Membrane composition and ion-permeability in extremophiles

Abstract: Protons and sodium ions are the only used coupling ions in energy transduction in Bacteria and Archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high as compared to sodium ions. In some thermophiles, therefore, sodium is the sole energy coupling ion. Comparison of the proton- and sodium permeability of the membranes of variety of bacterial and archaeal species that differ in their optimal growth temperature reveals that the permeation processes of protons and sodium ions must occur by different mechanisms. The proton permeability increases with the temperature, and has a comparable value for most species at their respective growth temperatures. The sodium permeability is lower than the proton permeability and increases also with the temperature, but is lipid independent. Therefore, it appears that for most bacteria the physical properties of the cytoplasmic membrane are optimised to ensure a low proton permeability at the respective growth temperature.     

Characterization of hepatic lactogen receptor. Subunit composition and hydrodynamic properties

The structure of the membrane-bound and Triton X-100-solubilized female rat liver prolactin receptor has been studied by affinity cross-linking/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and sucrose/H2O and sucrose/D2O density gradient centrifugation. Hydrodynamic characterization revealed that the 125I-human growth hormone receptor-detergent complex represents a molecular species with a Stokes radius of 61 A, a sedimentation coefficient of 5.0 s, and a calculated molecular weight of 158,000. The molecular weight of the receptor was calculated to be 92,000. Three lactogenic hormone-binding species with Mr values of 87,000, 40,000, and 35,000, respectively, were repeatedly found when detergent-solubilized preparations were analyzed using an affinity cross-linking technique. Estrogen treatment of female rats increased the intensity of these bands. Occasionally, an Mr 165,000 hormone-binding species was also found. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies (first dimension, nonreducing; second dimension, reducing) demonstrated that disulfide- and nondisulfide-linked hormone-binding species with Mr values of 40,000 and 35,000 are contained within the Mr 87,000 species. It is concluded that the Triton X-100-solubilized female rat liver prolactin receptor has a molecular weight of about 90,000. This molecular species contains Mr 40,000 and Mr 35,000 hormone-binding subunits. It cannot be determined whether these subunits are combined with each other or with as yet undetected subunit(s) to make up the Mr 90,000 species, or whether each one of these subunits is a proteolytic fragment of the Mr 90,000 species.     

The novel peptide composition of the seeds of Trichosanthes dioica Roxb

The seeds of Trichosanthes dioica contain a large amount of peptides in the range of 2-8 kD. These peptides can be resolved in a discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) system using Tricine as the trailing ion. The seed proteins contain a number of charge species as determined by isoelectric focusing (IEF) in polyacrylamide gels. The peptides were focused in the basic region as determined by two-dimensional electrophoresis involving IEF and SDS-PAGE. The seed peptides have the unique property of being resistant to the action of silver nitrate, a sensitive reagent commonly used to stain proteins. The seed contains haemagglutinating activity which is inhibited by galactose.     

Electrophoretic microheterogeneity and subunit composition of the 13S coupling factors of oxidative and photosynthetic phosphorylation.

Two electrophoretically distinguishable species of the 13S coupling factor of oxidative phosphorylation from Alcaligenes faecalis are detectable by standard polyacrylamide gel electrophoresis in the absence of urea, detergents, or any other protein-denaturing reagents. The slower species (type IA) can be converted into the faster species (type IB) by treatment with ATP, and the fast form converts into the slow form when aged at 4 degrees. The enzyme undergoes these conversions both when it is free in solution and when it is membrane bound. The ATP analog adenylyl imidodiphosphate (AMP-PNP) gives the conversion without being hydrolyzed and without causing any apparent change in the mass of the protein, which suggests that the conversion may be a ligand-induced conformational change. Types IA and IB can convert into three other electrophoretically distinguishable species (types IIA, IIB, and III) if the purification procedure involves chromatography on a DEAE-Sephadex column equilibrated in phosphate buffer. These conversions can be prevented if the column is eluted in morpholinoethanesulfonic acid (Mes) buffer and KCl. Type IIA is convertible into type IIB by ATP treatment. Types IA and IB will also convert into types IIA and IIB and finally into type III when aged for extended periods of time at 4 degrees, without a detectable change in mass. Coupling factor activity is lost when type I enzyme converts into type II enzyme, as is the ability of the enzyme to bind to the membrane. However, ATPase activity does not change significantly. The mitochondrial 13S coupling factor shows up to three electrophoretically distinguishable species. The use of phosphate buffer during DEAE-Sephadex chromatography gives conversion of slower species into faster species. ATP treatment does not give interconversions, and aging at 4 degrees gives only a slow dissociation of the enzyme into subunits. The chloroplast 13S coupling factor also shows up to three electrophoretic species. Incubation with ATP does not give interconversions, but a temperature-dependent conversion of the major species into a faster species occurs upon aging. The subunit composition of the three 13S enzymes is very similar by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the major difference being in the number of classes of small polypeptides.     

Effects of stimulation on the ultrastructure and Na, K, Cl composition of the fundus of the rat plantar sweat gland

Initial stimulation of the rat plantar sweat gland with pilocarpine caused a variable degree of distension of the apical membrane of the secretory cell. This appeared to be a process of filtration of secretory cell cytoplasm through the apical terminal web. Further stimulation resulted in luminal dilatation, cytoplasmic depletion, and morbidity of some cells. These morphological changes in the footpad gland, which thus can no longer be considered as eccrine, were accompanied by a fall in potassium and a rise in sodium concentration within the secretory cells. The mode of secretion induced by pharmacological stimulation was fundamentally the same as that in the glands of species responsive to thermal stimulation.     

Bidimensional immunoelectrophoretic study of the antigenic composition of the membrane in various mycoplasma strains]

Membrane antigenic composition of Acheloplasma laidlawii PG9, A. granularum BTS-39, and Mycoplasma fermentans PG 18(G) was determined by means of bidimensional immunoelectrophoresis in the presence of sodium desoxycholate 0.5%. Depending upon the mycoplasma species from which membranes were obtained, 7 to 15 antigens were evidenced. Using sodium desoxycholate presents the advantage over non-ionic detergents to dissolve better the mycoplasmic membrane antigenic complexes. A comparative study of five strains belonging to the above-noted species confirms the serological heterogeneity of the Mycoplasmateles order and shows variability at the membrane antigenic composition level of Acheloplasma laidlawii.     

Acidocalcisomes of trypanosomatids have species-specific elemental composition

The elemental composition and stoichiometric profile of elements present in acidocalcisomes of different genera of the Trypanosomatidae family (insect, plant, and mammalian parasites) submitted to parallel cultivation conditions were studied. X-ray microanalysis using transmission electron microscopy in conjunction with a morphometric approach was used to investigate the elemental content, number, distribution, and volumetric density of acidocalcisomes of different species. Microanalytical data showed that the different parasites possess the same elemental composition (oxygen, sodium, magnesium, phosphorus, calcium, iron, and zinc) in their acidocalcisomes. However, the relative concentrations of the elements varied among species, but not within acidocalcisomes of individual species. Iron was detected in acidocalcisomes of all species analyzed, characterizing this element as a constituent of these organelles. Taken together, the results strongly indicate a species-specific composition of acidocalcisomes in trypanosomatid parasites.     

Chlorophyll-protein complex composition during chloroplast development: A species comparison

Barley, maize, pea, soybean, and wheat exhibited differences in chlorophyll a/b ratio and chlorophyll-protein (CP) complex composition during the initial stages of chloroplast development. During the first hours of greening, the chlorophyll a/b ratios of barley, pea, and wheat were high (a/b8) and these species contained only the CP complex of photosystem I as measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. A decrease in chlorophyll a/b ratio and the observation of the CP complexes associated with photosystem II and the light-harvesting apparatus occurred at later times in barley, pea, and wheat. In contrast, maize and soybean exhibited low chlorophyll a/b ratios (a/b<8) and contained the CP complexes of both photosytem I and the light-harvesting apparatus at early times during chloroplast development. The species differences were not apparent after 8 h of greening. In all species, the CP complexes were stabilized during the later stages of chloroplast development as indicated by a decrease in the percentage of chlorophyll released from the CP complexes during detergent extraction. The results demonstrate that CP complex synthesis and accumulation during chloroplast development may not be regulated in the same way in all higher plant species.Abbreviations Chl chlorophyll - CP chlorophyll-protein - CPI P700 chlorophyll-a protein complex of photosystem I - CPa electrophoretic band that contains the photosystem II reaction center complexes and a variable amount of the photosystem I light-harvesting complex - LHC the major light-harvesting complex associated with photosystem II - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 10335 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601.     

Carbohydrate composition of sarcolemma of rat myocardium

The carbohydrate composition of SL prepared from rat ventricular muscle was examined and compared with those of Mw and FSR prepared from the same species. The total carbohydrate content of SL was 308.58 micrograms/mg of protein, which was greater than that of Mw (74.49 micrograms/mg of protein) and FSR (76.50 micrograms/mg of protein). The carbohydrate of SL was composed of hexose (35.0%), hexuronic acid (7.3%), sialic acid (6.1%), methyl pentose (7.4%), and hexosamine (44.2%). The peculiar PAS-positive glycoproteins of SL were observed as five bands on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and the molecular weight of the main band of these was 85.4 kDa. Thus, each of the cell fractions obviously shows a distinct carbohydrate composition and the results obtained in the present study may suggest that the presence of the PAS-positive glycoprotein (85.4 kDa) can be used for routine monitoring of the purity of the SL fraction.     

Subunit composition of the membrane glycoprotein complex of Semliki Forest virus

The quaternary structure of the membrane glycoproteins E1, E2 and E3 of Semliki Forest virus has been determined in intact virus and in the protein complexes obtained after Triton X100 solubilization. Intact and solubilized virus were treated with a cleavable cross-linking reagent and the covalently cross-linked glycoprotein complexes were isolated and characterized using antibodies specific for the E1 and E2 membrane glycoproteins. The isolation and characterization procedure was done in a low sodium dodecyl sulphate concentration which prevented non-covalent association between glycoprotein species, but did not abolish antigen-antibody binding.The major glycoprotein complex seen after cross-linking of either intact or Triton X100 solubilized virus was an approximately 100,000 molecular weight species composed of E1-E2 heterodimers only. These findings show that E1 and E2 form a complex in the virus and that this complex is retained after solubilization with Triton X100. The smallest membrane glycoprotein E3 was not cross-linked to the other proteins and was therefore lost in the isolation procedure. However, the presence of E3 together with E1 and E2 in complexes obtained after Triton X100 solubilization of intact virus suggests that an E1-E2-E3 trimer is present in the virus. It is likely that this trimer forms the spike-like structures seen on the surface of the virus.We have observed that antibody specific for one component of the virus glycoprotein complex can induce rearrangement of uncross-linked complexes in Triton X100 solubilized form. This fact should be considered when using specific antibody for characterization of protein complexes.     

Studies in the diagnosis of mineral deficiency; the composition of weed leaves in relation to potassium deficiency in barley

Leaves of five weed species (Brassica sinapis, Cirsium arvense, Polygonum aviculare, P. convolvulus and Potentilla reptans) from fertilizer trials on barley at four sites in Hampshire were analysed with a view to using their composition in forecasting the response of barley to fertilizers. The samples were gathered from as many of the 108 plots as possible, and were analysed spectrographically for calcium, iron, magnesium, manganese, potassium and sodium.
Marked differences in composition between the five species were recorded, the most noteworthy being the high sodium content of Brassica sinapis and Cirsium arvense and the high manganese content of Potentilla reptans. There were also marked differences between the four sites, but these were not uniform as between the different species, and often failed to agree with those observed for barley.
Superphosphate applications decreased the manganese content of the weeds in many cases, and increased their calcium content. Muriate of potash increased their potassium content, but tended to decrease that of magnesium and sodium. The only general effect of sulphate of ammonia on the composition of the weeds was a decrease in iron content.
Except in Cirsium arvense , the potassium content of weed leaves was correlated with that of barley on the same plot if differences within a site only were considered. Differences between sites were not correlated in this way. The correlation between potassium content of weed leaves and the response of barley to muriate of potash application was worthy of note only in Polygonum convolvulus , and even in this case the correlation of site differences did not reach significance. It is tentatively suggested that increases in the grain yield of barley as a result of muriate of potash application are likely to occur only where the leaves of P. convolvulus contain less than 1.83% potassium.     

Quantitative analysis of the composition of the native and scrambled ribonuclease A

Reversible conversion between the native and scrambled proteins can be applied to analyze the denaturation curve of a disulfide-containing protein. In the case of RNase A, scrambled species could not be well separated from the native species by HPLC to permit precise quantitative analysis of the extent of denaturation. Methods are developed here to overcome this problem. The methods exploit the difference of conformational stability between the native and scrambled RNase A. When a sample of partially denatured RNase A was placed under mild reducing conditions (0.2-1 mM dithiothreitol for 10 min), the disulfide bonds of the native RNase A remain intact, whereas those of scrambled isomers become fully reduced. The native and fully reduced species of RNase A can be completely separated by HPLC. Alternatively, a mixture of partially denatured RNase A can be treated with mild concentration of proteolytic enzymes (trypsin or thermolysin). In this approach, scrambled isomers of RNase A were totally fragmented and readily separated from the native RNase A. These methods allow analysis and construction of the denaturation curves of RNase A in the presence of urea, GdmCl and GdmSCN.     

The effect of surface sterilization and the type of sterilizer on the genus composition of lichen-inhabiting fungi with notes on some frequently isolated genera

Surface sterilization is generally used for isolating lichen-inhabiting fungi as well as endophytic fungi, and ethanol and sodium hypochlorite are commonly used as the sterilizer. However, there are few studies on whether the type of chemicals used for surface sterilization affects the isolation results of lichen-inhabiting fungi. In this study, the genus composition of the lichen-inhabiting fungi of two lichen species (Flavoparmelia caperata and Peltigera dilacerata) were investigated 1) to reveal how the isolation result changes before and after surface sterilization and 2) to examine the effect of the sterilizer (ethanol, sodium hypochlorite, or hydrogen peroxide) on the composition of the isolated fungi. We isolated 652 non-lichenized fungal isolates from the two-lichen species and identified 84 genera. It was found that 1) every sterilizer effectively removed the fungi on the lichen surface and that 2) the composition of isolated fungi varied depending on the type of surface sterilizer. It was also shown that, such as the genus Sarea, there were some lichen-inhabiting fungi which could not be isolated at all by surface sterilization with ethanol or sodium hypochlorite, which are commonly used. In addition, the genus Virgaria was detected as lichen-inhabiting fungi for the first time. Our results suggest that single surface sterilization alone may underestimate the genus composition of lichen-inhabiting fungi.     

Purification and subunit composition of acetohydroxyacid synthase I from Escherichia coli K-12.

Acetohydroxyacid synthase I from Escherichia coli K-12 has been purified to near homogeneity. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two polypeptides, one with a molecular weight of 60,000 and one with a molecular weight of 9,500. These two polypeptides were present in constant proportion to each other and to enzyme activity. The molar ratio of the two polypeptides (Mr 9,500:60,000), estimated from stained polyacrylamide gels, was 1. Antisera raised against the 60,000 Mr polypeptide precipitated both the 60,000 and the 9,500 Mr polypeptides from extracts of cells labeled with [35S]methionine. The addition of sodium dodecyl sulfate before immunoprecipitation eliminated the smaller polypeptide, and only the larger one was recovered. The hydrodynamic properties of the native enzyme confirmed a previous report that the largest enzymatically active species has a molecular weight of about 200,000; this species contains both the 60,000- and 9,500-molecular-weight polypeptides.     

An evaluation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the identification of microsporidia

Microsporidian spore polypeptides separated with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) can be used to identify isolates of microsporidia. The spore polypeptides separated with SDS/PAGE provided unique, reproducible electrophoretic profiles which were not influenced by host species or the temperature at which the host larvae were maintained for development. Furthermore, host proteins were not detected in electrophoretic profiles of the spore polypeptides. Spore mixtures of two microsporidian species can be detected when the spore polypeptides of either or both species have been previously separated with SDS/PAGE.     

Potential use of the monosaccharide composition of bacteria for their identification

The monosaccharide composition of cell hydrolysates can be used as a criterion for the chemical differentiation of gram-positive bacteria. The monosaccharide composition of six bacterial species belonging to the genus Bacillus has been determined using gas chromatography, mass spectrometry and computers. The qualitative composition was similar, glucose, galactose, ribose and glucosamine being the main components in all of the species. Some Bacillus species differed in their minor components. Although the monosaccharide composition appeared to be homogeneous, bacteria can be identified in terms of their carbohydrate profile using computers. To this end, the monosaccharide composition of bacterial cells is represented as a two-dimensional data file including the qualitative composition of components and the quantity of each component.     

A simple electrophoretic procedure for the determination of the polypeptide composition of the subunits of Fraction 1 protein

A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 m urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques.     

Proteolytic interconversion of electrophoretic variants of the enzyme rhodanese

It has been confirmed that the enzyme rhodanese, although a homogeneous single polypeptide chain protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is separable by electrophoresis under nondenaturing conditions into four species which differ in net surface charge (I-IV in the order of increasing positive charge). Limited proteolysis can interconvert these species. Chymotrypsin converts IV and III to II and forms a small amount of I. Carboxypeptidase B converts IV to III. The total protein among the species remains constant, and two-dimensional gels show that the change induced is below the resolution of the sodium dodecyl sulfate-polyacrylamide gel system. The suggestion that the products can be produced in the order IV, III, and II is supported by the results of sequential treatment of rhodanese first with carboxypeptidase B and then with chymotrypsin. It is concluded that there are covalent differences among the rhodanese species identified to date and an interconversion of forms can be triggered by proteolysis at the COOH-terminal end of the Mr = 33,000 single polypeptide chain which comprises the enzyme. This conclusion is strengthened by the close similarity between the amino acid composition of the peptide released by chymotrypsin and the composition expected on the basis of the known sequence. Furthermore, it appears that form IV is the primary in vivo product and the other species arise from it.     

Proximate,caloric, nitrogen and mineral composition of bodies of some tropical bats

Proximate (live mass, water, lipid, ash, non-fat organic), caloric, nitrogen, and mineral (sodium, potassium, calcium, magnesium, and iron) concentrations and total body content of individuals of 24 species of Neotropical and Paleotropical bats were determined. Mass-related, concentration patterns were found for all measured variables, except iron. Concentrations increase with size for nitrogen, calcium, and magnesium but are concave, opening upward, for sodium and potassium. These last two elements reach minimal concentrations in bats weighing about 22 and 28 g dry mass, respectively. Total body content of nitrogen and minerals was compared with amounts in similar-sized birds and tetrapodal mammals.     

Lipid composition of yeastlike cells of the fungusPullularia pullulans

AlthoughPullularia pullulans is a polymorphic fungus, cultures have been obtained consisting exclusively of yeastlike cells. These cells can be considered as “medium lipid content” yeasts (5.7%). Thirty percent of the total lipids are phosphoglycerides, the most abundant of which are phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The bulk of the nonpolar lipids is made up of unsaponifiable matter, sterols, and hydrocarbons. Eighteen fatty acid species have been detected, but the C₁₆ and C₁₈ species are by far the most abundant. The major unsaturated species is oleic acid. Linolenic acid is found in significant amounts only in triglycerides and esterified sterols. Fatty acid moieties associated with phosphatidylcholine and phosphatidylethanolamine are more unsaturated than those associated with phosphatidylserine or cardiolipin. Considerable proportions of the phosphoglycerides exist in the form of plasmalogen, which is unusual in yeasts.