Alfred Haug

Molecular aspects of aluminum toxicity

The focus in this review is directed to molecular aspects of aluminum toxicity in animal and plant cells. Unique thermodynamic features of Al(lII) ions impart biological specificity which may form the biochemical basis of aluminum interactions with cellular constituents. Current knowledge about aluminum‐specific, molecular interactions is rather scanty. Al(III) ions may coordinate with nucleotides or complex to phospholipids resulting in impaired genetic expression or aluminum‐induced membrane phase changes, respectively. Aluminum‐specific structures are formed with proteins involved in iron transport. and with calmodulin, a key regulatory protein involved in coordinating the actions of second messengers. A novel hypothesis is put forward that Al‐calmodulin may be a primary lesion that occurs in the broadly defined syndrome of aluminum toxicity. Pertinent research areas are discussed.

The effect of Al3+ on the physical properties of membrane lipids in Thermoplasmaacidophilum

Abstract Using Electron Paramagnetic Resonance Spectroscopy, Al3+ was shown to produce a dramatic decrease of membrane lipid fluidity on the microorganism Thermoplasma acidophilum at a pH > 2. The ability of Al3+ to alter lipid fluidity was enhanced with increasing pH (from 3 to 5). At pH 4, 10−2 M Al3+ increased the lower lipid phase transition by 39°C, and a detectable change was observed with AlCl3 concentrations as low as 10−5 M. The ability of Al3+ to increase the lower lipid phase transition temperature of T. acidophilum is the largest of any cation/lipid interaction yet reported.

Aluminum Uptake by Neuroblastoma Cells

Aluminum uptake studies in viable neuroblastoma cells were performed. Aluminum uptake was largely dependent on the pH of the suspension medium. At physiological pH values, cells were apparently unable to incorporate detectable amounts of aluminum in the absence of proper mediators. Aluminum uptake was enhanced as the pH decreased, attaining a plateau at about pH 6.0. In experiments with 2 × 106 cells/ml, pH 6.0, and 25 μM aluminum in the medium, aluminum incorporation reached saturation at 5 nmol of aluminum/mg of cellular protein, accounting for 60–70% of aluminum added. At pH 6.0, cells showed a large capacity for accumulating aluminum; about 70% of intracellular aluminum was associated with the post‐mitochondrial fraction. At neutral pH, application of apotransferrin seemed to facilitate aluminum translocation into cells via membrane receptors. Fatty acids were also capable of mediating aluminum uptake at neutral pH, probably by forming aluminum‐fatty acid complexes. Low molecular weight aluminum chelators, e.g., citrate, inhibited aluminum uptake. Treatment of cells with energy metabolism blockers had virtually no influence on aluminum uptake, indicative of passive mechanisms. The results suggest that aluminum uptake occurs via different modes dependent on growth conditions, such as medium pH.

Aluminum interaction with phosphoinositide-associated signal transduction

Concerning molecular and cellular mechanisms of aluminum toxicity, recent studies support the hypothesis that interactions of aluminum ions with elements of signal transduction pathways are apparently primary events in cells. In the case of the phosphoinositide-associated signalling pathway of neuroblastoma cells, guanine nucleotide-binding proteins (G proteins) and a phosphatidylinositol-4,5-diphosphate (PIP2)-specific phospholipase C are probable interaction sites for inhibitory actions of aluminum ions. Following interiorization of aluminum by the cell, metal interactions decrease the accumulation of inositol phosphates, especially that of inositol-1,4,5-triphosphate (IP3), concomitant with derangements of intracellular Ca2+ homeostasis. In the presence of high concentrations of Ca2+, formation of IP3 is also diminished in aluminum-pretreated cells, presumably involving a process not requiring Mg2+-dependent G proteins. At higher aluminum doses, metal-induced changes in the lipid milieu of the membrane-bound phospholipase may play a role. These types of primary interactions of aluminum ions with elements of cellular communication channels are probably crucial in the manifestation of the multifacetted aluminum toxicity syndrome. If present as a phosphate-like fluoroaluminate, a stimulatory role of aluminum ions is displayed in G protein-coupled transmembrane signalling.

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.

Regulation of membrane lipid fluidity in Acholeplasma laidlawii: Effect of carotenoid pigment content

Abstract Acholeplasma laidlawii (oral strain) cells were grown in a lipid-extracted medium supplemented with arachidic acid (C20:0). The carotenoid content of the plasma membrane was appreciably dependent upon the levels of acetate or propionate present in the growth medium. 1. 1. Membranes isolated from cells grown in propionate (5 g/l) contained a low level of carotenoids, approximately 11 wt% of the total lipids. Neither the fatty acyl composition nor the membrane lipid/protein ratio was altered. However, spin-labelling experiments demonstrated a greater lipid fluidity in those membranes, as compared to those from cells grown in an acetate containing growth medium. Membranes isolated from cells grown in acetate (5 g/l) were characterized by a higher buoyant density, higher osmotic fragility and lower glycerol permeability. 2. 2. Growing cells in a medium containing 20 g/l of propionate instead of acetate, the membrane carotenoid content decreased by 57-fold. For cells grown in an acetate medium (20 g/l), high levels of carotenoid pigments (about 38 wt% of the total lipids) were obtained; fewer arachidoyl and more unsaturated acyl groups were found in the membrane lipids. Spin-labelling experiments showed only a very slight difference in lipid fluidity between the two types of membrane. 3. 3. These results suggest that carotenoid pigments rigidify the Acholeplasma membrane and the organism maintains its membrane fluidity within a narrow range by modifying the fatty acyl composition of the membrane lipids.

Organic acids prevent aluminum-induced conformational changes in calmodulin

At a molar excess of 10:1 for [citrate]/[calmodulin], citrate can prevent aluminum binding to calmodulin when present in the protein solution in micromolar concentration, as determined by fluorescence and circular dichroism spectroscopy. In contrast, citrate is only partially effective in restoring calmodulin to its native structure once the aluminum-calmodulin complex (3:1) is formed, as measured by the alpha-helix content of the protein. Considering the magnitude of the stability constant of the citrate-aluminum chelate, citrate and perhaps other carboxylic acids may protect calmodulin, and thus cells, from toxic aluminum ions.

Purification and partial characterization of a procaryotic glycoprotein from the plasma membrane of Thermoplasma acidophilum.

The obligate, thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum, grows optimally at 56 degrees C and pH 2.0. Its plasma membrane possessed 21--22 protein bands that were resolved by polyacrylamide gel electrophoresis. One major membrane protein, molecular weight 152 000, which stained for carbohydrate with periodic acid-Schiff reagent, accounted for 32% (w/w) of the total membrane proteins. It was isolated and further purified by concanavalin A affinity chromatography. The carbohydrate content amounted to less than 10% (w/w) compared to that of the entire glycoprotein. The carbohydrate moiety consisted mainly of mannose residues with branched alpha 1 leads to 2 linkages at the non-reducing ends of the glycopeptide as determined by permethylation followed by gas chromatography-mass spectrometry analysis. The reducing end was an N-glycosidic linkage between asparagine and N-acetylglucosamine. The amino acid composition of this glycoprotein showed 62 mol% hydrophobic residues, while the acidic amino acid content contributed 9 mol% more than that of the basic amino acids. The existence of membrane glycoproteins in the procaryotic, wall-less T. acidophilum may provide a protective coat for the plasma membrane. The stereochemistry and the conformation of the carbohydrate chains, in conjunciton with water turgor, may contribute to the rigidity of the membrane and the cation binding.

A thermodynamic and electron paramagnetic resonance study of structural changes in calmodulin induced by aluminum binding

Bovine brain calmodulin binds 3 mol aluminum per mol protein with dissociation constants in range of 10(-7) to 10(-6) molar. EPR spectra of spin-labelled calmodulin provide data indicating that aluminum binding causes decreased probe immobilization as compared to the effects of calcium binding. This result of aluminum binding indicates that A1-calmodulin is a more random, open polypeptide relative to the structure of Ca2+-calmodulin. Calorimetric measurements of aluminum binding provide data showing that the first mol of aluminum bound is accompanied by the largest enthalpic change (-3.9 kcal mol-1), whereas binding of the second and third mol of aluminum are each entropically driven.

Control of membrane lipid fluidity in Acholeplasma laidlawii

Membrane fluidity seems to play an important role in cellular processes such as regulation of enzyme activity [1,2], permeability [3], transport of nutrients [4], lateral motion of membrane constituents [5], and osmotic stability of cells [6]. In this communication we report that the microorganism Acholeplasma laidlawaii is able to maintain a virtually constant membrane lipid fluidity at the growth temperature when the growth temperature and the exogenous fatty acid source are varied.