Evidence of transferrin binding sites on the surface of Leishmania promastigotes.

A glycoprotein of 78,000 molecular mass (78 kDa), associated with the membrane of Leishmania infantum promastigotes, was identified and immunopurified by monoclonal antibody (mAb) LD9 produced against isolated membrane preparations. mAb LD9 was subsequently found to bind to human transferrin, also of 78 kDa. Binding of LD9 to transferrin was completely abolished when the mAb was preabsorbed by Leishmania membranes, thereby indicating that the 78-kDa Leishmania membrane-associated glycoprotein and transferrin have common antigenic epitope(s). The 78-kDa Leishmania membrane-associated protein was released in soluble nonaggregated form by mild treatment with acetic acid saline. Anti-transferrin polyclonal antibodies, recognized both the membrane-associated and the soluble form of the 78-kDa glycoprotein. The 78-kDa soluble form was characterized further as an iron-containing protein. The above data combined with iron uptake by promastigotes as demonstrated by the Prussian blue reaction indicate that the 78-kDa Leishmania membrane-associated glycoprotein is transferrin. The binding of 125I-human transferrin to Leishmania-purified membrane preparations was then investigated. The results indicate the presence of a high affinity saturable binding site (Kd = 2.2 10(-8) M) that is specific for transferrin. We suggest that the 78-kDa glycoprotein recognized by mAb LD9 is transferrin that binds to the surface of Leishmania promastigotes via a transferrin receptor.     

Amastigote and epimastigote stage-specific components of Trypanosoma cruzi characterized by using monoclonal antibodies. Purification and molecular characterization of an 83-kilodalton amastigote protein

Stage-specific mAb have been produced to amastigotes and epimastigotes of Trypanosoma cruzi (Brazil strain). mAb C-1 through C-6 reacted specifically with T. cruzi strains; no cross-reactions were found with membranes of promastigotes or amastigotes of Leishmania species. One mAb produced against the epimastigote membranes (C-5) was found to be specific against this stage by radioimmune binding assay, immunofluorescence, and radioimmunoprecipitation. mAb C-5 recognized a novel epimastigote protein at Mr (greater than 200,000) on immunoprecipitation with radiolabeled epimastigotes. Three amastigote stage-specific monoclonal antibodies were produced against membrane-enriched preparations of T. cruzi (Brazil strain) amastigotes grown in axenic culture (C-1 through C-3). By indirect immunofluorescence assay, monoclonal antibody C-2 bound only to T. cruzi amastigotes; no reaction with either tissue culture-derived trypomastigotes or epimastigotes was observed. mAb C-1 and C-2 each specifically immunoprecipitated a single protein molecule with Mr 83,000 from [35S]-methionine-labeled amastigotes. mAb C-2 was also used to affinity purify an 83-kDa Ag that was recognized by human Chagasic sera from patients of endemic countries of Latin America in an enzyme immunoassay. Amino acid composition and preliminary sequence data of the 83-kDa protein are presented. These mAb and/or purified Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of Chagas' disease.     

1F7, a novel cell surface molecule, involved in helper function of CD4 cells

We have developed a monoclonal antibody, anti-1F7, that inhibits soluble Ag-driven T cell proliferation as well as PWM-driven IgG synthesis. Anti-1F7 antibody reacts with approximately 57% of unfractionated T cells, 62% of CD4+ cells, and 54% of CD8+ cells. Although the 1F7 Ag is widely distributed among lymphoid cells, this Ag on CD4+ cells is preferentially expressed on the CDw29(4B4+) helper population. Moreover, anti-1F7 antibody further subdivides the CD4+CDw29+ cell subset into CDw29+1F7+ and CDw29+1F7- populations. The CD4+CDw29+1F7+ population of cells maximally proliferates to recall Ag such as tetanus toxoid, whereas helper function for PWM-driven IgG synthesis by B cells belongs to both the CD4+CDw29+1F7+ and CD4+CDw29+1F7- population of cells. The most prominent structure defined by this antibody is a 110-kDa molecule that is different from the 135-kDa, 160-kDa, and 185-kDa glycoproteins identified by anti-CDw29 antibody and the 180-kDa glycoprotein identified by UCHL-1 antibody. It is, however, related to the molecule recognized by anti-Ta1, an activation Ag on T cells. Furthermore, although the Ta1 molecule is recognized by anti-1F7 mAb, the 1F7 family of structures also includes molecules distinct from Ta1.     

Structural analysis of rod GTP-binding protein, Gt. Limited proteolytic digestion pattern of Gt with four proteases defines monoclonal antibody epitope.

The epitope of monoclonal antibody (mAb 4A), which recognizes the alpha subunit of the rod G protein, Gt, has been suggested to be both at the carboxyl terminus (Deretic, D., and Hamm, H.E. (1987) J. Biol. Chem. 262, 10839-10847) and the amino terminus (Navon, S.E., and Fung, B.K.-K. (1988) J. Biol. Chem. 263, 489-496) of the molecule. To characterize further the mAb 4A binding site on alpha t and to resolve the discrepancy between these results limited proteolytic digestion of Gt or alpha t using four proteases with different substrate specificities has been performed. Endoproteinase Arg-C, which cleaves the peptide bond at the carboxylic side of arginine residues, cleaved the majority of alpha t into two fragments of 34 and 5 kDa. The alpha t 34-kDa fragment in the holoprotein, but not alpha t-guanosine 5'-O-(3-thiotriphosphate), was converted further to a 23-kDa fragment. A small fraction of alpha t-GDP was cleaved into 23- and 15-kDa fragments. Endoproteinase Lys-C, which selectively cleaves at lysine residues, progressively removed 17 and then 8 residues from the amino terminus, forming 38- and 36-kDa fragments. Staphylococcus aureus V8 protease is known to remove 21 amino acid residues from the amino-terminal region of alpha t, with the formation of a 38-kDa fragment. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin cleaved alpha t progressively into fragments of known amino acid sequences (38, then 32 and 5, then 21 and 12 kDa) and a transient 34 kDa fragment. The binding of mAb 4A to proteolytic fragments was analyzed by Western blot and immunoprecipitation. The major fragments recognized by mAb 4A on Western blots were the 34- and 23-kDa fragments obtained by endoproteinase Arg-C and tryptic digestion. Under conditions that allowed sequencing of the 15- and 5-kDa fragments neither the 34- nor the 23-kDa fragments could be sequenced by Edman degradation, indicating that they contained a blocked amino terminus. The smallest fragment that retained mAb 4A binding was the 23-kDa fragment containing Met1 to Arg204. Thus the main portion of the mAb 4A antigenic site was located within this fragment, indicating that the carboxyl-terminal residues from Lys205 to Phe350 were not required for recognition by the antibody. Additionally, the antibody did not bind the 38- and 36-kDa or other fragments containing the carboxyl terminus, showing that the amino-terminal residues from Met1 to Lys17 were essential for antibody binding to alpha t.     

Identification of B cell epitopes of dengue virus 2 NS3 protein by monoclonal antibody

Dengue virus is a major international public health concern, and there is a lack of available effective vaccines. Virus-specific epitopes could help in developing epitope peptide vaccine. Previously, a neutralizing monoclonal antibody (mAb) 4F5 against nonstructural protein 3 (NS3) of dengue virus 2 (DV2) was developed in our lab. In this work, the B cell epitope recognized by mAb 4F5 was identified using the phage-displayed peptide library. The results of the binding assay and competitive inhibition assay indicated that the peptides, residues 460–469 (U460-469 RVGRNPKNEN) of DV2 NS3 protein, were the B cell epitopes recognized by mAb 4F5. Furthermore, the epitope peptides and a control peptide were synthesized and then immunized female BALB/c mice. ELISA analysis showed that immunization with synthesized epitope peptide elicited a high level of antibody in mice, and immunofluorescent staining showed that the antisera from fusion epitope-immunized mice also responded to DV2 NS3 protein, which further characterized the specific response of the present epitope peptide. Therefore, the present work revealed the specificity of the newly identified epitope (U460-469) of DV2 NS3 protein, which may shed light on dengue virus (DV) vaccine design, DV pathogenesis study, and even DV diagnostic reagent development.     

Involvement of protein serine and threonine phosphorylation in human sperm capacitation.

The involvement of serine and threonine phosphorylation in human sperm capacitation was investigated. Anti-phosphoserine monoclonal antibody (mAb) recognized six protein bands in the 43-55-kDa, 94 +/- 2-kDa, 110-kDa, and 190-kDa molecular regions, in addition to a faint band each in the 18-kDa and 35-kDa regions. Anti-phosphothreonine mAb recognized protein bands in six similar regions, except that the 18-kDa, 35-kDa, and 94 +/- 2-kDa protein bands were sharper and thicker, and an additional band was observed in the 110-kDa molecular region. In the 43-55-kDa molecular region, there was a well-characterized glycoprotein, designated fertilization antigen, that showed a further increase in serine/threonine phosphorylation after exposure to solubilized human zona pellucida. In a cell-free in vitro kinase assay carried out on beads or in solution, four to eight proteins belonging to similar molecular regions, namely 20 +/- 2 kDa, 43-55 kDa, 94 +/- 2 kDa, and 110 +/- 10 kDa, as well as in 80 +/- 4 and 210 +/- 10 kDa regions, were phosphorylated at dual residues (serine/tyrosine and threonine/tyrosine). Capacitation increased the intensity of serine/threonine phosphorylation per sperm cell, increased the number of sperm cells that were phosphorylated, and induced a subcellular shift in the serine/threonine-specific fluorescence. These findings indicate that protein serine/threonine phosphorylation is involved and may have a physiological role in sperm capacitation.     

cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen

The Ku antigen is a DNA-associated nuclear protein recognized by sera from patients with autoimmune diseases. It consists of two polypeptides of 86 and 70 kDa. cDNA clones encoding the 86-kDa subunit of the Ku antigen were isolated by probing lambda gt11 recombinant cDNA expression libraries with a monoclonal antibody specific for this protein. The amino acid sequence deduced from the cDNA comprises 732 amino acids and corresponds to a protein with molecular weight of 81.914. Nineteen residues at the NH2 terminus determined by protein sequencing corresponded to the sequence deduced from the cDNA. The predicted amino acid sequence contains a region with repeating leucine residues similar to the "leucine zipper" structure observed in the c-myc, v-myc, and c-fos oncogene products. The largest cDNA hybridized to 2.7- and 3.4-kilobase poly(A)+ mRNAs from HeLa cells. The cDNA clones expressed fusion proteins immunoreactive with the monoclonal antibody and sera from patients with autoimmune diseases.     

Purification and characterization of maturation-promoting factor in fish.

Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13suc1-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.     

Naegleria fowleri: enolase is expressed during cyst differentiation

Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri.     

A cell surface glycoprotein of rat basophilic leukemia cells close to the high affinity IgE receptor (Fc epsilon RI). Similarity to human melanoma differentiation antigen ME491.

A monoclonal antibody (mAb), AD1, was isolated that recognized a cell surface protein on rat basophilic leukemia cells (RBL-2H3). At high concentration, this antibody inhibited IgE-mediated but not calcium ionophore-induced histamine release (49% inhibition at 100 micrograms/ml). The mAb AD1 did not inhibit the binding of IgE or of several antibodies directed to the high affinity IgE receptor (Fc epsilon RI). Likewise, IgE did not inhibit mAb AD1 binding. However, several anti-Fc epsilon RI antibodies did inhibit mAb AD1 binding as intact molecules but not as Fab fragments. Therefore, the sites on the cell surface to which mAb AD1 binds are close to Fc epsilon RI. The mAb AD1 immunoprecipitated a broad, 50-60-kDa band from 125I-surface-labeled RBL-2H3 cells that upon peptide N-glycosidase F treatment was transformed into a sharp 27-kDa band. A similar 27-kDa protein was immunoprecipitated from surface-radiolabeled cells after culture with tunicamycin. Thus, the protein recognized by mAb AD1 is highly glycosylated with predominantly N-linked oligosaccharides. The N-terminal sequence of 43 amino acids was found to be different from any subunit of Fc epsilon RI but nearly identical to that of the human melanoma-associated antigen ME491. Therefore, mAb AD1 binds to a surface glycoprotein on RBL-2H3 cells sterically close to the Fc epsilon RI but distinct from the recognized subunits of the receptor.     

The primary structure of the 32-kDa subunit of human replication protein A

Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit. The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit. No significant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.     

Identification of the heterogeneous nuclear ribonucleoprotein A2/B1 as the antigen for the gastrointestinal cancer specific monoclonal antibody MG7

MG7 is an early gastrointestinal cancer specific monoclonal antibody. It can detect gastric cancer with high sensitivity and specificity. However, the target antigen for MG7 has not been identified. Western blot analysis revealed that the MG7 antibody reproducibly recognized two approximately 35 kDa proteins in the total cell lysates of human gastric carcinoma cell lines KATO III and MKN-45. Using a proteomic approach, we identified these MG7 immunoreactive proteins as the human heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). Western blot analysis of nuclear and cytosolic fraction of KATO III cells using either MG7 or hnRNP A2/B1 antibodies confirmed that the target antigen is located exclusively in the nucleus. With the use of archival samples, we also found that the level of hnRNP A2/B1 protein was increased in gastric cancer tissues (4 out of 5 patients), when compared to their corresponding matching normal stomach tissue.     

Combination Therapy Using Monoclonal Antibodies against Respiratory Syncytial Virus (RSV) G Glycoprotein Protects from RSV Disease in BALB/c Mice

Therapeutic options to control respiratory syncytial virus (RSV) are limited, thus development of new therapeutics is high priority. Previous studies with a monoclonal antibody (mAb) reactive to an epitope proximal to the central conserved region (CCR) of RSV G protein (mAb 131-2G) showed therapeutic efficacy for reducing pulmonary inflammation RSV infection in BALB/c mice. Here, we show a protective effect in RSV-infected mice therapeutically treated with a mAb (130-6D) reactive to an epitope within the CCR of G protein, while treatment with a mAb specific for a carboxyl G protein epitope had no effect. Combined treatment with mAbs 130-6D and 131-2G significantly decreased RSV-associated pulmonary inflammation compared to either antibody alone. The results suggest that anti-RSV G protein mAbs that react at or near the CCR and can block RSV G protein-mediated activities are effective at preventing RSV disease and may be an effective strategy for RSV therapeutic treatment.     

Comparison of the 34,000-Da pp60src substrate and a 38,000-Da phosphoprotein identified by monoclonal antibodies

One of the cellular targets of the pp60src tyrosine kinase is a phosphoprotein with a Mr = 34,000 and an isoelectric point of approximately 7.5 (Radke, K., Gilmore, T., and Martin, G. S. (1980) Cell 21, 821-828; Erikson, E., and Erikson, R. L. (1980) Cell 21, 829-836). We report here the preparation of monoclonal antibodies to partially purified 34-kDa protein and to a heretofore unrecognized phosphoprotein that is not a pp60src target. Two antibodies were initially obtained that recognized phosphoproteins in the Mr = 34,000-39,000 range. One of these antibodies immunoprecipitated a 34,000-Da protein which, on the basis of its molecular mass, phosphorylation state, and isoelectric point, was determined to be the 34-kDa pp60src substrate. The second monoclonal antibody bound to a 38,000-Da nucleolar associated protein, which appeared not to be a target of the pp60src kinase and was found by tryptic analysis to be structurally unrelated to the 34-kDa protein. The monoclonal antibody to the 34-kDa protein coupled to Sepharose CL-4B was used to purify the pp60src substrate to homogeneity in milligram quantities. Both the purified 34-kDa protein and the monoclonal antibody are currently being used in studies aimed at elucidating the structure and function of this pp60src target.     

Clonal T lymphocyte recognition of the fine structure of the HLA-A2 molecule

A human alloimmune cytotoxic T lymphocyte (CTL) clone (4E4) was generated against the HLA-A2 molecule. Lysis of 51Cr-labeled HLA-A2 target cells was blocked by monoclonal antibodies (mAb), including mAb PA2.1 (anti-HLA-A2), mAb BB7.2 (anti-HLA-A2), mAb 4B (anti-HLA-A2-plus-A28), mAb MA2.1 (anti-HLA-A2-plus-B17), and mAb W6/32 (anti-HLA-A,B,C), which are directed against different serologic epitopes on the HLA-A2 molecule. However, HLA-A2 mutant lines lacking the serologic epitope recognized by mAb BB7.2 (anti-HLA-A2) were efficiently lysed by CTL 4E4. Thus, although mAb may block cytolysis, the HLA-A2 epitope recognized the 4E4 CTL clone is distinct from the HLA-A2-specific epitope recognized by serologic reagents. Moreover, analysis of HLA-A2 population variants revealed that only the predominant HLA-A2.1 subtype molecule was recognized by CTL 4E4. No cross-reactivity on other, biochemically related HLA-A2 population subtypes was observed, including HLA-A2.2 cells (Hill, CVE, ZYL, M7), HLA-A2.3 cells (TENJ, DK1), or HLA-A2.4 cells (CLA, KNE). This CTL clone appears to recognize a single epitope and, like monoclonal antibody counterparts, can be used to discriminate among immunogenic cellular and serologic epitopes on closely related HLA-A2 molecules. On the basis of the known sequence changes in mutant and subtype HLA-A2 molecules, it appears that the sequence spanning residues 147 to 157 may be critical for cellular recognition of this Class I MHC molecule.     

Use of a heat-stable microtubule-associated protein class-specific antibody to investigate the mechanism of microtubule binding.

Members of the heat-stable family of microtubule-associated proteins (MAPs), MAP 2, tau, and MAP 4, contain three or four tandem imperfect repeated sequences close to their carboxyl termini. These sequences lie within the microtubule-binding domains of the MAPs; they have been proposed to be responsible for microtubule binding and the ability of these MAPs to lower the critical concentration for microtubule assembly. Their spacing may reflect that of the regularly arrayed tubulin subunits on the microtubule surface. We here characterize the 32- and 34-kDa chymotryptic microtubule-binding fragments of MAP 2 identified in earlier work. We identify the primary chymotryptic cleavage site in high molecular weight MAP 2 as between Phe1525 and Lys1526, within 13 amino acids of the known MAP 2 splice junction. We have raised a monoclonal antibody to the 32- and 34-kDa fragments and find that it reacts with all members of the heat-stable MAPs class. To determine where it reacts, we sequenced immunoreactive subfragments of the 32- and 34-kDa fragments, selected several cDNA clones with the antibody, and tested for antibody reactivity against a series of synthetic MAP 2 and tau peptides. We identify the epitope sequence as HHVPGGG (His-His-Val-Pro-Gly-Gly-Gly). The antibody also recognized several other MAP 2 and tau repeats. Despite reacting with this highly conserved element, we find that the antibody does not block microtubule binding, but binds to the MAPs and co-sediments with microtubules. These results suggest that there are other regions besides the repeated elements which are essential for microtubule binding.     

Isolation and characterization of goldfish cdk2, a cognate variant of the cell cycle regulator cdc2.

This paper reports the nucleotide and predicted amino acid sequences of the goldfish cdk2, a cognate variant of the cell cycle regulator cdc2. The predicted protein sequence shows strong homology to the other known cdk2 (88% for Xenopus and 90% for human). A monoclonal antibody against the C-terminal sequence of goldfish cdk2 recognized a 34-kDa protein in extracts from various goldfish tissues. The protein level was high in such tissues as testis and ovary containing actively dividing cells. Protein cdk2 binds to p13sucl, the fission yeast suc1+ gene product, but not to cyclin B, with which cdc2 forms a complex. The kinase activity of cdk2 increased 30-fold when oocytes matured, although its protein level did not remarkably change. Anti-cdk2 immunoprecipitates from 32P-labeled mature oocyte extracts contained a 47-kDa protein, which was not recognized by either anti-cyclin A or anti-cyclin B antibody, indicating complex formation of cdk2 with a protein other than cyclins A or B.     

Functional expression of NADPH oxidase components (alpha- and beta-subunits of cytochrome b558 and 45-kDa flavoprotein) by intrinsic human glomerular mesangial cells.

Recently, we have shown that human glomerular mesangial cells (HMCs) release oxygen radicals from the plasma membrane in response to cytokines. Now we have used diphenylene iodonium, a covalent binding inhibitor of activated 45-kDa flavoprotein, in neutrophils radiolabeled with 125I and could identify a 45-kDa protein band in a separated HMC plasma membrane fraction. Low temperature difference spectroscopy showed a peak absorbance at 428 and 558 nm. Direct potentiometry of HMC membranes (-340 to -160 mV) showed the presence of a low potential cytochrome (76 pmol/mg to HMC membrane protein) identified as cytochrome b558. In slot blots, mouse monoclonal antibody (mAb) 7D5, specific for the extracellular domain of the alpha-subunit, showed a positive reaction with HMCs. In Western blots, mAb 449, directed against the cytoplasmic epitope of the alpha-subunit, identified a 23-kDa protein; and mAb 48, raised against the large (beta) subunit of cytochrome b558 of human neutrophils (Verhoeven, A. J., Bolscher, B. G. J. M., Meerhof, L. J., van Zwieten, R., Keijer, J., Weening, R. S., and Roos, D. (1989) Blood 73, 1686-1694), detected a smear between 75 and 100 kDa in denatured HMC membrane protein. These data determined with HMCs, suggest for the first time the expression of three essential components of NADPH:O2- oxidoreductase in mesenchymal cells.     

The phosphorylation of human link proteins

Three link proteins of 48,44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain 32P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO4/mole link protein.     

A monoclonal antibody that recognizes mammalian cortical granules and a 32-kilodalton protein in mouse eggs

The fertilization-induced exocytosis of egg cortical granules (CGs) is responsible for a block to polyspermy, crucial to the viability of many species. The contents of mammalian CGs have been an elusive target for analysis because of picogram quantities of CG proteins. By using media enriched in secreted CG contents from calcium ionophore-induced eggs as an immunogen, a monoclonal antibody was raised that immunolocalized to structures in the mouse egg cortex with all the hallmarks of CGs. These structures were the correct size, absent from the region over the metaphase II spindle, and greatly reduced after fertilization. Double-labeling experiments confirmed that the antibody recognized the same population of CGs as those recognized by Lens culinaris agglutinin. On Western blots, the antibody primarily recognized a 32-kDa protein (and secondarily one at approximately 25 kDa) in mouse eggs. Analysis of biotin-labeled secreted proteins from activated eggs confirmed that CGs release only a small number of major proteins (45, 34, 32, 28, and approximately 20 kDa by SDS-PAGE). We therefore propose that the 32-kDa protein identified by this antibody is likely to correspond to the 32-kDa protein released from activated eggs and that it may be involved in the block to polyspermy. These methods should make it possible to generate additional antibodies to study the structure of CG components as well as their roles in the polyspermy block and CG biogenesis.