Cardiac alpha-crystallin

A major component of the soluble fraction of rat heart is a homopolymer (Mr about 400-650 k) of a small protein (Mr about 20 k). This cardiac protein, which is highly homologous to alpha-B-crystallin, was isolated in its native state and visualized by electron microscopy. A homogeneous population of globular particles with an average diameter of about 14-16 nM could be seen using either negative staining or rotary shadowing procedures. The structures were globular in nature with a central depression (torus-like structures). Polyclonal antibodies, raised against the cardiac crystallin, were used for the immunocytochemical localization of the macromolecular complexes. Using fluorescent secondary antibodies, a clear and sharp striation of fixed and permeabilized rat heart myocytes could be observed, similar to that observed with anti-desmin antibodies and characteristic for the central region of the I-band. Cardiac crystallin was not found associated with F-actin in preparations of rat heart myofibrils. On the other hand, it was a major contaminant of cardiac desmin preparations. These observations suggest that cardiac crystallin is involved in the organization of cytoskeletal filaments of the Z-lines.     

Tissue and species conservation of the vertebrate and arthropod forms of the low molecular weight (16–18000) proteins of gap junctions

Summary Gap junctions have been isolated from four murine tissues, from rat and Xenopus laevis liver, and from Nephrops norvegicus (Norway lobster) hepatopancreas. The preparations of gap junctions from each vertebrate tissue contain a single major protein, M_r 16000, and those from Nephrops hepatopancreas a protein, M_r 18000. Immunocytochemical studies using affinity-purified antibodies raised against gap junctions from Nephrops show the junctional origin of the 18k protein. Immunological studies using Western blotting and biochemical studies using tryptic peptide mapping show no significant differences between the 16k junctional proteins of mouse and hence provide no evidence of tissue variation. These studies also suggest that the mouse, rat, and Xenopus 16 k proteins and the Nephrops 18 k protein share some common structural features.     

Immunological characterization of rat cardiac gap junctions: Presence of common antigenic determinants in heart of other vertebrate species and in various organs

Summary Antibodies to the following synthetic peptide, SALGKLLDKVQAY, were purified by affinity chromatography and characterized by ELISA and immunoblotting. These antibodies, shown to be specific to the major protein constitutent of isolated rat heart junctions: connexin 43, cross-reacted with a homologous protein in immunoreplicas of whole heart fractions of trout, frog, chicken, guinea pig, mouse and rat, suggesting a phylogenic conservation of connexin 43 in vertebrates. By immunoblotting of whole organ fractions it was also demonstrated that these antibodies cross-reacted with major proteins ofM _r 32 and 22 kD in rat and mouse liver, ofM _r 41 kD in rat cerebellum, ofM _r 43 kD in uterus, stomach and kidney of rat, ofM _r 46 and 70 kD in rat lens, suggesting that these proteins share common or related epitopes with the synthetic peptide and connexin 43.     

Proteolysis of cardiac gap junctions during their isolation from rat hearts

Summary Gap junctions (GJ) isolated from rat hearts in presence of the protease inhibitor phenylmethylsulfonylfuoride (PMSF) contain a M_r 44,000 to 47.000 major polypeptide and have a urea-resistant layer of fuzz on their cytoplasmic surfaces, whereas junctions isolated without PMSF are proteolyzed to a M_r 29.500 polypeptide by a serine protease and have smooth cytoplasmic surfaces (C.K. Manjunath, G.E. Goings & E. PageAm. J. Physiol. 246:H865–H875, 1984). Rat liver GJ isolated with or without PMSF contain a M_r 28,000 polypeptide and have smooth cytoplasmic surfaces. Here we examine the origin, type and inhibitor sensitivity of the heart protease; why similar proteolysis is absent during isolation of rat liver gap junctions; and whether the M_r 44.000 to 47,000 cardiac GJ polypeptide is the precursor of the M_r 29,500 subunit. We show that the M_r 44,000 to 47,000 polypeptide corresponds to the unproteolyzed connexon subunit; that proteolysis of this polypeptide occurs predominantly during exposure to high ionic strength solution (0.6m KI) which releases serine protease from mast cell granules; that this protease is inhibitable with PMSF and (less completely) soybean trypsin inhibitor and chymostatin; and thatin vivo degranulation of mast cells by injecting rats with compound 48/80 fails to prevent breakdown of cardiac GJ during isolation. The results support the concept that GJ from rat heart and liver differ in protein composition.     

Development of monoclonal antibodies to 3-hydroxy-3-methylglutaryl-coenzyme A reductase

This report describes the development of a series of monoclonal antibodies to rat liver 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR). Sera from hybridoma tumor-bearing mice were used to remove and characterize HMGR activity from a mixture of rat liver proteins. Two IgG₂ monoclonal antibodies removed separately greater than 80% HMGR activity while non-immune mouse or negative hybridoma-derived sera were ineffective. Radiolabeled immunoprecipitates of enzyme preparations resolved in one- and two-dimensional SDS-PAGE showed two predominant subunits at M_r 52,000 and 54,000. Our results indicate that in these preparations of rat liver proteins HMGR exists as a heteropolymer with at least two distinct subunits of different molecular weights.     

Purification and characterization of three major hemolymph proteins of an insect,Calpodes ethlius (lepidoptera,hesperiidae)

Like many other Lepidoptera, fifth-stage Calpodes larvae have three major hemolymph proteins. Their molecular weights were estimated by 3-15% nondenaturing polyacrylamide gel electrophoresis (N-PAGE) as 470,000 (arylphorin; Ar), 580,000 (storage protein 2; SP2) and 720,000 (storage protein 1; SP1). Carbohydrate is associated with all three, but only Ar has lipid. The three proteins have been purified by preparative N-PAGE and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. On 3-15% SDS gels, Ar dissociated into 82,000 M_r subunits, SP2 into 86,000 M_r subunits, and SP1 into both 86,000 and 90,000 M_r subunits. The 470,000 M_r protein is identified as Ar because it is rich in aromatic amino acids. The 580,000 and 720,000 M_r proteins are rich in glycine and are called storage proteins. Electron microscopy of negatively stained preparations shows that each polymer has a different geometrical arrangement of subunits. SP1 is a cube made from eight subunits. SP2 is a hexamer in the form of a pentahedral prism. Ar is probably an octahedron made from six subunits. All three geometrical arrangements could permit the presence of a central carrying space.     

Pancreatic B-cells secrete a range of novel peptides besides insulin

Insulin secretion by a transplantable rat islet B-cell tumour is accompanied by the release of two putative proinsulin cleavage intermediates, four peptides of M_r 9000–12 000 (excluding proinsulin) and peptides of M_r 21 000, 34 000 and 60 000. Granule-enriched subcellular preparations contain major peptides of identical M_r values. Of these peptides seven at least coincide in molecular weight with peptides secreted by isolated rat islets and thus may be constituents of the normal insulin secretory granule.     

Immunolocalization of the 110,000 molecular weight cytoskeletal protein of intestinal microvilli

The core structures of microvilli from absorptive cells of the intestinal epithelium are primarily composed of calmodulin (M_r 16,000), actin (M_r 43,000), villin (M_r 95,000) and a protein of M_r 110,000. We have isolated this protein and raised antibodies against it. The antibodies interact specifically with villin and M_r 110,000 polypeptides present in isolated microvilli or brush borders. However, after absorption on an immobilized villin preparation, these antibodies still immunoprecipitate the M_r 110,000 protein but not villin. Thus, these two proteins appear to share some antigenic determinants but also contain other determinants specific for each protein. Immunolocalization studies have been performed using specific antibodies against the M_r 110,000 protein. Immunofluorescent studies on thin frozen sections of intestinal cells show that this protein is located in the brush border and at the basolateral faces of these polarized cells. Immunoferritin studies on rat brush borders demembranated with the detergent Triton X-100 show the association of the M_r 110,000 protein with core filaments of microvilli, as well as with some filaments localized in the terminal web network.Using sealed, right-side-out vesicles prepared from pig intestinal mucosa in the presence of Ca²⁺ and Mg²⁺, a polypeptide of M_r 140,000 was found to be a major component of the Triton X-100 insoluble pellet. This protein is a minor component of an equivalent pellet obtained from isolated microvilli prepared in the presence of EDTA. The significance of this M_r 140,000 polypeptide associated with the core residue of intestinal microvilli is discussed.     

Cyclic GMP-dependent phosphorylation of an endogenous protein from rat heart

An endogenous substrate protein (M_r = 70K) for cGMP-dependent protein kinase (G-PK) was found in the cytosol of the rat heart. This protein was specifically phosphorylated by G-PK, with a K_a for cGMP of 0.08 μM, compared with that for cAMP of 2 μM. At least two substrate proteins (M_r = 110K and 26K) specific for cAMP-dependent protein kinase (A-PK) were also noted. The finding in heart of an endogenous substrate protein for G-PK distinguishable from those for A-PK provides additional evidence suggesting an independent role for cGMP in the regulation of cardiac function.     

The protein sequence homology of γ-crystallins among major vertebrate classes and their DNA sequence homology to heat-shock protein genes

A systematic characterization of lens crystallins from five major classes of vertebrates was carried out by exclusion gel filtration, cation-exchange chromatography and N-terminal sequence determination. All crystallin fractions except that of γ-crystallin were found to be N-terminally blocked. γ-Crystallin is present in major classes of vertebrates except the bird, showing none, or decreased amounts, of this protein in chicken and duck lenses, respectively. N-Terminal sequence analysis of the purified γ-crystallin polypeptides showed extensive homology between different classes of vertebrates, supporting the close relatedness of this family of crystallin even from the evolutionarily distant species. Comparison of nucleotide sequences and their predicted amino acid sequences between γ-crystallins of carp and rat lenses and heat-shock proteins demonstrated partial sequence homology of the encoded polypeptides and striking homology at the gene level. The unexpected strong homology of complementary DNA (cDNA) lies in the regions coding for 40 N-terminal residues of carp γ-II, rat γ2-1, and the middle segments of 23,000- and 70,000-M _r heat-shock proteins. The optimal alignment of DNA sequences along these two segments shows about 50% homology. The percentage of protein sequence identity for the corresponding aligned segments is only 20%. The weak sequence homology at the protein level is also found between the invertebrate squid crystallin and rat γ-crystallin polypeptides. These results pointed to the possibility of unifying three major classes of vertebrate crystallins into one α/β/γ superfamily and corroborated the previous supposition that the existing crystallins in the animal kingdom are probably mutually interrelated, sharing a common ancestry.     

Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein,in mammal heart and brain

Summary According to the sequence of connexin 43, a cardiac gap junctional protein, the domain contained within residues 314–322 is located 60 amino acids away from the carboxy-terminus. Antibodies raised to a peptide corresponding to this domain label a unique 43-kD protein on immunoblots of both purified gap junctions and whole extracts from rat heart. Immunofluorescence investigations carried out on mammal heart sections reveal a pattern consistent with the known distribution of intercalated discs. Immunogold labeling performed with ultrathin frozen sections of rat heart or partially purified rat heart gap junctions demonstrate that antigenic determinants are associated exclusively with the cytoplasmic surfaces of gap junctions.The antibodies were shown to cross-react with a 43-kD protein on immunoblots of whole extracts from human, mouse and guinea pig heart. However, no labeling was seen when heart of lower vertebrates such as chicken, frog and trout, was investigated. These results, confirmed by immunofluorescence investigations, were interpreted as a loss of antigenic determinants due to sequence polymorphism of cardiac connexin 43.Proteins ofM _r 43 and 41 kD, immunologically related to cardiac connexin 43, were detected in immunoblots of mouse and rat brain whole extracts. mRNAs, homologous to those of cardiac connexin 43 and of the same size (3.0 kb), are also present in brain. Immunofluorescence investigations with primary cultures of unpermeabilized and permeabilized mouse neural cells showed that the antigenic determinants recognized by the antibodies specific for connexin 43 are cytoplasmic and that the labeling observed between clustered flat cells, is punctate, as expected for gap junctions. Double labeling experiments demonstrated that the immunoreactivity is associated with GFAP-positive cells, that is to say, astrocytes.     

Expression of 5α-reductase in bacteria as a trp E fusion protein and its use in the production of antibodies for immunocytochemical localization of 5α-reductase

A cDNA encoding a full-length rat 5α-reductase was isolated using female rat liver mRNA and the polymerase chain reaction, and fused to the Escherichia coli trp E gene in a pATH expression vector. The trp E-5α-reductase fusion protein expressed in bacteria and a synthetic oligopeptide corresponding to the C-terminus of rat 5α-reductase were used as antigens to produce rabbit polyclonal antibodies to 5α-reductase. Antibodies to the 5α-reductase portion of the fusion protein and to the peptide were purified by affinity chromatography. Antibodies against the 5α-reductase fusion protein reacted with a single component of rat liver microsomes with M_r 26,000 on Western blots, consistent with the size of 5α-reductase predicted from its cDNA, and with a M_r 23,000 component on Western blots of detergent extracts of rat ventral prostate nuclei; other rat ventral prostate cellular fractions (mitochondrial, microsomal, cytosol) bound little or no antibody. Antibody against the synthetic peptide reacted with a M_r 26,000 component of rat liver microsomes as well as with several components in various cellular fractions of rat ventral prostate. With anti-5α-reductase fusion protein antibodies, specific immunocytochemical staining was observed in the epithelial cell nuclei of the rat ventral prostate, seminal vesicle, epididymis and other accessory sex glands. This nuclear staining was specific, since antibodies from non-immunized rabbits did not give nuclear staining and preincubation of the anti-5α-reductase fusion protein antibodies with the trp E-5α-reductase fusion protein eliminated nuclear staining. Incubation of antibodies with trp E (without the 5α-reductase fusion) had no effect on nuclear staining. Specific staining was not detected in the cytoplasm of these epithelial cells. Little or no specific staining was observed in stromal cells in these rat tissuess. Human prostate was also immunocytochemically stained with this antibody. Specific staining was found in both epithelial and stromal cell nuclei.     

Detection of 180 kDa proteins in electroplax sodium channel preparations

• 1.1. Co-isolating proteins (M_r 170,000–220,000) from sodium channel preparations made from the electric organ of the electric eel (Electrophorus electricus) were detected on Western blots using monoclonal a antibodies.
• 2.2. Similar protein patterns were seen on immunoblots containing immunoprecipitated protein from eel muscle and brain tissues but not heart.
• 3.3. These co-isolating proteins could be separated from the mature TTX-sensitive channel protein (M_r 280,000) using a lentil lectin-Sepharose column.
• 4.4. The 180 kDa proteins do not appear to be channel-related and can be detected as contaminants in electroplax sodium channel preparations using the monoclonal antibodies described here.

     

Intermediate filament proteins in echinoderm coelomocytes

The presence and organization of intermediate filament (IF) proteins in petaloid coelomocytes from two species of echinoderms, the sea urchin Strongylocentrotus droebachiensis and the sea cucumber Cucumaria frondosa, were studied. Two monoclonal antibodies (IFA and Ah6) and one polyclonal antibody (W3-1) that together recognize invertebrate as well as vertebrate IF proteins were used to probe coelomocytes by immunofluorescence and immunoblotting methods. All three antibodies cross-reacted with a single M_r 68 000 sea urchin lamin, as well as two putative lamin isoforms of approximately M_r 70 000 and 68 000 in sea cucumber coelomocytes. Both IFA and Ah6 labeled granular material in the cytoplasm of sea urchin coelomocytes; by contrast, IFA labeling revealed a striking network of reticular material irregularly arrayed within the central regions of the sea cucumber coelomocyte cytoplasm. In addition, foci of Ah6-positive material were present in coelomocyte nuclei from both species. Comparison of immunoblotting patterns among whole cell and isolated nuclear preparations suggest that the cytoplasmic IF-like material is composed of M_r 46 000 and 58 000 polypeptides, while M_r 215 000 and 185 000 proteins are candidates for the immunoreactive nuclear foci. Further study of the functions of these non-filamentous arrays of IF proteins may furnish valuable insights into the evolution of IF function within vertebrate cells, particularly with respect to certain cytoplasmic and nuclear regulatory functions with which IF proteins have been speculated to be involved.     

Cytoplasmic network arrays demonstrated by immunolocalization using antibodies to a high molecular weight protein present in cytoskeletal preparations from cultured cells

Antisera were raised in rabbits against the two major components of intermediate filament preparations from glia-derived C6 cells, polypeptides of M_r around 300 000 and 58 000 (vimentin). These, and a third antiserum raised against microtubule proteins from hog brain, were shown to be specific for their respective immunogens. The assay employed involved the separation of components of crude cell extracts or filament preparations by SDS-polyacrylamide gel electrophoresis, and their subsequent transfer to and immobilization on nitrocellulose sheets. Cross-reacting counterparts of the immunogens were found in various cell lines, including C6, BALB/c 3T3, SV101, CHO, HeLa and PtK2 cells. In indirect immunofluorescence studies, antibodies to the high-M_r polypeptide component stained dense cytoplasmic network arrays of seemingly short, irregularly oriented fibres and lines of dots, in fibroblasts and in HeLa cells, but not in PtK2 cells. In well spread cells these networks were clearly distinguishable in morphology from the fibres decorated by antibodies to either microtubule protein or vimentin. The network arrays were resistant towards treatments with Triton X-100 and colcemid. By double immunofluorescence microscopy of single cells, using an additional antibody preparation to vimentin raised in guinea pigs, it was shown that after prolonged colcemid treatment of BALB/c 3T3 cells both; vimentin filaments and the structures stained by antibodies to the high-M_r component, accumulated in corresponding areas of the cytoplasm. The possibilities are discussed that this novel network-like structure is of the intermediate filament type and that it might function as a cross-linker of cytoplasmic—in particular cytoskeletal—elements. To signify its fluorescent localization and its possible linking role it is proposed to call the high-M_r component of intermediate filament preparations from cultured cells ‘plectin’.     

Regulation of rat cardiac nuclei-associated Mg2+-NTPase by phosphorylation

A nucleoside triphosphatase (NTPase) activity appeared to be associated with a highly purified nuclear preparation from rat cardiac ventricles. Different nucleoside triphosphates (UTP > GTP > ITP > CTP) supported this enzymic activity, which was stimulated by Mg` but not by Call. The nuclear NTPase activity could be down regulated by endogenous phosphorylation of a 55,000 M_r protein. Maximal phosphorylation of the 55,000 M_r protein occurred in the presence of Mg²⁺-ATP. Addition of cAMP, cGMP, Ca²⁺, Ca²⁺/phospholipid, Ca²⁺/calmodulin, and catalytic subunit of cAMP-dependent protein kinase was not associated with any further phosphorylation of the 55,000 M_r protein. However, in the presence of Ca²⁺/calmodulin or the catalytic subunit of the cAMP-dependent protein kinase additional proteins became phosphorylated, but these had no effect on the Mg²⁺-NTPase activity. These results indicate that a protein with M_r 55,000 may be involved in the regulation the Mg²⁺-NTPase activity associated with rat cardiac nuclei.Abbreviations Hg Hemoglobin - GAR Goat Anti-Rabbit antibody - SR Sarcoplasmic Reticulum - NTP Nucleoside Triphosphate - TCA Trichloroacetic acid - PAGE Polyacrylamide gel electrophoresis     

Rat heart gap junctions as disulfide-bonded connexon multimers: Their depolymerization and solubilization in deoxycholate

Summary Unproteolyzed gap junctions isolated from rat heart and liver were analyzed for the presence of inter-subunit disulfide bonds by sodium dodecylsulfate polyacrylamide gel electrophoresis. Rat cardiac junctions contained multiple disulfide bonds connecting theM _r 47,000 subunits of the same connexon and of different connexons. Inter-subunit disulfide bonds were absent in liver junctions. Unproteolyzed rat heart gap junctions were resistant to deoxycholate in their oxidized state, but dissolved readily in the detergent when the disulfide bonds were cleaved with -mercaptoethanol. Disulfide bonding in proteolyzed cardiac junctions was limited to pairs ofM _r 29,500 subunits. These junctions were not soluble in deoxycholate even in the presence of -mercaptoethanol. These results show that heart and liver junctions differ in their quarternary organization.     

Phenylalanine 4-monooxygenase from bovine and rat liver: Some physical and chemical properties

Phenylalanine 4-monooxygenase was purified from bovine liver using a modification of the procedure developed for the rat liver enzyme (Shiman, R., Gray, D. W., and Pater, A. 1979. J. Biol. Chem. 254:11300–11306). The enzyme preparation appeared essentially homogeneous on polyacrylamide gel electrophoresis under non-denaturing conditions. Electrophoresis in the presence of dodecyl sulfate revealed that about 95% of the protein had a mobility, corresponding to M_r=51,000. The remaining 5% was recovered in two minor bands corresponding to M_r of about 35,000 and 15,000 and is likely to result from limited proteolysis of the native enzyme with dissociation of the fragments on denaturation by detergent. The enzyme comigrated with the rat liver enzyme on polyacrylamide gel electrophoresis in both systems studied. No significant difference was observed between the amino acid composition of the bovine and rat liver enzyme, in the reactivity of their sulfhydryl groups or in their iron content (i.e. 1.5–3.0 iron atoms per peptide chain of M_r=50,000). Both enzymes contained less than 0.01 copper atom per peptide chain. The enzymes were inhibited in a similar manner by the chelator bathophenanthroline disulfonate (selective for iron and copper), but not by bathocuproine disulfonate (specific for copper). The results indicate that the bovine and rat liver enzymes are closely similar and that iron, but not copper, is essential for enzyme activity. High performance size-exclusion liquid chromatography revealed that both native enzymes exist in different oligomeric forms, but further studies are required to understand the physicochemical basis for this phenomenon.Abbreviations Bathophenanthroline 4,7-diphenyl-1, 10-phenanthroline - bathocuproine 2,9-dimethyl-4,7-diphenyl-1, 10-phenanthroline - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - dansyl 1-dimethylaminonaphthalene-5-sulfonyl - DMPH₄ 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine - M_r relative molecular mass     

Production and Characterization of Monoclonal Antibodies to Wall-Localized Peroxidases from Corn Seedlings

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a M_r 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a M_r 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.     

Tobacco embryogenesis: Storage-protein-accumulating cells of embryo, suspensor, and endosperm are able to undergo cytokinesis

Summary It is widely accepted that seed storage proteins accumulate only in cells which have entered the cell expansion phase and do not continue to divide. Here we present data indicating that the accumulation of storage globulins in tobacco begins already during early embryogenesis in a period of sustained mitotic activity. Western blot analysis revealed that polypeptides of the legumin-like 12S globulins (M_r 60000, 40000, 20000) appear at mid/late globular stage, whereas the vicilin-like 7S globulin (M_r 50000) follows during the transition from heart to torpedo stage. The accumulation of legumin-like polypeptides begins first in the endosperm during the mid globular stage followed in the embryo-suspensor complex during the heart-shaped stage. The vicilin-related fraction appears first in the endosperm and three days later in the embryo. Examination of individual cells from squash preparations revealed that protein bodies are not confined to intermitotic cells, but are also present in cells undergoing mitosis. Protein bodies of dividing cells situated outside the mitotic apparatus are not metabolized during cytokinesis. The only cell type which loses its protein bodies completely prior to the first mitotic division is the primary hypophysis cell. Our finding that storage proteins can occur in dividing cells independent of their origin and developmental capacity indicates that the cell expansion hypothesis of storage protein accumulation has to be revised.