The halophilic properties of pyruvate kinase fromVibrio costicola,a moderate halophile

Summary Pyruvate kinase fromVibrio costicola, a moderate halophile, appears to be adapted to functioning in the presence of salt. Its stability depends on the ionic strength of the medium. The amino acid composition resembles that of other halophilic enzymes. It is proposed that the halophilic pyruvate kinase utilizes preferentially the Mn⁺⁺ cofactor which forms more stable complexes in the presence of physiological concentration of salt.     

On the regulatory properties of a halophilic malic enzyme from Halobacterium cutirubrum.

The NADP-linked malic enzyme from Halobacterium cutirubrum is strongly inhibited by acetyl-CoA and NADH, and rather weakly inhibited by oxaloacetate and glyoxylate, in the presence of very high KCl concentrations (3 M), considered physiological for the extremely halophilic bacteria.     

Partial sequence of the gene for a serine protease from a halophilic archaeumhaloferax mediterranei R4, and nucleotide sequences of 16S rRNA encoding genes from several halophilic archaea

A part of the gene coding for a halophilic serine protease from a halophilic archaeumHaloferax mediterranei R4 was amplified by PCR and its 672 nucleotide sequence was determined. Tentative translation to the amino acid sequence suggested that the enzyme was quite similar to halolysin produced by another halophilic archaeum strain 172P1. Nucleotide sequences of 16S rRNA encoding genes from 9 halophilic archaea were determined. Alignment of 19 sequences known so far showed that there are more than 20 positions carrying bases or deletions specific for each halobacterial genus:Halobacterium, Haloarcula, Haloferax, andHalococcus.     

Ecology and physiology of phototrophic bacteria and sulfate-reducing bacteria in marine salterns

Marine salterns are habitats for a large variety of halophilic bacteria. In the anoxic zones, halophilic sulfur bacteria develop mainly at the sediment surface, but only a few of them have so far been isolated from such environments. Among the phototrophic sulfur bacteria that sometimes form purple layers underneath the green cyanobacterial layers, members of the generaEctothiodhodospira, Chromatium (C. salexigens), Thiocapsa (T. halophila) were isolated. They grow by using sulfide as an electron donor. In the marine salterns, sulfide originates from active sulfate reduction. Among the halophilic sulfate-reducing bacteria, onlyDesulfovibrio halophilus andDesulfohalobium retbaense have so far been isolated. The ecology and physiology of both kinds of bacteria are discussed in this paper.     

On the regulatory properties of a halophilic malic enzyme fromHalobacterium cutirubrum

Summary The NADP-linked malic enzyme fromHalobacterium cutirubrum is strongly inhibited by acetyl-CoA and NADH, and rather weakly inhibited by oxaloacetate and glyoxylate, in the presence of very high KCl concentrations (3,M), considered physiological for the extremely halophilic bacteria.This work was supported by grants from the consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, and the Consejo de Investigaciones de la Universidad Nacional de Rosario. JJC and MCV are members of the Carrera del Investigador Científico of the former and the latter institutions, respectively.     

Dealing with active oxygen intermediates: A halophilic perspective

Although oxygen tensions in the halophilic environment are diminished, they are nevertheless sufficient for the generation of active oxygen intermediates as byproducts of metabolism. Therefore, like all other aerobes, halophilic bacteria are compelled to possess the means to detoxify potentially lethal active oxygen intermediates. This review examines the superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen scavenging capacity of the halophiles,Halobacterium spp. Specifically, it looks at the potential of the bacteria to generate active oxygen intermediates and then examines the roles of superoxide dismutase, catalase, peroxidase and catalase-peroxidase. It also looks at some non-enzymatic means of neutralizing potentially lethal active oxygen intermediates.     

Glycerol production byDunaliella

Summary Species of the unicellular algaDunaliella possess outstanding tolerance of a wide range of salinities. They can adapt to grow in salt media which range from less than 0.5 M to saturated salt solutions and withstand enormous osmotic shocks through a unique osmotic adaptation. The osmoregulating mechanism depends on photosynthetic production of glycerol, whose intracellular concentration varies in direct proportion to the extracellular salt concentration and reaches values in excess of 50% of the total dry weight of the cells.Dunaliella, and another halotolerant glycerol producing alga,Asteromonas gracilis, osmoregulate biochemically by controlling glycerol biosynthesis and degradation. 3 new enzymes, NADPH-dihydroxyacetone-reductase, dihydroxyacetone kinase and glycerol-1-phosphatase seem to be involved in the osmoregulatory response via glycerol inDunaliella andAsteromonas. A hypothetical scheme of glycerol metabolism in these algae utilizing these enzymes is presented. Growth studies ofDunaliella indoors and outdoors showed that salt concentrations favoring maximal glycerol productivity are not identical with those required for maximal algal productivity. Maximal yield of glycerol occurred around 2 M NaCl while maximal algal productivity occurred below 0.5 M NaCl. Observed yields of glycerol inDunaliella culture outdoors are compared with theoretically calculated maximal yield.     

Effect of salt concentration on binding of proteins to a non-ionic adsorbent

Summary The effect of NaCl concentration on the adsorption of several proteins to palmityl-substituted Sepharose 4B has been investigated. It has been observed that the degree of adsorption first decreases and then increases with increasing salt concentrations, followed by total immobilization. The results are qualitatively explained by the simple theory of Debye-Hückel-Kirkwood, as applied to poly-electrolytes in the presence of salt.Acknowledgments. The authors wish to thank Ms H. Keshavarzi, Ms J. Nobakht and Mr F. Mohanazadeh for their technical assistance.     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

In smooth muscle the Mr 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of Mr 17,000 that binds 4 moles of Ca2+; and a larger protein of Mr circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca2+. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of Mr 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of Mr 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca2+-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

Influence of medium amino acids on the ouabain sensitive and insensitive 86Rb+-fluxes in HeLa cells

Components of the 86Rb+-influx in HeLa cells were investigated in Joklik minimal essential medium, or in Earle's balanced salt solution with and without medium amino acids. The presence of amino acids led to the stimulation of the ouabain sensitive 86Rb+-uptake and inhibition of the diuretic-sensitive and residual 86Rb+-fluxes. These results show that the presence of amino acids is an important regulator of the K+/Rb+-fluxes under normal conditions in growth medium.     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

Summary In smooth muscle the M_r 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of M_r 17,000 that binds 4 moles of Ca²⁺; and a larger protein of M_r circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca²⁺. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of M_r 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of M_r 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca²⁺-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

Spermine protects protein kinase C from phospholipid-induced inactivation

Phosphatidylserine (PS), an activator of protein kinase C (PKC) in the assay of protein phosphorylation, inhibited this enzyme in a time-dependent manner following preincubation in the absence of Ca²⁺. The phospholipid-induced inactivation of kinase activity was dependent on the PS content and on the charge density of liposomes. This inactivation of PKC could be reduced, but not completely eliminated, by addition of Ca²⁺. In the present work the effect of a naturally occurring polyamine (spermine) on the PS-induced inactivation of PKC was investigated. The presence of spermine during preincubation without Ca²⁺ was effective in suppressing the PS-induced inactivation of PKC over the period (20 min) required for PS to inhibit the enzyme by 95%. PKC exists in two membrane-bound states: a reversible one which can be dissociated by Ca²⁺ chelators (membrane-associated form) and an irreversible one which is chelator-stable (membrane-inserted form). Gel filtration experiments on the PKC-PS complex formed in the presence of Ca²⁺ indicated that less insertion of enzyme into liposomes occurred in the presence of spermine and that the kinase activity of the reversibly membrane-associated PKC was protected from PS inactivation.     

An improved assay for UDPglucose pyrophosphorylase and other enzymes that have nucleotide products

UDPglucose pyrophosphorylase catalyses the interconversion UDPglucose plus pyrophosphate and glucose 1-phosphate plus UTP. Several assay methods for this enzyme have been described but the only one that can be used to investigate the specificity with respect to various UDPsugars is based on coupling to UTP formation. This assay employs phosphoglycerate kinase to catalyse the formation 1,3-bisphosphoglycerate which is then used to oxidise NADH in the presence of glyceraldehyde 3-phosphate dehydrogenase. We have found that the activity of phosphoglycerate kinase towards UTP is low which limits the usefulness of the assay to very low rates, in agreement with the published recommendation of Hansen et al.⁵. Here it is shown that the dynamic range of the assay is increased by more than five fold on addition of nucleoside diphosphate kinase and ADP, which convert UTP to the preferred phosphoglycerate kinase substrate, ATP. It is also shown that the improved assay is suitable for enzymes with other nucleotide triphosphate products.     

Role of vitamin B 12 in the incorporation of ribonucleosides into the DNA of various Periplaneta americana cell lines]

Hemocytes and non hemocytes in vitro cell lines from Periplaneta americana, have been tested for their ability to incorporate, in the presence of vitamin B 12, various nucleic acid precursors into their DNA. Evidence was shown for enhanced transformation of all ribonucleotides into deoxyribonucleotides in the presence of vitamin B 12. Moreover, in hemocyte cell lines, it was demonstrated that vitamin B 12 produced an activation of thymidine kinase activity.     

Zur Einbettung biologischer Objekte in Acrylpolymerisaten

Summary Inhibition of vinyl polymerization by embedding biological specimens is due to the presence of biogenic amines. This inhibition is eliminated by smooth acylation and salt formation, respectively, by means of acid anhydrides.     

Action of an ATP analog, adenosine 5'-hyophosphophosphate in the reactions catalysed by hexokinase and fructose-6-phosphate kinase]

Adenosine 5'-hypoposphate phosphorylates glucose and fructose 6-phosphate in the presence of hexokinase and fructose 6-phosphate kinase respectively. It behaves as a competitive inhibitor versus ATP in the hexokinase reaction. Its affinity for the two enzymes is similar to that of ATP, the maximal velocities being however much lower.     

The inability of nuclear dehydrogenating clostridia to oxidize bile salt hydroxyl groups.

In a survey of the intracellular bile salt oxidoreductase activity in fecal bacteria, 16 strains of nuclear dehydrogenating clostridia and 2 strains of non-nuclear dehydrogenating C. paraputrificum were demonstrated unable to oxidize cholate at any of the 3 OH groups. Since nuclear dehydrogenation at the delta-1 and delta-4 position requires a 3-oxo precursor steroid, it appears that these organisms require the presence of a 3 alpha-hydroxysteroid dehydrogenating organism for nuclear dehydrogenation.     

Separation of modulator-dependent protein kinase (type I) from cyclic GMP-dependent protein kinase in mouse testes

A new type of enzyme, modulator-dependent protein kinase (type I) (M-PKI), was successfully isolated from the cytosol fraction of mouse testes. It was eluted slightly after the peak of cyclic GMP-dependent protein kinase (G-PK) by Sephadex G-200 gel filtration. Unlike either cyclic AMP-dependent protein Kinase (A-PK) or G-PK, its maximal activity depended exclusively on the presence of crude protein kinase modulators (PKM) or partially purified stimulatory modulator (PKMs).     

Compatible solutes of halophilic eubacteria: molecular principles,water-solute interaction,stress protection

Compatible solutes are best described as organic osmolytes responsible for osmotic balance and at the same time compatible with the cells' metabolism. A comprehensive survey (using HPLC and NMR methods) on halophilic/halotolerant eubacteria has revealed the full diversity of compatible solutes employed in nature. Molecular principles derived from the spectrum of compounds found in the bacterial world may be summarized as follows. Compatible solutes are polar, highly soluble molecules and uncharged at physiological pH. With the exception of proline (a proteinogenic amino acid) they are characterized as amino acid derivatives of the following types: betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine. Using nearinfrared spectroscopy we have also been able to demonstrate that compatible solutes are strong water-structure formers and as such probably excluded from the hydration shell of proteins. This preferential exclusion probably explains their function as effective stabilizers of the hydration shell of native proteins (protection against heating freezing and drying). Hence these typical products of halophilic eubacteria have a considerable potential as stabilizing/protecting agents on both molecular and whole-cell level. Thorough understanding of common structural principles and fundamental water-solute interactions will ultimately enable us to design novel highly efficient stress protectants and stabilizers of biomolecules.     

Migration inhibition of mammary epithelial cells by Syk is blocked in the presence of DDR1 receptors

The non-receptor tyrosine kinase Syk is a well-characterized hematopoietic signal transducer, which is also expressed in non-hematopoietic cells. In epithelial cells, the function of Syk is not wholly known. It interacts with the receptor tyrosine kinase DDR1 and is frequently lost from metastatic mammary tumors. Here, using genetic tracing, we demonstrate Syk expression in murine mammary epithelium, myoepithelium and skin epithelium, but not in intestinal or lung epithelia. Investigating possible functions of Syk, we found a substantial suppression of cell mobility that depended on Syk kinase activity in trans-well migration and wounding assays. Co-expression of DDR1 resulted in constitutive interaction and strong activation of Syk kinase. Most importantly, Syk-mediated migration inhibition was blocked in the presence of DDR1, while conversely DDR1 knockdown restored migration inhibition. Our study identifies Syk as a potent inhibitor of epithelial migration and describes a first functional consequence of the interaction with the collagen receptor DDR1.