Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains.

This study examined the distribution of the major outer sheath proteins (MOSP) in several Treponema denticola strains and reports the isolation of a 64-kDa protein from the outer sheath of human clinical isolate T. denticola GM-1. The outer sheath was isolated by freeze-thaw procedures, and the distribution of outer sheath proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). T. denticola GM-1, MS25, SR-5, and three low-passage clinical isolates possessed an MOSP with a relative molecular mass of 60 to 64 kDa. This MOSP was absent in T. denticola ATCC 35404 (TD-4) and clinical isolate SR-4. The latter possessed an MOSP of 58 kDa. 125I labeling revealed both MOSP to be dissociated forms of higher-molecular-mass oligomeric units between 116 and 162 kDa. Two-dimensional SDS-PAGE confirmed the modifiability of these MOSP. Isoelectric focusing of the 64-kDa MOSP indicated a pI of 6.7. Immunoblots with antiserum to GM-1 whole cells revealed the 64-kDa protein to be immunogenic and not cross-reactive with the MOSP of TD-4 or SR-4, and monospecific antibody to the 64-kDa protein recognized common epitopes on the high-molecular-weight oligomeric protein. These antibodies did not react with any component of TD-4 whole cells in immunoblots or in immunogold electron microscopy. Fab fragments inhibited the adherence of T. denticola GM-1 to human gingival fibroblasts by 78% (1:1,600; 0.72 micrograms of protein per ml), while TD-4 adherence was not inhibited. Amino acid analysis revealed a slightly acidic protein, devoid of cysteine, with 36% hydrophobic residues. Cyanogen bromide fragmentation of the 64-kDa protein revealed that a 42-kDa fragment contained a T-L-D-L-A-L-D segment which was 100% homologous with an integrin alpha subunit of a human leukocyte adhesion glycoprotein p 150,95.     

The kidney as a novel target tissue for protein adduct formation associated with metabolism of halothane and the candidate chlorofluorocarbon replacement 2,2-dichloro-1,1,1-trifluoroethane.

Hydrochlorofluorocarbons (HCFCs) have been identified as chemical replacements of the widely used chlorofluorocarbons (CFCs) that are implicated in stratospheric ozone depletion. Many HCFCs are structural analogues of the anesthetic agent halothane and may follow a common pathway of biotransformation and formation of adducts to protein-centered and other cellular nucleophiles. Exposure of rats to a single dose of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) or of the candidate CFC substitute HCFC 123 (2,2-dichloro-1,1,1-trifluoroethane) led to the formation of trifluoroacetylated protein adducts (CF3CO-proteins) not only in the liver, but also in the kidney as a novel target tissue for protein trifluoroacetylation. CF3CO-proteins in the kidney amounted to about 5% of those formed in the liver of the same animal. The amount of CF3CO-proteins formed within the kidney was roughly reflected by the capacity of metabolism of halothane or HCFC 123 by rat kidney microsomes in vitro which amounted to about 10% of that observed with liver microsomes. By immunohistochemistry, CF3CO-proteins in the kidney were mainly localized in the tubular segments of the cortex. In the liver, the density of CF3CO-proteins decreased from the central vein towards the portal triad. In vitro incubation of rat liver microsomes with halothane or HCFC 123 resulted in extensive formation of CF3CO-proteins and reproduced faithfully the pattern of liver CF3CO-proteins obtained in vivo. CF3CO-proteins generated in vitro were immunochemically not discernible from those generated in vivo. Glutathione (5 mM) and cysteine (5 mM) virtually abolished CF3CO-protein formation; the release of Br- from halothane and Cl- from HCFC 123 was reduced to much lesser a degree. S-Methyl-glutathione, N-acetyl-cysteine, methionine, and N-acetyl-methionine only slightly affected the formation of CF3CO-proteins or metabolism of either substrate. The data suggest that metabolism and concomitant CF3CO-protein formation of halothane or of candidate CFC replacements like HCFC 123 is not restricted to the liver but also takes place in the kidney. Furthermore, an in vitro system for CF3CO-protein formation has been developed and used to show that protein-centered and glutathione-centered nucleophilic sites compete for intermediates of metabolism of halothane or of HCFC 123.     

Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16 alpha-carbonitrile-inducible rat cytochromes P-450.

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.     

An immunoreactive form of erythrocyte protein 4.9 is present in non-erythroid cells

Using immunoblots and an affinity-purified antibody prepared against human erythrocyte protein 4.9, we have demonstrated and quantified the presence of an immunoreactive form of this protein in avian and bovine brain and lens tissues, avian heart, as well as in human platelets and mammalian, avian, piscine, and amphibian erythrocytes. Both the 48 kDa and the 52 kDa variants were observed in human erythrocytes, whereas 50 kDa and 54 kDa immunoreactive forms were observed in human platelets. As reported for erythroid protein 4.9, platelet protein 4.9 was phosphorylated in response to treatment with phorbol ester. Bovine brain showed five cross-reactive polypeptides in the 47 to 52 kDa range while avian brain and avian and bovine lens exhibited predominantly a 49-kDa band. Cross-reactivity was not observed in a number of cell lines and tissues including leukocytes, liver, kidney, pancreas, and skeletal muscle. Immunofluorescence indicated that protein 4.9 was present in cortical fiber cells of avian lens and in neurons of avian cerebrum.     

Epitopes of group A streptococcal M protein shared with antigens of articular cartilage and synovium

Rabbit antisera evoked by purified pepsin-extracted group A streptococcal M proteins were screened for the presence of joint cross-reactive antibodies by indirect immunofluorescence using thin sections of mouse knee joints. Pep M1, M5, and M18 antisera contained antibodies that cross-reacted with chondrocytes, cartilage, and synovium. Immunofluorescence inhibition assays showed that some of the joint cross-reactive epitopes were shared among the three heterologous serotypes of M protein. The pep M5 joint cross-reactive epitopes were localized to three different synthetic peptides of the C-terminal region of pep M5. Immunoblot analyses showed that the M5 joint cross-reactive antibodies recognized two proteins of human synovium and cartilage of molecular mass 56 and 58 kDa. The cross-reactive antibodies binding to the 56-kDa protein were inhibited by purified vimentin in immunoblot inhibition experiments. M protein-specific antibodies from patients with acute rheumatic fever were also shown to cross-react with joint tissue in a pattern similar to the rabbit antisera. Rabbit and human M protein-specific antibodies that were bound to articular cartilage activated significant levels of complement when compared to control serum, suggesting that M protein joint cross-reactive antibodies could potentially be involved in the pathogenesis of ARF and arthritis.     

A monoclonal antibody against chorion proteins of the sea bass Dicentrarchus labrax (Linnaeus, 1758): studies of chorion precursors and applicability in immunoassays

The monoclonal antibody DLE7 was obtained against 44- to 50-kDa polypeptides solubilized from the vitelline envelope of the Mediterranean sea bass Dicentrarchus labrax. In Western blot analysis of chorion lysates it recognized cross-reactive bands at 44 kDa, 48 kDa, and 110 kDa. Previous affinity blotting with concanavalin-A showed that most of solubilized bands were glycosylated. Enzymatic deglycosylation of chorion proteins followed by Western blot analysis with DLE7 showed that the 48-kDa and 110-kDa antigens were differentially affected by endoglycosidase-F treatment. When DLE7 was employed in immunofluorescence analysis, isolated chorions and ovarian cryosections stained intensely. Positivity was also observed in liver cryosections of spawning females but not in liver of males and nonspawning females. To study the origin and delivery of chorion proteins, DLE7 was used in Western blot analysis of liver homogenates and blood serum of spawning females. Cross-reacting bands were detected in liver (90 kDa) and serum (180 kDa, 50 kDa). DLE7 was also used for the first time to set up an indirect ELISA assay to detect egg antigens in the blood of egg-producing females, raising the possibility of using DLE7 as a female-specific marker of spawning for sea bass.     

Physicochemical characterization and monoclonal and polyclonal antibody recognition of Baylisascaris procyonis larval excretory-secretory antigens

Baylisascaris procyonis larval excretory-secretory (ES) antigens consisted of complex glycoproteins ranging from 10 kDa to over 200 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and lectin binding. Five monoclonal antibodies (Bapr1-Bapr5) produced against B. procyonis ES antigens were assayed by western blotting with larval ES antigens from B. procyonis, Baylisascaris melis, Baylisascaris transfuga, Ascaris suum, and Toxocara canis. Bapr1 and Bapr2 recognized periodate-sensitive epitopes on 14-kDa ES components of B. procyonis, B. melis, and B. transfuga, whereas Bapr4 and Bapr5 recognized periodate-resistant epitopes present on 55-kDa ES components of B. procyonis and B. melis. Bapr3 primarily recognized periodate-resistant epitopes on 33-45-kDa components of B. procyonis and B. melis ES. Heterologous rabbit antisera cross-reacted with many B. procyonis ES antigens on western blots, but recognition of the 33-45-kDa components was genus-specific. Normal human sera and T. canis-positive human sera also cross-reacted with many B. procyonis ES antigens, including those of 33-45 kDa. However, periodate oxidation markedly decreased cross-reactions and allowed for differential immunodiagnosis of B. procyonis versus T. canis. These studies demonstrated that antibody recognition of carbohydrate epitopes on ES components is an important cause of cross-reactions in antibody detection assays. Recognition of periodate-resistant (protein) epitopes on the 33-45-kDa B. procyonis ES components appears to be useful for genus-specific immunodiagnosis of larva migrans caused by Baylisascaris spp.     

Immunodetection and localization of protein(s) related to retinal S-antigen (arrestin) in kidney.

S-antigen (arrestin) is a cytosolic protein which regulates phototransduction in retinal rods. A protein immunologically related to S-antigen was identified in fractions from soluble extract of bovine kidney enriched by gel filtration or by immunoaffinity chromatography using a polyclonal antibody to retinal S-antigen. On immunoblots, this protein was recognized by a panel of monoclonal antibodies (mAbs S2D2, S1A3 and S9E2) directed against different S-antigen epitopes and displayed the same apparent molecular mass (48 kDa) as retinal S-antigen. All three mAbs revealed a specific immunoreactivity by indirect immunocytochemical technique on rat kidney sections. The three mAbs recognized some but not all glomerular cells, identified as epithelial cells by immunoelectron microscopy using the mAb S9E2. Both mAbs S2D2 and S1A3 gave a diffuse cytoplasmic staining in all tubule cells. Proximal tubule cells exhibited a weak immunoreactivity, whereas distal and collecting tubule cells were strongly labeled. In contrast, the mAb S9E2 immunoreaction was restricted to a cell subpopulation from distal and collecting tubules corresponding to intercalated cells identified by immunoelectron microscopy. With the mAb S9E2, the labeling of proximal tubule cells was localized in the apical region of the cytoplasm. These results suggest that two or more 48-kDa proteins immunologically cross-reactive with retinal S-antigen are present in kidney. The observed pattern of distribution is in keeping with the hypothesis that such proteins could play a role in the regulation of G-protein-related receptors present in renal glomerulus and tubule epithelial cells.     

Endoplasmic reticulum of rat liver contains two proteins closely related to skeletal sarcoplasmic reticulum Ca-ATPase and calsequestrin

Rat liver endoplasmic reticulum (ER) membranes were investigated for the presence of proteins having structural relationships with sarcoplasmic reticulum (SR) proteins. Western immunoblots of ER proteins probed with polyclonal antibodies raised against the 100-kDa SR Ca-ATPase of rabbit skeletal muscle identified a single reactive protein of 100 kDa. Also, the antibody inhibited up to 50% the Ca-ATPase activity of isolated ER membranes. Antisera raised against the major intraluminal calcium binding protein of rabbit skeletal muscle SR, calsequestrin (CS), cross-reacted with an ER peptide of about 63 kDa, by the blotting technique. Stains-All treatment of slab gels showed that the cross-reactive peptide stained metachromatically blue, similarly to SR CS. Two-dimensional electrophoresis (Michalak, M., Campbell, K. P., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 1317-1326) of ER proteins showed that the CS-like component of liver ER, similarly to skeletal CS, fell off the diagonal line, as expected from the characteristic pH dependence of the rate of mobility of mammalian CS. In addition, the CS-like component of liver ER was released from the vesicles by alkaline treatment and was found to be able to bind calcium, by a 45Ca overlay technique. From these findings, we conclude that a 100-kDa membrane protein of liver ER is the Ca-ATPase, and that the peripheral protein in the 63-kDa range is closely structurally and functionally related to skeletal CS.     

Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation.     

Characterization of a female-specific hepatic mitochondrial cytochrome P-450 whose steady-state level is modulated by testosterone

Polyclonal antibody to mitochondrial P-450c27/25 reacted with two proteins of apparent molecular masses of 52 kilodaltons (kDa) and 50 kDa from the female rat liver mitochondrial proteins bound to an omega-octylaminoagarose column. The two proteins were purified to greater than 85% homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography, and both were found to be P-450 as judged by dithionite-reduced CO difference spectra. Both of the P-450 forms required mitochondrial-specific ferredoxin and ferredoxin reductase for in vitro reconstitution of enzyme activities, suggesting that they are mitochondrial forms. The 52-kDa P-450 exhibited the properties of mitochondrial 27/25-hydroxylase with respect to high vitamin D3 25-hydroxylase activity [1.4 nmol (nmol of P-450)-1 min-1] and N-terminal amino acid sequence. The 50-kDa P-450, on the other hand, lacked significant vitamin D3 25-hydroxylase activity, but showed 17 beta-reductase [0.380-0.400 nmol (nmol of P-450)-1 min-1] and 17 beta-oxidase [0.1-0.16 nmol (nmol of P-450)-1 min-1] activities with both androgens and estrogens as substrates. Immunoblot analysis of proteins using a monoclonal antibody specific for P-450c27/25 showed a 2-3-fold higher level of this enzyme in the female liver mitochondria than in the males. Similarly, use of a polyclonal antibody in the immunoblot analysis showed that the 50-kDa P-450 is female-specific. The relative level of P-450c27/25 was reduced significantly in castrated females, while the level of the female-specific 50-kDa P-450 was increased. However, the levels of both enzymes were increased in castrated males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat-shock response in Legionella pneumophila

The heat-shock response of Legionella pneumophila was examined by radiolabelling bacterial cell proteins with [35S]methionine following a temperature shift from 30 to 42 degrees C. Five heat-shock proteins were identified as having molecular masses of 17, 60, 70, 78, and 85 kilodaltons (kDa). The 85- and 60-kDa proteins were equally distributed between supernatant and pellet fractions following ultracentrifugation at 100,000 x g, the 70- and 78-kDa proteins were found primarily in the supernatant, and the 17-kDa protein was found primarily in the pellet. Synthesis of subsets of the heat-shock proteins could be stimulated by novobiocin, patulin, or puromycin. Ethanol, an effector of the heat-shock response in other microorganisms, had little effect on L. pneumophila, even at the highest concentration tolerated by the bacterial cells (1.9%). Finally, the 60-kDa heat-shock protein of L. pneumophila was immunologically cross-reactive with a polyclonal antibody prepared to the Escherichia coli groEL protein. However, a mouse monoclonal antibody reactive with the 60-kDa protein of all legionellae tested did not cross-react with the E. coli groEL protein, suggesting that the Legionella 60-kDa protein contains common and unique epitopes.     

Biosynthesis, processing, and subcellular localization of rat spermbeta-D-galactosidase

During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm beta-D-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid beta-D-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that beta-D-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of beta-D-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of beta-D-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of beta-D-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of beta-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of beta-D-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.     

Immunopurification and structural analysis of a putative epithelial Cl- channel protein isolated from bovine trachea.

We have purified to homogeneity a 38-kDa protein (called p38) from bovine tracheal epithelium. This protein, when reconstituted into liposomes, mediates stilbene disulfonate-sensitive 125I- conductive uptake. On nonreduced or partially reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this protein associates into a doublet of 62-64 kDa. In some experiments a multimer of 141 kDa was also observed. Rabbit polyclonal anti-P38 antibodies have been produced and used to immunopurify the native transporter. Upon reconstitution of the immunoaffinity-purified protein into liposomes, a 260-fold enhancement of 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate and valinomycin-sensitive 125I- uptake was observed as compared to proteoliposomes containing unseparated material. On Western blots of total solubilized tracheal membrane proteins or semipurified fractions, the antibody recognized the 62-64-kDa doublet much better than the original 38-kDa antigen. Similar protein bands were detected in T84 and CFPAC cells as well. However, if apical membrane proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, the antibody recognized major bands at 140 and approximately 240 kDa. Upon partial reduction, immunolabeling of these proteins diminished with the concomitant appearance of the 62-64-kDa doublet. Upon complete reduction, the appearance of 32- and 38-kDa proteins was evident with the disappearance of the 62-64-kDa doublet. We hypothesize that the native Cl-channel is a heteromer containing at least four subunits connected by S-S bridges.     

Molecular mimicry between the human immunodeficiency virus type 1 gp120 V3 loop and human brain proteins.

Immunologically cross-reactive proteins in the human brain that resemble the V3 loop of human immunodeficiency virus type 1 (HIV-1) gp120 have been identified. When several homogenized tissues from normal brains were used, a monoclonal antibody raised against amino acids 308 to 320 of the V3 loop reacted with three prominent human brain proteins (HBP) of 35, 55, and 110 kDa. Among the three, the 55-kDa HBP appears to be specific to the central nervous system. These results indicate that the V3 loop of HIV-1 gp120 shares an epitope with HBP. An immune response to the V3 loop that generates cross-reactive antibodies to cellular proteins may be an autoimmune mechanism by which HIV-1 can damage the central nervous system.     

Insulin and insulinlike growth factor 1 (IGF-1) receptors during central nervous system development: expression of two immunologically distinct IGF-1 receptor beta subunits.

Insulin and insulinlike growth factor 1 (IGF-1) receptors are present in brain, yet their function remains obscure. Expression of these tyrosine kinase-bearing growth factor receptors during rat brain development was examined by using three antipeptide antibodies directed against epitopes in the beta subunits (AbP2, AbP4, and AbP5). All three antibodies recognized both insulin and IGF-1 receptors. Membranes were prepared from fetal brains (14 to 21 days of gestation), neonatal brain (postnatal day 1), and adult brain. Immunoblot analyses using AbP4 and AbP5 revealed a 92-kilodalton (kDa) protein that corresponded to the beta subunit of the insulin and IGF-1 receptors. Densitometric scanning of immunoblots indicated that receptor proteins were 4- to 10-fold more abundant in fetal brain membranes than in membranes from adult brain. Expression was highest during 16 to 18 days of gestation and declined thereafter to the relatively low level found in adult brain. Immunoblot analyses with AbP2 as well as ligand-activated receptor autophosphorylation revealed an additional protein of 97 kDa. This protein was phosphorylated in response to IGF-1 and was not directly recognized by AbP4 or AbP5. The covalent association of the 97-kDa protein with the 92-kDa beta subunit was indicated by the ability of AbP4 and AbP5 to immunoprecipitate both proteins under nonreducing conditions but only the 92-kDa protein after reduction. In contrast, AbP2 immunoprecipitated both proteins regardless of their association. This immunospecificity remained unchanged after deglycosylation of the isolated proteins. Two-dimensional tryptic phosphopeptide analysis showed that the 92- and 97-kDa subunits of the IGF-1 receptor are related but distinct proteins. Taken together, the data suggest that the 92- and 97-kDa subunits differ in primary amino acid sequence. Thus, two distinct beta subunits may be present in a single IGF-1 receptor in brain. These subunits have in common an epitope recognized by an antibody to the tyrosine kinase domain (AbP2) but differ in regions thought to be important in receptor kinase regulation and signal transduction.     

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.     

Analysis of mammalian dynein using antibodies against a polypeptides of sea urchin sperm flagellar dynein

Two different affinity-purified polyclonal antibodies were prepared against A polypeptides of dynein 1 extracted from sea urchin sperm. These antibodies, named AD1 and AD2, reacted exclusively with the alpha and beta heavy chains of dynein 1. Using these antibodies, we analyzed their cross-reactivity with dynein of mammalian cells. Immunohistochemically, both AD1 and AD2 stained dynein-related structures such as cilia of rabbit tracheal epithelia and flagella of rat spermatozoa. Immunoblots of the proteins extracted from mammalian cilia and flagella revealed the presence of A polypeptide-like proteins which cross-reacted with AD1 and AD2. Immunoblot analysis showed that the cross-reactive proteins were localized to the 370-kDa band of rabbit cilia and the 390- and 350-kDa bands of rat sperms. The reaction patterns showed that there were some differences between the two antibodies. On ciliary protein immunoblots, AD1 recognized about half of the broad band region which reacted with AD2, and AD1 also recognized only the 350-kDa band of the flagella extract, suggesting that the antibody reveals only a beta-like polypeptide. Immunoprecipitation studies using the ciliary proteins and AD2 confirmed that the immunoreactive protein had ATPase activity. Given these results, we have characterized mammalian dyneins previously reported by other laboratories.     

Characterization and purification of the hyaluronan-receptor on liver endothelial cells.

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.     

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.