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.     

Separation of membrane proteins in an undenatured form by isoelectric focusing

Conditions are described which allow good resolution of membrane proteins in an undenatured form by isoelectric focusing in thin polyacrylamide gels in Triton X-100. High voltages and deionization of the acrylamide are essential. Streaking is grossly reduced by sample application in Bio-Gel P-60, by deionization of the Triton, and dialysis of membrane samples against low ionic strength buffer at slightly alkaline pH. The latter step also greatly improves solubilization of the membrane components. Reproducible isoelectric focusing patterns of proteins from red cell, thyroid, and lymphocyte membrane are presented.     

Free-solution isoelectric focusing for the purification of Staphylococcus aureus enterotoxin C1

A free-solution isoelectric focusing protocol was developed for the preparative purification of Staphylococcus aureus enterotoxin C1 (SEC1). A toxin consisting of a single isoelectric species, pI 8.8, was purified. Thirty-nine milligrams of SEC1 was recovered from 3 liters of culture supernatant. This significantly improved purification scheme utilized ammonium sulfate precipitation and the Bio-Rad Rotofor isoelectric cell to complete isolation in 2 days, thereby avoiding the protein degradation prevalent when published procedures are used. The purification protocol developed here for SEC1 is used to illustrate the utility of Rotofor fractionation in the general purification of bacterial exotoxins.     

Excretion of cytoplasmic proteins (ECP) in Staphylococcus aureus

E xcretion of c ytoplasmic p roteins (ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in Staphylococcus aureus very similar to that of Sbi, an IgG‐binding protein, which is secreted via the Sec‐pathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan‐binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non‐excreted Ndh2, a soluble NADH:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism.     

Device for miniscale isoelectric focusing of proteins

A simple device is developed for mini-scale electrofocusing of proteins. The main apparatus consists of only two glass tubes joined by a small tubing. No special cooling system, stopcocks, stands, etc., are needed. Even the need for a peristaltic pump for fractionation is eliminated. The apparatus does not require very high voltages and the amount of Ampholines is drastically reduced. The model can be used for analytical as well as semi-quantitative purposes.     

Comparative study of sturgeon oocyte soluble proteins by isoelectric focusing

1. Soluble caviar (oocyte) proteins of four sturgeon species from the Caspian sea, Acipenser stellatus (Sevrouga), Acipenser guldenstadti (Ossietre), Acipenser nudiventris (Chipe) and Huso huso (Belouga) were studied by isoelectric focusing. 2. Isoelectric focusing patterns of these proteins differ from one species to another and allow the identification of the specific origin of caviar. 3. Moreover, this technique allowed the discrimination of a subspecies, Acipenser guldenstadti persicus from the species. 4. This biochemical characterization of caviar proteins allowing identification of sturgeon species producing caviar could be added to data used in fraud tests.     

Study of thermostable proteins in potatoes by isoelectric focusing]

With the aid of the method of isoelectric focusing eight thermostable proteins with isoelectric points of 3.6, 3.9, 4.u, 5.1, 5.8, 6.6, 7.2 and 10.0 were found in the extract from potato tubers of the Lyubimets variety. All the proteins had inhibitory activity in relation to alpha-chymotrypsin and trypsin.     

Analysis of pulmonary surfactant apoproteins by isoelectric focusing

Apoproteins of Mr 38 000, 32 000 and 26 000 are found in surfactant isolated from rat lungs. The surfactant isolated from monkey lungs, on the other hand, contains the 38 kDa apoprotein and not the 32 and 26 kDa apoproteins. These preparations of pulmonary surfactant contain, in addition, several serum proteins. We have used a combination of salt- and sucrose-density gradient centrifugations to isolate and further purify surfactant from the washings of rat lungs. Thus, a preparation of pulmonary surfactant was obtained which contained exclusively the 38, 32, 26 and 10-12 kDa apoproteins, and which was rich in phosphatidylcholine and phosphatidylglycerol. Using an immunoassay and an immunoblotting technique, it was established that the 38, 32 and 26 kDa apoproteins are not serum proteins. The surfactant apoproteins of rat and monkey were further subjected to the high-resolution of isoelectric focusing. Thus, rat surfactant apoproteins resolved into 11 bands in the pH range 4.64-5.53. A second-dimensional electrophoresis in a sodium dodecyl sulfate system led to the migration of the 11 bands, separated by first-dimensional isoelectric focusing, into three distinct groups with apparent molecular weights of 38 000, 32 000 and 26 000, respectively. Upon isoelectric focusing, the apoproteins from monkey lung surfactant also separated into several bands in the pH range 5.18-5.82. After electrophoresis in the second dimension as above, these bands migrated as a single group with an apparent molecular weight of 38 000. Neuraminidase treatment of rat surfactant apoproteins, and subsequent IEF, led to the disappearance of several low-pI variants with a concomitant increase in the amounts of higher-pI variants. Thus, the sialic acid content of surfactant apoproteins accounts for, in large part, the observed charge heterogeneity.     

Current trends in capillary isoelectric focusing of proteins

Isoelectric focusing (IEF) in thin capillaries is reviewed here. After an introduction on the genesis and chemistry of the carrier ampholyte buffers, different approaches to IEF are discussed and evaluated. The classical approach consists on IEF under conditions of suppressed electroosmotic (EOF) flow, usually obtained by covalently bonding hydrophilic polymers to the inner capillary wall. The other approach consists of IEF in dynamically (and partially) coated capillaries, so as to allow a reduced EOF flow to coexist with the IEF process, so that focusing and transport of the train of stacked bands occurs simultaneously. The various experimental parameters: focusing, elution and detection steps, pI measurements, as well as typical drawbacks, such as isoelectric precipitation are evaluated. The review ends with some examples of analytical separations, at the moment mostlyl limited to focusing of native hemoglobins (normal and point mutants). These separations are compared with those obtained by slab-gel IEF and in immobilized pH gradients.     

Synthetic ampholytes for the isoelectric focusing of proteins

Products of the copolymerization of acrylic acid with oligoethylene oligoamines were used to establish natural pH gradients for the isoelectric fractionation of protein mixtures by polyacrylamide gel electrophoresis. The unfractionated synthesized ampholytes were compared with the commercially available ampholytes with respect to the number of ampholyte components, the pH gradient and the resolution of protein mixtures. The performance of the ampholytes prepared employing the more highly substituted ethylene amines was found to be comparable to that of the commercial ampholytes over the pH range 4 to 8.     

An isoelectric focusing procedure for erythrocyte membrane proteins and its use for two-dimensional electrophoresis

Procedures are described and evaluated for one-dimensional isoelectric focusing of erythrocyte membrane dissolved in lysine, urea, and Triton X-100 without using sodium dodecyl sulfate (SDS) and for two-dimensional electrophoresis with SDS in the second dimension. The membrane was completely dissolved, most of the proteins including the anion porter(s) entered the focusing gel, and complex, well-resolved patterns were seen. Ampholines, 2-mercaptoethanol, or SDS in the applied sample each seriously reduced focusing resolution and phenylmethylsulfonyl fluoride blurred the patterns. The two-dimensional patterns showed more and sharper spots than did patterns obtained from membrane initially dissolved with SDS. Anion porter spots were seen with both procedures. However, major cytoskeletal proteins were much less well recovered with the former procedure than with the latter.     

Antigenic characterization of flavivirus structural proteins separated by isoelectric focusing.

Isoelectrofocusing of nonionic-detergent-disrupted flaviviruses separated the envelope glycoprotein of 53,000 to 58,000 daltons and the nucleocapsid protein of 14,000 daltons. The envelope protein and nucleocapsid protein were isolated at isoelectric points of pI 7.8 and 10.3, respectively. The antigenic determinants of St. Louis encephalitis, Japanese encephalitis, and dengue virus envelope and nucleocapsid proteins were examined by solid-phase competition radioimmunoassay. By the appropriate selection of antiserum and competing proteins, it was possible to distinguish type-specific, complex-reactive and flavivirus group-reactive antigenic determinants. The envelope glycoproteins of St. Louis encephalitis, Japanese encephalitis, and dengue viruses were found to contain each of these three classes of antigenic determinants. Most of the determinants on the envelope protein were type specific, some were complex reactive, and a small fraction were flavivirus group reactive. The nucleocapsid protein contained only flavivirus group-reactive antigenic determinants.     

An isoelectric focusing study of plasma membrane actin.

Plasma membranes were purified from secondary chick embryo fibroblasts labeled with [³⁵S]methionine for 1 or 18 h. The total cell homogenate, postnuclear supernatant and plasma membrane fractions were analyzed by two-dimensional electrophoresis (isoelectric focusing followed by SDS-slab gel electrophoresis). The α, β, and γ isoelectric variants of actin were present in similar proportion in membranes, supernatant, and cell homogenate as determined by incorporation of ³⁵S into each species of actin. These results indicate that the plasma membrane actin of chick fibroblasts is heterogeneous and that no isoelectric variant of actin is unique to the plasma membrane.     

Nuclear and cytoplasmic genome relationships in the genus Avena: Analysis by isoelectric focusing of ribulose biphosphate carboxylase subunits

Comparisons of the isoelectric points of small and large subunits of ribulose biphosphate carboxylase extracted from a number of diploid, tetraploid, and hexaploid Avena species have been used to obtain information on the nuclear and cytoplasmic genome relationships within the genus. All species tested had small subunits with similar isoelectric points, so their analysis provided no information of taxonomic value. Three types of large subunits could be distinguished by this method, and the distribution of each among the available species provides strong evidence against the involvement of a C genome diploid (such as A. ventricosa) as the maternal parent in the formation of either tetraploid or hexaploid species. One type of large subunit was confined to the perennial tetraploid, A. macrostachya, and its position in the genus and possible origin are discussed. The value of this approach in studying genome relationships within the genus Avena and related genera is assessed.