Precursors in the biosynthesis of vasopressin and oxytocin in the rat. Characteristics of all the components in high-performance liquid chromatography.

A reverse-phase high performance liquid-chromatography (h.p.l.c.) protocol has been developed, whereby all the major known posterior-pituitary components that are derived from the processing of pro-oxytocin and pro-vasopressin can be separated one from another. Thus, in a single chromatographic step, it has been possible to separate vasopressin (VP), oxytocin (OT), oxytocin-neurophysin (rOT-Np), vasopressin-neurophysin (rVP-Np) and vasopressin-glycopeptide (rVP-GP) from acid extracts of the neurointermediate lobes of rat pituitary glands. All these peptides except rVP-GP were labelled in the neural lobe by 24h after a hypothalamic injection of [35S]cysteine, whereas all except VP were labelled by 24h after a similar injection of [3H]leucine. Three major labelled proteins were isolated from 20 min [35S]cysteine-injected rats when extracts of the supraoptic nucleus were subjected to Sephadex G-75 chromatography, h.p.l.c. and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Immunoprecipitation with antisera raised against rat neurophysins, VP and OT revealed 21000- and 19000-mol.wt. common precursors to VP and rVP-Np and a 15000-mol.wt. common precursor to OT and rOT-Np. Some immunoreactive rVP-Np could occasionally be detected in the Vo of Sephadex G-75 chromatograms of Wistar rat supraoptic-nucleus extracts, but no evidence of [35S]neurophysin in this fraction was obtained from h.p.l.c. fingerprinting of its S-carboxymethylated tryptic digests. Radioimmunoassay for rVP-Np and rOT-Np revealed that about 70-80% of the total recovered immunoreactive neurophysin (IR-Np) in the supraoptic nucleus eluted from Sephadex G-75 and h.p.l.c. in the positions of rVP-Np and rOT-Np. Evidence is presented for an approx. 20000-mol.wt. rOT-Np in both Wistar and Brattleboro rats and for an approx. 20000-mol.wt. component in the Brattleboro rat that is recognized by vasopressin-neurophysin antisera.     

Tumour calcitonin. Interaction with specific calcitonin receptors.

The human epidermoid bronchial carcinoma (BEN) cell line has been shown to have specific membrane binding sites for calcitonin and to secrete high-molecular-weight forms (ranging from 40000 to 10000) of immunoreactive calcitonin. Synthetic salmon and human calcitonins and a thyroid extract of porcine calcitonin have been shown to displace 125I-labelled salmon calcitonin from the receptors in a dose-related fashion. The binding to these receptors of calcitonins derived from the BEN cell line and a medullary thyroid carcinoma with molecular weights ranging from 28000 to 3500 (both separated by gel-filtration chromatography) has been investigated. Neither major peaks of BEN-cell-line calcitonin showed receptor binding activity. Only one form of medullary thyroid carcinoma calcitonin, that which co-eluted with synthetic calcitonin monomer on gel-filtration chromatography, caused any significant displacement of labelled hormone from the receptors.     

A 70000-molecular-weight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure.

The glycoprotein of pig gastric mucus has been isolated free of non-covalently bound protein as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and equilibrium density-gradient centrifugation. After reduction with 0.2 M-mercaptoethanol, protein was released from the glycoprotein, which consisted of a major 70000-mol.wt. component and a minor 60000-mol.wt. component. The 70000-mol.wt. protein fraction was separated from the reduced glycoprotein by either density-gradient centrifugation in CsCl or by gel filtration. Analysis of the 70000-mol.wt. protein fraction showed that, within the limits of the analysis, it was non-glycosylated, and its amino acid analysis was quite different from that of the reduced glycoprotein, which is high in serine, threonine and proline. There was a ratio of one 70000-mol.wt. protein per native glycoprotein molecule of 2 X 10(6) mol.wt. Dissociation of the native glycoprotein into glycoprotein subunits (5 X 10(5) mol.wt.) by reduction or proteolysis results in the release or hydrolysis respectively of the 70000-mol.wt. protein. A similar 70000-mol.wt. protein is demonstrated in human gastric mucus glycoprotein. A structural role for the proteins in these mucus glycoproteins is proposed.     

Purification of the 22000- and 20000-mol.wt. forms of human somatotropin and characterization of their binding to liver and mammary binding sites.

Quantitative data concerning the binding of 22000-mol.wt. human somatotropin and its 20000-mol.wt. variant are described using pregnant-rabbit liver and mammary-gland receptors. The purification and the complete chemical characterization of both human somatotropin and its 20000-mol.wt. variant is also presented. Contamination of the 20000-mol.wt.-variant preparation by 22000-mol.wt. hormone was found to be 0.5% by weight as measured in radioimmunoassay using monoclonal antibody. Labelling of human somatotropin and its 20000-mol.wt. variant using the Iodogen method is described as well as the characterization of the binding to pregnant-rabbit liver and mammary-gland receptor preparations. The maximum binding capacity of the 125I-labelled human somatotropin was between 50 and 60% to liver particulate receptor, whereas that of the 20000-mol.wt. variant was 30%. The specificity of binding of both forms to rabbit hepatic and mammary-gland receptor was found to be similar for both proteins in the same system. The affinity constants and capacity were respectively 0.7 X 10(10)M-1 and 815 fmol/mg of protein for human somatotropin and 0.6 X 10(10)M-1 and 1.250 fmol/mg of protein for the 20000-mol.wt. variant. These data suggest that both proteins behave as partial agonists to the receptors studied.     

Release of two thioesterase domains from fatty acid synthetase by limited digestion with trypsin.

Limited digestion, with trypsin, of the fatty acid synthetase from rat mammary gland releases an enzymically active thioesterase component that, under denaturing conditions, consists of two major species of mol.wts. 35000 and 17500 and a minor species, mol.wt. 15,000. The 17500- and 150000-mol.wt. species are shown to originate from the 35000-mol.wt. species as a result of nicking by trypsin. The nicked polypeptides are enzymically active. The fatty acid synthetase is inhibited by [1,3-14C]di-isopropyl phosphorofluoridate, which is shown to bind to, and inactivate, two thioesterase active sites. When the [1,3-14C]di-isopropyl phosphate-labelled fatty acid synthetase is subjected to limited digestion with trypsin, all of the radioactivity is recovered in the isolated thioesterase component, i.e. in the 35000-mol.wt. polypeptide and its nicked products. Since the isolated thioesterase is shown to bind only one di-isopropyl phosphate residue per 35000-mol.wt. polypeptide, we conclude that the fatty acid synthetase has two thioesterase domains, both of which are removed by limited trypsin treatment.     

Preparation of hepatic gap (communicating) junctions. Identification of the constituent polypeptide subunits

1. Gap (communicating) junctions are plasma-membrane specializations of characteristic morphology that form transmembrane channels allowing direct communication between cells. Their preparation is described starting from mouse liver plasma membranes and the constituent polypeptides are deduced. 2. Gap junctions co-purify with collagen fibres when the plasma-membrane residues insoluble in N-dodecyl sarcosinate are fractionated on sucrose gradients. Sucrose-density perturbation by relipidation of isolated gap junctions or the use of urea to remove non-junctional membranes both failed to diminish the collagen content of fractions. 3. Removal of collagen by treatment with purified collagenase preparations yielded morphologically satisfactory gap-junction fractions. Analysis by polyacrylamide-gel electrophoresis of the polypeptides present in gap junctions prepared by procedures omitting or using collagenases indicated two non-glycosylated polypeptides, a major component of apparent mol.wt. 38000 and a minor 40000-mol.wt. component. These two polypeptides were also present in plasma membranes and the intermediate fractions. 4. Proteolysis of the gap-junction polypeptides yielding components of mol.wt. 34000, 25000 and below 20000 occurred when iodinated gap junctions were subject to prolonged collagenase treatment, thus explaining the variable polypeptide composition of gap junctions reported by others. 5. The morphological properties of the isolated gap junctions prepared by the various procedures are described.     

Comparison of pathways of copper metabolism in aorta and liver. A functional test of metallothionein.

Soluble fractions from chick liver and aorta were examined for copper-binding proteins. In liver a zinc-binding thionein appeared to be the major binding protein for copper. Aortic tissue contained only traces of this thionein protein. Unlike liver, moderate amounts of soluble copper in aorta showed no association with macromolecules. Chicks fed on copper-deficient diets for 8 days had one-third the liver copper concentrations of controls. Aortic copper concentration was decreased only slightly, but the activity of lysyl oxidase, a copper-dependent enzyme in aorta, was decreased significantly. Treating the deficient chicks with CuSO4 (1 mg/kg) restored liver copper rapidly. The increase correlated with the binding of copper to a 10 000-mol.wt. component in the soluble fraction. Aortic copper concentrations responded much less to the CuSO4 treatment, but lysyl oxidase activity was again measurable in the tissue. Radioactive isotopes of copper bound almost exclusively to the 10 000-mol.wt. component in liver and to components of mol.wt. 30 000 or above in aorta. Hardly any of the administered radioactivity appeared with the 10 000-mol.wt. components in aorta, and none was found with unbound copper. The 30 000-mol.wt. components in aorta showed superoxide dismutase activity that was sensitive to NaCN. They also showed the highest specific activity of copper of any other aorta component. A clear distinction was seen between the metabolism of copper in liver and aortic tissues. Whereas a copper thionein, metallothionein, was a major component in the liver pathway, it is doubtful that this protein plays a major role in the intracellular metabolism of copper in aortic tissue.     

Proteinase inhibitors of bovine nasal cartilage.

Extracts from bovine nasal cartilage with 1 M-guanidinium chloride were fractionated by ultrafiltration. Gel chromatography of the low-molecular-weight material resolved three distinct fractions with inhibitory activity against (a) collagenases (22000 mol.wt.), (b) thiol proteinases cathepsin B and papain (13000 mol.wt.), and (c) trypsin and other serine proteinases (7000 mol.wt.).     

Absence of two membrane proteins containing extracellular thiol groups in Rhnull human erythrocytes.

Rhnull human erythrocytes lack all the antigens of the Rhesus blood-group system and are associated with mild chronic haemolytic anaemia. These erythrocytes have an abnormal shape and increased osmotic fragility. Labelling studies with the impermeant maleimide N-maleoylmethionine [35S]sulphone show that Rhnull erythrocytes lack two extracellular thiol-group-containing membrane components of apparent mol.wts. 32 000 and 34 000. Immunoprecipitation with mouse monoclonal antibody R6A (which reacts with all normal erythrocytes, but fails to react with Rhnull erythrocytes) specifically precipitates the 34 000-mol.wt. component from normal erythrocytes. Similar studies with human anti-Rh(D) serum shows that this antibody reacts with the 32 000-mol.wt. component. The results suggest that the R6A-binding polypeptide and the Rh(D) polypeptide may be involved in the maintenance of the shape and viability of the human erythrocyte.     

Characterization of molecules involved in protein translocation using a specific antibody

The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane- bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived. The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover, immunoprecipitation of elastase extracts alkylated with [14C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of mol wt 72,000.     

Characterization and partial sequence of di-iodosulphophenyl isothiocyanate-binding peptide from human erythrocyte anion-transport protein.

We investigated the presumed anion-binding domain of the anion-transport protein from human erythrocyte membranes, using 2,6-di-iodo-4-sulphophenyl isothiocyanate, an inhibitor of anion transport. The 125I-labelled reagent binds covalently to the protein with a half-maximal inhibitory concentration of 86 microM. Treatment of unsealed erythrocyte 'ghosts' with chymotrypsin yielded a membrane-bound fragment (mol.wt. 14 500 +/- 1000) that contained all the protein-bound radioactivity. The binding of the inhibitor to this peptide gave a pattern very similar to that obtained for the effect of the compound on phosphate transport into erythrocytes. The peptide is therefore presumed to be intimately involved in the mediation of anion exchange. Cleavage of the 14 500-mol.wt. transmembrane fragment with CNBr resulted in the production of two peptides with apparent molecular weights of 8800 and 4700. The 4700-mol.wt. peptide is the N-terminal portion of the 14 500-mol.wt. peptide. The attachment site for 2,6-di-iodo-4-sulphophenyl isothiocyanate is situated near the C-terminal of the 8800-mol.wt. peptide. This locates the inhibitor-binding site near the chymotrypsin cleavage point at the extracellular surface of the membrane. A partial sequence (residues 1--38) of the 8800-mol.wt. peptide was obtained.     

Secretion of immunoreactive calcitonin by human breast carcinomas.

Twenty-three out of 28 patients with metastatic breast carcinoma and one out of 13 patients with localised disease had raised levels of plasma immunoreactive calcitonin. Monolayer cultures of breast carcinomas maintained for up to 10 weeks released immunoreactive calcitonin, and a primary breast carcinoma passaged in "nude" mice for over a year contained material immunologically and chromatographically resembling the monomeric form of human calcitonin. These studies indicate that breast carcinomas can produce calcitonin and that plasma calcitonin measurements may be useful in staging patients with breast carcinomas.     

Polymeric structure of pig small-intestinal mucus glycoprotein. Dissociation by proteolysis or by reduction of disulphide bridges.

Pig small-intestinal mucus glycoprotein, of molecular weight 1.72 X 10(6), is cleaved by Pronase digestion into glycoprotein subunits of molecular weight 4.5 X 10(5). Of the protein component of the native glycoprotein 29% by weight was lost on Pronase digestion, with no loss of carbohydrate. The non-glycosylated region of the protein that was lost with proteolytic digestion had a broad spectrum of amino acid residues, in contrast with the glycosylated region of the protein, which was resistant to proteolysis and was rich in serine, threonine and proline residues. Reduction with 0.2M-mercaptoethanol dissociated the Pronase-digested glycoprotein subunits into smaller glycoprotein subunits of molecular weight 2.7 X 10(5). On reduction, the native glycoprotein was dissociated into subunits of molecular weight 2.4 X 10(5), a similar size to those obtained from reduction of the Pronase-digested glycoprotein. On reductive dissociation of the native glycoprotein, in addition to glycoprotein subunits, protein was also released principally as a component of 90000 molecular weight. This protein was separated quantitatively from the reduced glycoprotein in amounts compatible with one 90000-mol.wt. protein molecule per 1.72 X 10(6)-mol.wt. native glycoprotein molecule. No 90000-mol.wt. protein was released on reduction of the isolated Pronase-digested glycoprotein. Pig small-intestinal mucus glycoprotein is therefore a covalent polymer of glycoprotein subunits joined by disulphide bridges. This polymeric structure differs in important respects from that previously shown for gastric mucus, in particular with respect to the size and number of component subunits per native molecule.     

The molecular integrity of chloroplast ribosomal ribonucleic acid

Instability of chloroplast rRNA has been observed with essentially all chloroplast RNA preparations. This paper describes experiments that show that, under normal conditions of preparation and fractionation, only the heavy chloroplast component (mol.wt. 1.1x10(6)) is unstable, the light chloroplast rRNA (mol.wt. 0.56x10(6)) and the cytoplasmic rRNA species (mol.wt. 1.3x10(6) and 0.70x10(6)) being stable. The stability of the 1.1x10(6)-mol. wt. molecule varies with different plant species, as also does the size and the number of fragments produced. Cleavages in three particular regions of the molecule are very frequent within the range of tissues studied. The 1.1x10(6)-mol.wt. rRNA is, however, stabilized by the presence of Mg(2+) during the preparation and fractionation of the RNA.     

Studies on the assembly of the rat lens capsule. Biosynthesis and partial characterization of the collagenous components.

1. Isolated rat lens capsules synthesized hydroxy[3H]proline-containing polypeptides when incubated with [3H]proline. 2. The collagenous polypeptides synthesized during a 2 h incubation were analyzed by both gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and shown to have an apparent mol.wt. of approx. 180,000. 3. No evidence was obtained for conversion of these polypeptides into a lower-molecular-weight species in experiments where capsules were labelled for 2 h and chased with non-radioactive proline for up to 22 h. However, a time-dependent incorporation of the 180,000-mol.wt. species into a larger collagenous component was observed and this could be prevented by the inclusion of beta-aminopropionitrile in the incubation medium. 4. The radioactive components synthesized by the capsules correspond to subunits of the intact lens capsule and the direct incorporation of the polypeptide of mol.wt. 180,000 into deoxycholate-insoluble basement membrane was demonstrated.     

A possible role for 5-hydroxytryptamine as a mediator for calcitonin actions on the gastrointestinal tract and pancreas in rats.

Administration of pig calcitonin (10 M.R.C. units/kg body wt.) produced an immediate decrease in 5-hydroxytryptamine content in the antrum-duodenum region and ileum. In contrast, the hormone treatment rapidly increased the concentration of 5-hydroxytryptamine in the blood and pancreas. Serum immunoreactive gastrin did not change over a period of 3h after calcitonin administration. The results suggest that the release of 5-hydroxytryptamine probably mediates calcitonin actions on the gastrointestinal tract and pancreas.     

Effects of heat shock on the expression of thrombospondin by endothelial cells in culture

Heat-shock proteins from confluent primary cultures of bovine aortic endothelial cells were analyzed by SDS-polyacrylamide gels. In addition to the increased synthesis of the classical heat-shock proteins, there is an increase of a 180,000-mol wt polypeptide in the growth media of heat-shocked cells. Immunoprecipitation with specific antiserum indicates that the 180,000-mol wt polypeptide is thrombospondin. Assay of mRNA levels coding for thrombospondin after brief hyperthermic treatment (45 degrees C, 10 min), followed by a recovery of 2 h at 37 degrees C, results in a twofold increase in mRNA abundance. In contrast, the activation level of the 71,000-mol wt heat-shock protein mRNA occurs at an earlier time than for thrombospondin mRNA. Immunofluorescence microscopy was used to study the intracellular and extracellular distribution of thrombospondin. Thrombospondin is localized to a prominent pattern of granules of intracellular fluorescence in a perinuclear distribution in cells not exposed to heat. Upon heat treatment, the pattern of granules of intracellular fluorescence appears more pronounced, and the fluorescence appears to be clustered more about the nucleus. There are at least three pools of extracellular forms of thrombospondin: (a) the fine fibrillar extracellular matrix thrombospondin; (b) the punctate granular thrombospondin; and (c) the thrombospondin found in the conditioned medium not associated with the extracellular matrix. When bovine aortic endothelial cells are exposed to heat, the extracellular matrix staining of a fibrillar nature is noticeably decreased, with an increase in the number and degree of fluorescence of focal areas where the punctate granule thrombospondin structures are highly localized. No gross morphological changes in extracellular matrix staining of fibronectin was noted. However, the intermediate filament network was very sensitive and collapsed around the nucleus after heat shock. We conclude that the expression of thrombospondin is heat-shock stimulated.     

Calcitonin biosynthesis: evidence for a precursor.

Calcitonin biosynthesis has been studied in chicken ultimobranchial glands incubated in vitro in the presence of radioactive amino acids. The results obtained suggest the existence of a biosynthetic precursor of higher molecular weight or procalcitonin. This precursor has been identified by pulse-chase experiments, molecular weight determinations, biological activity measurements and analysis of tryptic peptides. Its molecular weight is about 13000 (calcitonin, about 3500) as determined by polyacrylamide gel electrophoresis. Procalcitonin is present in small amounts in chicken ultimobranchial glands and it is biologically active in rats.     

The calcitonin receptor on T 47D breast cancer cells. Evidence for glycosylation.

The glycosyl nature of the receptor for the peptide hormone calcitonin has been investigated in a human breast cancer cell line, T 47D. Studies have been carried out to assess the ability of various lectins and of the antibiotic tunicamycin to inhibit specific binding of calcitonin to the cells, to reduce cross-linking of photoactive calcitonin to a macromolecular receptor component and to influence calcitonin stimulation of cyclic AMP. Pre-incubation of cells with low concentrations of tunicamycin for 72 h resulted in a reduction of total specific binding by approx. 80% and a 40% reduction in calcitonin-stimulated adenylate cyclase; formation of the cross-linked receptor component was also inhibited. Wheat-germ lectin showed the most marked inhibition of total specific binding and cyclic AMP production. However, cross-linking of photoactive calcitonin to receptor component was totally inhibited by this lectin. Soya-bean lectin brought about very little reduction in total specific binding but had more profound effects on calcitonin-stimulated cyclic AMP production and cross-linking of photoactive calcitonin. Concanavalin A and lentil lectin showed some inhibition of all parameters. The data indicate that the calcitonin receptor in T 47D cells is associated with glycosyl moieties, the major contributors of which are N-acetyl-D-glucosamine residues, but N-acetyl-D-galactosamine and mannose residues are also associated.     

Isolation and preliminary characterization of epithelial-specific messenger ribonucleic acids and their products during embryonic tooth development.

Experiments were designed to identify and characterize tissue-specific proteins involved in the process of tooth organogenesis. Epithelial and mesenchymal proteins were extracted from intact molar organs or mechanically separated tissues obtained from 25-day New Zealand White rabbit embryos. Labelling experiments with [35S]methionine followed by radioautography or gel electrophoresis and fluorography showed the presence of label only in epithelial proteins. Most of these proteins range from 43 000 mol.wt. and higher, except for one band of approx. 16 000 mol.wt. A mRNA fraction of 16--26S was isolated by ultracentrifugation on sucrose gradients. When translated in a reticulocyte-lysate cell-free system, the mRNA obtained from intact molar organs resulted in the synthesis of three proteins, of mol.wts. 65 000, 58 000 and 43 000. A similar mRNA fraction obtained from dental-pulp mesenchyme gave only the 43 000-mol.wt. protein, indicating that the 65 000- and 58 000-mol.wt. proteins are derived from epithelial cells.