Sucrose Concentration Gradient Research Articles

Effect of milk variant, sugar and voltage gradient on physico‐chemical, rheology and sensory properties of basundi prepared by ohmic heating

The study aimed to optimise the process of basundi manufacture by ohmic heating (OH) instead of thermal processing. The factors considered were the type of milk (cow milk, buffalo milk and mixed milk), sucrose concentration (5, 7.5, and 10% [w/w] of milk) and voltage gradient (12.5, 13.33, 14.29, 15.38 and 16.66 V/cm). The parameters considered were physico‐chemical, rheological and sensory quality. The optimised product was prepared with standardised buffalo milk (BM), 5% (w/w of milk) added sucrose and processed at 13.33 V/cm. It significantly (P < 0.05) affected the physico‐chemical properties of the optimised product and basundi showed pseudoplastic behaviour.

The influence of sucrose on soil nitrogen availability – A root exudate simulation using microdialysis

Root exudates are thought to promote nitrogen (N) availability via rhizosphere interactions, but empirical evidence is difficult to obtain given the scale and temporary nature of these processes. Microdialysis has potential to simulate root exudation patterns and quantify the effects on N availability simultaneously, but this has so far not been attempted. In a conceptual root exudation study, we used sucrose as a simple C source to investigate if microdialysis could detect the effects of continuous localised C supply on soil inorganic N fluxes. Through retrodialysis we released sucrose and simultaneously monitored diffusive soil N fluxes over one week, followed by a further seven days without sucrose. Based on current understanding of rhizosphere N dynamics, we hypothesised that N fluxes are inversely related to sucrose release, and upon ceasing release, N fluxes would increase. Using a 5 mM sucrose perfusate, C releases resulted in decreased N fluxes, but contrary to our hypothesis, N fluxes did not increase after ceasing sucrose release (c.f. control soil). Diffusive sucrose efflux from microdialysis probes increased in soils amended with N-rich litter suggesting that microbial activity and associated sucrose consumption altered sucrose concentration gradients. The fluxes of sucrose breakdown products glucose and fructose were greatest in litter treatments receiving sucrose, indicative of increased invertase activity in the presence of fresh organic matter. In the short term (days), sucrose release did not prompt an increase in inorganic N availability, possibly because of stimulated microbial growth and increased N demand under C-rich conditions. Our study confirms that microdialysis allows time-sensitive insight into the dynamic interactions of carbon and N in the rhizosphere.

Impact of fat crystallization on the resistance of W/O/W emulsions to osmotic stress: Potential for temperature-triggered release

Water-in-oil-in-water (W/O/W) emulsions can be designed to encapsulate, protect, and release both hydrophilic and hydrophobic functional compounds. In this study, we examined the impact of crystallizing the fat phase on the resistance of W/O/W emulsions to osmotic stress, with the aim of developing osmotic-responsive systems. Polyglycerol polyricinoleate (PGPR) was used as a hydrophobic surfactant to stabilize the inner water droplets, while Quillaja saponin and whey protein isolate (WPI) were used as hydrophilic surfactants to coat the oil droplets. The impact of fat crystallization was examined by using either a liquid (soybean oil, SO) or semi-solid (hydrogenated soybean oil, HSO) fat as the oil phase. An osmotic stress was generated by establishing a sucrose concentration gradient between the internal and external water phases. Alterations in the droplet size, morphology, and stability of the W/O/W emulsions was measured when the sucrose concentration gradient was changed. The W/O droplets in the SO-emulsions swelled/shrank when the external sucrose concentration was below/above the internal sucrose concentration, which is indicative of water diffusing into/out of the droplets. Conversely, there was no change in the size of the W/O droplets in the HSO-emulsions under the same conditions, which was attributed to the mechanical strength of the fat crystal network resisting swelling or shrinking. HSO-emulsions did exhibit swelling when they were heated above a critical temperature, due to melting of the fat crystals and disruption of the crystal network. Our results demonstrate that crystallization of the oil phase of W/O/W emulsions can prevent water transport due to osmotic stress, which may be useful for developing temperature-triggered delivery systems for application in foods, cosmetics, pharmaceuticals, or personal care products.

DEVELOPMENT OF A TEST-SYSTEMS FOR THE IDENTIFICATION OF THE SOIL BORNED TOBACCO RATTLE VIRUS BASED ON THE USE OF ELISA

On the base of the isolate TRV PV-0361 of the tobacco rattle virus from the commercial collection of the DSMZ company (Germany) have been developed methods for maintaining the virus in vitro culture on N. clevelandii plants, propagation the virus in the spring-summer period in greenhouse conditions, “soft” isolation of a purified virus preparation by clarifying leaf juice with low-speed centrifugation and treatment with non-ionic detergent Triton-X-100 followed by precipitation of the virus with polyethylene glycol-6000 and three-fold ultracentrifugation using sucrose cushion, sucrose concentration gradient and differential centrifugation. The purified virus preparation was used for producing rabbit antiserums according to the scheme we worked out. The obtained antiserum had the following titers - specific 1: 5 · 105, non-specific 1: 8 · 103. Based on antibodies isolated from this antiserum, coating antibodies and peroxidase conjugates were obtained, which made it possible to create ELISA test systems for determining TRV with sensitivity of about 12-16 ng/ml. The resulting test systems can be used in practical work on quality control and certification of seed potatoes.

Exploratory Analysis Applied for the Evaluation of Yerba Mate Adulteration (Ilex paraguariensis)

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to evaluate the potential to detect different sucrose concentrations added as adulterants in yerba mate (Ilex paraguariensis). For this purpose, the data acquired from 15 yerba mate samples were analyzed by hierarchical cluster analysis (HCA) and principal component analysis (PCA). Sucrose solutions from 5 to 30% of the total yerba quantity were added to each sample and analyzed in conjunction with the raw yerba mate sample. The fingerprint region (1300–800 cm−1) was extracted from the resulted spectra, smoothed by the Savitzky Golay method, normalized, average spectra of the triplicates and first derivative. Discrimination between the different sucrose concentrations in the samples was achieved by the exploratory analysis, demonstrating a sucrose concentration gradient in the PC1 × PC2 score plot. In this way, the results suggest a potential industrial use of ATR-FTIR alongside with chemometrics to avoid a frequent sugar adulteration in a simple, fast, and reliable methodology.

Comparison of Root Characteristics and Sugar Components in Root and Leaf at Early Growth Phase of Sweet Potato Varieties with Significant Difference in Valid Storage Root Number

Starch sweet potato varieties Shangshu 19 and Jixu 23 differing in valid storage root number per plant significantly were used to investigate root characteristics, sugar components in root and leaf at early growth phase and their relationship with the formation of storage root per plant at top cover stage. The results showed that, Shangshu 19 with higher valid storage root number regrew quickly with more new roots after seedling transplanting, developed fiberous roots mainly in the whole early growth phase, and formed a stable rate of top biomass/total root system biomass (T/TR) at 15 and 30 days after planting. Jixu 23 achieved the steady number of adventitious root and valid storage root at 15 and 30 days after planting, respectively. Meanwhile, Jixu 23 showed the lower rate of top biomass/storage root biomass (T/SR) at top cover stage (45 days after planting). On the other hand, in point of metabolism of sucrose and hexoses, and the formation of stored polysaccharide polymers, Shangshu 19 had sig- nificantly lower rate of sucrose/hexoses in the whole early growth phase, formed the greater sucrose concentration gradient be- tween leaves and roots, and had 1-kestose and nystose in roots at 15 and 30 days after planting. Its starch content in leaf was sig-

Effect of exogenous sucrose on growth and active ingredient content of licorice seedlings under salt stress conditions

Licorice seedlings were taken as experimental materials, an experiment was conducted to study the effects of exogenous sucrose on growth and active ingredient content of licorice seedlings under NaCl stress conditions. The results of this study showed that under salt stress conditions, after adding a certain concentration of exogenous sucrose, the licorice seedlings day of relative growth rate was increasing, and this stress can be a significant weakening effect, indicating that exogenous sucrose salt stress-relieving effect. The total flavonoids and phenylalanine ammonia lyase (PAL) activity were significantly increased, the exogenous sucrose can mitigated the seedling roots under salt stress, the licorice flavonoid content in the enhanced growth was largely due to the activity of PAL an increased, when the concentration of exogenous sucrose wae 10 mmol x L(-1), PAL activity reaching a maximum, when the concentration of exogenous sucrose was 15 mmol x L(-1), PAL activity turned into a downward trend, the results indicating that this mitigation has concentration effect. After applying different concentrations of exogenous sugar, the contents of liquiritin changes with the change of flavonoids content was similar. After applying different concentrations of exogenous sucrose, the content of licorice acid under salt stress was higher than the levels were not reached during salt stress, the impact of exogenous sucrose concentration gradient of licorice acid accumulation was not obvious.

Difference and Related Reason for Assimilate Distribution of Sweetpotato Varieties with Different Root Tuber Yields

A field experiment was conducted at agricultural experiment station of Shandong Agricultural University, Tai'an, Shandong Province in two growth seasons of 2011 and 2012. Six typical varieties of edible sweetpotato in North China were used, including Longshu 9, Hongxiangjiao, Taizhong 6, Sushu 8, Yizi 138, and Beijing 553, to study the difference and reason for assimilate distribution among varieties with different yield levels. The distribution characteristics of assimilate was studied by 13 C labeling during storage roots development; sucrose content of different stem parts and stem base diameter were measured as well. And the relationship between the above traits and harvest index, root tuber yield was also analyzed. The results showed that the root tuber yield of high-yielding varieties(Longshu 9, Hongxiangjiao, Taizhong 6, and Sushu 8) was about 60 t hm–2, whereas that of intermediate and low-yielding varieties(Yizi 138 and Beijing 553) was less than 50 t hm–2. And the harvest index of high-yielding varieties was significantly higher than that of intermediate and low-yielding varieties. The root tuber of high-yielding varieties became assimilate distribution center at early bulking stage, while for intermediate and low-yielding varieties that was at late bulking stage; and high-yielding varieties also had significantly higher 13 C distribution rate than that of intermediate and low-yielding varieties, indicating that high-yielding varieties had relatively higher ability to transport assimilate from functional leaves to root tubers. Sucrose content of stem base was higher than that of stem top, and the sucrose concentration gradient of intermediate and low-yielding varieties was higher than that of high-yielding varieties. Low-yielding varieties' stem base thickened faster than high-yielding varieties in the development of sweetpotato. The correlation analysis showed that root tuber yield had significantly positive correlation with harvest index(r = 0.98**, 2011; r = 0.93**, 2012). Root tuber yield and harvest index had significantly positive correlation with 13 C distribution rate of root tuber(0.87* and 0.91**, 2011; 0.92** and 0.99**, 2012); they also had significantly negative correlation with stem base diameter during middle and late growth stages(–0.78* and –0.83*; –0.88** and –0.95**). Therefore, high-yielding varieties got higher harvest index mainly due to less time taken by their root tuber to become assimilate distribution center and stronger ability of assimilate transportation from functional leaves to root tubers; and low-yielding varieties got lower abilities of assimilate transportation mainly because assimilate unloading in root tubers was not smoothly.

Carbon unloading in roots in relation to root senescence in Cercis chinensis seedlings under drought stress

When Cercis chinensis seedlings suffered from drought treatment, net photosynthetic rates had been significantly reduced at the end of the drought treatment. Compared with the control, the activities of acid invertases in roots had increased 5 and 11 days after drought treatment. Seventeen days after drought treatment, the activities of acid invertases in roots were significantly decreased, while activities of alkaline invertases in roots had also been significantly reduced. As the moisture in culture media decreased, so the activities of sucrose synthases in leaves decreased slightly. In roots, their activities had significantly increased 5 and 11 days after drought treatment. The contents of fructose in roots reduced as the moisture in culture media decreased and 11 and 17 days after drought treatment the reduction was significant. The content of glucose in roots clearly did not change as drought stress occurred further, but was still less than that in the control seedlings. Similarly, the content of sucrose reduced as the moisture in culture media decreased. At the beginning of the drought stress, the content of sucrose was significantly higher than that in the control and afterwards there were no differences between drought-treated seedlings and the control. The gradient of the sucrose content between leaves and roots was 0.0982 mg·g−1 FW 17 days after drought treatment, while the gradient of the seedlings under normal condition was 1.3832 mg·g−1 FW. The sucrose concentration gradient reduced by 92.9%. The reduction in the sucrose content gradient under drought stress decreased the sucrose partitioning in roots. Therefore, our results support the hypothesis of ‘shared control’.

Mass transfer in osmotically dehydrated apple stored at temperatures above zero

Material dehydrated in sucrose solution by osmosis for 3 h at 30 °C and for 1 h at 70 °C was stored for 24, 72 and 144 h at 5, 9, 15 and 20 °C. Internal gradients of dry matter and sucrose concentration caused mass transfer in the material. Dry matter content in the surface layers was close to 40%, while sucrose concentration was between 20% and 23%. At the distance of 10 mm from the mass transfer surface dry matter content was still higher than that in raw apple, but sucrose concentration was that of raw apple. Concentration profiles of sucrose changed with time and storage temperature. The higher was the temperature the faster was the diffusion of sucrose. In material osmosed at 30 °C for 3 h and stored for 72 h at 5, 9 and 15 °C sucrose concentration gradients were still present. However, after 144 h at 9 °C the concentration of sucrose became independent on the distance from the mass exchange surface. Diffusion of sucrose in material osmosed at 70 °C for 1 h was much faster than that observed in samples osmosed at lower temperature. The effective diffusion coefficient of sucrose was dependent on temperature and time of storage, and distance from the mass transfer surface as well. Since distance was related to concentration a relationship between sucrose concentration and effective diffusion coefficient was found. The effective diffusion coefficient was of the order of 10 −9–10 −12 m 2/s.

Microhydraulic Actuation Using Biological Ion Transporters Reconstituted on Artificial BLM

ABSTRACTPlants and animals have the natural ability to exhibit force through controlled pressurization of cellular compartments. The mechanism through which force is generated is powered by biological fuels. The process involves moving ions against an established concentration gradient expending free energy from bio-fuels like Adenosine-tri-phosphate (ATP), kinesin etc., Materials exhibiting deformation using biological processes are called Nastic materials. The functional component in mass transfer across the cell boundary is the ion transporter embedded in cell membranes. The ion transporters which are complex protein molecules, move ions and water molecules for an applied chemical or electrical stimulus. The bio-inspired microhydraulic actuator uses the same functional component in plant cells reconstituted on a planar bilayer lipid membrane (BLM) formed from purified lipids. The protein transporters pump ions and fluid into an enclosed cavity to cause deformation. The controlled fluid transport through AtSUT4(proton-sucrose co-transporter extracted from Arabidopsis thaliana) reconstituted on a 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt) (POPS), 1-Palmitoyl-2-Oleoyl-sn-Glycero- 3-Phosphoethanolamine (POPE) BLM on porous lead silicate glass plate driven by a proton gradient demonstrated the ability to move fluid across the membrane. This article discusses a prototype microhydraulic actuator that increases in thickness for an applied pH and sucrose concentration gradient.

Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.

Ultrastructure of Chloroplasts in Phloem Companion Cells and Mesophyll Cells as Related to the Stimulation of Sink Activity by Cytokinins

Changes in the chloroplast ultrastructure and starch and lipid content in the mesophyll and phloem companion cells of the phloem were studied after induction of source and sink functions in leaf tissues. A detached sugar-beet leaf, one half of which was treated with water (source part) and the other half of which was treated with 10–4 M benzyladenine (BA) (acceptor part), was used as a model. After 65-h exposure to diffuse light, starch disappeared and lipid content increased in the source part of the leaf, with simultaneous disorganization of the chloroplast structure, which was most pronounced in the companion cells. Changeover from the source to sink function, induced by BA treatment, did not lead to marked destructive changes in the chloroplast structure of companion cells and resulted in the appearance of starch and in further increase in the level of lipids. Smaller amounts of starch also appeared in the mesophyll chloroplasts in the sink part of the leaf. We suppose that: (1) BA promotes the storage of assimilates, which are imported from the source part of the leaf to the companion cells, in the form of starch and lipids within chloroplasts; and this storage contributes to the maintenance of the sucrose concentration gradient in the conducting system between donor and sink parts of the leaf and, thus, activates metabolite inflow and (2) a barrier exists in the sink part of the leaf for assimilates destined to mesophyll cells, which restricts their export from the phloem.

Plasmodesmata and solute exchange in the phloem

Photoassimilates and other substances accumulate against concentration gradients in the phloem, a process known as loading. In mature leaves, the sieve element–companion cell complexes (SE–CCCs) of minor veins, where loading occurs, are connected to surrounding cells by plasmodesmata. These pores appear to participate in loading in plants that translocate raffinose and stachyose, but in sucrose- and polyol-translocating species their function is less certain. Indeed, large numbers of plasmodesmata between the SE–CCCs and surrounding cells should cause a dissolution of the concentration gradient unless the size exclusion limit of the pores is small enough to retain accumulated solute species. In leaves of willow, Salix babylonica L., a sucrose-translocating plant with a high degree of symplastic connectivity into the minor vein phloem, the sucrose concentration gradient is absent between mesophyll and phloem, leading to the conclusion that phloem loading does not occur. Once inside the SE–CCC, solute may be able to pass freely between sieve elements and companion cells since they are also symplastically connected. However, due to net flux into the sieve tubes in source leaves, this should cause a continual drain of metabolic intermediates out of companion cells. It is proposed that this transport step is regulated in minor veins to prevent continual loss of needed solute molecules to the translocation stream.

Naloxone administration following operant training of sucrose/water discrimination in the rat.

The suppression of food intake observed following naloxone administration has often been ascribed to palatability or taste. Unfortunately, many confounds become apparent when attempts are made to isolate such factors in the investigation of ingestive behaviors. In the present study, rats (two groups) were trained to discriminate either a 10% or 5% sucrose solution from water (0.1 ml). These mildly food deprived subjects (95% of free-feeding weight) were trained to press the appropriate lever in a two-lever operant chamber following sampling of sucrose or water; successful responding was reinforced by delivery of a 45 mg grain food pellet. Following random exposure to reduced sucrose concentrations tested under extinction, a sucrose concentration gradient (1.0, 0.5, 0.1, 0.05, 0.01 and 0.005% sucrose solution) was established for both training groups under i.p. saline administration. Data collected under i.p. saline were then compared to those collected following random i.pf1p4loxone administration (3.0, 1.0, 0.3 and 0.1 mg/kg). No significant differences were observed between the sucrose concentration gradients obtained under saline and those obtained under naloxone, suggesting that the anorectic effect of naloxone is not primarily determined by discrimination of sweet taste.

Sucrose Concentration Gradients along the Post-Phloem Transport Pathway in the Maternal Tissues of Developing Wheat Grains.

Sucrose concentrations were measured in serial frozen sections of the post-phloem transport pathway in developing wheat (Triticum aestivum L.) grains. In normally importing grains, there was an approximately linear concentration gradient along the pathway, with a difference between the ends of the pathway of about 180 mM. This indicates an unusually low resistance for cell-to-cell transport, due perhaps to the large size-exclusion limit for the pathway. However, the existence of concentration gradients raises presently unresolvable questions about the relative contributions of diffusion versus bulk flow to transport within the symplast. The concentration gradient disappeared when sucrose movement ceased (i.e. in excised grains or when endosperm cavities of attached grains were perfused with p-chloromercuribenzene sulfonate [PCMBS] or with 1660 mOsm sorbitol). PCMBS appeared to block solute release into the endosperm cavity, whereas the sorbitol treatment, previously shown to cause localized plasmolysis in the chalaza, appeared to block movement across the chalaza. Sieve element/companion cell unloading appears to be an important control point for assimilate import. The sucrose concentration gradient and, probably, turgor and osmotic gradients are extremely steep there. PCMBS blocked import without affecting the sucrose concentration in the vascular parenchyma around the phloem. Thus, blockage of unloading was more complex than a simple "backing up" of solutes in the vascular parenchyma.

Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A1 but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A1 vanadate and azide. Kimetic studies of PPase activity gave an apparent Km, for PP3 of 18 μM. Identical distributions of bafilomycin‐ and NO3‐sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin‐sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin‐sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35‐day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP‐ and PP1‐dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H+ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H+/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP1) stimulation of [14C]‐sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p‐chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy‐requiring uptake mechanisms at the tonoplast.

Internodal Compartmentation of Stem‐Infused [14C]Sucrose in Sweet and Grain Sorghum

Stem infusion was evaluated for quantifying downward movement, intracellular allocation, and interorgan partitioning of assimilate in sweet and grain sorghum [Sorghum bicolor (L.) Moench] stems during mid‐grain fill and post‐black layer. Sucrose concentration did not vary below the site of infusion with [14C]sucrose, although gradients of intracellular 80%‐ethanol soluble and insoluble 14C‐components were established in the stem. Variation in gradient steepness between cultivars resulted from aspects of parenchymal uptake or phloem uptake of sucrose and appears related to sucrose‐concentrating ability. The introduced [14C]sucrose concentration gradient in stem tissue is useful for studying stem mechanisms that controls ink strength.

Cloning and bacterial expression of the CYS3 gene encoding cystathionine gamma-lyase of Saccharomyces cerevisiae and the physicochemical and enzymatic properties of the protein

By screening a yeast genomic library, we isolated and characterized a gene rescuing the cysteine requirement in a "cys1" strain of Saccharomyces cerevisiae. Except for four residues in the open reading frame composed of 1,182 nucleotides, the DNA sequence was the same as that for the CYS3 (CYI1) gene, encoding cystathionine gamma-lyase (EC 4.4.1.1), and isolated previously as a cycloheximide-induced gene (B. Ono, K. Tanaka, K. Naito, C. Heike, S. Shinoda, S. Yamamoto, S. Ohmori, T. Oshima, and A. Toh-e, J. Bacteriol. 174:pp.3339-3347, 1992). S. cerevisiae "cys1" strains carry two closely linked mutations; one (cys1) causes a defect in serine O-acetyltransferase (EC 2.3.1.30), and another, designated cys3, impairs cystathionine gamma-lyase activity. Rescue of the cysteine requirement by the gene encoding cystathionine gamma-lyase is consistent with both defects being responsible for the cysteine auxotrophy. In an effort to further determine the physicochemical and enzymatic properties of this enzyme, a coding fragment was cloned into an Escherichia coli expression plasmid, and the protein was produced in the bacteria. The induced protein was extracted by sonication and purified to homogeneity through one course of DEAE-cellulose column chromatography. The yield of the protein was approximately 150 mg from cells cultured in 1 liter of L broth. The protein showed molecular weights of approximately 194,000 and 48,000 (for the subunit), suggesting a tetrameric structure. An s20,w value of 8.8 was estimated by centrifugation in a sucrose concentration gradient. No sulfhydryl groups were detected, which is consistent with the absence of cysteine residues in the coding sequence. The isoelectric point was at pH 5.2. The protein showed a number of cystathionine-related activities, i.e., cystathionine beta-lyase (EC 4.4.1.8), cystathionine gamma-lyase, and cystathionine gamma-synthase (EC 4.2.99.9) with L-homoserine as substrate. In addition, we demonstrated L-homoserine sulfhydrylase (adding H2S) activity but could find no detectable serine O-acteyltransferease activity. In this paper, we compare the enzymatic properties of the protein with those of homologous enzymes previously reported and discuss the possibility that this enzyme has a physiological role as cystathionine Beta-lyase and cystathionine gamma-synthase in addition to its previously described role as cystathionine gamma-lyase.

Purification of viruses by electrophoresis in sucrose concentration gradients. 1972.

Zone electrophoresis (ZE) in concentration gradients of sucrose proved to be a powerful technique in the purification of viruses although it is seldomly used as a single step for the separation of the infective agent from extraneous matter but usually as a final procedure. No difficulties were experienced in the purification of plant viruses with ZE possibly because of the resistance of the agents to the chloroform butanol treatment which the impure plant viruses received prior to the ZE. Likewise no difficulties were encountered during purification of human and animal picorna viruses because they also are resistant to chloroform or chloroform-butanol treatment prior to ZE. Purification of animal and possibly human reoviridae ZE as a final step is involved as they are associated with or even attached to the host cell membranes which are lipoidal in nature. Because the lipoidal material on the virus has an affinity for organic solvents the virus particles are caught in the emulsion when an extract of the crude virus material is shaken with the organic solvent. With these viruses polyethylene glycol M(r) 6000 (PEG) is often helpful as a means of displacing most of the host proteins as a step in the partial purification prior to ZE. Neurotropic African horse sickness virus which presented many problems to overcome was rendered free of extraneous proteins to the extent that electron micrographs could be made of it. Neurotropic Rift Valley fever virus was similarly difficult to purify as it was always associated with mouse brain components. This virus was finally purified from infected mouse blood in which the virus particles were not associated with host cell debris and other extraneous proteins. Zone electrophoresis experiments were not conducted on viruses with large particle sizes e.g. myxoviruses because they could be purified without a great deal of effort using polymer displacement, and thin layer centrifugation. Zone electrophoresis as a means of final purification was vividly displayed with the purification of five viruses of the larvae of the pine emperor moth, Nudaurelia cytheria cytheria.