INFLUENCE OF METHOD FOR REMOVAL OF SESTON ON THE DISSOLVED ORGANIC MATTER1

Comparisons of various methods and method modifications for treating water samples to render them free of seston prior to analysis of dissolved organic matter have corroborated a number of suspected sources of error. Among the more important points arising from this study arc: 1. All cellulose ester filters must be washed to remove elutable carbon. 2. In some instances filtration to dryness may produce artifacts resulting from cell injury. 3. A significant difference in filter retention can result between 0.45 and 0.22 μ membranes. 4. Among the methods most satisfactory are wet filtration through 0.22 μ pre-washed Millipore membranes and continuous-flow centrifugation at ca. 10,000 x g and 100 cc/min flow rate, both of which have their inherent weaknesses and limitations. 5. Regular centrifugation does not remove some planktonic organisms which have considerable buoyancy, or organic substances may somehow be released by cells without producing morphological damage. The newly developed bio dialysis technique for dissolved organic matter collection consistently yielded lower values than continuous-flow centrifugation. In contrast, biodialysis yielded lower values for pond water and higher values for Scenedesmus cultures than the best filtration method. Evidence suggests that biodialysis will be useful as both a supplementary and, in some zuays, more accurate method in studies of dissolved organic matter.     

Localization of 3,5,3'-L-triiodothyronine receptor in rat kidney mitochondrial membranes

Binding of triiodothyronine(T₃) to submitochondrial fractions from rat kidney was studied. Both inner and outer mitochondrial membranes were purified by sucrose gradient centrifugation. Both membranes had specific binding sites for T₃. Scatchard analysis of T₃ binding by membranes gave different affinity constants between inner and outer membranes. In studies with gel filtration of soluble T₃ receptors, four main T₃ binding activities in outer membranes and two main T₃ binding activities in inner membranes were isolated. The results indicate that both inner and outer mitochondrial membranes have specific binding sites for T₃ and that each membrane has a specific structure in T₃ receptor.     

A STUDY OF THE MOLECULAR SIZES AND STABILITY OF CHOLINE ACETY LTRANSFER ASE

Aggregation of the enzyme acetyl-CoA: choline-O-acetyltransferase (ChAc, EC 2.3.1.6) which appears to be homogeneous has been observed. The molecular weight of the most abundant form of ChAc was estimated by gel filtration and sucrose gradient centrifugation to be in the range 58,000-62,000. The most frequently encountered aggregates were much larger and eluted in the void volume from Sephadcx® G-100 and G-150 indicating molecular weights in excess of 400,000. In fact, they were subsequently found to be 1.2 × 10⁶ and 1.9 × 10⁶ by sucrose gradient centrifugation. The percentage of activity associated with high molecular weight ChAc increased with purification, but these aggregates disappeared after storage for 2-3 weeks at ?20°C. The loss occurred independently of any fall in enzymic activity in the preparations examined.     

Observations on the separation of Theileria sporozoites from ticks

Hyalomma anatolicum anatolicum ticks infected with Theileria annulata were partially fed on rabbits and then ground up with tissue culture medium. The ground up ticks were treated by centrifugation at 100 g, filtration through membranes of 8 μm pore diameter and centrifugation on a discontinuous density gradient of Percoll. Counts of sporozoites and tick debris were made from Giemsa stained slides of samples at each stage of the separation. Debris was removed during light centrifugation and filtration at a greater rate than sporozoites. After filtration approximately 41% of the original sporozoites remained in the suspension. After density gradient centrifugation most sporozoites were found in a distinct zone, at approx. 1·08 g/cm³ density, separate from most dense debris and light debris and soluble contaminants. After this final centrifugation approximately 24% of the original sporozoites remained in the recovered suspension.     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.     

Evaluation of options for harvest of a recombinant E. Coli fermentation producing a domain antibody using ultra scale‐down techniques and pilot‐scale verification

Ultra scale‐down (USD) methods operating at the millilitre scale were used to characterise full‐scale processing of E. coli fermentation broths autolysed to different extents for release of a domain antibody. The focus was on the primary clarification stages involving continuous centrifugation followed by depth filtration. The performance of this sequence was predicted by USD studies to decrease significantly with increased extents of cell lysis. The use of polyethyleneimine reagent was studied to treat the lysed cell broth by precipitation of soluble contaminants such as DNA and flocculation of cell debris material. The USD studies were used to predict the impact of this treatment on the performance and here it was found that the fermentation could be run to maximum productivity using an acceptable clarification process (e.g., a centrifugation stage operating at 0.11 L/m² equivalent gravity settling area per hour followed by a resultant required depth filter area of 0.07 m²/L supernatant). A range of USD predictions was verified at the pilot scale for centrifugation followed by depth filtration. © 2016 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 32:382–392, 2016     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

ALTERATIONS IN THE BINDING OF [3H]LEUCINE-ENKEPHALIN TO STRIATAL MEMBRANES BY ENDOGENOUS FACTORS

—Saturation binding studies with [³H]leu-enk ([tyrosyl-3, 5-³H(N)]⁵leu-enkephalin) revealed the presence of high and low affinity binding sites in a paniculate fraction derived from rat striatum. The binding of [³H]leu-enk to the high affinity component (K_D= 2.0 ± 0.3 nM) was sensitive to morphine and levorphanol, while the binding to the low affinity component (K_D= 21 ± 2 nM) was not. Incubation of the membranes, prior to assay for 30 min at 37°C, followed by centrifugation at 27, 000 g for 20 min in order to pellet the membranes allowed the detection of a factor, present in the high speed supernatant, which caused a dose-dependent inhibition of the binding of [³H]leu-enk to the morphine-sensitive and insensitive binding components. Investigations into the nature of the morphine-insensitive binding component demonstrated that it was an artifact since it was not detectable when bound and free ligand were separated by centrifugation. Furthermore, [³H]leu-enk bound to Whatman glass fiber filters, used to collect bound ligand, in a morphine-insensitive manner, and under conditions where the binding of [³H]leu-enk to the morphine sensitive component diluted proportionally with serial dilutions of the membranes, the binding to the morphine-insensitive component did not. The factor present in the high speed supernatant did not dialyze and its effects were mimicked by either trypsin or soybean trypsin inhibitor, but not by bovine serum albumin. The apparent inhibition of the binding of [³H]leu-enk to these binding components is probably not of biological significance, but the fact that the artifactual morphine insensitive binding component of striatal membranes has been shown to decrease by 20–30% following lesions of the substantia nigra suggests that the influence of this endogenous factor must be controlled for.     

Influencing Factors and Applicability of the Viability EMA-qPCR for a Detection and Quantification of Campylobacter Cells from Water Samples

In recent years, increasing numbers of human campylobacteriosis cases caused by contaminated water have been reported. As the culture-based detection of Campylobacter is time consuming and can yield false-negative results, the suitability of a quantitative real-time PCR method in combination with an ethidium monoazide pretreatment of samples (EMA-qPCR) for the rapid, quantitative detection of viable Campylobacter cells from water samples was investigated. EMA-qPCR has been shown to be a promising rapid method for the detection of viable Campylobacter spp. from food samples. Application of membrane filtration and centrifugation, two methods frequently used for the isolation of bacteria from water, revealed a mean loss of up to 1.08 log₁₀ cells/ml from spiked samples. Both methods used alone lead to a loss of dead bacteria and accumulation of viable bacteria in the sample as shown by fluorescence microscopy. After filtration of samples, no significant differences could be detected in subsequent qPCR experiments with and without EMA pretreatment compared to culture-based enumeration. High correlations (R² = 0.942 without EMA, R² = 0.893 with EMA) were obtained. After centrifugation of samples, qPCR results overestimated Campylobacter counts, whereas results from both EMA-qPCR and the reference method were comparable. As up to 81.59% of nonviable cells were detected in pond water, EMA-qPCR failed to detect correct quantities of viable cells. However, analyses of spiked tap water samples revealed a high correlation (R² = 0.863) between results from EMA-qPCR and the reference method. After membrane filtration, EMA-qPCR was successfully applied to Campylobacter field isolates, and results indicated an advantage over qPCR by analysing defined mixtures of viable and nonviable cells. In conclusion, EMA-qPCR is a suitable method to detect viable Campylobacter from water samples, but the isolation technique and the type/quality of the water sample impact the results.     

Isolation, characterization and protein and polar lipid compositions of Paracoccus denitrificans outer membrane

Sucrose density gradient centrifugation of Paracoccus denitrificans strains ATCC 13543 and ATCC 17741 cell envelopes plus poly-β-hydroxybutyrate, isolated from organisms broken using a French pressure cell, revealed three bands of densities: I, 1.16 g/ml; II, 1.19 g/ml; III, 1.24 g/ml. On the basis of chemical and enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the bands were identified as: I, cytoplasmic membrane; II, poly-β-hydroxybutyrate; III, outer membrane plus poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate was removed by increased low-speed centrifugation before deposition of cell envelopes. Density gradient centrifugation of cell envelopes gave a simple pattern of two bands, cytoplasmic and outer membranes. In both strains outer membranes showed a broad protein band at M_r 70 000–83 000 upon SDS-polyacrylamide gel electrophoresis of samples solubilized at 25°C, which was not present in samples solubilized at 100°C, where a single major band was present of M_r 32 000 in strain ATCC 13543 and 35 000 in strain ATCC 17741. The major outer membrane protein stained positively for lipid in both strains, as did an M_r 70 000 protein, which was the second major protein in strain ATCC 17741. The second major outer membrane protein of stain ATCC 13543 had an M_r of 20 000 in unheated samples but 23 000 in heated samples. This protein was not present in strain ATCC 17741. Quantitative data on the polar lipid compositions of cell envelope fractions are presented.     

5′-Nucleotidases of retinal rod membranes

5′-Nucleotidase (EC 3.1.3.5) was solubilized from rod membranes with Ammonyx LO and purified by chromatographic methods. A highly sensitive radioassay was developed. The purified enzyme behaved as a homogeneous protein of 75,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as a protein of 79,000 in gel filtration. Thus, the enzyme does not contain subunits. The K_m values obtained were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin. Rabbit muscle G-actin formed a complex with the enzyme and inhibited its activity. The catalytic site of the enzyme was localized on the internal surface of the disk which, in terms of membrane sidedness, corresponds to the cell surface. A soluble 5′-nucleotidase was extracted from rod membranes with Tris buffer (pH 8.0) containing EGTA in the dark; less enzyme was extracted if the membranes had been exposed to light or incubated with Ca²⁺. The extracted enzyme was partially purified. The enzyme was unstable and lost 50% of its activity in 3 days at 3 °C. The K_m values were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by G-actin. A role for the soluble enzyme in the regulation of 5′-GMP in the rod outer segment was suggested.     

A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria

Aims: Escherichia coli is the pre‐eminent microbiological indicator used to assess safety of drinking water globally. The cost and equipment requirements for processing samples by standard methods may limit the scale of water quality testing in technologically less developed countries and other resource‐limited settings, however. We evaluate here the use of ambient‐temperature incubation in detection of E. coli in drinking water samples as a potential cost‐saving and convenience measure with applications in regions with high (>25°C) mean ambient temperatures. Methods and Results: This study includes data from three separate water quality assessments: two in Cambodia and one in the Dominican Republic. Field samples of household drinking water were processed in duplicate by membrane filtration (Cambodia), Petrifilm? (Cambodia) or Colilert^® (Dominican Republic) on selective media at both standard incubation temperature (35–37°C) and ambient temperature, using up to three dilutions and three replicates at each dilution. Matched sample sets were well correlated with 80% of samples (n = 1037) within risk‐based microbial count strata (E. coli CFU 100 ml^?1 counts of <1, 1–10, 11–100, 101–1000, >1000), and a pooled coefficient of variation of 17% (95% CI 15–20%) for paired sample sets across all methods. Conclusions: These results suggest that ambient‐temperature incubation of E. coli in at least some settings may yield sufficiently robust data for water safety monitoring where laboratory or incubator access is limited. Significance and Impact of the Study: Ambient‐temperature incubation of E. coli may be a promising option for reducing the complexity and costs associated with water safety monitoring for faecal indicator bacteria such as E. coli in a field context in resource‐limited settings, as are often encountered in developing countries and after disasters.     

Composition of soluble cuticular lipids and water permeability of cuticular membranes from Citrus leaves

Water permeability and composition of soluble cuticular lipids of isolated cuticular membranes from leaves of Citrus aurantium L. were investigated for 3 successive years. The average water permeability coefficient determined using 169 cuticular membranes was 1.09·10^–7 cm s^–1 with a standard deviation of 0.78·10^–7 cm s^–1. There were no significant differences in water permeability between years. Cuticular membranes are characterized by a great variability in water permeability both within and between years. Both water permeability of individual membranes and variability between membranes are shown to be determined by soluble cuticular lipids contained within the cuticular membranes. The soluble cuticular lipids of Citrus leaves are composed of fatty acids, primary alcohols, esters, and hydrocarbons. They occur in amounts of 9.84 g cm^–2, which represents approx. 3% of the total mass of isolated cuticular membranes. The specific weight of cuticular membranes (365.4 g cm^–1) and total amount of soluble cuticular lipids did not vary significantly between years. Significant differences were observed for the amounts and composition of the constituent classes of lipids. Six homologues comprise 86% of the fatty acids (C₁₆; C₁₈; C₁₉; C₂₁; C₂₄; C₂₆), 83% of the primary alcohols (C₂₄; C₂₆; C₂₈; C₃₀; C₃₂; C₃₄) and 88% of the esters (C₃₆; C₃₈; C₄₀; C₄₁; C₄₂; C₄₄). Eleven major homologues amount only to 62% of the total hydrocarbons (C₁₆; C₁₇; C₁₈; C₂₀; C₂₆; C₂₇; C₂₉; C₃₀; C₃₁; C₃₂; C₃₃). Variability in the composition of soluble cuticular lipids between years was much smaller than variability of water permeability and, therefore, no relation between composition of soluble cuticular lipids and water permeability could be found. It is suggested that this may be due to the fact that the lipid composition observed represents the averages of 20 to 30 membranes analyzed so that differences between individual membranes may have been leveled out.Abbreviations CM cuticular membranes - MX polymer matrix - P_d permeability coefficient for diffusion of water - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid     

The action of carboxyl modifying reagents on the ryanodine receptor/Ca2+ release channel of skeletal muscle sarcoplasmic reticulum

Summary

In this work we show that ryanodine binding to junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor (RyR) is inhibited in a time — and concentration-dependent fashion by prior treatment with the carboxyl reagent dicyclohexylcarbodiimide (DCCD). Exposure of the membrane-bound RyR to the water soluble carboxyl reagents 1-ethyl-3 (3-(dimethylamino) propyl carbodiimide (EDC) or N-ethyl-pheny-lisoxazolium-3 -sulfonate (WRK) only slightly affects their ryanodine binding capacity. The amphipathic reagent N-ethoxy cabonyl-2-ethoxy-1, 2-dihydroquinaline (EEDQ) inhibited ryanodine binding at relatively high concentrations. DCCD-modifica-tion of the SR decreased the binding affinities of the RyR for ryanodine and Ca²⁺ by about 3- and 18-fold, respectively.

The single channel activity of SR membranes modified with DCCD and then incorporated into planar lipid bilayers is very low (5–8%) in comparison to control membranes. Application of DCCD to either the myoplasmic (c/s) or luminal (trans) side of the reconstituted unmodified channels resulted in complete inhibition of their single channel activities. Similar results were obtained with the water soluble reagent WRK applied to the myoplasmic, but not to the luminal side. The DCCD-modified non-active channel is re-activated by addition of ryanodine in the presence of 250_üM Ca²⁺ and is stabilized in a sub-conductance state. With caffeine, ryanodine re-activated the channel in the presence of 100_üM of Ca^2+. The results suggest that a carboxyl residue(s) in the RyR is involved either in the binding of Ca²⁺, or in conformational changes that are produced by Ca²⁺ binding, and are required for the binding of ryanodine and the opening of the Ca²⁺ release channel.     

Guinea Pig Brain Histamine N-Methyltransferase: Purification and Partial Characterization

Abstract: Histamine N-methyltransferase (EC 2.1.1.8) was purified 4400–fold in 12% yield from guinea pig brain. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE-cel-lulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous by gel electrophoresis and was stable for at least 3 months at 80°C. It had an apparent molecular weight of 29 ,000 ± 1000 as determined by both gel filtration through Sephadex G-100 and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the protein was found to be 5.3. The pH optima for methylation of histamine were determined to be 7.5 and 9.0; the K_ms for histamine and S-adenosyl-l-methionine were 13.57 ± 0.74 μM and 6.1 ± 0.12 μM, respectively; the K_i for S-adenosyl-l-homocysteine was 24.5 ± 1.45 μM.     

Solubilization of active (H+ + K+)-ATPase from gastric membrane

(H⁺ + K⁺)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg²⁺-ATPase and p-nitrophenylphosphatase activities (68 ± 9 μmol P_i and 2.9 ± 0.6 μmol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K⁺ (up to 0.69 ± 0.09 nmol P_i incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1–2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 × g × 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K⁺-stimulated ATPase and p-nitrophenylphosphatase activities and K⁺-sensitive phosphorylation: Mg²⁺-ATPase (μmol P_i/mg protein per h) 32 ± 9 (basal) and 86 ± 20 (K⁺-stimulated); Mg²⁺-p-nitrophenylphosphatase (μmol p-nitrophenol/mg protein per h) 2.6 ± 0.5 (basal) and 22.2 ± 3.2 (K⁺-stimulated); Mg²⁺-phosphorylation (nmol P_i/mg protein) 0.214 ± 0.041 (basal) and 0.057 ± 0.004 (in the presence of K⁺). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H⁺ + K⁺)-ATPase in a still active structure.     

Usefulness of the hydrogen sulfide test for assessment of water quality in Bangladesh

Aim: To evaluate the usefulness of the hydrogen sulfide (H₂S) test for assessing water quality in Bangladesh. Methods and Results: We tested 382 water samples from a variety of sources using locally produced H₂S test kits and laboratory‐based membrane filtration for the detection of Escherichia coli. Compared with membrane filtration, H₂S tests, when incubated for 24 h, had both a sensitivity and positive predictive value (PPV) of <40% when analysis was restricted to water samples with E. coli levels below 100 colony forming units (CFU) per 100 ml. In contrast, for E. coli levels from 1000 to 9999 CFU per 100 ml, sensitivity was 94% and PPV 88%; specificity was 97% and negative predictive value was 99%. Conclusions: The hydrogen sulfide test, when incubated at 24 h, is a promising alternative for assessing water quality where E. coli levels may be high. An improved understanding of the incremental impact of contamination level on health is needed to better determine its usefulness. Significance and Impact of the Study: The hydrogen sulfide test is inexpensive, easy to use and portable. Its use may allow rapid assessment of water quality in situations where cost or logistics prevent use of other testing methods, such as in remote areas or during floods and other natural disasters.     

The influence of alkali and heat treatment on yeast protein

The soluble components in disintegrated cells of Saccharomyces cereivisiae have been characterized by means of extraction, centrifugation, dialysis, and gel filtration. The influence of alkali and heat treatment on the protein and RNA in the soluble fraction from disintegrated yeast cells and on functional properties of protein concentrates have been studied. After water extraction and centrifugation at 100000 g 42% of the nitrogen containing components of the disintegrated cells were recovered in the supernatant. By extraction at pH 11.5 an additional 31% of the nitrogen was solubilized. Half of the water-soluble nitrogen-containing components has a molecular weight lower than 5000. In the water- and alkali-soluble fractions about 80% of each amino acid was recovered The water-soluble protein was separated into 3 fractions by gel filtration on Sephadex G 200. The major portion of the protein had a molecular weight about 100,000. The amount of protein in this fraction was decreased after treatment at increasing pH and temperature. No degradation of protein to low molecular peptides occurred. The amount of RNA in the soluble fraction was only slightly influenced by alkali treatment and by heat treatment at pH 7.5 in the presence of 5% NaCl. RNA was not degraded to low molecular components of the treatments. The solubility of protein concentrates decreased after treatment at alkaline pH and after heat precipitation.     

Development of an improved procedure for isolation and purification of exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483

A method was developed for the isolation and purification of exopolysaccharide (EPS) produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483 that can be adapted for industrial-scale operation. Hydrolyzed milk medium, which was ultrafiltered to remove molecular species larger than 2.5×10⁵ Da, was found to be a suitable growth medium for the bacteria, which produced approximately 400 mg EPS/l . Optimal isolation of EPS was achieved using centrifugation, filtration and ethanol precipitation methods. Insoluble and soluble EPS fractions were obtained. The soluble fraction was purified using a series of ethanol precipitations to achieve approximately 98% (w/w) purity. This fraction consisted of galactose, glucose, rhamnose and mannose in the ratio of approximately 5:1:0.6:0.5, with traces of glucosamine.     

Preparation and polypeptide composition of plasma membrane and other subcellular fractions from wild oat (Avena fatua) aleurone

Plasma membranes can be isolated from a variety of plant tissues by first preparing a post-mitochondrial membrane fraction enriched in plasma membranes, by differential centrifugation, and partitioning this on a dextran-polyethylene glycol two-phase system. With wild oat aleurone, however, we observed that differential centrifugation could not be used to produce a microsomal fraction enriched in plasma membrane. Approximately 70% of the plasma membrane in aleurone homogenates was pelleted by sequential centrifugation at 100 g× 10 min and 1000 g× 10 min. The remainder sedimented at 112 000 g× 1 h. All the material that was pelletable by centrifugation was, therefore, subjected to dextran-polyethylene glycol two-phase partitioning. The plasma membrane marker enzymes glucan synthase II (GSII, EC 2. 4. 1. 34) and UDP-glucose:sterol glucosyltransferase (SGT, EC 2. 4. 1.) were enriched in the upper phase, whereas cytochrome c oxidase activity (EC 1. 9. 3. 1), a mitochondrial marker enzyme, was depleted. The presence of endoplasmic reticulum (ER) and protein body membranes in the phase system was assessed by probing western blots, of SDS-PAGE separated proteins, with polyclonal antiserum either to binding protein (BiP, an ER marker) or to tonoplast intrinsic protein (TIP, a protein body membrane marker). BiP and TIP were present in the lower phase, but were not detected in the upper phase. In addition, the polypeptide patterns of material in the upper and lower phases were very different. These observations suggested that high purity aleurone plasma membrane had been isolated. Although the procedure for isolating plasma membranes was applicable to both aleurone protoplasts and layers, the polypeptide patterns of plasma membranes prepared from these sources were very different. The major protein components of wild oat aleurone were 7 S and 12 S storage globulins. These proteins were present in the lower phase, but not in the plasma membrane enriched upper phase, after aqueous two-phase partitioning. Differential centrifugation studies showed that it was necessary to homogenise aleurone in a buffer of pH 6. 0 or less if a soluble protein fraction, essentially devoid of storage globulins, was to be obtained. The use of these fractionation techniques is discussed in relation to photoaffinity labelling of gibberellin (GA)-binding proteins in aleurone.