Purification and characterization of a hormone-like factor which inhibits cholera secretion

Sera raised against the alpha-, beta- and gamma-subunits of the mouse 7 S NGF were used to characterize translation products coded by submaxillary gland mRNAs microinjected into Xenopus oocytes. Anti-beta NGF sera did not cross-react with any material. In contrast, the precursors of the alpha- and gamma-subunits, as well as that of renin were identified. Use of tunicamycin, and a comparison of the translation products obtained in oocytes or in the reticulocyte lysate indicated that oocytes achieved the cleavage of signal sequences, the glycosylation of the alpha- and gamma-precursors, and the subsequent secretion of the 3 proteins. In the submaxillary gland, however, the mature forms of alpha NGF, gamma NGF and renin are composed of peptides of smaller size than those produced by the oocytes. These latter appear to lack specific proteases involved in the terminal processing of the submaxillary gland proteins.     

Microtubule-binding domain of tau proteins

Limited chymotryptic digestion of whole tau proteins produced a fragment of Mr 14,000 (CT14), which was able to bind to microtubules reconstituted from tubulin alone in the presence of taxol. This fragment was also found to persist in microtubules when microtubules consisting of tau proteins and tubulin were digested by chymotrypsin. Analysis of amino acid composition revealed that CT14 was rich in lysine and proline residues, suggesting unique structure of microtubule-binding domain of tau proteins. Amino-terminal sequence of CT14 was determined to be Ser-Ser-Pro-Gly-Ser-Pro-Gly-Thr-Pro-Gly-Ser-Arg-Ser-Arg-X-Pro-Ser-Leu-Pr o. No heterogeneity was detected in this amino-terminal sequence of 19 residues. Five species of polypeptides consisting of tau proteins were separated from each other by gel electrophoresis and subjected to chymotryptic digestion. CT14 was produced from each of the tau polypeptides by chymotryptic digestion, indicating that all tau polypeptides have a common microtubule-binding domain.     

Cross-linked proteins associated with a specific mRNA in the cytoplasm of HeLa cells

Cytoplasmic messenger RNAs of eukaryotic cells are distributed between polysomal and post-polysomal fractions (free) as protein-bound complexes. These studies were designed to determine whether a specific mRNA isolated from different subcellular compartments is complexed with the same family of polypeptides. As a first approach we have examined the proteins associated with mRNA which codes for histone H4. To perform these experiments HeLa cells were exposed to ultraviolet light to cross-link in vivo polypeptides which are closely associated with nucleic acid. To identify the polypeptides associated with mRNA specific for histones a genomic probe for histone H4 mRNA was immobilized on epoxy-cellulose. By hybrid selection specific mRNPs containing histone mRNA were isolated. Our results reveal the existence of a number of polypeptides associated with both polysomal and post-polysomal histone mRNAs. In polysomal histone mRNA two polypeptides of Mr = 49 000 and 52 500 were the major components. In contrast polypeptides of Mr = 43 000 and 57 000 were the major polypeptide components of post-polysomal (or free) histone mRNA. Furthermore, these results also suggest that the polypeptides associated with either polysomal or free H4 histone mRNA represent a subset of proteins found in poly(A)-free fractions or poly(A)-rich mRNA fractions.     

Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs

Taxol was used to prepare microtubules from unfertilized eggs of sea urchins Lytechinus pictus, Strongylocentrotus droebachiensis , and Strongylocentrotus purpuratus. By electron microscopy, these microtubules possessed normal morphology and were decorated with projections. The polypeptides present were tubulin plus microtubule-associated proteins (MAPs) which included various high molecular weight polypeptides, and a Mr = 80,000 polypeptide. These MAPs were extracted from the microtubules by differential centrifugation in high ionic strength buffers, yielding a pellet of microtubules which were not decorated with projections. The Mr = 80,000 and high molecular weight MAPs were separated using Bio-Gel A-1.5 m chromatography, and shown to bind taxol-stabilized microtubules assembled from purified bovine brain tubulin. A dynein-like MgATPase activity is present in sea urchin egg extracts. 10-20% of this MgATPase co-pelleted with the taxol-assembled microtubules, under conditions where greater than 90% of the tubulin pelleted. During subsequent fractionation of the microtubules, by (i) high salt extraction followed by gel filtration or sucrose density gradient fractionation or (ii) ATP extraction, the MgATpase co-purified with high Mr MAPs. The MgATPase which remained in the microtubule-depleted egg extract was partially purified by (NH4)2SO4 fractionation, followed by Bio-Gel A-5 m and hydroxylapatite chromatography. The high Mr MAP MgATPase and the hydroxylapatite MgATPase both contained a prominent polypeptide (Mr approximately 350,000), which co-migrated on sodium dodecyl sulfate gels with the major heavy chain of dynein extracted from sperm axonemes. Our data suggest that this Mr approximately 350,000 polypeptide is cytoplasmic dynein.     

Storage protein precursor polypeptides in cotyledons of Pisum sativum L. Identification of, and isolation of a cDNA clone for, an 80000-Mr legumin-related polypeptide

An 80 000-Mr polypeptide, which bound to anti-legumin IgG, was detected among labelled polypeptides from cotyledons at late stages of development. When poly(A)-containing RNA from similar cotyledons was translated in a cell-free protein-synthesizing system, an 80 000-Mr polypeptide was also detected. Immunoprecipitation of translation products with anti-legumin IgG showed that, in addition to the major legumin precursor polypeptides of Mr approximately 60 000, the 80 000-Mr polypeptide was specifically immunoprecipitated. A cDNA clone, pCD32, was found to select an RNA coding for an 80 000-Mr polypeptide in hybrid-selection experiments. Additional minor polypeptides of Mr 63 000 and 65 000 were present in translation products of RNA selected by pCD32; all three polypeptides were immunoprecipitated by anti-legumin IgG. Thermal elution of RNAs bound to pCD32 showed that the affinity of pCD32 to the RNA coding for the 80 000-Mr polypeptide was greater than to the RNAs coding for the 63 000-Mr and 65 000-Mr polypeptides. In similar hybrid-selection experiments, another cDNA clone, pCD40, selected RNAs coding predominantly for polypeptides of Mr 63 000 and 65 000. A minor polypeptide of Mr 80 000 was also detected among these products; again all three polypeptides were immunoprecipitated by anti-legumin IgG. Peptide mapping revealed close similarities between the 80000-Mr polypeptide and the 63 000-Mr/65 000-Mr polypeptides obtained by translation of RNAs selected by pCD32. There were similarities also between maps obtained from translation products of RNA selected by pCD32 and those obtained from anti-legumin IgG immunoprecipitates of total translation products of poly(A)-containing RNA.     

Characterization of calcium-dependent membrane binding proteins of brain cortex.

Proteins of Mr 68 000, 34 000 and 32 000 were selectively extracted by EGTA from brain cortex. The three proteins that were extracted along with calmodulin were acidic, monomeric, and did not exhibit structural homology, as demonstrated by one-dimensional peptide mapping. The Mr-68 000 protein was purified to homogeneity and had a Stokes radius of 3.54 nm and S20,W value of 5.1S. Purified calmodulin, Mr-68 000 protein and two proteins of Mr 34 000 and Mr 32 000, interacted with the brain particulate fraction, with half-maximal binding occurring at 3.5 microM, 8.3 microM and 150 microM-Ca2+ respectively. Proteins were bound independently of each other and calmodulin. Pretreatment of the particulate fraction with trypsin prevented the Ca2+-dependent binding of calmodulin; however, the binding of the Mr-68 000 protein or the Mr-32 000 and -34 000 proteins was unaffected. The Mr-68 000 protein of bovine brain did not cross-react immunologically with Mr-67 000 calcimedin from chicken gizzard.     

Characterization of maize microtubule-associated proteins, one of which is immunologically related to tau

Microtubule-associated proteins (MAPs) are identified as proteins that copurify with tubulin, promote tubulin assembly, and bind to microtubules in vitro. Higher plant MAPs remain mostly unknown. One example of non-tubulin carrot proteins, which bind to neural microtubules and induce bundling, has been reported so far [Cyr, R. J., & Palewitz, B. A. (1989) Planta 177, 245-260]. Using taxol, we developed an assay where higher plant microtubules were induced to self-assemble in cytosolic extracts of maize cultured cells and were used as the native matrix to isolate putative plant MAPs. Several polypeptides with an apparent molecular masses between 170 and 32 kDa copolymerized with maize microtubules. These putative maize MAPs also coassembled with pig brain tubulin through two cycles of temperature-dependent assembly-disassembly. They were able to initiate and promote MAP-free tubulin assembly under conditions of nonefficient self-assembly and induced bundling of both plant and neural microtubules. One of these proteins, of about 83 kDa, cross-reacted with affinity-purified antibodies against rat brain tau proteins, suggesting the presence of common epitope(s) between neural tau and maize proteins. This homology might concern the tubulin-binding domain, as plant and neural tubulins are highly conserved and the plant polypeptides coassembled with brain tubulin.     

Cell-free translation of proline-rich protein mRNAS from human submandibular gland

RNA was isolated from a human submandibular gland and separated into poly A-enriched and poly A-deficient fractions by chromatography on oligo (dT) cellulose. Both of these RNA fractions stimulated methionine incorporation into polypeptides in a reticulocyte lysate translation system. Two in vitro translation products templated by poly A-enriched mRNA were isolated by immunoprecipitation with immune serum directed against human salivary anionic proline-rich protein I. These polypeptides were shown to be precursors of proline-rich proteins on the basis of Mr, affinity for the antiserum, and preferential incorporation of proline. This study is the first to demonstrate cell-free translation of the mRNAs for human proline-rich salivary protein precursors.     

Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.     

Identification and characterization of plasma membrane proteins that bind to microtubules in pollen tubes and generative cells of tobacco

The organization and function of microtubules in plant cells are important in many developmental stages. Connections between microtubules and the endomembrane system of plant cells have been discovered by microscopy, but the molecular characteristics of these relationships are mostly unknown except for a few cases. Using two antibodies raised against microtubule-associated proteins (MAPs) from maize, we have identified two polypeptides that share properties of the MAP family in the pollen tube of Nicotiana tabacum. The two polypeptides (with an apparent Mr of 161 and 90 kDa) bind efficiently to animal and plant microtubules and are found in association with the cellular membranes of the pollen tube, from which they can be solubilized with a zwitterionic detergent. One of these proteins has been purified and shown to promote the assembly of tubulin and, to a lesser extent, the bundling of microtubules. Subcellular fractionation indicated that the two proteins are associated with the plasma membrane compartment. The two proteins are found to co-localize in situ with cortical microtubules in the vegetative cytoplasm of tobacco pollen tubes; co-localization is also evident in the generative cell. According to these data, both the 161 and 90 kDa polypeptides are likely to mediate the interactions between the plasma membrane and microtubules in pollen tubes. In addition, functional data indicate that these MAP-like proteins take part in the process of microtubule assembly and reorganization occurring during cell growth. The evidence that both proteins associate with different cellular compartments also suggests a broad-spectrum role in mediating the dynamic relationships between microtubules and plant cell membranes.     

Regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase and its subunits in the flagellate Chlorogonium elongatum. Different levels of translatable messenger RNAs for the large and the small subunits in autotrophic and heterotrophic cells as determined by immunological techniques

Poly(A)-rich and poly(A)-free RNAs were isolated from autotrophic and heterotrophic cells of the phytoflagellate Chlorogonium elongatum and translated in an mRNA-depleted reticulocyte lysate system. Immunoprecipitation methods were improved to detect large and small subunits of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase synthesized in vitro. Large-subunit polypeptides were shown to be the translation products of poly(A)-free RNA having the same molecular weight as large subunits made in vivo. Small-subunit polypeptides were synthesized when poly(A)-rich RNA was used as a template. They were made in vitro as a precursor, with an Mr about 6000 larger than mature small subunits. Cells growing heterotrophically in the dark with acetate are provided with lower levels of mRNA activities for the large and the small subunits is at least partially controlled by the amounts of translatable mRNAs.     

Biosynthesis and processing of legumin-like storage proteins in Lupinus angustifolius (lupin).

Synthesis, secretion and post-translational proteolysis of the storage proteins in cotyledons of Lupinus angustifolius L. (lupin) have been examined in vivo and in vitro by using a combination of pulse-chase experiments with [3H]- or [35S]-labelled amino acids, subcellular fractionation and cell-free translation from poly(A)+ (polyadenylylated) RNA or membrane-bound polyribosomes. Related polypeptides were identified by immunoprecipitation, separation on sodium dodecyl sulphate/polyacrylamide gels and fluorography. The synthesis and processing of two proteins were compared. Conglutin alpha, the 11 S protein, was found as a family of precursor polypeptides of Mr 68000-88000 when translated from poly(A)+ RNA under conditions where signal segments were not cleaved, and Mr 64000-85000 both when sequestered into the endoplasmic reticulum and when accumulated in the protein bodies. Pulse-chase labelling showed that cotyledons from early stages of development were completely incapable of further proteolysis of these precursors. Nevertheless, in the same juvenile cotyledons, the precursors of the minor storage protein conglutin gamma, two polypeptides with Mr 50000-51000, were proteolytically cleaved to mature subunits of Mr 32000 and 17000 within 2 h. Further cleavage of the precursors of conglutin alpha into families of mature subunits of Mr 21000-24000 and 42000-62000 was detected in more mature cotyledons. A model is proposed which suggests that the mature subunits are produced by a single proteolytic cleavage of each of the three major precursors of conglutin alpha and also suggests that a close similarity exists between these subunits and those of other legumin-like proteins. The enzyme responsible for this cleavage, which appears at a specific stage in the middle of cotyledonary development, seems to be an integral part of the programmed developmental sequence in these pods.     

Precursors of storage proteins in Lupinus angustifolius.

The proteins that are synthesized during differentiation and development in the cotyledons of Lupinus angustifolius L. were characterized both in situ and after purification. The proteins present in situ were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and subjected to 'Western'-blot analysis to identify immunologically related polypeptides. The major storage proteins of the lupin, conglutins alpha and beta, were both present in juvenile tissue only as higher Mr precursors. For conglutin beta, a family of at least three polypeptides of Mr 66 000-72 000 accumulated during the earliest phases of protein synthesis in the developing cotyledon (20-28 days after flowering). Later in development each of these polypeptides disappeared and there was the concurrent appearance in the cotyledon of the lower-Mr fragments characteristic of mature conglutin beta. For conglutin alpha, an equivalent family of precursor polypeptides of Mr 60 000-83 000 was detected. Multiple internal sites for proteolytic cleavage of all these precursors appeared to be present. However, processing of the precursors was sufficiently slow to allow them to accumulate to over 50% of total soluble protein in juvenile tissue. The precursors were purified by column chromatography under non-dissociating conditions and shown by ultracentrifugation to be multimeric proteins with Mr values in the range 150 000-200 000.     

Nicotine-stimulated proteins in mouse cells are distinct from heat-shock proteins.

Treatment of mouse tissue-culture cells with nicotine concentrations of 1 mM or less had no significant effects on cell viability, morphology or protein synthesis, but higher concentrations resulted in both altered cell morphology (rounding and vacuolization) and alterations in [3H]leucine-labelled protein profiles on sodium dodecyl sulphate/polyacrylamide gels. The synthesis of a Mr-70 000 protein was increased more than 2-fold relative to that of other major cellular proteins in 3T3 and L929 cells treated with 5 mM-nicotine and in B16 cells treated with 10 mM-nicotine, and this protein appeared to be a soluble cytoplasmic polypeptide. The radiolabelling of several additional polypeptides (Mr 62 000 in 3T3 cells, and Mr 45 000 and 38 000 in B16 cells) was also stimulated by nicotine. The nicotine-enhanced Mr-70 000 protein was distinct, however, from a major cell stress/heat-shock protein whose synthesis was stimulated after incubation of cells at 43.5 degrees C for 20 min.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel

Brain ankyrin was purified from pig brain membranes in milligram quantities by a procedure involving affinity chromatography on erythrocyte spectrinagarose. Brain ankyrin included two polypeptides of Mr = 210,000 and 220,000 that were nearly identical by peptide mapping and were monomers in solution. Brain ankyrin and erythrocyte ankyrin are closely related proteins with the following properties in common: 1) shared antigenic sites, 2) high-affinity binding to the spectrin beta subunit at the midregion of spectrin tetramers, 3) a binding site for the cytoplasmic domain of the erythrocyte anion channel, 4) a binding site for tubulin, 5) a similar domain structure with a protease-resistant domain of Mr = 72,000 that contains the spectrin-binding activity and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg of membrane protein in demyelinated membranes based on radioimmunoassay with antibody raised against brain ankyrin and affinity purified on brain ankyrin-agarose. Brain spectrin tetramers are present at 30 pmol/mg of membrane protein. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes. Brain ankyrin also may attach microtubules to membranes independently of spectrin and has the potential to interconnect microtubules and spectrin-associated actin filaments.     

Identification and spatial arrangement of high molecular weight proteins (Mr 300 000-330 000) co-assembling with microtubules from a cultured cell line (rat glioma C6)

The distribution of three high molecular weight proteins, MAP-1 (Mr 330 000), MAP-2 (Mr 300 000) and plectin (Mr 300 000) in various fractions obtained in cycles of temperature-dependent polymerization/depolymerization of microtubules from rat glioma C6 cells was studied. Using gel electrophoresis and immunoautoradiography/immunoblotting all three proteins were found to codistribute only partially with tubulin because considerable parts remained in the cold-insoluble fractions. Moreover, the proteins, particularly MAPs, were proteolytically degraded during cycling. By contrast, when microtubules were polymerized with taxol after isotonic cell lysis a considerable enrichment of MAP-1 and MAP-2 was achieved; again, plectin co-distributed only partially. In this procedure too, MAPs, especially MAP-2, were found to be highly subject to proteolysis, unless free Ca2+-ions were rigorously avoided. Proteolytic fragments generated from MAP-2 were of similar size independent of whether temperature- or taxol-dependent polymerization procedures were used, suggesting the occurrence of a MAP-2-specific protease. When the spatial arrangement of the high Mr proteins on taxol-polymerized C6 cell microtubules was directly visualized using gold-immunoelectron microscopy, a periodical, apparently helical, decoration of microtubules was found for MAP-1 and MAP-2; plectin was irregularly arrayed. A predominantly helical arrangement of both MAPs was demonstrated also for microtubules reconstituted from mammalian brain.     

Formation of a 22S mRNA X rRNA X protein complex during translation of globin messenger RNA

Poly(A)-rich RNA was isolated from rabbit reticulocyte polyribosomes by affinity chromatography on oligo(dT)-cellulose and fractionated in sucrose gradients under non-denaturing conditions. Most of the translatable RNA sedimented in sucrose gradients both as free 9S mRNA and as a 22S complex containing 18S ribosomal RNA and a protein of Mr 66 000. The complex was characterized by identification of the translation products. Experiments with both labelled globin mRNA and Mr-66 000 protein indicate that the complex is not an artefact, but rather that it is formed during the initiation of protein synthesis. The Mr-66 000 protein appears to be a component of the 48S pre-initiation complex and recycles before 80S complex formation.     

Localization and Characterization of Tubulin-Like Proteins Associated with Brain Mitochondria: The Presence of a Membrane-Specific Isoform

A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the alpha- and beta-tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin-like proteins were then visualized in mitochondrial membranes by protein A-gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two-dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of beta-tubulin but the isoelectric point of alpha-tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with alpha-tubulin and may result from a posttranslational modification of that subunit.