions in the yolk lipoprotein complex from Xenopus laevis were examined by introducing a series of lipid spin-labels into the complex and observing the electron spin resonance spectra as a function of the position of the label along the lipid chains, temperature, pH, and charge on the lipid polar head group. Analyses of the spectra show that, in addition to the expected component arising from lipid associated with protein, a second component with increased segmental flexibility and the greater temperature dependence characteristic of lipid/lipid interactions is observed. These spin-labeling data and supporting compositional data indicate that much of the lipid is organized into a lipid-rich region or pool, consistent with the earlier model derived from electron microscopy and diffraction data and with companion 31P and 2H nuclear magnetic resonance data reported in the preceding paper [Banaszak, L. J., & Seelig, J. (1982) Biochemistry (preceding paper in this issue)]. The bilayer-like component exhibits a greater restriction of motion compared to vesicles of the isolated lipids at the same temperature, as would be expected for a relatively small lipid pool. Phospholipids exchange between the two motionally distinguishable environments. The equilibrium binding undergoes a shift between these two environments as a function both of pH and of the charge on the phospholipid polar head group. This shift in average binding affinity is opposite in direction to that reported for membrane proteins and implicates negatively charged groups on the protein that repel negatively charged phospholipids. This effect is greatly reduced by alkaline phosphatase treatment, suggesting that some of the lipid binding sites are in close proximity to phosphorylated residues on the protein.

The heat stabilization resulting from specific association of serine proteases with either of two multiheaded protease inhibitors, chicken ovoinhibitor or lima bean protease inhibitor, was determined at pH 6.7 in a differential scanning calorimeter. The 2:1 complex of either bovine alpha-chymotrypsin or subtilisin BPN' with ovoinhibitor showed two major denaturation endotherms; each 1:1 complex showed one major endotherm. Association with ovoinhibitor increased the kinetic thermal stabilities over those of the free chymotrypsin or subtilisin. Association with lima bean protease inhibitor stabilized bovine beta-trypsin greater than porcine beta-trypsin greater than bovine alpha-chymotrypsin. Complexes having different proteases bound to the same inhibitor, such as chymotrypsin . ovoinhibitor . subtilisin (1:1:1) or trypsin . inhibitor . chymotrypsin (1:1:1), denatured like mixtures of the 1:1 complexes. These results show more clearly that 2:1 association with multiheaded inhibitors stabilizes the two bound protease molecules independently. Each bound protease and the domain(s) of the inhibitor influenced by specific binding of this protease are denatured as a unit. Thus, 2:1 complexes comprise at least two new denaturing units. The extent of heat stabilization appears roughly proportional to the Kassoc determined by other methods. The results are consistent with other evidence that binding sites for proteases on multi-headed inhibitors are relatively independent in structure and function.

In a study of lipid-protein interactions in egg yolk, it was found that L-alpha-dipalmitoyl lecithin gave two distinct noncovalent complexes (A and B) with apovitellenin I, an apoprotein in the major yolk lipoprotein. Interaction took place under widely varied conditions, and yolk lecithin gave similar complexes. Complex A, which was formed within minutes, consisted of round particles of about 9 nm diameter. Complex B, which was formed more slowly, consisted of larger particles, possibly resembling curved discs, with diameter of 30-40 nm. The preparation and some properties of these complexes are described. It is suggested that they may be suitable for an extensive study of phospholipid-protein interactions in yolk.

A method is described for the chromatographic separation of mixtures of egg-yolk proteins of low solubility by using a hydrophobic column (phenyl-Sepharose) and eluting with increasing concentrations of aqueous urea at low pH. The resolving power of the method was established by tests on proteins and protein fragments of known sequence. The theoretical basis for the method remains, however, unclear. Factors such as the aggregation of the protein often appeared to be more important than its hydrophobicity in determining the urea concentration needed for elution. The method was applied to the mixture of apoproteins from the low-density lipoprotein (density about 0.95 g/ml) of avian egg yolk. For the previously studied apoproteins from egg yolk of the hen (Gallus domesticus), hydrophobic chromatography provided a new and convenient method for isolating the main apoproteins (hen apovitellenins I-VI). For the hitherto unexplored apoproteins from egg yolk of the duck (Anas platyrhynchos) the method has now been used to isolate three new proteins, two of which were not readily separated by methods based on molecular size. The elution pattern obtained with duck egg-yolk apoproteins is not the same as that of the hen egg-yolk apoproteins, although we suggest a relationship for the three new apoproteins based on their amino acid compositions and other properties. Possible roles for the apoproteins in avian egg yolk are described.

We have isolated cDNA clones and determined the gene structure of chicken ovoinhibitor, a seven domain Kazal serine proteinase inhibitor. Using RNA blot hybridization analysis, the gene was identified initially as a region 9-23 kilobases upstream of the gene for the related inhibitor ovomucoid. Ovoinhibitor RNA appears in oviduct and liver. cDNA clones were identified by screening an oviduct cDNA library with a nick-translated DNA restriction fragment which contained an exon of the gene. The mature protein sequence derived from a cDNa clone is in excellent agreement with that which we obtained from direct sequencing of purified ovoinhibitor. The protein-sequencing strategy is reported. The P1 amino acids of the Kazal domains are consistent with the known broad inhibitory specificity of ovoinhibitor. The gene is about 10.3 kilobases in length and consists of 16 exons. Each Kazal domain is encoded by two exons. Like ovomucoid, introns fall between the coding sequences of the ovoinhibitor domains, an arrangement which may have facilitated domain duplication. The intradomain intron occurs in an identical position in all of the ovoinhibitor and ovomucoid Kazal domains, suggesting that this intron was present in the primordial inhibitor gene. We discuss the location of the intradomain intron in relation to the known structure of four Kazal inhibitors and suggest a scheme for the evolution of the ovoinhibitor gene.

In Xenopus laevis four estrogen-responsive genes are expressed simultaneously to produce vitellogenin, the precursor of the yolk proteins. One of these four genes, the gene A2, was sequenced completely, as well as cDNAs representing 75% of the coding region of the gene. From this data the exon-intron structure of the gene was established, revealing 35 exons that give a transcript of 5,619 bp without the poly A-tail. This A2 transcript encodes a vitellogenin of 1,807 amino acids, whose structure is discussed with respect to its function. At the nucleic acid as well as at the protein level no extensive homologies with any sequences other than vitellogenin were observed. Comparison of the amino acid sequence of the vitellogenin A2 molecule with biochemical data obtained from the different yolk proteins allowed us to localize the cleavage products on the vitellogenin precursor as follows: NH2 - lipovitellin I - phosvitin (or phosvette II - phosvette I) - lipovitellin II - COOH.

As part of a comparative study of egg yolk from different avian species, the major lipoprotein and its mixed apoproteins from the egg yolk of the chinese goose (Anser cygnoides) have been prepared. From the apoprotein mixture, two new proteins, of molecular weight approximately 10000 and 22000 according to gel electrophoresis in detergent, have been isolated by gel-filtration chromatography in urea. The protein of lower molecular weight corresponds in amino acid sequence to apovitellenin I, a protein previously isolated from other avian species. As a comparison with other members of the same avian family (Anatidae), the amino acid sequence of apovitellenin I from the pekin duck (Anas platyrhynchos) was re-investigated and that of the muscovy duck (Cairina moschata) investigated. These were found to be identical to the sequence of goose's apovitellenin I. The second new protein is similar in composition, molecular weight, and solubility to apovitellenin II, a protein present in small amount in hen's egg yolk. A protein corresponding to apovitellenin II could not, however, be detected in the egg yolk of either species of duck.

Modifications of the method of Wallace et al. [Can. J. Biochem. 44, 1647-1655 (1966)] for phosvitin isolation from vertebrate eggs were devised to enhance the method's general effectiveness. Phosvitins which do not precipitate on dilution from solutions of their lipovitellin complexes may be selectively adsorbed onto, and desorbed from, DEAE-cellulose. Phosvitins which are too small for dialysis or ultrafiltration may be concentrated or desalted by precipitation with stoichiometric amounts of ferric ions, followed by iron removal with EDTA on gel filtration. Since phosvitin distribution among ammonium sulfate fractions depends on initial protein and salt concentrations in a species-specific manner, pilot experiments are needed to establish conditions for optimal fractionation.

Artemia trypsin-like proteinase has been reported previously to be highly inhibited in the embryo (B. Ezquieta and C.G. Vallejo (1985) Comp. Biochem. Physiol. 82B, 731-736). We report now that Artemia lipovitellin, the major storage protein complex, inhibits the proteinase. We have carried out an in vitro study of the characteristics of the inhibition. Lipovitellin, a glycolipoprotein of high molecular mass (650 kDa), behaves initially as a substrate but after a limited proteolysis becomes an inhibitor of the proteinase. The enzyme although inhibited in the hydrolysis of the protein substrate retains activity toward low molecular weight substrates. The residual activity on the protein substrate is inhibited by small inhibitors of the proteinase. These features of lipovitellin inhibition are reminiscent of the trap mechanism of alpha 2-macroglobulin inhibition, previously proposed as suitable for regulating proteolytic processes involved in development. Inhibition by lipovitellin is greater at low temperatures and has been determined at 17 and 37 degrees C, in the lower and higher part of the viable temperature range of Artemia development. At high temperature the proteinase hydrolyzes the inhibitor quite efficiently and the inhibition is lower. The inhibition by lipovitellin appears specific for Artemia trypsin-like proteinase when compared with other control pairs protein/proteinase. The results may provide support for an additional role of storage proteins as developmental inhibitors of proteinases.

Single crystals of a yolk lipoprotein from two species of lamprey oocytes have been prepared and shown to belong to the monoclinic space group C2. The unit cell dimensions combined with molecular weight determinations support the idea that the lipoprotein is a symmetrical dimer centered on a crystallographic 2-fold symmetry axis. The lipid content is estimated to be about 15% (w/w) and the crystals are suitable for structural studies by single crystal X-ray analysis. The X-ray results are correlated with those obtained by electron diffraction.

Electron diffraction of in vitro-grown lipoprotein crystals from cyclostome eggs (Ichthyomyzon unicuspis) has revealed lattice parameters (a = 19.4 nm, b = 8.9 nm, c = 8.9 nm, beta = 103.4 degrees; apparent symmetry C2 at 3 nm resolution) that were very close to those determined by X-ray diffraction in the same specimen and to others determined by the same electron microscope method in five cyclostome species (in-vivo crystals). Electron imaging has shown all investigated lipoprotein crystals to present similar views projected down the principal crystal axes. Electron diffraction of embedded and heavy-metal-treated lipoprotein crystals is a rapid procedure and a useful low-angle extension to X-ray diffraction.

1. The interaction of chicken ovoinhibitor with pancreatic trypsin, chymotrypsin and elastase I was studied in the absence of substrate by affinity chromatography, electrophoresis and gel filtration. 2. It was found that ovoinhibitor has, at least, five separate and non-overlapping binding sites: two for trypsin, two for chymotrypsin and one for elastase. Most likely, all five sites may be occupied simultaneously. 3. Electrophoretic studies indicated that two binding sites for trypsin and probably also for chymotrypsin differ in their affinity toward the respective enzyme.

Interaction of porcine elastase II with native and modified chicken egg-white ovoinhibitor was studied by determining the residual activity of the partially inhibited enzyme and by direct measurement of the stoichiometry of interaction using affinity chromatography, electrophoresis and gel filtration. It was found that the chymotrypsin binding site that is not modified by mild oxidation with N-chlorosuccinimide (Shechter et al., Biochemistry, (1977) 16, 992-997) is capable of binding elastase II as well. The binding of chymotrypsin and elastase II is mutually exclusive and the affinity of chymotrypsin is stronger. Binding of 2 mol trypsin as 1 mol elastase I by ovoinhibitor does not interfere with the binding of elastase II. There is also an indication that the second binding site for chymotrypsin is capable of forming a complex with an additional molecule of elastase II, but the binding is so weak that it could be detected only by electrophoresis.

Yolk-platelet crystals in the teleosts Pelvicachromis pulcher and Noemacheilus barbatulus and the amphibians Xenopus laevis, Rana temporaria, R. esculenta, and Triturus sp. have been studied by electron diffraction and imaging using a standardized processing (glutaraldehyde-osmium tetroxide fixation, glutaraldehyde-urea embedding, thin-section staining), by X-ray microanalysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their constituents. The crystal lattice is orthorhombic having--following standardized processing--in three amphibians a = 9.0 nm, b = 17.6 nm, c = 19.2 nm, and in the two teleosts a = 8.9 nm, b = 17.6 nm, c = 20.0 nm (averages). These values are very close to X-ray data from wet crystals (Xenopus laevis). Crystal images in the three axial projections point to the presence of space group P212121 (or an approximation of it since the lipovitellin dimers cannot be fully equivalent in some cases), to differences between the phosvitins of the two teleosts, and to a highly unusual stain exclusion from large crystal constituents interpreted as representing lipovitellin dimers. Microanalysis in ultrathin cryosections and other preparations revealed K and Cl to be the prominent ions in the crystals (and in the superficial layer of the platelet). Gel electrophoresis (including data of cyclostomes) showed considerable molecular variations despite a closely similar crystal architecture, emphasizing a physiological significance of the architecture, which may have remained conserved for nearly 400 million years according to paleontologic views.

The X-ray crystallographic structure of the lipid-protein complex lipovitellin-phosvitin has been determined with the multiple isomorphous replacement method using four heavy-atom derivatives. Lamprey yolk lipovitellin-phosvitin is a dimeric molecule of molecular weight 352,000. The monomer consists of three polypeptide chains. The smallest is known as phosvitin and has an extremely high phosphoserine content. The monomeric unit also contains about 16% (w/w) of non-covalently bound lipid, probably in a monolayer or bilayer-like configuration. Within each monomer is a "cavity" or region of low electron density. The cavity has a volume of about 68,000 A3 and is believed to contain the lipid in a presumably disordered state. The cavity is roughly conical in shape and is lined on two sides by seven and eight-stranded antiparallel beta-sheets. The base of the cavity opens away from the intersubunit interface, but appears partially closed off from solvent regions by additional antiparallel beta-sheet structure. The beta-sheets lining the sides of the cavity are surrounded by a shell of two curved layers of 16 interconnected helices. The helices in either layer of the shell are all roughly parallel to each other and antiparallel to all of the helices of the other layer. The connectivity of the helices resembles a "superhelix" and is different from the connectivities seen in proteins containing four-helix bundles. There are an estimated 1300 amino acids in lamprey lipovitellin-phosvitin and almost 1000 alanine residues have been modeled into electron density. The remaining residues are assumed to be disordered.