Urea and guanidinium chloride denature protein L in different ways in molecular dynamics simulations.

In performing protein-denaturation experiments, it is common to employ different kinds of denaturants interchangeably. We make use of molecular dynamics simulations of Protein L in water, in urea, and in guanidinium chloride (GdmCl) to ascertain if there are any structural differences in the associated unfolding processes. The simulation of proteins in solutions of GdmCl is complicated by the large number of charges involved, making it difficult to set up a realistic force field. Furthermore, at high concentrations of this denaturant, the motion of the solvent slows considerably. The simulations show that the unfolding mechanism depends on the denaturing agent: in urea the beta-sheet is destabilized first, whereas in GdmCl, it is the alpha-helix. Moreover, whereas urea interacts with the protein accumulating in the first solvation shell, GdmCl displays a longer-range electrostatic effect that does not perturb the structure of the solvent close to the protein.

Detecting oxidative post-translational modifications in proteins.

Oxidative stress induces various post-translational modifications (PTM); some are reversible in vivo via enzymatic catalysis. The present paper reviews specific procedures for the detection of oxidative PTM in proteins, most of them including electrophoresis. Main topics are carbonylated and glutathionylated proteins as well as modification of selected amino acids (Cys, Tyr, Met, Trp, Lys).

Zea mays L. protein changes in response to potassium dichromate treatments.

The plant metabolic response to heavy metal stress is largely unknown. The present investigation was undertaken to examine the influence of different concentrations of potassium dichromate on the Zea mays L. plantlets. A clear effect of chromium on maize plantlets growth and seed germination was observed strating from 100-300 ppm up to 1500 ppm. In this concentration range, chromium uptake was dependent on the concentration in the medium. Metallothioneins, involved in heavy metal binding, were measured by capillary electrophoresis (CE), and showed a dose-response induction. Protein profile analyzed by two-dimensional gel electrophoresis showed differential expression of several proteins. Identification of spots of upregulated proteins was performed by MALDI mass spectrometry. Results showed that proteins induced by heavy metal exposure are principally involved in oxidative stress tolerance or in other stress pathways. Induction of proteins implicated in sugar metabolism was also observed. Identification of factors involved in plant response may lead to a better understanding of the mechanisms involved in cell protection and tolerance. This information could be used to improve agricultural production and environmental quality.

Role of alpha1 acid glycoprotein in the in vivo resistance of human BCR-ABL(+) leukemic cells to the abl inhibitor STI571.

BACKGROUND: Chronic myeloid leukemia is caused by a chromosomal translocation that results in an oncogenic fusion protein, Bcr-Abl. Bcr-Abl is a tyrosine kinase whose activity is inhibited by the antineoplastic drug STI571. This drug can cure mice given an injection of human leukemic cells, but treatment ultimately fails in animals that have large tumors when treatment is initiated. We created a mouse model to explore the mechanism of resistance in vivo. METHODS Nude mice were injected with KU812 Bcr-Abl(+) human leukemic cells. After 1 day (no evident tumors), 8 days, or 15 days (tumors >1 g), mice were treated with STI571 (160 mg/kg every 8 hours). Cells recovered from relapsing animals were used for in vitro experiments. Statistical tests were two-sided. RESULTS: Tumors regressed initially in all STI571-treated mice, but all mice treated 15 days after injection of tumor cells eventually relapsed. Relapsed animals did not respond to further STI571 treatment, and their Bcr-Abl kinase activity in vivo was not inhibited by STI571, despite high plasma concentrations of the drug. However, tumor cells from resistant animals were sensitive to STI571 in vitro, suggesting that a molecule in the plasma of relapsed animals may inactivate the drug. The plasma protein alpha1 acid glycoprotein (AGP) bound STI571 at physiologic concentrations in vitro and blocked the ability of STI571 to inhibit Bcr-Abl kinase activity in a dose-dependent manner. Plasma AGP concentrations were strongly associated with tumor load. Erythromycin competed with STI571 for AGP binding. When animals bearing large tumors were treated with STI571 alone or with a combination of STI571 and erythromycin, greater tumor reductions and better long-term tumor-free survival (10 of 12 versus one of 13 at day 180; P:<.001) were observed after the combination treatment. CONCLUSION: AGP in the plasma of relapsed animals binds to STI571, preventing this compound from inhibiting the Bcr/Abl tyrosine kinase. Molecules such as erythromycin that compete with STI571 for binding to AGP may enhance the therapeutic potential of this drug.

Electrophoretic analysis of horse tissue ferritins at different pH values.

Electrophoretic mobilities of ferritins from horse heart, liver and spleen were compared in the pH range 3.5-10. Electrophoretic titrations and continuous buffer electrophoreses were used. The order of anionic mobilities was heart > liver > spleen at alkaline pH and this order was reversed in the neutral to acidic range. This order of mobilities, and the intermediate behavior of liver in respect to the other two ferritins correlate with the known subunit composition of the three ferritins, and strongly support the idea of different amino acid residues being exposed on the protein shell surfaces. From the analyses of differential mobility around the pK values of the various ionizable groups it was concluded that heart and spleen ferritins have a very similar number of acidic amino acid residues on their surfaces, whereas they differ in basic residues. Heart seems to have about 15% more Lys and Arg, and twice as many His as spleen.

Binding of polyanions to carrier ampholytes in isoelectric focusing.

The binding of carrier ampholytes to polyanions is markedly pH-dependent: it is very strong at pH 3, rather weak at pH 5 and abolished at pH 7. Binding is affected by the type of negative charge, its density and spatial orientation on the polyanion. On the basis of the type of negative charge, the binding strength decreases in the following order: polyphosphate greater than polysulphate greater than polycarboxylate. Given the same type of negative charge, the binding is dependent on charge density and its space orientation: thus polyglutamic acid forms stronger complexes than polygalacturonic acid. The minimum length of the polyanion eliciting a measurable binding appears to be of the order of about six negative charges, as demonstrated with hexametaphosphate.

Isoelectric focusing of heparin. Evidence for complexing with carrier ampholytes.

The basis for heparin fractionation into 21 components by isoelectric focusing has been shown to be a strong interaction between the polysaccharide and different amphoteric species in the Ampholine mixture. This was demonstrated by altering the heparin/Ampholine ratio, by loading the sample either before focusing or to a prefocused gel slab and by re-running single heparin bands. The complexes exhibiting apparent pI values in the pH range 3.2--4.5 appear to be particularly stable, probably because an optimal amount of amino groups (4 to 5) in the Ampholine molecules are protonated. When stained with Toluidine blue, the heparin/Ampholine complexes precipitated in the gel exhibited different degrees of metachromasia, reflecting competition of the dye and individual components of Ampholine with respect to binding sites of heparin: at least three colours, violet, blue and indigo, are distinguishable. Ampholine, when added to a heparin . Toluidine blue complex in solution displaces the dye from the polysaccharide.

Affinity titration curves: determination of dissociation constants of lectin-sugar complexes and of their pH-dependence by isoelectric focusing electrophoresis.

By performing electrophoresis perpendicular to a stationary pH gradient (pH-mobility curves) in polyacrylamide gels containing a specific ligand either covalently fixed or entrapped in the gel matrix, it is possible to measure dissociation constants (Kd) and their pH-dependence in the pH range 3.5-10. The present technique, called 'affinity titration curves', is an extension of 'affinity electrophoresis'. This system has been applied to the study of the interaction between lectins and sugars: lectin from Ricinus communis seeds and alpha-D-galactose, and lectin from Lens culinaris seeds and alpha-D-mannose. The pH-dependence of Kd values indicated a more rapid decrement of affinity of both lectins for their ligands at acidic pH as compared to alkaline pH. For both lectins, maximum affinity was found in the pH range 7-8. Since the ionic strength of focused carrier ampholytes is 100-200-times lower than in conventional electrophoresis, the Kd values found by the present method are generally lower than the same values obtained by affinity electrophoresis.

Interaction of alpha 2-HS-glycoprotein with immobilized triazine dyes.

We studied the interaction of alpha 2-HS-glycoprotein with immobilized Cibacron blue F3-GA (Blue A) and Procion red HE-3B (Red A). When whole plasma was applied on the Blue A, alpha 2-HS-glycoprotein remained unbound, together with other plasma proteins. In contrast, when this fraction was applied on the Red A, alpha 2-HS-glycoprotein was shown to bind tightly and was eluted with a linear sodium chloride gradient between 0.5 and 0.8 M. This proved to be a useful two-step technique for the purification of alpha 2-HS-glycoprotein. Further characterization revealed that the protein appeared homogeneous by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis with yields greater than 30%. A small (less than 5%) but significant fraction of alpha 2-HS-glycoprotein with a same molecular weight as the native protein was consistently found in the wash of the Red A column, and may correspond to alpha 2-HS-glycoprotein bound to a yet unidentified ligand.

Interaction of group-specific component (vitamin D-binding protein) with immobilized Cibacron blue F3-GA.

Group-specific component (vitamin D-binding protein) was purified to homogeneity from human plasma by a three-step procedure involving pseudo-ligand affinity chromatography on immobilized Cibacron blue F3-GA followed by gel filtration and ion-exchange chromatography. Upon pseudo-ligand chromatography, Gc globulin was separated into two peaks. The first, which represented approx. 4% of the total Gc globulin, was eluted together with other alpha-globulins of similar Mr and/or pI, and the second (96% of Gc globulin) was clearly retarded. Collection of the latter provided a fraction 10-fold enriched in Gc globulin, with yields higher than 90%. Incubation of plasma with trace amounts of radioactively-labeled 25-OH vitamin D3 showed that the radioactivity coeluted with the first peak. In addition, after saturation with 25-OH vitamin D3, all the Gc globulin was eluted in the first peak. This indicates that the two peaks correspond to the holo and the apo forms of the protein, respectively, and suggests that either the interaction of the apo form with the Cibacron blue dye involves the binding site for vitamin D metabolites, or that the holo-protein undergoes a conformational change as a consequence of formation of the complex.

Mycobacterial Cpn10 promotes recognition of the mammalian homologue by a mycobacterium-specific antiserum.

Self-tolerance, a key feature of the immune system, is still a matter of intense debate. We give here evidence for a peculiar behavior of an antiserum against Mycobacterium tuberculosis chaperonin 10 (m-Cpn10), which could have implications for the mechanism of self-recognition by antibodies against non-self. We show that this antiserum can interact in terms of both inhibition of biological activity and physical association (immunoprecipitation), with the mammalian homologue of m-Cpn10, but only if the bacterial protein is present. Several lines of evidence led us to exclude that the two proteins physically associate to form heterocomplexes: (1) the behavior of the antiserum was not shared by a monoclonal antibody against m-Cpn10; (2) a matrix selective for human Cpn10 (h-Cpn10) did not co-purify m-Cpn10; (3) the distribution pattern in non-denaturing isoelectric focusing of labeled m-Cpn10 was not altered by the presence of the unlabeled h-Cpn10. We conclude therefore that the antiserum against M. tuberculosis Cpn10 also recognizes mammalian Cpn10, with an affinity/avidity regulated by the mycobacterial protein, or by the promotion of hetero-oligomerization. This emergence of self-recognition in the presence of M. tuberculosis Cpn10 could imply a breaking of self-tolerance in situations of infection or vaccination.

Divergent pathways regulate ligand-independent activation of ER alpha in SK-N-BE neuroblastoma and COS-1 renal carcinoma cells.

The alpha-estrogen receptor (ER alpha) transcriptional activity can be regulated either by binding to the cognate ligand or by intracellular signaling pathways responsive to a variety of factors acting through cell membrane receptors. Studies carried out in HeLa and COS-1 cells demonstrated that the cross-coupling between estrogen and growth factor receptors is mediated by p21ras and requires phosphorylation of a specific serine residue (Ser 118 in the human ER alpha and Ser 122 in mouse ER alpha) located in the ER alpha N-terminal activation function 1 (AF-1). Likewise, in the SK-N-BE neuroblastoma cell line p21ras is involved in the cross-coupling between insulin and ER alpha receptors. However, in this cell line Ser 122 is not necessary for insulin-dependent activation of unliganded ER alpha. In addition, after insulin activation, the electrophoretic mobility associated to serine hyperphosphorylation of ER alpha in SK-N-BE and in COS-1 cells is different. Our study rules out the possibility of tyrosine phosphorylation in unliganded ER alpha activation by means of transactivation studies of ER alpha tyrosine mutants and analysis of Tyr phosphorylation immunoreactivity. The two cofactors for steroid receptors RIP 140 and SRC-1 do not seem to be specifically involved in the insulin-induced ER alpha transactivation. The present study demonstrates the possibility of an alternative, cell-specific pathway of cross-coupling between intracellular and membrane receptors, which might be of importance for the understanding of the physiological significance of this mode of activation in the nervous system.

Oligonucleotide squelching reveals the mechanism of estrogen receptor autologous down-regulation.

Antisense oligos complementary to the 5'-end, but not to the 3'-end, of the estrogen receptor (ER) messenger RNA caused a paradox accumulation of ER protein in MCF-7 cells. The same effect was observed after treatment of the cells with the corresponding sense oligos. The oligos interfering with ER down-regulation were demonstrated to specifically bind the ER with affinities in the nanomolar range. It is, therefore, proposed that the ER up-regulation induced by the oligos might be due to squelching of the ER (or ER-inducible proteins) from their binding site located in the 5'-end of the ER gene. We also report that transcriptionally inactive ER mutants can undergo down-regulation, and that in denaturing gels, the migration profile of ER-oligo and ER-estrogen-responsive element complexes are dissimilar. We, therefore, propose that ER can interact with DNA in different ways and at different binding sites. These observations might have important pharmacological consequences, since specific drugs could be devised to induce the ER conformation necessary to perform only selected tasks of the ER transcriptional repertoire.

Acute-phase proteins before cerebral ischemia in stroke-prone rats: identification by proteomics.

BACKGROUND AND PURPOSE: A high degree of proteinuria has been reported in stroke-prone spontaneously hypertensive rats (SHRSP). We studied the effect of salt loading on the detailed protein pattern of serum and urine in 3 rat strains: Wistar-Kyoto, spontaneously hypertensive rats, and SHRSP, an inbred animal model for a complex form of cerebrovascular disorder resembling the human disease. METHODS: Rats were given a permissive diet and received 1% NaCl in drinking water. The protein pattern in body fluids was assessed over time by 2-dimensional electrophoretic analysis. Brain alterations were monitored by MRI and histology. RESULTS: Several proteins were excreted in urine after weeks of treatment and in advance of stroke: transferrin, hemopexin, albumin, alpha(2)-HS-glycoprotein, kallikrein-binding protein, alpha(1)-antitrypsin, Gc-globulin, and transthyretin. Markers of an inflammatory response, including very high levels of thiostatin, were detected in the serum of SHRSP at least 4 weeks before a stroke occurred. CONCLUSIONS: In SHRSP subjected to salt loading, an atypical inflammatory condition and widespread alterations of vascular permeability developed before the appearance of anomalous features in the brain detected by MRI. Urinary concentrations of each of the excreted serum proteins correlated positively with time before stroke occurred.

Glycosylation pattern of platelet glycoprotein IIb and IIIa in type IIa hypercholesterolemia.

The microheterogeneity and polymorphism of platelet membrane glycoproteins IIb and IIIa from hypercholesterolemic type IIA patients and from normolipidemic controls were compared by isoelectric focusing followed by immunological detection. Their size and the extent of glycosylation were investigated by polyacrylamide gel electrophoresis and carbohydrate affinity stain. No qualitative or quantitative differences could be detected, either within each test group or between the two groups. This finding rules out the hypothesis that altered molecular forms of the glycoproteins forming the fibrinogen receptor might be involved in the pathogenesis of platelet hyperaggregability in hypercholesterolemic patients.

Human globin chain separation by isoelectric focusing in ultrathin polyacrylamide gels.

We describe three basic modifications of our previous method for thalassemia screening by isoelectric focusing of heme-free globin chains: the gel thickness is reduced from 2 mm to 240 microns; the level of the detergent Nonidet P-40 in the gels is decreased from 3% to 0.5%; the polyacrylamide slab is covalently fixed to the supporting glass plate by treatment with silane A-174. By the present method the entire focusing process, starting from gel moulding up to gel destaining and drying, is completed within 4 h, a fraction of the time needed in our previous technique. More than a 100 samples can be analyzed per working day. The present technique also affords increased sensitivity: less than 1 microgram protein/band is detected and less than 200 picomol of hemoglobin are needed for each analysis. Band sharpness and resolution in our ultrathin gels is also considerably increased.

Electrophoretic separation of A gamma and G gamma human globin chains in Nonidet P-40.

Electrophoresis in cellulose acetate in the presence of 3% Nonidet P-40 can resolve two neutral genetic variants, A gamma and G gamma human fetal globin chains. The ratio of these two chains, determined by densitometry of the electrophoretic strips, is in excellent agreement with the Gly-Ala ratio obtained by chemical analysis of the cyanogen bromide fragment gamma CB3. It is suggested that the detergent binds preferentially to the hydrophobic amino acid segment 133-141 in the A gamma chain, thus masking either a Lys or an Arg residue at the two extremes.

Isoelectric focusing as a tool for the investigation of post-translational processing and chemical modifications of proteins.

It has been demonstrated that good agreement may be observed between computed and experimental isoelectric point (pI) values when proteins of known sequence are focused under denaturing conditions on immobilized pH gradient IPG slabs, at least in the pH range 4-7.5. Hence, discrepancies between expected and found in this experimental set-up may be reliably ascribed to some kind of post-transcriptional processing, or chemical modification, having taken place in the sample. This evaluation is made easier when the comparison is set between the pI of a parent molecule and that (or those) of one to several of its derivatives as resolved in a single experiment (for instance, as a spot row in two-dimensional maps); no previous knowledge is required in these cases about the amino acid composition of the primary structure. The effects on protein surface charge are discussed in this review mainly for two biologically relevant processes, glycosylation and phosphorylation. Then, the pI shifts are analysed for some protein modifications that may occur naturally but can also be artefactually elicited, such as NH2 terminus blocking, deamidation and thiol redox reactions. Finally, carboxymethylation and carbamylation are used to exemplify chemical treatments often applied in connection with electrophoretic techniques and involving charged residues. Procedures to be applied in order to verify whether a given modification has occurred, and often relying on the focusing of a treated specimen, are detailed in each section. Numerical examples on model proteins are also discussed. As an important field of application of the above concepts may be genetic engineering, an exhaustive bibliographic list dealing with pI evaluation and structural assessment on recombinant proteins is included.

The use of dye-ligand affinity chromatography for the purification of non-enzymatic human plasma proteins.

Literature data are analysed in this review on the use of immobilized triazine dyes for the characterization, isolation and purification of non-enzymatic human plasma proteins in both conventional and high-pressure liquid chromatography systems. Attention is focused on the mode of interaction between the dyes and these proteins, as well as on the advantages over previously reported techniques. Future developments are discussed.

Isoelectric focusing following by electrophoresis of proteins for visualizing their titration curves by zymogram and immunofixation.

After isoelectric focusing followed by electrophoresis at right angles in the same gel slab, it is possible to visualize the titration curve of proteins by zymograms or immunofixation even of an unpurified sample. This information can be very useful for the selection of the proper purification strategy by charge-dependent methods, e.g. ion-exchange chromatography, zone and disc electrophoresis and isotachophoresis. The titration curve also gives information on the stability of the protein as a function of the prevailing pH of the medium, in the pH 3-10 range. A region of instability is found for most proteins in acidic conditions, below pH 4.5, while most proteins are stable in the alkaline pH region, at least up to pH 10. The best method for developing zymograms and immunoprints appears to pH 10. The best method for developing zymograms and immunoprints appears to be the 'sandwich technique', by which a thin agarose slab, cast on an hydrophilic polyester sheet, and impregnated with appropriate reagents, is left in contact with a polyacrylamide gel thin layer used to generate the titration curves.