Isoelectric focusing and isoelectric points of aminoacyl-tRNA synthetases

Isoelectric points and isoelectric focusing behaviour of 10 highly purified eukaryotic aminoacyl-tRNA synthetases from 3 sources, Saccharomyces cerevisiae, Euglena gracilis and Phaseolus vulgaris were examined. The pI-values measured on polyacrylamide gels under native conditions are situated between pH 5.0-7.5. A microheterogeneity was observed for 9 enzymes appearing otherwise homogeneous on gel electrophoresis. A compilation of the isoelectric points of aminoacyl-tRNA synthetases is given and literature data are compared with our experimental results.     

Isoelectric focusing of high-molecular-weight protein complex under native conditions using agarose gel

The isolation and characterization of protein complexes are essential steps toward understanding cellular functions. A method for separating and characterizing high-molecular-weight protein complexes using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with native agarose gel isoelectric focusing (IEF) is described. Using this method, fractions containing high-molecular-weight protein complexes were analyzed. The advantages of using native agarose gel IEF include the ability to concentrate the protein complexes and the ease of handling when performing 2D separations. Although limited with respect to the size of molecules and particles that may be separated, this method is useful for the isolation and characterization of high-molecular-weight protein complexes.     

Rhodopsin phosphorylation occurs at metarhodopsin II level

Photolyzed rhodopsin was phosphorylated in bovine rod outer segments incubated at –10 C. In the experiment in which urea-treated outer segments and rhodopsin kinase were incubated with ATP in the presence of 30% glycerol, the extent of phosphate incorporation at –10 C was about 30% of that at 37 C. Separation of phosphorylated rhodopsin by isoelectric focusing indicated that a limited number of sites were phosphorylated at –10 C. The partially phosphorylated pigment incorporated more phosphates when the temperatures was raised to 37 C. This was partly due to decreased inhibition of phosphorylation by glycerol at higher temperature. Since the maximum phosphorylation at –10 C (at which metarhodopsin II is stable) occurred at a pH value (6.0) lower than the pKa for metarhodopsin I-metarhodopsin II transition, metarhodopsin II was suggested to be the preferred substrate for rhodopsin kinase at –10 C. Limited proteolysis with thermolysin of rhodopsin phosphorylated at 37 C released peptides containing about 50% of the total phosphate incorporated. In contrast, proteolytic digestion of rhodopsin phosphorylated at –10 C released negligible amounts of phosphate-containing peptides. The results were taken to suggest that the incorporation of phosphates at metarhodopsin II level under the present condition occurred in the residues other than those removed by thermolysin digestion.Based on material presented at the Fifth International Congress of Eye Research, Eindhoven, October 1982     

The arrestin-bound conformation and dynamics of the phosphorylated carboxy-terminal region of rhodopsin

Visual arrestin binds to the phosphorylated carboxy-terminal region of rhodopsin to block interactions with transducin and terminate signaling in the rod photoreceptor cells. A synthetic seven-phospho-peptide from the C-terminal region of rhodopsin, Rh(330-348), has been shown to bind arrestin and mimic inhibition of signal transduction. In this study, we examine conformational changes in this synthetic peptide upon binding to arrestin by high-resolution proton nuclear magnetic resonance (NMR). We show that the peptide is completely disordered in solution, but becomes structured upon binding to arrestin. A control, unphosphorylated peptide that fails to bind to arrestin remains highly disordered. Specific NMR distance constraints are used to model the arrestin-bound conformation. The models suggest that the phosphorylated carboxy-terminal region of rhodopsin, Rh(330-348), undergoes significant conformational changes and becomes structured upon binding to arrestin.     

Purification of plant cell walls: Isoelectric focusing of CaCl2 extracted enzymes

Summary A preparative procedure for cell wall isolation and purification was developed. The purity of the isolated cell walls was judged biochemically by the lack of activity of cytoplasmic marker enzymes and morphologically by examination at both the light and electron microscope levels. The purified cell walls were extracted with various salt treatments and the molecular weight range of most of the extracted proteins was between 14 and 31 kDa. The salt extracted hydrolytic enzymes were basic in nature (pI>7.0) compared to their cytosolic counterparts (pI<7.0). Some enzymes were readily extracted from cell walls (-glucosidase and -NAcglucosaminidase) with high salt treatment while most of the -mannosidase activity associated with purified cell walls could not be removed even with sequential high salt treatments.     

Methods of isoelectric focusing

The method of isoelectric focusing has been avoided by many workers because of expense, technical difficulty, and problems of interpretation. Inexpensive, easy, and interpretable results are possible using equipment and reagents commonly available. Methods which allow these results are presented and explained.     

Isoelectric focusing and ELISA evaluation of a <Emphasis Type="Italic">Blastomyces dermatitidis</Emphasis> human isolate

Blastomyces dermatitidis is a dimorphic fungal organism and the causative agent of blastomycosis. This organism is endemic east of the Mississippi river as is the fungal organism Histoplasma capsulatum. This study was performed to determine if sensitive and specific antigens from the B. dermatitidis yeast phase lysate (human isolate 592) could be separated using isoelectric focusing (IEF) to eliminate antigens that are cross-reactive with H. capsulatum. Indirect enzyme linked immunosorbent assays were performed to test for reactivity and cross-reactivity and indicate that certain fractions (4–6) were highly reactive. Fraction 16 exhibited a high degree of cross-reactivity with H. capsulatum. This study indicates that IEF may be a useful method for the separation of B. dermatitidis proteins.     

Isoelectric focusing in polyacrylamide-agarose

A procedure using a 2.5% acrylamide-0.5% agarose gel for slab or tube isoelectric focusing is described. This composite gel is durable and enables a rapid focusing of high-molecular-weight compounds.     

Analysis of Staphylococcus aureus cytoplasmic membrane proteins by isoelectric focusing

Cytoplasmic membranes were isolated from late-exponential phase Staphylococcus aureus 6538 P and the membrane proteins examined under non-denaturing conditions by thin-layer isoelectric focusing (TLIEF) in a pH 3.5–9.5 gradient. Isolated membrane preparations retained protein integrity as judged by the demostration of membrane bound adenosine triphosphatase (ATPase) activity in addition to four solubilzed membrane enzyme markers. Membranes were effectively solubilized with 2.5% Triton X-100 (final concentration). Examination of Triton X-100 solubilized membrane preparations established the presence of 22 membrane proteins with isoelectric points between 3.7 and 6.0. The focused proteins displayed the following enzymatic activities and isoelectric points by zymogram methods: ATPase (EC 3.6.1.3), 4.20; malate dehydrogenase (EC 1.1.1.37), 3.90; lactate dehydrogenase (EC 1.1.1.27), 3.85; two membrane proteins exhibited multiple bands upon enzymatic staining: NADH dehydrogenase (EC 1.6.99.3), 4.25, 4.35; succinate dehydrogenase (EC 1.3.99.1), 4.85, 5.10, 5.35.     

Preliminary analysis of root exudates of in vitro-micropropagated Alnus glutinosa clones

Biochemical analysis at Alnus glutinosa (L.) Gaertn. root exudates was carried out in order to investigate the probable role of these exudates in the establishment of the Alnus-Frankia symbiosis. Sufficient amounts of sterile root exudates were collected from plants originating from five in vitro-micropropagated A. glutinosa clones (AG1, AG2, AG3, AG4, AG8) grown under aerobic conditions in nutrient solution with Ballotini glass beads. Cytological controls showed good preservation of roots at the end of the collecting period. The total protein and sugar contents of crude root exudates were determined and related to the dry root weight. These results showed a provenance effect. Soluble high molecular-weight fractions (i.e. the mucilage) were isolated following rigorous purification of the crude root exudates and within this fraction proteins were characterized by isoelectric focusing. The protein pattern of the five clones tested showed major bands in common and some minor specific ones. These experimental data will serve as a useful basis for future physiological investigations.     

A microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin II and bacteriorhodopsin-like properties

Rhodopsins possess retinal chromophore surrounded by seven transmembrane α-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photoreceptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacteriorhodopsin (BR). The other, named middle rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory rhodopsins. MR may be a missing link in the evolution from type 1 rhodopsins (microorganisms) to type 2 rhodopsins (animals), because it is the first microbial rhodopsin known to have 11-cis-retinal similar to type 2 rhodopsins.     

Parallel isoelectric focusing chip

Fast isoelectric focusing (IEF) is becoming a key method in modern protein analysis. We report here the theory and experimental results of new parallel isoelectric devices (PID) for fast IEF. The main separation tool of any PID is a dielectric membrane with conducting channels filled by immobiline gels of varying pH. The pH value of the surrounding aqueous solution is not equal to the pH of any of the channels. The membrane is held perpendicular to the applied electric field. Proteins are collected (trapped) in the channels whose pH values are equal to the pI of the proteins. The fast particle transport between different channels takes place due to convection in the aqueous solution. We developed a mathematical model for PID. Experiment duration is shown to be proportional to the number of different bands N (the peak capacity in standard IEF) in contrast with N(2) for usual IEF devices. This model was validated with experimental results. Parallel IEF accelerates the fractionation of proteins by their pI values (down to several minutes) allowing a more desirable collection efficiency to be achieved. The main theoretical limitation of PID resolution is the sensitivity of proteins to pH change due to the Coulomb blockade effect. The existence of a minimal pH change deltapH(min) for each type of protein is shown: deltapH(min) approximately r(-1) for globular molecules with radius r.     

Analysis of single erythrocytes by injection-based capillary isoelectric focusing with laser-induced native fluorescence detection

A modified version of capillary isoelectric focusing (cIEF) was developed to separate hemoglobin variants contained within single human erythrocytes. Laser-induced native fluorescence with 275 nm excitation was used to detect the separated hemoglobins. In this method, baseline fluctuations were minimized and detection sensitivity was improved by using dilute solutions of anolyte, catholyte, and carrier ampholytes (with methylcellulose). Since electrosmotic flow was used for mobilization of the focused bands, separation and detection were integrated into a single step. The capillary was first filled with only ampholyte solution, and the cell (or standard) was injected as in capillary zone electrophoresis. The ∼90 fl injection volume for individual cells is 7×10⁴ times lower than those previously reported. Adult (normal and elevated A₁), sickle (heterozygous), and fetal erythrocytes were analyzed, with the amounts of hemoglobins A₀, A_1c, S and F determined. The pH gradient for cIEF was linear (r² = 0.9984), which allowed tentative identification of Hb F_ac. Variants differing by as little as 0.025 pI units were resolved.     

Isoelectric focusing of human head hair proteins. Preliminary research

The examination of human hair keratin to obtain genetic information, which may be useful also in forensic sciences, has been carried out with the use of isoelectrophoretic procedure obtaining considerable evidence for the existence of specific-species patterns. In this paper the keratins extracted from hairs of 280 subjects belonging to Sardinian people (113 males, 167 females, aged from 1 to 89, belonging to 52 families) were analyzed using IEF in thin-layer polyacrylamide gel (0.5 mm) in the pH range 2.5–7.0, followed by the silver staining method. Number, position and colour of the bands were the same in all the analyzed samples but a large individual variability was revealed for the relative intensity of some bands. Differences for a long time storage were not revealed as well hair's sample as protein extract: Neither were differences in the number and position of the bands analyzing samples of hair from several sites of the head of the same individual revealed. The results obtained are a useful indication to continue this research considering the numerous fields of application of this analysis system.     

Spectral tuning in sensory rhodopsin I from Salinibacter ruber

Organisms utilize light as energy sources and as signals. Rhodopsins, which have seven transmembrane α-helices with retinal covalently linked to a conserved Lys residue, are found in various organisms as distant in evolution as bacteria, archaea, and eukarya. One of the most notable properties of rhodopsin molecules is the large variation in their absorption spectrum. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) function as photosensors and have similar properties (retinal composition, photocycle, structure, and function) except for their λ(max) (SRI, ～560 nm; SRII, ～500 nm). An expression system utilizing Escherichia coli and the high protein stability of a newly found SRI-like protein, SrSRI, enables studies of mutant proteins. To determine the residue contributing to the spectral shift from SRI to SRII, we constructed various SRI mutants, in which individual residues were substituted with the corresponding residues of SRII. Three such mutants of SrSRI showed a large spectral blue-shift (>14 nm) without a large alteration of their retinal composition. Two of them, A136Y and A200T, are newly discovered color tuning residues. In the triple mutant, the λ(max) was 525 nm. The inverse mutation of SRII (F134H/Y139A/T204A) generated a spectral-shifted SRII toward longer wavelengths, although the effect is smaller than in the case of SRI, which is probably due to the lack of anion binding in the SRII mutant. Thus, half of the spectral shift from SRI to SRII could be explained by only those three residues taking into account the effect of Cl(-) binding.     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.     

Isoelectric focusing of the human TSH receptor A subunit

The water soluble A subunit of the human TSH receptor has been shown to have an isoelectric point of 5. As both TSH and TSH receptor antibodies have isoelectric points in the region of 8–10, charge-charge interactions must be of major importance in the binding of hormone or antibody to the TSH receptor A subunit.     

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.     

Spirocamallanus cricotus (Nematoda): isoelectric focusing and spectrophotometric characterization of its hemoglobin and that of its piscine host, Micropogonias undulatus

The hemoglobins of Spirocamallanus cricotus, a reddish-colored, camallanid nematode, and its Atlantic croacker fish host, Micropogonias undulatus, were characterized with spectrophotometry and isoelectric focusing. Hemoglobin from female parasites' perienteric fluid and homogenized male parasites gave Spectrophotometric peaks at 412, 539, and 575 nm, whereas female worms drained of perienteric fluid and homogenized differed by having a Soret peak of 408 nm. Changing the ionic strength of the buffer from 0.1 to 0.01 M shifted the Soret peak to 406 nm for the female parasites' perienteric fluid and ground male parasites and 404 nm for homogenized female parasites. In all cases, the β band had a higher absorption than the α band suggesting a high O₂ affinity for the parasite hemoglobin. Host hemoglobin had peaks of 406, 437, and 577 nm. Isoelectric focusing not only confirmed the Spectrophotometric evidence that host and parasite hemoglobins differed, but also showed that the parasite's analyzed hemoglobin fractions differed from one another by having different isoelectric points.