A monoclonal antibody to the estrogen receptor inhibits in vitro criteria of receptor activation by an estrogen and an anti-estrogen

The effect of an IgM class monoclonal antibody (B36) (Greene, G. L., Fitch, F. W., and Jensen, E. V. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 157-161) raised against the calf uterine estrogen receptor was tested in vitro on certain parameters of estrogen receptor activation by estradiol or 4-hydroxytamoxifen, a potent anti-estrogen. The following results were obtained. The antibody prevented the decrease in the dissociation rate of the receptor-estradiol complex which results from activation of the complex, whereas it did not affect the dissociation rate of the receptor-4-hydroxytamoxifen complex, which remains unchanged upon activation. The antibody also increased the dissociation rate of the preactivated receptor-estradiol complex. The antibody protected the naked estrogen receptor against heat-inactivation. B36 partially inhibited the binding of the estradiol- and 4-hydroxy-tamoxifen-receptor complexes to DNA adsorbed onto cellulose, but did not reverse the receptor-DNA binding. This inhibition was not overcome by higher DNA concentrations and was more pronounced for the receptor interacting with estrogen than with anti-estrogen. All these effects were specific since they were related to antibody/antigen recognition and were dose-dependent. These results indicate that the binding of the antibody to the estrogen-activated receptor induces a conformational change in the receptor and that the antibody can prevent and overcome the effect of activation whatever its mechanism. They also confirm that the conformations of the estrogen receptor differ when bound to estradiol or to 4-hydroxytamoxifen.     

Chick heat-shock protein of Mr = 90,000, free or released from progesterone receptor, is in a dimeric form

A monoclonal antibody (BF4) has been used to characterize and purify the heat-shock protein of Mr approximately 90,000 (hsp 90) present in the chick oviduct. In low salt cytosol, the sedimentation coefficient of hsp 90 is approximately 6.8 S, the Stokes radius approximately 7.1 nm, and the calculated Mr approximately 204,000, thus suggesting a dimeric structure. In 0.4 M KCl cytosol, only slightly smaller values were determined (approximately 6.5 S, approximately 6.8 nm, and approximately 187,000). Following purification by ion exchange and immunoaffinity chromatography, hsp 90 migrated as a single silver-stained band at Mr approximately 90,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sedimentation coefficient 6.2 S, the Stokes radius approximately 6.8 nm, and the Mr approximately 178,000 confirmed the dimeric structure. However, in both antigen or antibody excess conditions, only one molecule of monoclonal antibody could be bound to the hsp 90 dimer. Whether steric hindrance in a homodimer or the presence of two different 90-kDa proteins in a heterodimer explains this result cannot yet be decided. The dimer is not dissociated by high salt (1 M KCl) or the chaotropic agent (0.5 M NaSCN), but is disrupted by 4 M urea, suggesting a stabilization of the structure by hydrogen bonds. The molybdate-stabilized progesterone receptor hetero-oligomer form of approximately 8 S sedimentation coefficient was purified, and its hsp 90 component was then released by salt treatment. It was found to sediment at approximately 5.8 S and have a Stokes radius approximately 7.1 nm, giving Mr approximately 174,000. This observation is consistent with a previous report suggesting from specific activity determination, scanning of polyacrylamide gels, and cross-linking experiments that each purified nontransformed progesterone receptor molecule includes one progesterone binding unit per two 90-kDa protein molecules (Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., and Baulieu, E. E. (1984) Biochemistry 23, 6016-6023). This work brings direct evidence that both free hsp 90 and the non-hormone binding hsp 90 component released from the nontransformed steroid receptor in the cytosol are in a dimeric form.     

Hsp56: a novel heat shock protein associated with untransformed steroid receptor complexes

The recently-described p59 protein has been shown to be associated with untransformed steroid receptors present in rabbit uterus and rat liver cytosols (Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275; Renoir, J.-M., Radanyi, C., Faber, L. E., and Baulieu, E.-E. (1990) J. Biol. Chem. 265, 10740-10745), while a smaller version of this protein (p56) interacts with glucocorticoid receptors in human IM-9 cell cytosols (Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990) Biochemistry 29, 5145-5152). In addition to interacting with glucocorticoid receptors, the p56 protein of IM-9 cell cytosol is also found as part of a large heteromeric complex that contains both the 70-kDa and 90-kDa heat shock proteins (hsp70 and hsp90, respectively). Given this association of p56 with the two major stress proteins, I have speculated that p56 may itself be a heat shock protein. In this paper, the effect of heat stress on the rate of synthesis of p56 is determined. Intact IM-9 cells were exposed to 37 or 43 degrees C for 4 h, followed by pulse-labeling with [35S]methionine. Analysis of whole cytosolic extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveal an increased rate of radiolabeling for hsp70, hsp90, hsp100, ad hsp110, but no heat-inducible protein of smaller relative molecular mass is detected. However, immune-purification of p56 from normal and heat-stressed cytosols with the EC1 monoclonal antibody results in the presence of a 56-kDa protein that exhibits an increased rate of synthesis in response to heat stress. The results of two-dimensional gel Western blots employing the EC1 antibody demonstrate that this heat-inducible protein is indeed the EC1-reactive p56 protein and that the induction effect is not due to unequal yields of p56 during immune-purification. Heat stress has no effect on the composition of the p56.hsp.70.hsp90 complex, except that the complex derived from heat shocked-cells contains both the constitutive and heat-inducible forms of hsp70. Induction of p56 also occurs in IM-9 cells subjected to chemical stress (sodium arsenite). It is proposed that p56 is a steroid receptor-associated heat shock protein which can now be termed hsp56. Like hsp90, hsp56 likely serves in some vital cellular role apart from any specific function it provides in steroid receptor action.     

Immunologic analysis of human breast cancer progesterone receptors. 1. Immunoaffinity purification of transformed receptors and production of monoclonal antibodies

A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors [Sullivan, W. P., Beito, T. G., Proper, J., Krco, C. J., & Toft, D. O. (1986) Endocrinology (Baltimore) 119, 1549-1557] cross-reacts with the Mr 120,000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D human breast cancer cells. Single-step immunoaffinity chromatography results in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with [3H]promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step. A second purification step by diethylaminoethyl chromatography gives further enrichment to 3720 pmol/mg of protein (or 44% purity) to yield essentially two proteins, 120-kilodalton (kDa) B receptors and a 76-kDa non-steroid binding protein, each in approximately equivalent amounts. B receptors purified under these conditions are transformed and biologically active. They were maintained as undegraded 120-kDa doublets and retained both hormone and DNA binding activities. Isolated B receptors were free of the 90-kDa non-steroid binding protein observed to be associated with 8S untransformed receptors in other systems and were free also of the non-hormone binding 105-108-kDa B antigen described previously to copurify with chick PR. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgG1), B-64 (IgG1), and B-11 (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG1), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displace the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react only with receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology. These new MAbs are valuable reagents for further studies of human receptor structure and function and for clinical immunodetection of PR in breast tumors.     

Composition, assembly and activation of the avian progesterone receptor

When isolated from chick oviduct cytosol by antibody adsorption, the inactive progesterone receptor is associated with the two heat shock proteins, hsp90 and hsp70, plus three additional proteins termed p54, p50, and p23 according to their molecular weights. While their functions remain unknown, all of these receptor associated proteins are dissociated upon receptor activation in intact cells. To better understand the assembly and activation mechanisms of progesterone receptor complexes, we have developed a cell-free system for studying receptor interactions with hsp90 and hsp70 and have used this system to examine requirements for hsp90 binding to the receptor. Purified receptor, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: (1) absence of progesterone, (2) elevated temperature (30°C), (3) presence of ATP, and (4) presence of Mg²⁺. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl₂ and an ATP regenerating system. ATP depletion of lysate by dialysis or ATPase addition blocks hsp90 binding to the receptor. When progesterone is added to pre-formed receptor complexes in reticulocyte lysate it promotes activation and the dissociation of hsp90. This process is also dependent upon ATP. Thus, both the assembly, and activation of the progesterone receptor can be accomplished in the reticulocyte lysate system.     

Characterization of the adenovirus E3 protein that down-regulates the epidermal growth factor receptor. Evidence for intermolecular disulfide bonding and plasma membrane localization.

We have characterized the biosynthesis and processing of a 91 amino acid hydrophobic integral membrane protein encoded by human group C adenoviruses which down-regulates the EGF receptor (Carlin, C. R., Tollefson, A. E., Brady, H. A., Hoffman, B. L., and Wold, W. S. M. (1989) Cell 57, 135-144). Previous studies have shown that two immunologically related proteins are produced in vivo, a 13.7-kDa protein encoded by E3 message f and a 11.3-kDa protein derived from 13.7 kDa by proteolysis (Hoffman, B. L., Ullrich, A., Wold, W. S. M., and Carlin, C. R. (1990) Mol. Cell. Biol. 10, 5521-5524; Tollefson, A. E., Krajcsi, P., Yei, S., Carlin, C. R., and Wold, W. S. M. (1990) J. Virol. 64, 794-801). We report here that the 13.7- and 11.3-kDa proteins form intermolecular disulfide bonds cotranslationally at Cys-31 and tend to migrate as high molecular weight aggregates under nonreducing conditions. Both proteins are also present at the cell surface, as evidenced by specific immunoprecipitation from intact monolayers enzymatically labeled with 125I. Moreover, an antiserum specific for a putative extracellular epitope recognizes the same viral proteins as antibodies directed against a C-terminal synthetic 15-mer. The 13.7- and 11.3-kDa proteins are detected at early time points during pulse-chase radiolabeling of infected cells, do not undergo any further changes in molecular weight, and focus at their predicted isoelectric points (7.4 and 7.2, respectively). Identical results are obtained in stable transfectants constitutively expressing only 13.7 and 11.3 kDa, suggesting that biosynthesis and processing is not dependent on other viral proteins. These results have been incorporated into a computer-based model to predict the orientation of 13.7 and 11.3 kDa in the lipid bilayer. This model provides a basis for testing predictions regarding the topology of the viral proteins, as well as putative interactions with heterologous proteins in the microenvironment of the plasma membrane that cause down-regulation of the epidermal growth factor receptor.     

Monoclonal antibody to the hen oviduct progesterone receptor produced following in vitro immunization

The progesterone receptor of the hen oviduct is composed of two non-identical hormone-binding polypeptide subunits, A (Mr = 79,000) and B (Mr = 108,000). We used a highly purified preparation of B to immunize mouse spleen cells in vitro. After 5 days in culture, the cells were fused with SP2/0-Ag 14 myeloma cells. The resultant hybridomas were screened using an enzyme linked immunosorbent solid phase assay, and those hybridomas producing antibodies binding to the immunogen were cloned by limiting dilution. One such clone, 9B3-12, secreted an antibody of immunoglobulin class IgM, which binds to B. This was indicated by the ability of the antibody to increase the rate of sedimentation coefficient of the B subunit. Further, when the proteins in the B preparation were separated by electrophoresis and blotted onto nitrocellulose filters the antibody bound to a protein of 108,000 daltons. The antibody produced by 9B3-12 also reacted with subunit A and with the human progesterone receptor but failed to bind to the chick liver glucocorticoid receptor or to progesterone in the absence of its receptor.     

Steroid hormone-dependent interaction of human progesterone receptor with its target enhancer element

We investigated the requirement of steroid hormone for the specific binding of progesterone receptor to its cognate progesterone responsive element (PRE) in cell-free experiments. We prepared unfractionated nuclear extracts from human breast cancer (T47D) cells which are rich in progesterone receptors and used a gel retardation assay to monitor receptor-DNA complex formation. Exposure of receptor to either progesterone, R5020, or the antiprogestin RU38 486 in vivo or in vitro led to the formation of two protein-DNA complexes (1 and 2) which were not detected in nuclear extracts unexposed to hormone. Similar treatment with cortisol or estradiol failed to induce the formation of these complexes. The complexes were specific for PRE, since they could be competed efficiently in binding competition experiments by oligonucleotides containing PRE. A monoclonal antibody which recognizes both A and B forms of human progesterone receptor, interacted with both complexes 1 and 2 and shifted them to slower migrating forms. Another antibody which only recognizes the B form interacted with only complex 1 but not with complex 2, establishing that the complexes 1 and 2 were indeed formed by progesterone receptor forms B and A, respectively. We conclude from the above studies that in vivo or in vitro treatment of nuclear progesterone receptor with either progesterone or R5020 or RU38 486 alone can lead to detection of high affinity complexes formed between the PRE and the receptor present in unpurified nuclear extracts.     

Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM

Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain.     

ZK98299, a novel antiprogesterone that does not interact with chicken oviduct progesterone receptor

Steroid antagonists, at receptor level, are valuable tools for elucidating the mechanism of steroid hormone action. We have examined and compared the interaction of avian and mammalian progesterone receptors with progestins; progesterone and R5020, and a newly synthesized antiprogesterone ZK98299. In the chicken oviduct cytosol, [3H]R5020 binding to macromolecule(s) could be eliminated with prior incubation of cytosol with excess radioinert steroids progesterone or R5020 but not ZK98299. Alternatively, [3H]ZK98299 binding in the chicken oviduct was not abolished in the presence of excess progesterone, R5020, or ZK98299. In the calf uterine cytosol, [3H]R5020 or [3H]ZK98299 binding was competeable with progesterone, R5020 and ZK98299 but not estradiol, DHT or cortisol. Furthermore, immunoprecipitation and protein A-Sepharose adsorption analysis revealed that in the calf uterine cytosol, the [3H]R5020-receptor complexes were recognized by anti-progesterone receptor monoclonal antibody PR6. This antibody, however, did not recognize [3H]ZK98299-receptor complexes. When phosphorylation of progesterone receptor was attempted in the chicken oviduct mince, presence of progesterone resulted in an increased phosphorylation of the known components A (79 kDa) and B (110 kDa) receptor proteins. Presence of ZK98299 neither enhanced the extent of phosphorylation of A and B proteins nor did it reverse the progesterone-dependent increase in the phosphorylation. The avian progesterone receptor, therefore, has unique steroid binding site(s) that exclude(s) interaction with ZK98299. The lack of immunorecognition of calf uterine [3H]ZK98299-receptor complexes, suggests that ZK98299 is either interacting with macromolecule(s) other than the progesterone receptor or with another site on the same protein. Alternatively, the antisteroid binds to the R5020 binding site but the complex adopts a conformation that is not recognized by the PRG antibodies.     

Binding of heat shock proteins to the avian progesterone receptor.

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.     

Developmentally regulated antigen associated with calcium crystals in tobacco anthers

We have found and characterized an antigen associated with crystal-containing cells in the stomium and connective tissue of the anthers of Nicotiana tabacum L. (tobacco). The antigen, defined by the monoclonal antibody NtF-8B1, localizes to subcellular regions surrounding the crystals. At the light-microscope level, the antigen is detectable just after the first appearance of crystals in the connective tissue of the anther, and at approximately the same time as the appearance of crystals in the stomium. The antigen is not detectable on a Western blot, and gave inconclusive results on a test of periodate sensitivity. It is not the crystals themselves, nor is the presence of the crystals required for antibody recognition. The antigen is sensitive to heat and protease treatment, indicating that it is a protein. The antigen is not tightly membrane-bound, in spite of its localization closely surrounding the crystals. Chemical tests indicate that the druse crystals in the stomium are calcium oxalate.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate This research was supported by a National Science Foundation postdoctoral fellowship to B.L.H., by National Science Foundation grants DMB-87-15799 and to W.E.F. BSR-88-18035, and by U.S. Department of Agriculture grant GAM-89-01056. The authors thank Phillip T. Evans (Louisiana State University, Baton Rouge, USA), Wilma L. Lingle, Harry T. Horner, Jr. (Iowa State University), and A. Jack Fowler, Jr., for advice and helpful discussions.     

A heat shock protein complex isolated from rabbit reticulocyte lysate can reconstitute a functional glucocorticoid receptor-Hsp90 complex.

When unliganded glucocorticoid receptor that has been stripped free of associated proteins is incubated with rabbit reticulocyte lysate, the receptor becomes associated with the 70- and 90-kDa heat shock proteins (hsp70 and hsp90), and the untransformed state of the receptor is functionally reconstituted [Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., & Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400]. Recently, an hsp70-containing protein complex (200-250 kDa) purified from rabbit reticulocyte lysate was shown to maintain a fusion protein bearing the mitochondrial matrix-targeting signal in a state that is competent for mitochondrial import [Sheffield, W. P., Shore, G. C., & Randall, S. K. (1990) J. Biol. Chem. 265, 11069-11076]. In this work, we show that this partially purified mitochondrial import-competent fraction contains both hsp90 and hsp70. When the purified fraction is immunoadsorbed with a monoclonal antibody specific for hsp90, a significant portion of the hsp70 is co-immunoadsorbed, suggesting that hsp90 and hsp70 are present together as a complex. The partially purified fraction maintains a hybrid precursor protein containing the mitochondrial matrix-targeting signal of rat pre-ornithine carbamyl transferase in an import-competent state. Incubation of immunopurified glucocorticoid receptor with this fraction of reticulocyte lysate results in ATP-dependent association of the receptor with both hsp70 and hsp90, and the resulting complexes are functional as assessed by return of the receptor to the high-affinity steroid binding conformation. The glucocorticoid receptor hetero-complex reconstituting activity of the lysate fraction is low relative to its mitochondrial import activity. Importantly, however, this is the first demonstration of the functional and structural reconstitution of the untransformed state of any steroid receptor utilizing a partially purified system.     

Localization of sites of photoaffinity labeling of the large subunit of Escherichia coli ribosomes by arylazide derivative of puromycin

Previous work (Nicholson, A. W., Hall, C. C., Strycharz, W. A., and Cooperman, B. S. (1982) Biochemistry 21, 3797-3808) showed that [3H]p-azidopuromycin photoaffinity labeled 70 S Escherichia coli ribosomes and that photoincorporation into 50 S subunit proteins was in the order L23 greater than L18/22 greater than L15. In the present work we report on immunoelectron microscopic studies of the complexes formed by p-azidopuromycin-modified 50 S subunits with antibodies to the N6,N6-dimethyladenosine moiety of the antibiotic. The p-azidopuromycin-modified 50 S subunits appear to be identical to unmodified control subunits in electron micrographs. Complexes of modified subunits with antibodies to the N6,N6-dimethyladenosine moiety of p-azidopuromycin were visualized in micrographs. Individual subunits with a single bound antibody (monomeric complexes) and pairs of subunits cross-linked by a single antibody (dimeric complexes) were separately evaluated and showed similar results. Two regions of p-azidopuromycin photoincorporation were identified. The primary site, seen in about 75% of the complexes, is between the central protuberance and small projection, on the side away from the L7/L12 arm, in a region thought to contain the peptidyltransferase center. The secondary site, of unknown significance, is at the base of the subunit maximally distant from the arm. These placements are essentially identical to those we observed in analyses of puromycin photoincorporation (Olson, H. M., Grant, P. G., Cooperman, B. S., and Glitz, D. G. (1982) J. Biol. Chem. 257, 2649-2656) and quantitatively similar to evaluations of monomeric puromycin-50 S subunit complexes. The data support the placement of proteins L23, L18/22, and L15 at or near the peptidyltransferase center at the primary site and suggest, in addition, that the secondary site includes a genuine area of puromycin affinity.     

Assessment of structural similarities in chick oviduct progesterone receptor subunits by partial proteolysis of photoaffinity-labeled proteins

Partial proteolytic fragmentation of the two chick oviduct progesterone receptor subunits was used to identify structural features shared by the two proteins. Both subunits can be photoaffinity labeled at their hormone-binding sites (Birnbaumer, M., Schrader, W. T., and O'Malley, B. W. (1983) J. Biol. Chem. 258, 1637-1644) using the radioactive steroid [methyl-3H] 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione. Native subunits A (Mr = 79,000) and B (Mr = 108,000) were partially purified, photoaffinity-labeled, and then subjected to various mild proteolytic digestions. Labeled fragments were analyzed by fluorography after electrophoresis of the digests under denaturing conditions. Digestion patterns were characteristic for each protease tested. However, fragments from both A and B were indistinguishable for all peptides of less than Mr = 60,000. Time course studies demonstrated the sequential production of progressively smaller discrete fragments in a manner consistent with a precursor-product relationship among them and established the existence of similar structural domains resistant to proteolysis in both proteins. Autoradiographic peptide maps were obtained by 125I-labeling of pure A and B protein isolated by two-dimensional gel electrophoresis followed by exhaustive tryptic digestion and two-dimensional separation. These studies revealed that a significant proportion of the smaller A protein differs in its primary sequence from that of the B protein which excludes the possibility of their sharing a precursor-product relationship. We conclude that B and A subunits are separate proteins with common structural features in the native state, but with considerable amino acid sequence differences. The simplest hypothesis consistent with these findings is that B and A are the products of two separate genes which have diverged to give rise to two different but related proteins that fold in such a manner as to be almost indistinguishable by proteolytic attack of their native conformation.