Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis

Porphyromonas gingivalis, a putative pathogen in human periodontal disease, possesses a 60-kDa heat shock protein (hsp60, GroEL). The GroEL homologs are known to be key molecules in auto-immune reactions because of the sequence similarity with human hsp60. In this study, B-cell epitopes on P. gingivalis GroEL (PgGroEL) were analyzed by both Western immunoblotting with truncated PgGroEL and by the multi-pin synthetic peptide approach. To examine auto-antibody production in periodontitis patients, Western immunoblotting with human gingival fibroblasts was performed. Deletion mutants were constructed from the cloned PgGroEL gene (P. gingivalis groEL), and four C-terminal truncated PgGroEL and one N-terminal truncated PgGroEL were prepared from the deletants. Sera from periodontitis patients reacted with all truncated PgGroEL used in this study. The results suggest that the B-cell epitopes were overlaid throughout PgGroEL. To determine the detailed locations of the B-cell epitope, 84 decapeptides covering the entire PgGroEL were synthesized and the serum IgG response to the peptides was examined. Epitope mapping using the synthetic peptides confirmed that the B-cell epitopes were overlaid throughout the length of PgGroEL and revealed that highly conserved peptides between PgGroEL and human hsp60 were recognized by the serum antibodies. Immuno-reactivity against human gingival fibroblasts was examined with sera from 30 periodontitis patients and 10 periodontally healthy subjects. IgG antibody against the 65-kDa antigen in human gingival fibroblasts (same molecular mass as human hsp60) was detected in two patients. Although IgG production against human hsp60 may be rare case in periodontitis patients, the results of epitope mapping demonstrated the potential of PgGroEL to cause the cross-reactions with human hsp60.     

Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.     

Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.     

Immunochemical characterization of an IgG-binding protein of Streptococcus suis

Several bacterial species express surface proteins with affinity for the constant region (F_c) of immunoglobulin (Ig) of different animal species. Previous studies from our group have reported the presence of an IgG-binding protein in various serotypes of Streptococcus suis . This molecule was also shown to bind in a non-immune fashion chicken IgY and to our knowledge this characteristic is unique. In the present study, by dot-blotting, we showed that the native protein, obtained by affinity chromatography, reacted more strongly with IgG from various animal species than the denatured material. Using a competitive enzyme-linked immunosorbent assay the affinity of the native 60-kDa protein (previously identified as a 52-kDa protein) towards IgG of various animal species was compared to pig IgG. Bovine, goat and human IgG were able to compete effectively with pig IgG whereas chicken IgY constituted a poor competitor. Peptide mapping analysis using denatured protein indicated that pig and bovine IgG recognized the same proteolytic fragment whereas chicken IgY did not. The smallest proteolytic fragment that retained the binding activity towards the IgG of the different animal species tested had a molecular mass of approximately 40 kDa. Fragments with M _r<40 kDa showed specific binding activities. That is, the smallest fragment binding pig and bovine IgG had a M _r of 30 kDa whereas for goat and human IgG a fragment of less than 16 kDa still showed binding activity. Finally, we observed that antisera raised against a heat-shock protein of Pseudomonas aeruginosa reacted with the 60-kDa S. suis protein indicating that the S. suis 60-kDa protein is a member of the 60-kDa hsp family that possesses the characteristic of binding in a non-immune way mammalian IgG and chicken IgY.     

The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish,Carassius auratus

Goldfish Carassius auratus were acclimated to either 10 or 30°C for a minimum of 5 weeks. A 65-kDa protein specific to warm-temperature-acclimated fish was extracted from the gel with 70% formic acid after two-dimensional electrophoresis of the muscle cytoplasmic protein fraction. The 65-kDa protein thus prepared to homogeneity was used to raise specific antibodies in rabbit by conventional methods. The antibody produced exhibited specific reaction with a protein having the same molecular weight from brain and liver tissue, suggesting that the 65-kDa protein is a ubiquitous cytosolic component in warm-acclimated goldfish. When water temperature was increased from 20 to 30°C over a 20-h period, a prominent amount of the 65-kDa protein was observed in muscle tissue extracts within 5 days of additional rearing; this was demonstrated by immunoblotting with the specific antibody. The N-terminal amino acid sequence of the 65-kDa protein was determined as Asp-Glu-Pro-Gln-Gly-His-Gln-His (or Asp)-Glu-Leu, differing from that of a family of known heat-shock proteins having about 70 kDa in molecular mass (hsp 70). No interaction between ATP and the 65-kDa protein revealed by ATP-agarose affinity chromatography further confirmed the different properties of the 65-kDa protein from those of hsp 70.Abbreviations ATP adenosine 5-triphosphate - hsp heat-shock protein(s) - IgG immunoglobulin G - mRNA messenger ribonucleic acid - PMSF phenylmethylsulphonyl fluoride - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Analysis of protein binding to heat shock protein 70 in pancreatic islet cells exposed to elevated temperatures or interleukin 1 beta

To elucidate a role for heat shock proteins in islet function, isolated pancreatic islets were labeled with [35S]methionine after control, heat shock, or interleukin 1 beta (IL-1 beta) treatment, extracted in the presence of detergent, and then passed over affinity columns with antibodies against heat shock protein 70 (hsp 70), hsp 70 itself, or ATP conjugated to the columns. In control or IL-1 beta-treated islets, the antibody column efficiently absorbed hsp 70 together with two other proteins of molecular masses 46 and 53 kDa. In extracts from heat-shocked cells, the binding of cellularly synthesized hsp 70 to the antibody column was inefficient but improved by the addition of unlabeled partially purified hsp 70 to the extracts. When assessing the binding of proteins in the extracts to the hsp 70 column, hsp 70 and the 46- and 53-kDa proteins among others all bound to the column. No differences in the patterns of binding to the hsp 70 column between extracts from the different islet exposures were noticed. The 46-kDa protein was identified as actin by immunoblot analysis. ATP-agarose column chromatography revealed a pattern of binding similar to that of the hsp 70 column. It is concluded that hsp 70 contains at least two functional domains, one adjacent to the epitope recognized by the antibody and active in restoring cellular function after heat shock, whereas the other has the ability to bind the 46- and 53-kDa and possibly other proteins. Furthermore, the stress induced by heat shock differs significantly from that after IL-1 beta treatment with respect to the functional behavior of hsp 70.     

The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate.

The 70-kDa heat shock protein hsp70 and its constitutively expressed cognate, hsc70, are abundant proteins implicated in a number of cellular processes. When a permeabilized cell system for examining the transport of proteins into the nucleus is depleted of hsc70 and hsp70, either by affinity chromatography on ATP-agarose or with antibodies against these proteins, nuclear transport activity is lost. Full activity is restored by the addition of HeLa proteins that bind to ATP-agarose. hsc70 and hsp70 are the active factors, since activity is also fully restored by the addition of either recombinant hsc70 or hsp70 which has been bacterially expressed and highly purified. The restoration of activity is saturable. The transport system requires other cytosolic factors as well, including at least one protein that is sensitive to inactivation by N-ethylmaleimide, but neither hsc70 nor hsp70 is the sensitive protein.     

Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes.

We characterized a 24-kDa protein associated with matrix hsp70 (mt-hsp70) of Neurospora crassa and Saccharomyces cerevisiae mitochondria. By using specific antibodies, the protein was identified as MGE, a mitochondrial homolog of the prokaryotic heat shock protein GrpE. MGE extracted from mitochondria was quantitatively bound to hsp70. It was efficiently released from hsp70 by the addition of Mg-ATP but not by nonhydrolyzable ATP analogs or high salt. A mutant mt-hsp70, which was impaired in release of bound precursor proteins, released MGE in an ATP-dependent manner, indicating that precursor proteins and MGE bind to different sites of hsp70. A preprotein accumulated in transit across the mitochondrial membranes was specifically coprecipitated by either antibodies directed against MGE or antibodies directed against mt-hsp70. The preprotein accumulated at the outer membrane was not coprecipitated by either antibody preparation. After being imported into the matrix, the preprotein could be coprecipitated only by antibodies against mt-hsp70. We propose that mt-hsp70 and MGE cooperate in membrane translocation of preproteins.     

Tetranectin-like protein in vertebrate serum: a comparative immunochemical analysis

The glycoprotein tetranectin (TN) found in human serum is a 90-kDa homotrimeric C-type lectin binding Ca²⁺, heparin and plasminogen kringle 4. TN is suggested as being implicated in tissue remodelling. The antigenic reactivity of putative TN was examined in serum from 14 different animal species using three sandwich enzyme immunoassays for human TN. Crab-eating macaque serum showed the strongest reaction, followed by horse and cat. Serum from cow, goat, pig, mouse and chicken reacted weakly, while dog, trout, and the amphibian and the reptile species did not react. The TN-like protein from macaque, horse and cat serum bound heparin and showed the same dependence on Ca²⁺ for interaction with the monoclonal antibodies as human TN. Gel filtration of sera from the three animal species showed that the TN-like protein eluted as single peaks with a M_r of 70–90 kDa. Western blotting of horse and cat TN-like protein electrophoresed under reducing conditions showed that the antibodies against human TN reacted with a single band with an approximate M_r of 30 kDa, indicating that the TN-like protein is also a homotrimer. Horse and cat TN-like protein interacted with human kringle 4-sepharose. Most likely, the reacting protein represents crab-eating macaque, horse and cat homologues of human TN.     

Immunization with heat shock protein 70 from methylcholanthrene-induced sarcomas induces tumor protection correlating with in vitro T cell responses

The cytosolic members of the heat shock protein 70 (hsp70) family have in recent years been shown to elicit protective immunity mediated via binding to antigenic peptides in tumor and viral animal models. In this study we have used the methylcholanthrene (MC)-induced sarcomas MC57S and MC57X, previously shown to express individually distinct MHC-I associated peptides recognized by tumor necrosis factor-alpha (TNF-alpha) producing CD8(+) T cells. With hsp70 purified from tumor or liver tissue, we were able to confirm that tumor-derived hsp70 elicited in vivo protection against a challenge with the same tumor as that used for hsp70 isolation. We also observed that immunizing with hsp70 isolated from tumor tissue resulted in a significantly better protection than immunizing with hsp70 isolated from the liver tissue of healthy mice. In two out of three experiments however, immunization with liver-derived hsp70 as well as tumor-derived hsp70 resulted in a significantly delayed tumor outgrowth as compared to saline-injected controls. In vitro, T cell lines from mice immunized with tumor-derived hsp70 could recognize tumor cells from the same MC57 tumor as that used for the hsp70 purification, resulting in TNF-alpha production. In sera from hsp70-immunized mice we observed undetectable or only very low levels of anti-hsp70 antibodies, suggesting that it is possible to repeatedly immunize the mice with no significant interference from neutralizing antibodies. We therefore conclude that hsp70 immunization can lead to protection against the tumor it was purified from, with a low risk of eliciting neutralizing antibodies that could affect further immunizations.     

The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies

Immunization with extracellular sexual stages of the malaria parasites can induce the production of antibodies which block the development of the parasites in the midgut of a mosquito after a blood meal. We have generated a number of monoclonal antibodies against gametes and zygotes of the human malaria Plasmodium falciparum. Two monoclonal antibodies (mAb) reacting with a 230-kDa gamete surface protein (mAb 1B3 and 2B4 both isotype IgG2a) were found to block transmission of P. falciparum to mosquitoes. Blocking was complement dependent and this was verified in vitro by the rapid lysis of newly formed gametes and zygotes in the presence of the mAb and active complement. Both mAb reacted by immunofluorescence with the surface of gametes and zygotes from isolates of P. falciparum from various geographical areas. Each mAb immunoprecipitated a 230-kDa protein from 125I-labeled surface proteins of newly formed gametes and zygotes and immunoblotted a protein doublet of about molecular mass 260 and 230 kDa from gametocytes and gametes of P. falciparum. Only the 230-kDa protein is expressed on the surface of newly formed macrogametes and zygotes. The 230-kDa gamete surface protein forms a molecular complex with two proteins of 48 and 45 kDa. The 48- and 45-kDa gamete surface proteins have previously been shown to be targets of mAb which block infectivity of P. falciparum to mosquitoes. The present study now demonstrates that antibodies against the 230-kDa gamete surface protein block transmission of P. falciparum to mosquitoes. The 230-kDa gamete protein is thus a potential candidate for a gamete vaccine.     

Monoclonal antibodies recognizing different epitopes of the 27-kDa gap junction protein from rat liver

Monoclonal antibodies (2-3E2, 6-3G11, and 7-3H6) against gap junction plaques purified from rat liver were prepared and characterized. Immunoblot analysis of liver gap junctions revealed that all three antibodies reacted with the 27-kDa protein, but not with the 22-kDa one. The 2-3E2 and 6-3G11 antibodies both reacted with the 27-kDa protein in gap junctions purified from livers of the rat, mouse, rabbit, and guinea pig; the 7-3H6 antibody, however, failed to react with the 27-kDa protein from guinea pig liver. The 7-3H6 antibody reacted strongly with the 24- to 26-kDa degradation products of the 27-kDa protein. Indirect immunofluorescence showed that the 6-3G11 and 7-3H6 antibodies both gave the same specific fluorescence labeling on rat liver cryosections, suggesting that these two antibodies recognized the cytoplasmic sites of the 27-kDa protein. Immunoblot analysis of protease-digested fragments from the 27-kDa protein revealed that the 7-3H6 antibody reacted with the 24- and 17-kDa fragments (including portions of the carboxyl-terminal domain of the 27-kDa protein) produced with endoproteinases Arg-C and Lys-C, respectively. Immunoblot analysis of CNBr fragments of the 27-kDa protein revealed that all three antibodies reacted with the 10-kDa fragment, which is thought to be the carboxyl-terminal domain of the 27-kDa protein. These results demonstrate that three monoclonal antibodies recognize different epitopes of the cytoplasmic sites (probably the carboxyl-terminal domain) of the 27-kDa liver gap junction protein.     

Detection of autoreactive antibodies to serum IgG and IgA in amoebic liver abscess cases.

Assessment of autoreactive antibodies in response to healthy human serum IgA and IgG was performed by indirect haemagglutination assay on serum samples from 81 amoebic liver abscess cases for IgA and 70 for IgG. Appropriate controls were taken simultaneously. IgA, IgG were isolated and purified from a healthy human serum through Sephadex G-200 and protein A CL 4B sepharose chromatography. These immunoglobulins were used for the detection of its own antibodies in amoebic liver abscess cases. This revealed that 43.20% and 48.50% of the cases were positive for IgA and IgG respectively, where as only 19.35% and 28.30% of the controls were in positive category (IgA and IgG respectively). The mean titres with standard deviation of the autoreactive antibodies to serum IgA both in ALA cases and controls shows a highly significant difference between tests and controls (P less than 0.001). Similarly the mean titres with standard deviation both in ALA and controls for the serum IgG differed significantly (P less than 0.001). This suggests the presence of autoreactive antibodies against serum IgA and IgG in amoebic liver abscess cases.     

SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein.

SSC1 is an essential member of the yeast HSP70 multigene family (E. Craig, J. Kramer, and J. Kosic-Smithers, Proc. Natl. Acad. Sci. USA 84:4156-4160, 1987). Analysis of the SSC1 DNA sequence revealed that it could encode a 70,627-dalton protein that is more similar to DnaK, an Escherichia coli hsp70 protein, than other yeast hsp70s whose sequences have been determined. Ssc1p was found to have an amino-terminal extension of 28 amino acids, in comparison with either Ssa1p, another hsp70 yeast protein, or Dnak. This putative leader is rich in basic and hydroxyl amino acids, characteristic of many mitochondrial leader sequences. Ssc1p that was synthesized in vitro could be imported into mitochondria and was cleaved in the process. The imported protein comigrated with an abundant mitochondrial protein that reacted with hsp70-specific antibodies. We conclude that Ssc1p is a mitochondrial protein and that hsp70 proteins perform functions in many compartments of the cell.     

Constitutive expression of heat-shock protein 70 in mammalian cells confers thermoresistance.

A 70-kDa heat-shock-protein (hsp 70) expression vector which contains the human hsp 70 gene linked to the human beta-actin promoter, was constructed and used to transfect CV1 monkey cells. Stably transfected CV1 clones were isolated which constitutively synthesized increased amounts of hsp70 at normal temperature. It is shown that these clones are resistant to elevated temperature. This finding indicates that hsp70 is involved in the protection of the cells against a lethal heat treatment and maybe responsible for the phenomenon of thermotolerance.     

The identification and partial characterization of merosin--a new specific protein of the basement membranes of certain tissues

A tissue-specific basement membrane-associated protein has been identified by the use of monoclonal antibodies prepared against a protein fraction of human placenta. In frozen sections of human tissues the monoclonal antibodies decorated basement membranes of Schwann cells, striated muscle, and trophoblast. In antibody-affinity chromatography of limited pepsin digests of human placenta, a 65-kDa polypeptide was bound by the monoclonal antibodies. Polyclonal antisera and new monoclonal antibodies were raised against the isolated 65-kDa polypeptide, and they stained human tissues identically to the original monoclonal antibodies. An 80-kDa polypeptide was detected by these antibodies in placental extracts prepared without proteolysis. The 65-kDa and 80-kDa polypeptides were immunologically distinct from laminin, type IV collagen, fibronectin and major serum proteins. These polypeptides are presumably derived from a novel basement membrane-associated protein which we named merosin. Several cDNA clones were isolated which code for a protein specifically recognized by polyclonal antibodies to the 65-kDa fragment. In developing mouse tissues, merosin was first detected at the newborn stage. The restricted tissue distribution and the late development appearance of merosin suggest that the protein has a tissue-specific function in highly differentiated cells.     

Immunofluorescence colocalization of the 90-kDa heat-shock protein and microtubules in interphase and mitotic mammalian cells

A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the water mold, Achlya ambisexualis, and that cross-reacts with the 90-kDa mammalian heat-shock protein (hsp90), and an antibody against tubulin were used to localize hsp90 and microtubules, respectively, in the same cultured rat endothelial and PtK1 epithelial cells by indirect immunofluorescence. AC88 and tubulin antibodies labeled the same structures in cells at all stages of the cell cycle, regardless of whether cells were permeabilized before or after fixation. Labeling of cell structures by both AC88 and anti-tubulin antibodies was identically affected by treating cells with colcemid. Double labeling with AC88 and anti-tubulin antibodies in interphase and mitotic cells is consistent with the conclusion that all microtubules are labeled and that no subclass of microtubules is preferentially labeled. Fluorescent labeling by AC88 was prevented by preabsorption of the antibody with purified rat hsp90 but was unaffected by preabsorption with purified 6S tubulin dimer. In contrast to AC88, fluorescent labeling by an anti-tubulin antibody was prevented by preabsorption with tubulin dimer but was unaffected by preabsorption with rat hsp90. Western-blot analysis demonstrated no cross-reactivity of AC88 for tubulin and no cross-reactivity of the anti-tubulin antibody for hsp90. A polyclonal antiserum fraction from a rabbit immunized with the 89-kDa heat-shock protein from chicken also labeled the mitotic apparatus in dividing cells and, somewhat less distinctly, fibrous structures in interphase cells. Labeling by hsp89 anti-serum was prevented by absorption with hsp90. AC88 also labeled microtubules in cultured mouse (L929 and 3T3), rat (endothelium and TRST), hamster (CHO) and primate (BSC, COS-1 and HeLa) cell lines. The demonstration of colocalization of hsp90 with microtubules should provide a valuable clue to eventual understanding of the cellular function of this ubiquitous, conserved and abundant stress-response protein.     

Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

The 56-59-kilodalton protein identified in untransformed steroid receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins

It has previously been shown that 9S, untransformed progestin, estrogen, androgen, and glucocorticoid receptor complexes in rabbit uterine and liver cytosols contain a 59-kDa protein [Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., & Faber, L. E. (1986) Biochemistry 25, 5269-5275]. In this work we show that the monoclonal antibody KN 382/EC1 raised against the rabbit 59-kDa protein reacts with 9S, untransformed glucocorticoid receptor complexes in cytosol prepared from human IM-9 lymphocytes but not with 4S salt-transformed receptors. The human protein recognized by the EC1 antibody is a 56-kDa protein (p56) of moderate abundance located predominantly in the cytoplasm by indirect immunofluorescence. There are at least six isomorphs of p56 by two-dimensional gel analysis. N-Terminal sequencing (20 amino acids) shows that p56 is a unique human protein. When p56 is immunoadsorbed from IM-9 cell cytosol, both the 70- and 90-kDa heat shock proteins are coadsorbed in an immune-specific manner. Neither heat shock protein reacts directly with the EC1 antibody. We conclude that p56 exists in cytosol in a higher order complex containing hsp70 and hsp90, both of which in turn have been found to be associated with untransformed steroid receptors.     

A calcium-binding protein with similarity to serum albumin localized to the ER-Golgi network and cell walls of spinach (Spinacia oleracea)

Using polyclonal antibodies raised against human serum albumin (HSA), a 70-kDa microsomal protein with an isoelectric point of approximately 6.5 was detected in spinach ( Spinacia oleracea L.). The protein was purified by selective ammonium sulfate precipitation and anion exchange HPLC. The protein shared 100% identity with the first 15 amino acids at the NH₂ terminus of HSA, including the X-X-H amino acid region, which was identified in HSA as being responsible for binding of copper, zinc, indole derivatives and calcium. Blue staining of the protein with the cationic carbocyanine dye 'Stains-all' and ⁴⁵Ca overlay following SDS-PAGE also suggest that the 70-kDa plant protein binds calcium. The protein reacted positively with carbohydrate specific thymol stain, and the carbohydrates associated with the protein were identified by gas chromatography-mass spectrometry (GC-MS) as galactose and galacturonic acid. The 70-kDa plant protein was present in the detergent-poor phase following Triton X-114 extraction of the microsomal proteins. Cell fractionation using continuous sucrose gradients showed that the protein is present in membrane fractions with high activity of endoplasmic reticulum (ER) and Golgi marker enzymes. Using nitrocellulose tissue prints probed with anti-HSA antibodies, we demonstrated that the protein is present in the apoplastic space of petioles, suggesting that the protein is secreted to the apoplast of cortex cells in plants. Localization and binding properties suggest that the plant protein identified in the present study may participate in secretion processes, possibly involved with the transport of precursors required for cell-wall synthesis.