Biochemical and genetic investigations on gap junctions from mammalian cells

Gap junction protein (26K) in mouse or rat liver has been studied using a rabbit antiserum directed against the sodium dodecylsulfate denatured 26K protein from mouse liver. The liver 26K protein has been localized in gap junction plaques of hepatic plasma membranes by immuno electron microscopy. Affinity purified anti-26K antiserum showed weak cross reactivity with mouse or bovine lens gap junction protein (MIP26). This result suggests some structural homology between the different gap junction proteins in liver and lens. After partial hepatectomy of young rats the liver 26K protein appears to be degraded and later resynthesized. A variant of established Chinese hamster fibroblastoid cells has been isolated and shown to be defective in metabolic cooperation via gap junctions.     

Biochemical and genetic investigations on gap junctions from mammalian cells

Gap junction protein (26K) in mouse or rat liver has been studied using a rabbit antiserum directed against the sodium dodecylsulfate denatured 26K protein from mouse liver. The liver 26K protein has been localized in gap junction plaques of hepatic plasma membranes by immuno electron microscopy. Affinity purified anti-26K antiserum showed weak cross reactivity with mouse or bovine lens gap junction protein (MIP26). This result suggests some structural homology between the different gap junction proteins in liver and lens. After partial hepatectomy of young rats the liver 26K protein appears to be degraded and later resynthesized. A variant of established Chinese hamster fibroblastoid cells has been isolated and shown to be defective in metabolic cooperation via gap junctions.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Immunocytochemical localization of the gap junction 26 K protein in mouse liver plasma membranes

Specific binding sites for anti-26 K antibodies directed against the liver gap junction protein (26 K) were localized by immunoelectron microscopy in gap junction plaques purified from hepatic plasma membranes. Using immunofluorescence microscopy we found discrete fluorescent spots on plasma membranes in cross sections of liver tissues after incubation with anti-26 K antibodies. This is consistent with the notion of specific binding to gap junction plaques. Quantitative binding of anti-26 K antibodies was indirectly measured by the protein A-gold technique. We found that urea/detergent-treated, purified gap junction plaques bind 30-fold more anti-26 K antibodies than preimmune serum. Anti-26 K antibodies also bind specifically to native gap junction plaques within hepatic plasma membranes although only about one fifth as efficiently as to purified plaques. Possibly the anti-26 K antibodies raised after injection of SDS-denatured 26 K protein into rabbits recognize the cytoplasmic face of urea/detergent-treated plaques better than that of native plaques. Some, if not most, of the vesicular structures in preparations of purified plaques appear to be derived from split gap junction plaques and are probably sheets of gap junction hemichannels. In some vesicles the former cytoplasmic face of the hemichannels is turned outside, other vesicles have the former cell surface turned outside. The anti-26 K antibodies do not recognize any 26 K protein on the sheets of partially split gap junction plaques, on the heterogeneous vesicular structures, or on non-junctional areas of hepatic plasma membranes. These results suggest that the conformation of the 26 K protein in plaques must be different from that of the 26 K protein in earlier biosynthetic steps of plaque assembly.     

Identification as Synapsin of a Synaptosomal Protein Immunoreacting with Anti-Myelin Basic Protein Antiserum

Rat brain proteins able to react with anti-myelin basic protein antiserum, raised under conditions to induce experimental allergic encephalomyelitis in rabbits, were examined by immunoblot methods after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Apart from the four forms of myelin basic protein present in rat brain, the antiserum detected other proteins of higher molecular weight. Subcellular fractionation shows that these high-molecular-weight proteins are relatively concentrated in a synaptosome-enriched fraction compared to a myelin fraction. A major protein fraction immunorelated to myelin basic protein migrated in the gels as a doublet with apparent molecular weights of approximately 80K and 86K; these proteins were tentatively identified as synapsin Ia and Ib. A purified synapsin preparation analyzed by immunoblot after two-dimensional gel electrophoresis also reacted with anti-myelin basic protein antisera. When the serum was purified by affinity chromatography on a myelin basic protein-conjugated Sepharose column the nonadsorbed material lost this activity whereas the eluted antibodies reacted with myelin basic protein and synapsin. In addition, sequence amino acid comparison of decapeptides showed some homology between these two proteins. A possible implication of immunological agents against myelin basic protein cross-reacting with extra-myelin proteins in the process of experimental allergic encephalomyelitis is considered.     

Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues

Rat heart and other organs contain mRNA coding for connexin43, a polypeptide homologous to a gap junction protein from liver (connexin32). To provide direct evidence that connexin43 is a cardiac gap junction protein, we raised rabbit antisera directed against synthetic oligopeptides corresponding to two unique regions of its sequence, amino acids 119-142 and 252-271. Both antisera stained the intercalated disc in myocardium by immunofluorescence but did not react with frozen sections of liver. Immunocytochemistry showed anti-connexin43 staining of the cytoplasmic surface of gap junctions in isolated rat heart membranes but no reactivity with isolated liver gap junctions. Both antisera reacted with a 43-kD polypeptide in isolated rat heart membranes but did not react with rat liver gap junctions by Western blot analysis. In contrast, an antiserum to the conserved, possibly extracellular, sequence of amino acids 164-189 in connexin32 reacted with both liver and heart gap junction proteins on Western blots. These findings support a topological model of connexins with unique cytoplasmic domains but conserved transmembrane and extracellular regions. The connexin43-specific antisera were used by Western blots and immunofluorescence to examine the distribution of connexin43. They demonstrated reactivity consistent with gap junctions between ovarian granulosa cells, smooth muscle cells in uterus and other tissues, fibroblasts in cornea and other tissues, lens and corneal epithelial cells, and renal tubular epithelial cells. Staining with the anti-connexin43 antisera was never observed to colocalize with antibodies to other gap junctional proteins (connexin32 or MP70) in the same junctional plaques. Because of limitations in the resolution of the immunofluorescence, however, we were not able to determine whether individual cells ever simultaneously express more than one connexin type.     

Immunological identification of four different polypeptides in 'subunit VII' of mammalian cytochrome c oxidase

Rat liver cytochrome c oxidase was separated by SDS-gel electrophoresis into 13 polypeptide bands. Monospecific antisera against the isolated polypeptides VIIa, VIIb and VIIc were raised in rabbits. Cytochrome c oxidase was blotted on nitrocellulose and incubated with the antisera. The antisera reacted only with their corresponding polypeptides, indicating no immunological relationship between polypeptides VIIa, VIIb and VIIc. The data also exclude that these polypeptides are proteolytic breakdown products of larger subunits.     

Immunofluorescent localization of 100K coated vesicle proteins

A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.     

Antigenic, molecular and functional heterogeneity of Clara cell secretory proteins in the rat

In an earlier publication we had reported the preparation of a rabbit antiserum specific for rat Clara cell secretory proteins. This rabbit anti-rat Clara cell serum was found to react with two proteins in rat lung lavage by crossed-immunoelectrophoresis. Immunoblotting of rat lung lavage proteins, after sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis, disclosed three bands of reactivity with anti-Clara cell serum. The relative molecular masses of these three proteins were about 200 (protein A) 55 (protein B) and about 12 kDa (protein C). Anti-Clara cell antibodies eluted from Sepharose-4B-linked protein C (as well as the antiserum raised by immunizing rabbits with protein C) reacted with proteins A and C. Anti-Clara cell antiserum unbound to proteins A and C (as well as antiserum raised by immunizing rabbits with protein B) reacted with protein B only. In non-SDS polyacrylamide gel electrophoresis, protein B migrated as a single band, slightly cathodic to albumin; protein C resolved into three bands, all anodic to albumin. Immunoblots of isoelectric focusing gels showed three bands (pI 5.2-5.7) that reacted with antibody to protein C, and four bands corresponding to protein B were seen in the pI range 4.6-5.0. As determined by immunoperoxidase staining of paraformaldehyde fixed methacrylate embedded 1 micron thick sections of rat lung, protein(s) A (and protein C) and protein B were present in the same cells and in the same granules. Protein B was resistant to trypsin digestion, whereas proteins A and C were readily degraded by trypsin. Rat Clara cell secretory proteins consist of at least two antigenic types that appear to be functionally distinct, and each antigenic type displays charge microheterogeneity.     

A polyclonal antiserum against the rabbit progesterone receptor recognizes the human receptor: biochemical characterization

Polyclonal antiserum was generated in guinea pigs immunized with the 116,000 Mr rabbit uterine progesterone receptor (PR). The PR antigen was partially purified by DEAE-cellulose chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and the 116,000 Mr band excised and injected into guinea pigs. The antiserum recognized on protein blots rabbit uterine PR of Mr 116,000 and 81,000. The antiserum was judged to be specific for PR from normal and malignant human tissues as determined by sedimentation shift on sucrose gradients, immunoprecipitation studies, protein blotting, and fluorographic analysis using photolabelled samples. Comparison of protein blots probed with this polyclonal antiserum or with a recently obtained monoclonal antibody to human PR indicated that similar PR structures were recognized in rabbit and human samples by both antisera. Characterization of the polyclonal antiserum has demonstrated its suitability for investigating the immunolocalization or PR in normal and malignant human tissues as well as the receptor structure detected on protein blots.     

Serological comparison of polyhedron protein and virions from four nuclear polyhedrosis viruses of plusiine larvae (Lepidoptera: Noctuidae)

Purified polyhedron proteins and purified, ultrasonicated virions of four nuclear polyhedrosis viruses (NPVs), separable into two morphologic groups of singly and multiply embedded virion types (SEVs and MEVs), were investigated by immunodiffusion and immunoelectrophoresis. The four viruses were Pseudoplusia includens SEV, Trichoplusia ni SEV, T. ni MEV, and Autographa californica MEV. In immunodiffusion, SEV polyhedron proteins formed two precipitin bands with antiserum to SEV polyhedron proteins, while MEV polyhedron proteins formed only one. All four proteins formed one precipitin band with antiserum to MEV polyhedron protein, with a spur between SEV and MEV proteins. In immunoelectrophoresis, mobilities of SEV proteins were significantly different from those of MEVs. Precipitin arc patterns were similar to those in immunodiffusion when electrophoresis was carried out at 4 C; at room temperature, a single arc of precipitation formed with all four proteins. SEV virions formed five possibly identical precipitin bands in immunodiffusion with antiserum to SEV virions. MEV virions formed three possibly identical precipitin bands when reacted with antiserum to MEV virions. Little or no cross-reactions were observed between SEV and MEV virions or between virions and polyhedron proteins. In immunoelectrophoresis, SEV virions formed three precipitin arcs in reactions with SEV antisera and none with MEV antisera; MEV virions formed two arcs with MEV antisera and none with SEV antisera. When antisera were subjected to electrophoresis, five arcs were formed by SEVs and three by MEVs in homologous systems, and none were formed in heterologous systems.     

Immunological properties of O2.- generating oxidase from bovine neutrophils

Two antisera have been prepared against the O2.- generating oxidase purified from bovine polymorphonuclear neutrophils (PMNs). The first antiserum was directed against the enzymatically active fraction obtained after isoelectric focusing (pI oxidase), which consisted of a major protein of Mr 65,000 [(1985) Biochemistry 24, 7231-7239]. The second antiserum was directed against the 65 kDa band excised from an SDS-polyacrylamide gel after electrophoresis of the pI oxidase preparation. The pI oxidase antiserum inhibited O2.- generation by PMN cells, PMN membranes and detergent-solubilized membranes. The 65 kDa band antiserum was virtually non-inhibitory against PMN cells; in contrast, it was nearly as potent as the pI oxidase antiserum on PMN membranes and detergent-solubilized membranes. Inhibition of O2.- generation by the pI oxidase antiserum was correlated with the immunoreactivity of four membrane-bound proteins of 65, 54, 18 and 16 kDa; the 65 kDa band antiserum reacted only with the two proteins of 65 and 54 kDa. It is concluded that the 18 and 16 kDa proteins, present in trace amounts in the pI oxidase preparation, are probably potent catalysts of the respiratory burst.     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. Phosphate-transport protein alpha and phosphate-transport protein beta (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes

Affinity-purified antibodies to mouse liver 26- and 21-kD gap junction proteins have been used to characterize gap junctions in liver and cultured hepatocytes. Both proteins are colocalized in the same gap junction plaques as shown by double immunofluorescence and immunoelectron microscopy. In the lobules of rat liver, the 21-kD immunoreactivity is detected as a gradient of fluorescent spots on apposing plasma membranes, the maximum being in the periportal zone and a faint reaction in the perivenous zone. In contrast, the 26-kD immunoreactivity is evenly distributed in fluorescent spots on apposing plasma membranes throughout the rat liver lobule. Immunoreactive sites with anti-21 kD shown by immunofluorescence are also present in exocrine pancreas, proximal tubules of the kidney, and the epithelium of small intestine. The 21-kD immunoreactivity was not found in thin sections of myocardium and adult brain cortex. Subsequent to partial rat hepatectomy, both the 26- and 21-kD proteins first decrease and after approximately 2 d increase again. By comparison of the 26- and 21-kD immunoreactivity in cultured embryonic mouse hepatocytes, we found (a) the same pattern of immunoreactivity on apposing plasma membranes and colocalization within the same plaque, (b) a similar decrease after 1 d and subsequent increase after 3 d of both proteins, (c) cAMP-dependent in vitro phosphorylation of the 26-kD but not of the 21-kD protein, and (d) complete inhibition of intercellular transfer of Lucifer Yellow in all hepatocytes microinjected with anti-26 kD and, in most cases, partial inhibition of dye transfer after injection of anti-21 kD. Our results indicate that both the 26-kD and the 21-kD proteins are functional gap junction proteins.     

Distribution and role of gap junctions in normal myocardium and human ischaemic heart disease

In the heart, individual cardiac muscle cells are linked by gap junctions. These junctions form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all the cells of the myocardium, ensuring their synchronous contraction. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera were raised to three synthetic peptides, each matching different cytoplasmically exposed portions of the sequence of connexin43, the major gap-junctional protein reported in the heart. The specificity of each antiserum for the peptide to which it was raised was established by dot blotting. New methods were developed for isolating enriched fractions of gap junctions from whole heart and from dissociated adult myocytes, in which detergent-treatment and raising the temperature (potentially damaging steps in previously described techniques) are avoided. Analysis of these fractions by SDS-polyacrylamide gel electrophoresis revealed major bands at 43 kDa (matching the molecular mass of connexin43) and at 70 kDa. Western blot experiments using our antisera indicated that both the 43-kDa and the 70-kDa bands represent cardiac gap-junctional proteins. Pre-embedding immunogold labelling of isolated gap junctions and post-embedding immunogold labelling of Lowicryl-embedded whole tissue demonstrated the specific binding of the antibodies to ultrastructurally defined gap junctions. One antiserum (raised to residues 131–142) was found to be particularly effective for cytochemical labelling. Using this antiserum for immunofluorescence labelling in combination with confocal scanning laser microscopy enabled highly sensitive detection and three-dimensional mapping of gap junctions through thick slices of cardiac tissue. By means of the serial optical sectioning ability of the confocal microscope, images of the entire gap junction population of complete en face-viewed disks were reconstructed. These reconstructions reveal the presence of large junctions arranged as a peripheral ring around the disk, with smaller junctions in an interior zone: an arrangement that may facilitate efficient intercellular transfer of current. By applying our immunolabelling techniques to tissue from hearts removed from transplant patients with advanced ischaemic heart disease, we have demonstrated that gap junction distribution between myocytes at the border zone of healed infarcts is markedly disordered. This abnormality may contribute to the genesis of reentrant arrhythmias in ischaemic heart disease.     

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. (1) Phosphate-transport protein and phosphate-transport protein β (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. (2) Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. (3) Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. (4) Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. (5) Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Protein heterogeneity in rabbit liver ferritin: two types of molecular dimers

Native pore-gradient polyacrylamide gel electrophoresis of rabbit liver ferritin reveals the usual single band of molecular monomers, but shows two bands at the position of molecular dimers. The proteins in these three bands were purified by excision from preparative slab gels. All three bands (1) contain considerable amounts of iron-rich ferritin when examined by electron microscopy, (2) show complete identity when reacted with anti-rabbit-ferritin antibodies, and (3) have similar amounts of H-type and L-type ferritin subunits with denaturing polyacrylamide gel electrophoresis. These results establish that there are two classes of ferritin molecular dimers. The larger dimer band uniquely also contains a polypeptide with Mr = 170,000. This unusual type of ferritin heterogeneity seems to be due to the presence of a non-ferritin protein associated only with one class of dimers.     

Insulin-like growth factor binding protein measurement: sodium dodecyl sulfate-stable complexes with insulin-like growth factor in serum prevent accurate assessment of total binding protein content by ligand blotting.

The possibility that sodium dodecyl sulfate (SDS)-stable complexes of insulin-like growth factor I (IGF-I) and its binding proteins (IGF-BP) exist in rat serum has been examined by using SDS-polyacrylamide gel electrophoresis (PAGE) followed by both [125I]IGF-I ligand blotting and immunoblotting with antisera directed against either IGF-BP3 or IGF-I. While ligand blotting of rat serum only revealed free IGF-BP subunits (Mr approximately 50, 35, and 30 kDa), immunoblotting with either the IGF-BP3 antiserum or IGF-I antiserum revealed major immunoreactive bands with higher molecular weights (greater than 110, approximately 100, and approximately 84 kDa). The IGF-BP3 antiserum also stained the 50-kDa form of the serum IGF-BP. Specifically stained protein bands were identified by comparison with control immunoblots incubated with normal rabbit serum. Treating the serum with 0.1 N HCl prior to electrophoresis reduced the amount of high molecular weight IGF-BP3 immunoreactive species, with a concomitant increase in the amount of the 50-kDa form. A similar result was obtained if the samples were boiled prior to electrophoresis. These data indicate that not all IGF-BP/IGF complexes may dissociate under normal SDS-PAGE conditions. Therefore, data obtained by using ligand blotting alone may underestimate the amount of total IGF-BP present, especially if the mixture being analyzed also contains large amounts of IGF.     

Isolation and identification of the third component of complement of Japanese quails

The identification of the third component of complement (C3) of Japanese quails was attempted by using rabbit antiserum prepared against quail serum-treated zymosan (ZX) as an initial reagent. This antiserum (anti-ZX) had agglutinating activity on rabbit erythrocytes reacted with quail antibody and quail complement (EACq) but not on EAq, and developed two precipitin lines against quail serum at beta- and gamma-regions in crossed immunoelectrophoresis. Subsequently, monospecific antisera to each of these precipitin lines were prepared in rabbits, and quail serum proteins reactive with these antisera were purified by salt precipitation followed by Sephadex gel filtration and DEAE cellulose column chromatography. One protein with a m.w. of 184,000 (184K) resembled mammalian C3 in that: 1) monospecific antiserum (anti-184K protein serum) agglutinated EACq but not EAq; 2) treatment of fresh quail serum with either inulin or zymosan resulted in the conversion of the precipitin line developed against 184K protein from gamma to beta in crossed immunoelectrophoresis; 3) the 184K protein was shown to consist of two polypeptide chains of 110K and 73K linked by disulfide bonds. Furthermore, the 184K protein in serum was cleaved through the incubation with inulin to 174K and 140K proteins that might correspond to C3b and C3c of human complement; 4) the 184K protein bound to zymosan was eluted with hydrazine or methylamine but not with Nonidet P-40, indicating that 184K protein binds to zymosan by a covalent bond but not by a hydrophobic one; and 5) by treatment of fresh quail serum with methylamine, complement reactivity was reduced, although its activity was restored by the addition of purified 184K protein. These results suggest the 184K protein is the quail's equivalent to mammalian C3. When quail serum was reacted with cells that had complement-activating capacity, quail C3 deposited on their membrane as in mammalians; however, no conversion of quail C3 was noted by the reaction with CVF. Antibody to quail C3 failed to cross-react with that in mammals.     

Biochemical and immunological characterization of the microfibrillar ecto-endoplasmic boundary in the ciliate Isotricha prostoma

The cytoplasm of the ciliated protozoan Isotricha prostoma is compartmented by a continuous fibrillar system made up of a double layer of 4 nm-diameter filaments: the microfibrillar ecto-endoplasmic boundary (EEB). Isolation of this structure after treatment of the cells in a buffer of low ionic strength in the presence of the detergent Triton X-100 evidenced connections linking the two filamentous layers. One dimensional electrophoresis on SDS-polyacrylamide gel of EEB fractions revealed several major proteins with apparent molecular weights between 11 and 23 K. Of these, two neighboring bands of MW22 and 23 K were removed from gels and used as antigens to obtain rabbit antibodies. The antiserum obtained reacted specifically with injected proteins as shown by the technique of immunological detection on nitrocellulose sheets using the peroxidase reaction product. Electron microscopy localization of the antigens with anti-IgG coupled with colloidal gold showed significant labeling of the EEB within the cortex of Isotricha permeabilized with Triton X-100. We hope that the 22-23 K antiserum will prove to be a useful tool for the comparative study of other non-actin filament systems in Protozoa.