High-voltage Electrophoresis Research Articles

Covalent bonding of protein to polyamine in the cyst coat of the protozoan Colpoda steinii.

1. High-voltage electrophoresis and chromatography before and after reaction with 5-dimethylaminonaphthalene-1-sulphonyl chloride have identified putrescine and spermidine in hydrolysates of cyst coat proteins from the protozoan Colpoda steinii. 2. Amounts present varied with putrescine up to 19.7 and spermidine up to 16.9 residues per 1000 amino acid residues. 3. The amines were not, in the main, removed by acid or alkaline extraction or by reprecipitation. They were present in hydrolysates of peptides isolated electrophoretically from acid-degraded coat protein. 4. Proteolysis of oxidised coat protein produced a soluble core polypeptide to which the major proportion of the amines were attached and which had a simple composition. It was composed almost entirely of glutamic acid or glutamine, glycine, serine and cysteic acid, these residues being present in the approximate ratio of 10:2:1:1. 5. When coat protein was treated with 5-dimethylaminonaphthalene-1-sulphonyl chloride and hydrolysed no fully substituted amines could be detected but putrescine with one group substituted and spermidine derivatives with one and two groups substituted were present.

Parkin Phosphorylation and Modulation of Its E3 Ubiquitin Ligase Activity

Mutations in the PARKIN gene are the most common cause of hereditary parkinsonism. The parkin protein comprises an N-terminal ubiquitin-like domain, a linker region containing caspase cleavage sites, a unique domain in the central portion, and a special zinc finger configuration termed RING-IBR-RING. Parkin has E3 ubiquitin-protein ligase activity and is believed to mediate proteasomal degradation of aggregation-prone proteins. Whereas the effects of mutations on the structure and function of parkin have been intensely studied, post-translational modifications of parkin and the regulation of its enzymatic activity are poorly understood. Here we report that parkin is phosphorylated both in human embryonic kidney HEK293 cells and human neuroblastoma SH-SY5Y cells. The turnover of parkin phosphorylation was rapid, because inhibition of phosphatases with okadaic acid was necessary to stabilize phosphoparkin. Phosphoamino acid analysis revealed that phosphorylation occurred mainly on serine residues under these conditions. At least five phosphorylation sites were identified, including Ser101, Ser131, and Ser136 (located in the linker region) as well as Ser296 and Ser378 (located in the RING-IBR-RING motif). Casein kinase-1, protein kinase A, and protein kinase C phosphorylated parkin in vitro, and inhibition of casein kinase-1 caused a dramatic reduction of parkin phosphorylation in cell lysates. Induction of protein folding stress in cells reduced parkin phosphorylation, and unphosphorylated parkin had slightly but significantly elevated autoubiquitination activity. Thus, complex regulation of the phosphorylation state of parkin may contribute to the unfolded protein response in stressed cells.

Speciation and adsorption of trace-level fission product of132Te,95Zr,99Mo and103Ru on inorganic materials

The potential uses of hydrous oxides in the treatment of radioactive wastes may be affected by the physical-chemical properties of these materials. The sorption behavior of trace level (less than 10 –14 g/ml) 235 U fission products in aqueous solutions was studied under static conditions on TiO2, MnO2 and SnO sorbents. A variety of anions, cations and neutral species of 132 Te, 95 Zr, 99 Mo and 103 Ru in aqueous solutions at 1, 3, 5, 7 and 10 pH values were identified and their retention values were calculated. High voltage electrophoresis was used to establish the chemical species of these radioisotopes formed in the solution.

Identification of G-protein-coupled receptor phosphorylation sites by 2D phosphopeptide mapping.

Reversible phosphorylation is important for G-protein-coupled receptor (GPCR) signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. This chapter describes a powerful analytical method for the direct identification of GPCR phosphorylation sites by two-dimensional (2D) phosphopeptide mapping. The GPCR of interest is isolated from 32P-labeled cells by immunoprecipitation and transferred to nitrocellulose membranes. In situ cleavage by trypsin releases phosphopeptides that are separated by a combination of high-voltage electrophoresis and chromatography. Phosphoamino acid analysis and Edman sequencing of isolated phosphopeptides reveals information that can lead to the direct identification of GPCR phosphorylation sites. Furthermore, the 2D phosphopeptide mapping technique allows the analysis of temporal and positional changes in the GPCR phosphorylation pattern under different physiological conditions.

MP069 Microbiological examinations of mediaeval soil samples for pathogenic spore formers. — Preliminary results

This chapter focuses on Lysine fermentation (Clostridium). The fermentation of lysine to fatty acids and ammonia by Clostridium sticklandii and certain other unidentified clostridial strains such as Clostridium M-E and Clostridium SB4 is a complex series of reactions. Growing cultures and suspensions of intact cells effect two types of cleavage of the carbon skeleton of lysine. In addition to the enzymes specific for L-α-lysine and D-α-lysine, a powerful racemase that interconverts these isomers is present in extracts of C. sticklandii and CIostridium M-E. The various diaminohexanoates that are interconverted in the lysine fermentation can be detected by a combination of chromatographic and electrophoretic procedures. When subjected to high voltage electrophoresis on Whatman 3 MM paper at pH 3.5, the basic amino acids migrate the following distances from the origin toward the cathode (cm): 3,6-diaminohexanoate (β-1ysine), 58.5; 3,5-diaminohexanoate, 53.5; and 2,6-diaminohexanoate (α-lysine), 49.5. At this pH, 2,5-diaminohexanoate has virtually the same mobility as α-lysine.

Purification of anthocyanins from species of Banksia and Acacia using high-voltage paper electrophoresis.

A new method has been developed for the isolation and rapid identification of anthocyanins from two floricultural crops based on the use of high-voltage paper electrophoresis with bisulphite buffer. Using this method, anthocyanin pigments were successfully purified as their negatively charged bisulphite-addition compounds from crude extracts of plant tissue. In conjunction with liquid chromatography-electrospray mass spectrometry, the method enabled the anthocyanins from the flowers of two Banksia species and the leaves of two Acacia species to be identified. The Banksia flowers contained both cyanidin and peonidin-based pigments, while the Acacia leaves contained cyanidin and delphinidin derivatives.

Mercury removal from aqueous and organo-aqueous solutions by natural Mexican erionite

The sorption of Hg(II) from aqueous and organo aqueous solutions was investigated by Mexican natural erionite. The mercury chemical species (anionic, cationic or neutral) were determined by high voltage electrophoresis, and the mercury chemical species present in the aqueous media were simulated by a program MEDUSA. The mercury sorption process was monitored during 48 hours. The mercury content was determined by neutron activation analysis. Mixtures of benzene/water[Hg(II)], toluene/water[Hg(II)] and ethanol/water[Hg(II)] were chosen as organo-aqueous media. It was found that both the mercury chemical species and the dielectric constant of solvents play an important role in the mercury sorption by erionite.

Bromine determination by neutron activation analysis and its distribution in the atmosphere

Bromine, one of the main participants in ozone layer destruction, is 10 to 100 times more effective than chlorine. There are two principal sources of methyl bromide emissions: the oceans and some pesticides that are used in farming. In this work, bromine was detected in “premium” and “magna sin” gasolines (2.86±0.96 and 1.54±0.38 ppm, respectively) as well as in condensed water found in exhaust pipes of vehicles. In addition, samples of rainwater were also analyzed to determine atmospheric bromine concentration. In water samples Br concentrations ranging from 2.09 to 0.06 ppm were found. The techniques utilised in our research were neutron activation analysis and high voltage electrophoresis, the latter to determine the chemical form of bromine in condensed water samples. Finally, suspended particles from rainwater were also analysed by scanning electron microscopy (SEM).

The emerging understanding of sickle cell disease.

The emerging understanding of sickle cell disease.

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells.

Neutral xyloglucan was purified from the cell walls of suspension-cultured rose (Rosa sp. 'Paul's Scarlet') cells by alkali extraction, ethanol precipitation and anion-exchange chromatography on 'Q-Sepharose FastFlow'. The procedure recovered 70% of the total xyloglucan at about 95% purity in the neutral fraction. The remaining 30% of the xyloglucan was anionic, as demonstrated both by anion-exchange chromatography at pH 4.7 and by high-voltage electrophoresis at pH 6.5. Alkali did not cause neutral xyloglucan to become anionic, indicating that the anionic nature of the rose xyloglucan was not an artefact of the extraction procedure. Pre-incubation of neutral [3H]xyloglucan with any of ten non-radioactive acidic polysaccharides did not cause the radioactive material to become anionic as judged by electrophoresis, indicating that stable complexes between neutral xyloglucan and acidic polysaccharides were not readily formed in vitro. The anionic xyloglucan did not lose its charge in the presence of 8 M urea or after a second treatment with NaOH, indicating that its anionic nature was not due to hydrogen-bonding of xyloglucan to an acidic polymer. Proteinase did not affect the anionic xyloglucan, indicating that it was not associated with an acidic protein. Cellulase converted the anionic xyloglucan to the expected neutral nonasaccharide and heptasaccharide, indicating that the repeatunits of the xyloglucan did not contain acidic residues. Endo-polygalacturonase converted about 40% of the anionic xyloglucan to neutral material. Arabinanase and galactanase also converted appreciable proportions of the anionic xyloglucan to neutral material. These results show that about 30% of the xyloglucan in the cell walls of suspension-cultured rose cells exists in covalently-linked complexes with acidic pectins.

Non-HPLC separation of water-soluble choline metabolites by two-dimensional high voltage electrophoresis and thin layer chromatography

In cholinergic neurons choline is directed to three main pathways; (1) conversion to phosphorylcholine (PCh) and cytidine diphosphate choline (CDP-choline) for the synthesis of phosphatidylcholine, (2) acylation to the neurotransmitter acetylcholine and (3) oxidation to betaine for the formation of methionine. Thus, the distribution of choline among the different metabolites is important for a better understanding of the regulation of these pathways in neurons. A non-HPLC method for the simultaneous separation of five choline metabolites found in neurons is described. High voltage electrophoresis (HVE) was combined with thin layer chromatography (TLC) to separate choline, PCh, CDP-choline, acetylcholine and betaine. This method is useful in studying the distribution of choline among its different metabolites in radiotracer experiments. Aqueous metabolites from leukemia inhibitory factor treated LA-N-2 cells labeled with [methyl- 3H]choline were separated by HVE followed by TLC in the same dimension. Although the separation appeared to be complete, some ‘tailing’ by PCh significantly elevated the radioactivity measured in CDP-choline. This tailing of PCh was confirmed by subjecting radiolabeled PCh alone to this multiple separation method. Contamination of CDP-choline by PCh was eliminated by subjecting the samples to HVE followed by TLC in the second dimension. This two-dimensional approach was consistently reproducible and achieved excellent resolution of all five metabolites. In addition, this technique also resolved a sixth choline-containing metabolite, glycerophosphorylcholine (GPC), a breakdown product of phosphatidylcholine.

Nicotianamine chelates both FeIII and FeII. Implications for metal transport in plants

Nicotianamine (NA) occurs in all plants and chelates metal cations, including FeII, but reportedly not FeIII. However, a comparison of the FeII and ZnII affinity constants of NA and various FeIII-chelating aminocarboxylates suggested that NA should chelate FeIII. High-voltage electrophoresis of the FeNA complex formed in the presence of FeIII showed that the complex had a net charge of 0, consistent with the hexadentate chelation of FeIII. Measurement of the affinity constant for FeIII yielded a value of 10(20.6), which is greater than that for the association of NA with FeII (10(12.8)). However, capillary electrophoresis showed that in the presence of FeII and FeIII, NA preferentially chelates FeII, indicating that the FeIINA complex is kinetically stable under aerobic conditions. Furthermore, Fe complexes of NA are relatively poor Fenton reagents, as measured by their ability to mediate H2O2-dependent oxidation of deoxyribose. This suggests that NA will have an important role in scavenging Fe and protecting the cell from oxidative damage. The pH dependence of metal ion chelation by NA and a typical phytosiderophore, 2'-deoxymugineic acid, indicated that although both have the ability to chelate Fe, when both are present, 2'-deoxymugineic acid dominates the chelation process at acidic pH values, whereas NA dominates at alkaline pH values. The consequences for the role of NA in the long-distance transport of metals in the xylem and phloem are discussed.

Evaluation of ck-mb isoform analysis for early diagnosis of myocardial infarction

Measurement of CK-MB and its isoforms by high-voltage electrophoresis has been proposed as a sensitive test for early detection of myocardial infarction (MI). We performed a prospective study of this test in 231 patients presenting to the Emergency Department with symptoms consistent with ischemic chest pain. Blood specimens were obtained at 0, 1, and 3 h following presentation, and plasma was immediately frozen and analyzed within 1 week by high-voltage electrophoresis for total CK-MB and isoforms. The test was considered positive whenever total CK-MB was elevated (>6 U/L) or the cardiac isoform MB 2 was relatively increased (MB 2 > 2 U/L and MB 2/MB 1 > 1.7). This test had a sensitivity of 68% overall and 55% for specimens collected within 3 h of symptom onset. It was positive within 3 h of presentation in 36/39 (92%) of patients with confirmed MI. Specificity was 92% overall and did not vary with time after symptoms. The CK-MB alone, at the cutoff of 6 U/L, had lower sensitivity overall (56%; p = 0.01) and within 3 h of onset (39%; p = 0.03), and higher specificity overall (98%; p < 0.001). Lowering the cutoff for CK-MB alone to match the sensitivity of the isoform test caused a greater loss of specificity. It is concluded that analysis of CK-MB by high-voltage electrophoresis is an effective method for rapid diagnosis of MI, with the isoform analysis enhancing early sensitivity.

60Co removal from aqueous solutions using charcoal

Charcoal, treated in different ways, can be used in the separation of60Co from aqueous solutions. The cobalt species involved in the process were identified by high voltage electrophoresis. The best results were obtained using thermally and chemically treated charcoal with a maximum retention of 1.24 mmol cobalt per gram. It was also found that the cobalt was not desorbed from the chemically treated charcoal when washed with distilled water. However, it was eluted from the charcoal with 2.3N hydrochloric solution. Samples containing60Co were analysed during the sorption process by gamma-ray spectrometry.

An on-column miniature conductivity cell and a photocouple separator for conductivity detection by capillary electrophoresis

A new miniature conductivity cell was developed as a conductivity detector in conjunction with the capillary electrophoresis system. The advantages of this cell were found to be the large electrode area, minimal dead volume, good resolution and high sensitivity. Moreover, the effect of common ground between the high voltage electrophoresis circuit and the detection system was eliminated effectively by a specially designed photocouple separator. This system was used successfully for the determination of amino acids in hydrolysed samples of human hair.

Identification ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid with a metabolic intermediate betweenS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine andS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid

S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid (I) was chemically synthesized in 15% yield by incubating a reaction mixture oftrans-urocanic acid and 3-fold excess of 3-mercaptopyruvic acid at 45°C for 6 days. The synthesized compound was characterized by fast-atom-bombardment mass spectrometry and high-voltage paper electrophoresis. CompoundI was identified with a product of an enzymatic reaction ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine (II) with rat liver homogenate in a phosphate buffer, pH 7.4. CompoundI was degraded toS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid (III), a compound previously found in human urine [Kinuta et al. (1994) Biochem J 297: 475–478], by incubation with rat liver homogenate. From these results, we suggest that compoundI is a metabolic intermediate for the formation of compoundIII from compoundII. The present pathway follows a formation of compoundII fromS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl] gluthathione [Kinuta et al. (1993) Biochim Biophys Acta 1157: 192–198], a proposed metabolite ofl-histidine.

Identification of Ser-Pro and Thr-Pro phosphorylation sites in chicken neurofilament-M tail domain.

The tail domain of the midsize chicken neurofilament polypeptide (NF-M) contains several different types of Ser-Pro and Thr-Pro putative phosphorylation sites. We determined which of these sites are actually phosphorylated in vivo. Chick sensory neuron cultures were incubated in [32P]phosphate, and the cytoskeletal fraction was mixed with a neurofilament fraction prepared from adult chicken brain. NF-M was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with chymotrypsin, and two large fragments were isolated. These were individually cleaved with trypsin, endoprotease Lys-C, or endoprotease Glu-C, and peptides separated by two-dimensional high-voltage electrophoresis and thin-layer chromatography. 32P-labeled phosphopeptides were eluted from the cellulose plates and subjected to microsequencing and mass spectometry. We found that of 21 potential Ser-Pro and Thr-Pro phosphoacceptor sites, at least 20 are phosphorylated in vivo: all four Lys-Ser-Pro sites and at least 16 of the 17 Lys-Xaa-Xaa-Ser/Thr-Pro repeats. In addition, a novel Ser-Pro site in the extreme carboxy terminus is phosphorylated. This site, which has no proximal Lys residue, is also found in mammalian NF-M, but has not been reported to be phosphorylated. Together with three casein kinase I sites we have found recently in the acidic amino-terminal segment of the tail, a total of 24 or 25 Ser and Thr phosphoacceptor sites have now been located in the chicken NF-M tail.

CREATINE KINASE SUBFORMS IN MARATHON RUNNERS 843

The lack of a rapid diagnostic marker specific to acute myocardial infarction (AMI) led to the development of a new high-voltage electrophoresis assay that separates creatine kinase (CK)-MB isoenzyme into its subforms. MB1 and MB2. The ability of the assay to detect early increases in the MB2/MB1 ratio in the blood and to reliably detect AMI within 6 hrs after the onset of chest pain has been shown to provide improved diagnosis of AMI. Positive clinical diagnosis is met when both the MB2 levels and subform ratios are positive (MB2≥2.6 U/L and ratio ≥ 1.7). Limited research has been conducted on the effectiveness of this assay on physically active individuals. Thus, the purpose of this study was to investigate the temporal patterns of MB subforms in plasma of marathon runners. Blood samples were obtained from 33 subjects (22 men, 11 women) prior to, immediately post, and 2.4, and 6 hrs post marathon race. Plasma MB2 activity, MB1 activity, MB2/MB1 ratio. CK-MB, and relative index (CK-MB/total CK) prior to the race were all within normal. Plasma MB2 activity, MB1 activity, and MB2/MB1 ratio immediately post race (3 to 5 hrs after race start) were elevated to clinically abnormal levels. Ninety% (30/33), 94% (31/33) and 100% of the subjects demonstrated a combined positive MB2 level and subform ratio immediately post, and 2 and 4 hrs post race. The kinetics of enzyme release in marathon runners is similar to that previously reported for AMI patients;acute acute skeletal muscle injury results in the release of tissue subforms of MB approx. 3 hrs after the onset of injury. These data suggest that the MB2/MB1 ratio cannot be used to differentiate between damage to the skeletal muscle vs the myocardium. Relative index values (<5%) across time suggest that skeletal muscle is the site of injury because values are less than that previously reported in AMI.

Overview of Protein Phosphorylation

This overview provides a history of protein phosphorylation research and provides the reader with an understanding of how and why labeling studies are performed. The various sites of protein phosphorylation are described along with the roles of the many kinases and phosphatases that regulate phosphorylation. Methods for detecting unlabeled phosphoamino acids, including high-voltage electrophoresis on thin-layer cellulose acetate plates, gel-shift assays, and the use of anti-phosphopeptide antibodies.

Phosphorylation of the Adenomatous Polyposis Coli Protein and Its Possible Regulatory Effects in Cells

The adenomatous polyposis coli ( APC ) gene is etiologically associated with familial adenomatous polyposis and gastrointestinal malignancies, but its cellular function and role in tumorigenesis are unclear. Recent reports indicate that wild-type, but not mutant, APC gene product (APC) is associated with and promotes the assembly of cytoskeletal microtubules in vitro , suggesting that this mechanism has importance in tumor development. Because other microtubule-associated proteins (MAPs) undergo phosphorylation in their normal functioning, we postulated that APC is a phosphoprotein. HCT116 cells, containing full-length APC protein, were [ 32P]-prelabeled, and a 300-kDa band corresponding to phosphorylated APC was immunoprecipitated using each of three different anti-APC antibodies. High voltage electrophoresis of [ 32P]-labeled APC showed the presence of phospho-serine and phospho-threonine residues. Further immunoprecipitation analyses showed phosphorylation of i) full-length APC in human lymphoblastoid cells and ii) carboxyl-truncated APC in SW480 and DiFi colon carcinoma cells. Thus, APC is probably a phosphoprotein in normal and malignant tissues. We hypothesize a mechanism whereby phosphorylation of APC may play a regulatory role in its interaction with microtubules. This may involve phosphorylation of (Ser/Thr)-Pro amino acid motifs in APC′s basic domain. We propose that deletion of this domain disrupts APC binding to microtubules, explaining how APC mutations are linked to cancer development.