Separation of proteins by high-speed pressure liquid chromatography

Two chromatographic systems for separation of proteins by high-speed pressure liquid chromatography are described. Molecular size exclusion chromatography was achieved by the use of porous silica deactivated by Carbowax-20M to prevent protein adsorption. Protein separations were successful provided the salt concentration in the eluting buffer was relatively high.The second system described is adsorption chromatography of proteins on deactivated Porasil. This technique involves elution of the proteins from the gel by means of a salt and pH gradient. In both systems the total time required for the chromatography is less than 1 hr.     

Erratum

Escherichia coli cells (unsaturated fatty acid auxotroph) have been adapted to grow on branched-chain fatty acids. Membrane vesicles were isolated from cells grown on a mixture of branched-chain fatty acids isolated from the lipids of Bacillus subtilis (E. coli (B. subtilis) membranes) and on a pure synthetic anti-isononadecanoic acid (E. coli (aC19) membranes).We have shown, using wide-angle X-ray diffraction and differential scanning calorimetry, that the ordered state of the lipids is perturbed in the case of E. coli (aC19) membranes. The perturbation leads to the presence of a large wide-angle X-ray diffraction at 4.25–4.3 Å, as opposed to the presence of a sharp 4.2 Å reflection in unperturbed systems.We have shown, using freeze-fracture electron microscopy, that a protein segregation exists in the case of E. coli (aC19) membranes (at low temperature the integral membrane proteins aggregate in the membrane domains containing the disordered lipids); we do not observe such segregation in the case of E. coli (B. subtilis) membranes. We conclude that in cases where the branching of the fatty acids introduces a perturbation of the lipid order, the integral membrane proteins can still be accommodated in membrane domains containing the ‘perturbed’ ordered lipids.Finally, we have determined the rate of β-galactoside transport in E. coli (aC19) and E. coli (B. subtilis) membranes as a function of temperature. We have shown that, in both cases, the Arrhenius representations display an increased slope in the region of the disorder-to-order transition. We conclude that such an increased slope may have different origins. In the case of E. coli (aC19) membranes, it is the result of the aggregation of the β-galactoside carriers together with other integral membrane proteins which may lead to the inactivation of the carriers; in the case of E. coli (B. subtilis) membranes, it is the result of the partial immobilisation of the carriers embedded in a lipid environment, of which the fluidity, despite the perturbation of its lipid order, is still much less than that associated with lipids in a totally disordered state.     

Lipoprotein lipase in rat lung. Effect of dexamethasone.

The effect of hormone administration on the activity of lipoprotein lipase in the lung was studied in the rat. The following hormones were administered: dexamethasone, L-thyroxine, estradiol-17beta and progesterone. In addition, lung lipoprotein lipase activity was studied in diabetic and lactating rats. Lipoprotein lipase activity was measured in dried, defatted preparations of rat lung using double labeled ([14C]palmitate, [3H]glycerol) chylomicron triacylglycerol as substrate. Dexamethasone administration caused a rise of 70% in the level of activity of lipoprotein lipase in acetone powders of lung and a 100% increase in the amount of enzyme released during heparin infusion into isolated, perfused lungs. Enzyme activity was higher in lungs of females than of male rats; however; the level of activity was unaffected by estrogen or progesterone administration to either male or ovariectomized rats. Diabetes, hyperthyroidism or lactation did not change lipoprotein lipase activity in the lung. The constant presence of lipoprotein lipase activity in the lung suggests that this organ is able to maintain a steady supply of triacylglycerol-fatty acids under a variety of physiological and pathological conditions. Stimulation of enzyme activity by dexamethasone could lead to increased uptake of triacylglycerol-fatty acids by the lung and may thus be a contributing factor to corticosteroid-induced enhanced surfactant synthesis.     

Hydrophobic carriers of vanadyl ions augment the insulinomimetic actions of vanadyl ions in rat adipocytes.

A novel family of vanadyl ion (VO2+, oxidation state +4) carriers is introduced. These carriers possess C2 symmetry, utilize two hydroxamate groups as ion binding sites, and optionally possess asymmetric carbons. Binding efficiencies and hydrophobicities are regulated by the use of a modular assembly. When applied to rat adipocytes, these carriers augment the potency of vanadyl ions to stimulate glucose metabolism. The complexes shift the dose-response curve to the left. Also, the maximal effect of vanadyl ions which is in the order of 20-30% of that of insulin is shifted toward maximal (100-115%) stimulation. Among several chelators studied, the order of synergistic potency was RL-252 greater than or equal to RL-262 greater than 1367. RL-239, RL-280, and RL-261 had smaller effects, whereas RL-282 had a negligible effect. The synergistic action of RL-252 (and other chelators as well) on VO2+ was already observed at a molar ratio of 1:0.01 of VO2+ to RL-252, respectively, and maximal augmentation occurred at a molar ratio of 1:0.1. The superiority of the hydrophobic chelators relative to the hydrophilic ones, together with the low molar ratio of chelator to VO2+ to achieve maximal effect, strongly suggests that these chelators act as vanadyl ionophores. This notion was confirmed by carrier-facilitated extraction of VO2+ from water into CHCl3 with the following order of decreasing efficacy: RL-262 greater than RL-252 greater than 1367 greater than RL-261. The chelators' potentiating effect may therefore be related to facilitated transport of VO2+ ions into the cells' interiors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excretion of glutamate from Corynebacterium glutamicum triggered by amine surfactants.

Corynebacterium glutamicum is used for the industrial production of glutamate. Excretion of the amino acid may be induced by various means. We have analyzed the characteristics of glutamate excretion induced by two amine surfactants, dodecylammonium acetate (DA) and dodecyltrimethylammonium bromide (DTA). Addition of these surfactants induced an immediate efflux of internal glutamate. It also induced a perturbation of the energetic parameters of the cell (decrease of delta mu H, decrease of the internal ATP concentration). The efflux was not the result of these perturbations: glutamate is taken up by the cells via an ATP-dependent unidirectional active transport system and no efflux took place as a consequence of an artificial decrease of the energetic parameters. In addition, amine surfactants also induced an excretion of other species, in particular potassium. We have tested the possibility that the effluxes result from a permeabilization of the lipid bilayer by analyzing the interactions between the surfactants and liposomes.     

On-line and in situ biosensors for monitoring environmental pollution

Efficient tools for on-line and in situ monitoring of environmental pollutants are required to provide early warning systems. In addition, such tools can contribute important information on the progress of various remediation treatments. One of the recently developed monitoring technologies involves the use of whole-cell biosensors. Such biosensors could be constructed to detect general toxicity or specific toxicity caused by one or more pollutants. Currently, a large spectrum of microbial biosensors have been developed that enable the monitoring of pollutants by measuring light, fluorescence, color or electric current. Electrochemical monitoring is of special interest for in situ measurements as it can be performed using simple, compact and mobile equipment and is easily adaptable for on-line measurements. Here we survey the potential application of electrochemical biosensors in monitoring of general toxicity as well as hydrocarbons and heavy metals.     

The lactose permease of Escherichia coli: evidence in favor of a dimer

Lactose permease from Escherichia coli T 206 was purified in octyl-beta-D-glucopyranoside (octyl-glucoside) according to Newman et al. [J. Biol. Chem. (1981) 256, 11804-11808]. In this detergent the protein has a very high tendency to aggregate nonspecifically. Therefore, exchange of octyl-glucoside was performed for another nonionic detergent, dodecyl octaethylene glycol monoether (C12E8), in which the protein is more stable. The amounts of bound C12E8 and phospholipids were measured using radioactive detergent and gas chromatography, respectively, and were found to be respectively 0.2 and 0.15 g/g protein. Analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium) and gel filtration (conventional and high performance liquid chromatography) experiments indicated that in this detergent the lactose permease existed mainly as a dimer. This result is at variance with the monomeric state of the protein reported by Wright et al. [FEBS Lett. (1983) 162, 11-15] in another nonionic detergent (dodecyl-o-beta-maltoside). We discuss the possible reason for this discrepancy and suggest that the dimeric state of association may well reflect the situation that prevails in the membrane.     

Reduced glutathione in chinese hamster ovary cells protects against inactivation of 3-hydroxy-3-methylglutaryl Coenzyme A reductase by 2-mercaptoethanol disulfide

When the disulfide of 2-mercaptoethanol (ESSE) is added to the medium of cultured Chinese hamster ovary (CHO) cells, a time and concentration dependent release of 2-mercaptoethanol to the medium is observed. The reduction of ESSE to 2-mercaptoethanol by cells is a saturable process, the rate being approximately 50 nmoles of 2-mercaptoethanol per mg cell protein for an hour upon exposure to 250 μM ESSE. Reduction rate of ESSE by cells attached to a substratum is independent of glucose and insulin for periods up to 4 hours. However, in detached cells, swirled in suspension, addition of glucose and insulin is necessary in order to obtain a linear reduction rate of ESSE. The rate limiting enzyme in the sterol biosynthetic pathway, 3-hydroxy-3-methyl-glutaryl Coenzyme A reductase (E.C. 1.1.1.34), is inhibited by ESSE when isolated from CHO cells but total nonsaponifiable lipids synthesis from [2?¹⁴C]-acetate in intact cells is not affected by ESSE at concentrations up to 500 μM. Cytosolic reduced glutathione can spontaneously exchange disulfide bonds with ESSE and thus prevent it from inhibiting the reductase. Cultured cells respond to ESSE administration by elevating their total and acid-soluble glutathione levels. The use of ESSE as a perturbant of the GSH Status in cells is discussed.