Adaptive response of Haloferax mediterranei to low concentrations of NaCl (< 20%) in the growth medium.

Halobacteria require 20-25% NaCl for optimal growth and lyse when the salt concentration falls below 10%. The response of Haloferax mediterranei cells to low concentrations of NaCl (< 20%) in the medium was studied. The cells adapted to and grew in concentrations of NaCl as low as 10% and survived in concentrations lower than 5%. The cells synthesised a red pigment, bacterioruberin, in response to stress caused by a low concentration of NaCl (< 20%).

Solution structure of glyceraldehyde-3-phosphate dehydrogenase from Haloarcula vallismortis.

The subunit molecular mass of glyceraldehyde-3-phosphate dehydrogenase from the extreme halophile Haloarcula vallismortis (hGAPDH) was determined by mass spectrometry to be 35990 +/- 80 daltons, similar to other GAPDHs. Complementary density, sedimentation and light scattering experiments showed the protein to be a tetramer that binds 0.18 +/- 0.10 gram of water and 0.07 +/- 0.02 gram of KCl per gram of protein, in multimolar KCl solutions. At low salt (below 1 M), the tetramer dissociated into unfolded monomers. This is the third halophilic protein for which solvent interactions were measured. The extent of these interactions depends on the protein, but all form an invariant particle, in multimolar NaCl or KCl solutions, that binds a high proportion of salt when compared to non-halophilic proteins.

Ketohexokinase (ATP:D-fructose 1-phosphotransferase) from a halophilic archaebacterium, Haloarcula vallismortis: purification and properties.

Ketohexokinase (ATP:D-fructose 1-phosphotransferase [EC 2.7.1.3]), detected for the first time in a prokaryote, i.e., the extreme halophile Haloarcula vallismortis, was isolated and characterized from the same archaebacterium. This enzyme was characterized with respect to its molecular mass, amino acid composition, salt dependency, immunological cross-reactivity, and kinetic properties. Gel filtration and sucrose density gradient centrifugation revealed a native molecular mass of 100 kDa for halobacterial ketohexokinase, which is larger than its mammalian counterpart. The enzyme could be labeled by UV irradiation in the presence of [ gamma-32P]ATP, suggesting the involvement of a phosphoenzyme intermediate. Other catalytic features of the enzyme were similar to those of its mammalian counterparts. No antigenic cross-reactivity could be detected between the H. vallismortis ketohexokinase and the ketohexokinases from different rat tissues.

Characterization of 1-phosphofructokinase from halophilic archaebacterium Haloarcula vallismortis.

1-Phosphofructokinase (EC 2.7.1.56) (1PFK) was purified and characterized for the first time from an archaebacterial halophile Haloarcula vallismortis. The purification procedure involving (NH4)2SO4 fractionation, (NH4)2SO4-mediated chromatography on Sepharose 4B, CM-cellulose chromatography, hydrophobic chromatography on phenyl Sepharose and adsorption chromatography on hydroxylapatite yielded a preparation with a specific activity of 128 and 100-fold purification. From gel filtration and sucrose density gradient ultracentrifugation, the apparent molecular mass of halobacterial 1PFK was found as 76 +/- 5 kDa. The halobacterial 1PFK appears to be monomeric and the possibility of an unstable phosphoenzyme intermediate during its catalysis could not be ruled out. As in the case of many halobacterial enzymes, the 1PFK was found to be halophilic and thermostable. Other catalytic features of halobacterial 1PFK were similar to its counterparts from eubacterial sources.

Characterisation and purification of ribulose-bisphosphate carboxylase from heterotrophically grown halophilic archaebacterium, Haloferax mediterranei.

The CO2-fixing enzyme of Calvin cycle ribulose-1,5-bisphosphate-carboxylase/oxygenase has been isolated from a halophilic bacterium, Haloferax mediterranei grown heterotrophically. A homogeneous preparation was obtained from sonicated extract of the cells by three steps, resulting in a specific activity of 52 nmol.min-1.mg protein-1. The physicochemical and catalytic properties of the enzyme were studied. The halobacterial ribulose-bisphosphate carboxylase is an oligomer of 54-kDa and 14-kDa subunits as detected by SDS/PAGE. By sucrose-density-gradient centrifugation, the molecular mass of the enzyme was estimated as approximately 500 kDa indicating a hexadecameric nature. No evidence for an additional form of the enzyme devoid of small subunits was obtained. The enzyme required Mg2+ for activity, KCl for activity and stability, and an optimal pH of 7.8. In contrast to many halophilic proteins, ribulose-bisphosphate carboxylase from H. mediterranei is not an acidic protein. From the comparison of amino acid composition of halobacterial enzyme with its counterparts from a few eukaryotic and eubacterial sources, the S delta Q values showed that these proteins share some compositional similarities.

Halophilic class I aldolase and glyceraldehyde-3-phosphate dehydrogenase: some salt-dependent structural features.

Aldolase and glyceraldehyde-3-phosphate dehydrogenase from the extremely halophilic archaebacterium Haloarcula vallismortis are stable only in high concentrations of KCl present within the physiological environment. Data concerning the structural changes in the two enzymes as a result of lowering of salt concentration and changes in pH were obtained by monitoring the intrinsic protein fluorescence in the presence of quenchers. When the KCl concentrations were lowered below 2 M or in the presence of 6 M guanidine hydrochloride, the emission maximum shifted to a longer wavelength, indicating enhanced exposure of tryptophyl residues to the solvent. The spectral characteristics of the two proteins in guanidine hydrochloride and 0.4 M KCl were identical. However, these denatured states appear to be different than those observed after acid denaturation. Further perturbation of fluorescence was observed due to I-, and application of the Stern-Volmer law showed that the total fluorescence was available to the quenchers only in 0.4 M KCl solutions. The unfolding of proteins in 0.4 M KCl was a gradual process which was accompanied by a time-dependent loss in enzyme activity. The activity loss was complete within 30 min for aldolase whereas in the case of GAPDH nearly 3 h was required for the destruction of activity. For both enzymes, inactivation and protein denaturation were strongly correlated. The data on activity and thermostability measurements of the two enzymes in varying concentrations of KCl and potassium phosphate revealed that though both proteins are halophilic, the forces in the maintenance of their stability could be different.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel technique for the preparation of osmotically stabilized and permeabilized cells of extremely halophilic bacteria.

The cells of Haloferax mediterannei were stabilized by cross-linking with 0.5% glutaraldehyde for 10 min. Such cells were found to be osmotically stable even when suspended in water. The stabilized cells could be permeabilized by treatment with chloroform without leakage of intracellular components. No significant difference in the properties of an intracellular enzyme aldolase was observed, using either cell-free extract or the osmotically stabilized and permeabilized cells. This novel technique can serve as a useful tool for studying in situ regulatory characteristics of intracellular functions in halobacteria and can also help in their re-use under more stabilized conditions for biotechnological applications.

Products of non-reductive CO2 assimilation in the halophilic archaebacterium Haloferax mediterranei.

The products of CO2 fixation by heterotrophically grown Haloferax mediterranei were analysed. The main 14C-labelled alpha-ketoacid detected following incubation with NaH14CO3 and pyruvate or propionate was pyruvate. In presence of these organic acids and NH4+, 14CO2 was incorporated into glutamic and aspartic acids and alanine.

An unusual class I (Schiff base) fructose-1,6-bisphosphate aldolase from the halophilic archaebacterium Haloarcula vallismortis.

An electrophoretically homogeneous class I (Schiff base) alsolase has been isolated for the first time from the archaebacterial halophile Haloarcula (Halobacterium) vallismortis. The aldolase was characterized with respect to its molecular mass, amino acid composition, salt dependency, immunological cross-reactivity and kinetic properties. The subunit mass of aldolase is 27 kDa, which is much smaller than other class I aldolases. By the gel filtration method, the molecular mass of the halobacterial enzyme was estimated as 280 +/- 10 kDa, suggesting a decameric nature. In contrast to many halobacterial proteins, the H. vallismortis aldolase, though a halophilic enzyme, did not show an excess of acidic residues. Unlike the eukaryotic aldolases, the activity of the halobacterial enzyme was not affected by carboxypeptidase digestion. The general catalytic features of the enzyme were similar to its counterparts from other sources. No antigenic similarity could be detected between the H. vallismortis aldolase and class I aldolase from eubacteria and eukaryotes or class II halobacterial aldolases.

Ribulose 1,5-bisphosphate dependent CO2 fixation in the halophilic archaebacterium, Halobacterium mediterranei.

The cell extract of Halobacterium mediterranei catalyses incorporation of 14CO2 into 3-phosphoglycerate in the presence of ribulose bisphosphate suggesting the existence of ribulose bisphosphate carboxylase activity in this halophilic archaebacterium.

Archaebacterial class I and class II aldolases from extreme halophiles.

Both, class I (Schiff-base forming) and class II (metal requiring) fructose biphosphate aldolases were found to be distributed among halophilic archaebacteria. The aldolase activity from Halobacteriium halobium, H. salinarium, H. cutirubrum, H. mediterranei and H. volcanii exhibited properties of a bacterial class II aldolase as it was metal-dependent for activity and therefore inhibited by EDTA. In contrast, aldolase from H. saccharovorum, Halobacterium R-113, H. vallismortis and Halobacterium CH-1 formed a Schiff-base intermediate with the substrate and therefore resembled to eukaryotic class I type. The type of aldolase did not vary by changes in the growth medium.

The purification and subunit structure of a membrane-bound ATPase from the Archaebacterium Halobacterium saccharovorum.

A membrane-bound ATPase from Halobacterium saccharovorum was solubilized using sodium deoxycholate and Zwittergent 3-10 and purified by hydrophobic and ammonium sulfate-mediated chromatography. The enzyme, which had a molecular mass of 350 kDa, was composed of two major (87 and 60 kDa) and two minor (29 kDa and 20 kDa) subunits. The halobacterial ATPases appear to be unlike any other ATPase described to date.

Muscle aldolase: the stress-dependent modification of catalytic and structural properties by rat muscle lysosomal cathepsin B.

Stress dependent variations in th properties of the rat muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) have been linked to the corresponding changes in the levels of proteolytic activities in rat muscle. Whole-body X-irradiation of rat was shown to result in loss of muscle aldolase activity towards fructose 1,6-bisphosphate by 50% while fructose 1-phosphate activity remained unchanged (Pote, M.S. and Altekar, W. (1980) Ind. J. Biochem, Biophys. 17, 255-262). Incubation of muscle extract of irradiated rat with that from control rat or rabbit muscle aldolase caused similar changes in aldolase activity. The changes are attributed to the action of catheptic enzymes possessing latency characteristics and capable of using aldolase as a substrate; the time course of their increase after irradiation corresponds to that of loss in muscle aldolase activities. Exposure of rats to stress resulted in an increase in the 'free' proteolytic activity, and the concomitant loss of 'bound' activity in muscle lysosomes indicates labilization of lysosomal membrane. The observed degradation of aldolase in vivo by muscle lysosomes is shown to be due to the action of cathepsin B (EC 3.4.22.1) present in the proteolytic enzymes released into cytosol under stress. Inactivation of rabbit muscle aldolase and rat muscle aldolase by rat muscle cathepsin B inhibited by leupeptin, antipain an iodoacetamide, but not be pepstatin. Inactivation is shown to be due to the release of C-terminal tyrosine if aldolase, required for its catalytic activity. Cathepsin B who acts as a rate-limiting enzyme in the degradation of aldolase. Such a proteolytic modification of aldolase in vivo could be relevant not only to the regulation of aldolase activity of glycolysis in muscle but also to the degradation of aldolase during stress conditions related to tissue damage and the maintenance of normal aldolase levels in the blood.