Intracellular turnover of stable and labile soluble liver proteins

The cytosol proteins of mouse or rat liver were separated on the basis of charge and characterized with respect to molecular size, turnover in vivo, and several chemical properties. The basic proteins, which were synthesized and degraded slower than acidic proteins, were generally smaller, as multimers and subunits, than the acidic proteins. Charge and size, therefore, did not appear to be independent characteristics for soluble liver proteins. The amino acid composition of the smaller, basic, stable proteins was very similar to that of the larger, acidic, labile proteins. The charge differences between these two protein fractions could be attributed to the ratio of acidic to basic amino acid residues rather than to carbohydrate, nucleic acid, or phosphate content. The percentage of hydrophobic amino acid residues in the two protein fractions was the same. The acidic labile proteins were more vulnerable to proteolysis and associated with the lysosomal-mitochondrial fractions somewhat more extensively in vitro than the basic, stable proteins. These studies indicate that the information determining the half-lives of soluble enzymes is in the protein moiety of those enzymes and that the factors that correlate with turnover may not be independent variables for soluble mammalian proteins.     

Identification of the milk fat globule membrane proteins: I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.     

Post-translational heterogeneity of the human vitamin D-binding protein (group-specific component)

The vitamin D-binding protein in human serum (the group-specific component) is an alpha 2-globulin which is genetically polymorphic in all populations studied. Previous work (J. Svasti and B. H. Bowman (1978) J. Biol. Chem. 253, 5188-5194, and J. Svasti, A. Kurosky, A. Bennett, and B. H. Bowman (1979) Biochemistry 18, 1611-1617) has shown that the electrophoretic variations of the proteins controlled by two allelic genes, Gc1 and Gc2, are due to at least three amino acid substitutions between Gc1 and Gc2 (Svasti et al. (1979] and to heterogeneity in the Gc1 phenotype arising from carbohydrate dissimilarities. Gc1 migrates electrophoretically as two protein bands, while Gc2 migrates cathodally as a single band. This study demonstrates a post-translational glycosylation difference occurring in a single area of the Gc1 sequence which accounts for the heterogeneity observed previously. The glycosylation site, a threonine residue, appears to be in a sequence which differs between Gc1 and Gc2. The O-glycosidic bond, which is typical of mucins, is rare in plasma proteins. The cyanogen bromide fragment containing the galactosamine-containing carbohydrate in Gc1 was partially sequenced through 20 residues from the amino terminus. No detectable galactosamine could be found in the homologous cyanogen bromide fragment in Gc2. A new purification procedure for the vitamin D-binding protein in human plasma has been developed. Three chromatographic steps provide purified protein.     

Preferential binding of two compatible solutes to the glycan moieties of Peniophora lycii phytase

Regulation of hydration behavior, and the concomitant effects on solubility and other properties, has been suggested as a main function of protein glycosylation. In this work, we have studied the hydration of the heavily glycosylated Peniophora lycii phytase in solutions (0.15-1.1 m) of the two compatible solutes glycerol and sorbitol. Osmometric measurements showed that glycerol preferentially binds to phytase (i.e., glycerol-glycoprotein interactions are more favorable than water-glycoprotein interactions resulting in a preferential accumulation of glycerol near the protein interface), while sorbitol is preferentially excluded from the hydration sphere (water-glycoprotein interactions are the more favorable). To assess contributions from carbohydrate and peptide moieties, respectively, we compared phytase (Phy) and a modified, yet enzymatically active form (dgPhy) in which 90% of the glycans had been removed. This revealed that both polyols showed a pronounced and approximately equal degree of preferential binding to the carbohydrate moiety. This preferential binding of polyols to glycans is in contrast to the exclusion from peptide interfaces observed here (for dgPhy) and in numerous previous reports on nonglycosylated proteins. Despite the distinct differences between peptide and carbohydrate groups, glycosylation had no effect on the stabilizing action provided by glycerol and sorbitol. On the basis of this, it was concluded that the carbohydrate mantle of Phy is equally accessible in the native and thermally denatured states, respectively (most likely fully accessible in both), and thus that its interactions with compatible solutes have little or no effect on conformational equilibria of the glycoprotein. For solubility and aggregation equilibria, on the other hand, the results suggest a polyol-induced stabilization of monomeric forms.     

Effects of E. coli chaperones on the solubility of human receptors in an in vitro expression system

The implementation of efficient technologies for the production of recombinant mammalian membrane receptors is an outstanding challenge in understanding receptor-ligand actions and the development of therapeutic antibodies. In order to improve the solubility of recombinant extracellular domains of human membrane receptors expressed in Escherichia coli, proteins were synthesized by an E. coli in vitro translation system supplemented with bacterial molecular chaperones, such as GroEL-GroES (GroEL/ES), Trigger factor (TF), a DnaK-DnaJ-GrpE chaperone system (DnaKJE), and/or a heat shock protein Hsp100, ClpB. The following three proteins that are prone to aggregation were examined: the extracellular domain (ECD) or the second immunoglobulin-like domain (IgII) of the human neurotrophin receptor TrkC (TrkC-ECD and TrkC-IgII), and the C-type lectin carbohydrate recognition domain of the human asialoglycoprotein receptor (ASGPR HI CRD). The cooperative chaperone system including GroEL/ES, DnaKJE and ClpB had a marked effect on the solubility of TrkC-ECD and TrkC-IgII, and the GroEL/ES-DnaKJE-TF chaperone system was more effective for TrkC-IgII. The GroEL/ES-DnaKJE-TF chaperone network increased the yield of soluble ASGPR HI CRD. The present findings demonstrate that E. coli molecular chaperones are useful in improving the yield of soluble recombinant extracellular domains of human membrane receptors in an E. coli expression system.     

Quantitation of oligosaccharides released by the β-elimination reaction

A quantitative micromethod has been described for monitoring the rate and extent of the β-elimination reaction as applied to O-glycosyl-glycoproteins utilizing alkaline tritiated borohydride. The procedure simultaneously labels the released oligosaccharides by their reduction to the corresponding tritiated alditols. The alkaline tritiated borohydride treatment also results in the labeling of the protein moiety of the glycoprotein and this can be quantitatively separated from the carbohydrate moiety on a cation exchange resin; the carbohydrate moiety is not adsorbed, while the protein moiety is adsorbed and then eluted with HCl. The radioactivity in the aqueous eluate of the resin is therefore a direct measure of the amount of oligosaccharides released by the β-elimination reaction. The sensitivity of the method is dependent on the specific activity of the tritiated sodium borohydride used. The stoichiometry of the reaction has been established by the use of N-acetylgalactosaminyl-O-glycoproteins, demonstrating that at the completion of the β-elimination reaction: (a) none of the radioactivity attributable to the protein moiety contaminates the carbohydrate moiety, (b) all the carbohydrate components of the glycoprotein are found in the aqueous eluate from the cationic exchange resin, (c) all the radioactivity in this aqueous eluate is associated with the sugar known to be at the reducing end of the oligosaccharide chain bound to serine or threonine of the glycoprotein (in the examples discussed, N-acetylgalactosamine), and (d) there is no additional hydrolysis of the oligosaccharide chains during the processing.     

tRNA concentration fine tunes protein solubility

Clusters of codons pairing to low-abundance tRNAs synchronize the translation with co-translational folding of single domains in multidomain proteins. Although proven with some examples, the impact of the ribosomal speed on the folding and solubility on a global, cell-wide level remains elusive. Here we show that upregulation of three low-abundance tRNAs in Escherichia coli increased the aggregation propensity of several cellular proteins as a result of an accelerated elongation rate. Intriguingly, alterations in the concentration of the natural tRNA pool compromised the solubility of various chaperones consequently rendering the solubility of some chaperone-dependent proteins.     

The apical lamina of the sea urchin embryo: Major glycoproteins associated with the hyaline layer

The hyaline layer (HL) surrounding the sea urchin blastula appears to dissolve in 1 M glycine. However, after this treatment, there persists over the surfaces of the blastomeres a layer of material, referred to here as the apical lamina (AL), that sloughs off as an adhesive convoluted bag upon gradual dissociation of the embryo. Isolated hyaline layers, referred to as HL-AL complexes, were analyzed by urea-SDS-polyacrylamide gel electrophoresis. A major protein of the HL-AL complex, hyalin, bands or precipitates in the stacking gel. Two other major proteins, both strongly PAS positive, migrate with apparent molecular weights of 175K and 145K daltons. As with intact embryos, the glycine wash removes the hyalin protein from the isolated HL-AL complex, leaving the undissolved AL which consists primarily of the 175K- and 145K-dalton proteins. The embryo's own perivitelline-localized cortical granule peroxidase heavily radioiodinates the proteins of the HL-AL complex, further verifying their apical, extracellular location. Unlike hyalin, the AL proteins do not precipitate with calcium ions. Compared to the entire HL-AL complex, the AL contains a greater percentage of carbohydrate. No sialic acid is associated with the HL-AL complex, but the AL contains some sulfate. In contrast to a published report based on ultrastructural staining, no biochemical evidence was found in this study for the presence of collagen or significant glycosaminoglycan within the HL-AL complex. No developmental differences were observed in AL proteins from 1-hr-old embryos compared to those from blastulae. However, there is evidence suggesting heterogeneity and developmental differences in hyalin. The possible organization of hyalin and the AL proteins into separate layers surrounding the embryo is discussed. The influence of the AL proteins in morphogenesis and cell adhesion is considered, and hypothetical roles attributed to the HL and hyalin are critically questioned.     

Interactions of a new antimitotic agent, NSC-181928, with purified tubulin

A series of biochemical analyses were carried out with keratoconus and normal corneas to determine the amount of stromal collagen, degree of posttranslational modification of collagen and the solubility of collagen. Our results revealed there was no obvious alteration in the degree of posttranslational modification of collagen in keratoconus corneas. However, the amount of collagen decreases and solubility of collagen increases in keratoconus corneas. It was also found that keratoconus corneas in organ culture produce substantially more collagenase and gelatinase activities than normal corneas. Our results suggest that keratoconus may represent a collagenolytic disease.     

Effect of glycosylation on the mechanism of renaturation of invertase from yeast

N-Glycosylation occurs cotranslationally as soon as the growing polypeptide chain enters the endoplasmic reticulum, before the final native-like folded state is reached. We examined the role of the carbohydrate chains in the mechanism of protein folding. The in vitro folding and association of yeast invertase are used as an experimental system. External invertase contains approximately 50% carbohydrate, whereas cytoplasmic invertase is not glycosylated. The functional native state of both proteins is a homodimer. At pH greater than or equal to 6.5 and at protein concentrations below 3 micrograms/ml, the kinetics of reactivation and the final yields are similar for the two invertases. For both proteins, reactivation is a sequential reaction with a lag phase at the beginning. The nonglycosylated protein tends to aggregate during reactivation at low pH and at protein concentrations above 3 micrograms/ml. After separation of inactive material, the renatured protein is indistinguishable from the original native state by a number of physicochemical and functional criteria. The results suggest that the carbohydrate moiety does not affect the mechanism of folding and association of invertase. However, glycosylation improves the solubility of unfolded or partially folded invertase molecules and hence leads to a suppression of irreversible aggregation. Such a protective effect may also be important for the in vivo maturation of nascent glycosylated protein chains.     

应用优化的双向电泳技术建立纤维堆囊菌So0157-2蛋白质组数据库

堆囊菌丰富的次级代谢产物是新药的重要来源,而蛋白质组学分析是研究代谢调控的有效方法．然而堆囊菌含有大量的胞外多糖以及黏液,干扰了蛋白质组学分析中蛋白质的溶解度、分辨率及重现性．为了高通量地筛选Sorangium cellulosum So0157-2表达的特异性蛋白,实验优化了S. cellulosum So0157-2双向电泳方法．首先,S. cellulosum So0157-2蛋白在裂解液中有更好的溶解度．pH 3～10非线性胶条和1 mg的蛋白上样量适用于第一向等电聚焦,分别提高了蛋白质点的分辨率和低丰度蛋白质的表达．15% SDS-PAGE 改善了S. cellulosum So0157-2蛋白分离的分辨率和重现性．最终,通过优化的双向电泳方法获得了S. cellulosum So0157-2 在M26培养基中培养3天的全蛋白质表达谱,并检测到552个蛋白质点．进而对表达蛋白通过MALDI-TOF-MS进行质谱鉴定,其中474个蛋白质得到鉴定,鉴定率85.9%．得到鉴定的蛋白质包括细胞结构和功能组分,以及细胞代谢合成酶类,其中8个蛋白质与糖类的转化和代谢相关,这有助于糖基化埃博霉素A的深入研究．该优化方法为进一步建立纤维堆囊菌So0157-2在各种培养条件下的蛋白质组表达数据库打下基础．     

Isolation and properties of four alpha-amylase isozymes from human submandibular saliva

Four isoamylases have been isolated from human submandibular secretions by gel filtration and isoelectric focusing. The isozymes (1A, 1B, 2A, 2B) were each purified about 8-fold and each yielded one major band on disc gel electrophoresis. In all cases the major protein band contained more than 95% of the protein and amylase activity recovered. The isoenzymes, in order of their relative positions on the polyacrylamide gels (from the anodal end), their isoelectric points, and percentage distribution in the submandibular secretion are as follows: isozyme 2A, pH 5.9, 9%; isozyme 1A, pH 5.9, 18%; isozyme 2B, pH 6.4, 63%; isozyme 1B, pH 6.4, 10%. Amino acid analyses showed that the protein compositions of the four isoamylases were essentially the same. Possible differences were noted in aspartic acid, serine, glutamic acid, and proline contents. Molecular weights, determined by SDS disc gel electrophoresis, were 57,000 for 1A and 1B, and 54,000 for 2A and 2B. This molecular weight difference is attributed mainly to the presence of bound carbohydrate on isozymes 1A and 1B. Gas Chromatographic analysis was used for determining the carbohydrate compositions. Molar ratios of sugars were similar for both glycoprotein amylases (moles sugar/mole enzyme): glucosamine, 3; mannose, 3; galactose, 2; fucose, 3. Isoamylase 1A, which had more carbohydrate than 1B, also contained about 2 moles of N-acetylneuraminic acid. Sialic acid was not detected in isozyme 1B.     

Enzyme-linked lectin assay (ELLA). II. Detection of carbohydrate groups on the surface of unfixed cells

An enzyme-linked lectin assay (ELLA) has been developed to detect specific carbohydrate units on the surface of unfixed cells. The assay may be read in standard ELISA plate readers, since the cell-bound enzyme-lectin conjugate is specifically eluted from the cells prior to development of the conjugate. ELLA, when read in an enzyme-linked immunosorbent assay (ELISA) plate reader, allows better detection and relative quantitation of specific surface carbohydrate units than is possible by standard immunofluorescence with fluorescein-conjugated lectins.     

A C-type lectin from the tunicate, Styela plicata, that modulates cellular activity

Previous studies have identified proteins from tunicates (invertebrate members of the Phylum Chordata) that have physicochemical and functional properties similar to those of the inflammatory cytokine, interleukin 1 (IL-1). Here we characterize one of those proteins from the tunicate, Styela plicata, that can stimulate tunicate and mammalian cell proliferation, activate phagocytosis, increase interleukin 2 (IL-2) secretion by mammalian peripheral blood mononuclear cells and enhance IL-2 receptor (IL-2R) expression by mammalian EL-4.IL-2 cells. Partial amino acid sequence data showed that the S. plicata protein resembles three C-type lectins (TC14, TC14-1 and TC14-2) from a closely related tunicate species, Polyandrocarpa misakiensis. Its similarity to carbohydrate recognition domains (CRDs) from P. misakiensis lectins suggests that the S. plicata protein modulates the activities of mammalian immunocompetent cells by interacting with carbohydrate moieties of glycosylated cell surface receptors.     

Is adrenal proenkephalin glcosylated?

Adrenal proenkephalin contains the sequence AsnSerSer that is a typical site for the attachment of asparagine-linked carbohydrate. The 5300- and 18,200-Da bovine adrenal proteins derived from proenkephalin contain this recognition sequence and were therefore analyzed for the presence of both amino and neutral sugars. Less than 0.05 mol of amino sugar and less than 0.1 mol of neutral sugar were found per mole of each protein. No amino sugar was detected in other high-molecular-weight adrenal [Met]enicephalin-containing proteins. Together these findings indicate that bovine adrenal proenkephalin does not contain asparagine-linked carbohydrate.     

Capillary affinity electrophoresis for the screening of post-translational modification of proteins with carbohydrates

Glycosylation is one of the most important post-translational events for proteins, affecting their functions in health and disease, and plays significant roles in various information traffics for intracellular and intercellular biological events (Hancock, W. S. J. Proteome Res. 2002, 1, 297). We have attempted to obtain the information on the numbers and amounts of carbohydrate chains. Interaction between carbohydrate chains and proteins that recognize them is a target to understand the biological roles of glycosylation. To date, there have been a few strategies for simultaneous analysis of the interactions between complex mixtures of carbohydrates and proteins. Here, we report an approach to categorize carbohydrate chains using a few glycoprotein samples as models for the studies on the analysis of post-translational modification of proteins with carbohydrates. A combination of some specific lectins was used as carbohydrate-binding proteins. The method is based on high-resolution separation of fluorescent-labeled carbohydrates by capillary electrophoresis with laser-induced fluorescent detection in the presence of carbohydrate-binding proteins at different concentrations. The present technique affords (1) simultaneous determination of carbohydrate chains, (2) binding specificity of the constituent carbohydrate chains to specific proteins, and (3) kinetic data such as the association constant of each carbohydrate. We found that the lectins employed in the present study could discriminate subtle difference in linkages and resolved the carbohydrate mixtures. The results will be useful, for example, to understand the biological events expressed with carbohydrate changes on the cell surface.     

利用EDA融合标签高效表达猪圆环病毒2型壳蛋白

原核生物作为宿主细胞被广泛应用于异源蛋白质的重组表达,并且为生物活性蛋白质的制备提供了一种高效、经济的方法,因而在分子生物学中得到普遍的应用。然而,病毒蛋白在使用原核重组表达系统进行重组表达时,会出现病毒蛋白溶解性差和表达量低等问题。因此,通过使用各种融合标签以增加目的重组蛋白的表达量和溶解性成为有效的方法。本研究通过使用3种融合标签（EDA标签、MBP标签和GST标签）以获得表达量高的可溶性重组表达猪圆环病毒2型壳蛋白;并比较3种融合标签对该蛋白表达量、溶解性和稳定性的影响。研究结果表明,EDA标签可以显著提高重组表达的猪圆环病毒2型壳蛋白表达量,并且能够增强该蛋白的稳定性;MBP标签可增强重组表达的猪圆环病毒2型壳蛋白表达量,但是不能改善该蛋白的稳定性;GST标签能够增强该重组表达蛋白的表达量,但是不能增强该蛋白的溶解性和稳定性。本研究将EDA作为PCV2-CP蛋白的融合标签,显著提高PCV2-CP-EDA重组蛋白的表达量和增强该重组蛋白的稳定性,为病毒蛋白的可溶性重组表达提供了一种新的融合标签。     

Synthesis,characterization and fluorescent properties of water-soluble glycopolymer bearing curcumin pendant residues

Curcumin is a potential natural anticancer drug with low oral bioavailability because of poor water solubility. The aqueous solubility of curcumin is enhanced by means of modification with the carbohydrate units. Polymerization of the curcumin-containing monomer with carbohydrate-containing monomer gives the water-soluble glycopolymer bearing curcumin pendant residues. The obtained copolymers (P1 and P2) having desirable water solubility were well-characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), UV–Vis absorption spectroscopy, and photoluminescence spectroscopy. The copolymer P2 with a molar ratio of 1:6 (curcumin/carbohydrate) calculated from the proton NMR results exhibits a similar anticancer activity compared to original curcumin, which may serve as a potential chemotherapeutic agent in the field of anticancer medicine.     

Glycogen in the proventriculus of the tsetse fly

Glycogen was detected in the proventriculus of the tsetse fly, Glossina morsitans morsitans, by ultrastructural, histochemical, and biochemical methods. This organ contained ten times or more glycogen on a dry weight basis than was found in the thoracic muscle. Proventriculi of male tsetse contained less glycogen than those of females belonging to the same age group and in teneral flies the amount of glycogen was about 50 per cent lower than in mature, fed flies of the same sex. Although the thoracic muscle of tsetse flies was considerably lower in glycogen than that of blowflies the amounts in the proventriculus of mature females of the two insect species were almost equal. It is suggested that this carbohydrate store may supply the energy required for secretory processes.     

Evaluation of applicability of tryptic peptide maps for "finger printing" mitochondrial membrane protein preparations

A background pattern of intense, polar and basic peptides is generated in a mixture of proteins which limits the applicability of “fingerprinting” by peptide maps as a method of establishing homologies among membrane proteins. In addition, it is observed that in such mixtures of proteins the peptide pattern in the “neutral” portion of the map is characterized by a few, weak, tailing peptides which appear on a smeared background of ninhydrin positive material. It is concluded that several types of control maps must be prepared along with maps of membrane fractions if real homologies are to be identified. Application of such control maps to analysis of a sample of mitochondrial membrane protein and a sample of quasicrystalline protein indicated that both of these preparations are disperse mixtures of proteins.