Characterisation of the organic matter pool in manures

In this research, different types of animal manure were evaluated with respect to organic matter (OM), total organic carbon (C(ot)), total N (N(t)), C(ot)/N(t) ratio, water-soluble organic carbon (C(w)), organic N (N(org)), carbohydrates, C(w)/N(org) ratio, humic acid-like carbon (C(ha)), fulvic acid-like carbon (C(fa)), humification index ((C(ha)/C(ot))x100) (HI) and the C(ha)/C(fa) and NH(4)(+)-N/NO(3)(-)-N ratios. In comparison with the limits set by the Spanish legislation for organic fertilisers, most of the manures had high OM contents, moderate N(org) concentrations (except in the case of the chicken and pig manures where this parameter was high) and C(ot)/N(t) ratios above the value stated in the legislation. The study of the different fractions of organic matter showed that the horse, pig and rabbit manures had the greatest content of C(ot). However, the fraction of easily-biodegradable organic compounds (C(w)) was significantly higher in the horse, goat and chicken manures. The study also showed that, in most cases, the percentage of fulvic acid-like C was greater than that of the humic acid-like C, indicating that the organic matter of these wastes is not completely humified. Values of HI ((C(ha)/C(ot))x100) and C(ha)/C(fa) ratio in the studied manures were not significantly different. Regarding the parameters related to the organic matter stability such as C(w), carbohydrates and the C(ot)/N(t), C(w)/N(org) and NH(4)(+)-N/NO(3)(-)-N ratios, it has been determined that the organic matter of these materials was not completely stabilised. The heterogeneity in OM composition of the studied manures did not allow the formulation of simple equations for evaluation of the composition of these wastes from easily-determined parameters.     

Enzyme electrodes based on organic metals

The parameters of enzyme electrodes based on organic metals are presented. Cytochrome b₂ (E.C. 1.1.2.3), glucose oxidase (E.C. 1.1.3.4), xanthine oxidase (E.C. 1.2.3.2) and peroxidase (E.C. 1.11.1.7) were used in electrodes sensitive to l-lactate, glucose, hypoxanthine and hydrogen peroxide. Electrocatalytic oxidation of NADH on organic metals and ethanol and acetaldehyde sensitive electrodes containing alcohol dehydrogenase (E.C. 1.1.1.1) were studied. Biocatalytic charge accumulation, the mechanism of electron exchange between the enzyme active centres and organic metals, and the future application of organic metals are discussed.     

An organic solvent-, detergent-, and thermo-stable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5

Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45 degrees C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over abroad range of temperatures (45-70 degrees C) and pH (8-10) range with an optimum activity at pH 10 and 65 degrees C. It was comparatively stable in the presence ofa relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45 degrees C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.     

An organic solvent-, detergent-, and thermostable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5

Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45°C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over a broad range of temperatures (45–70°C) and pH (8–10) range with an optimum activity at pH 10 and 65°C. It was comparatively stable in the presence of a relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45°C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.     

土壤有机碳动态:风蚀效应

土壤风蚀是引起土壤退化最广泛的形式和原因之一。土壤风蚀对土壤碳动态的影响机制一方面是土壤风蚀引起土壤退化使土壤生产力下降,输入土壤的碳数量减少;另一方面是富含有机碳的细粒物质直接移出系统。风蚀土壤碳的去向包括:(1)就近沉积,(2)沉积于水渠和河流,输入水体;(3)以粉尘形式运移,在远离风蚀区的地域沉积;(4)氧化释放至大气。风蚀引起土壤碳的迁移和沉积不仅导致土壤有机碳在地域间的再分布,使土壤性状的空间异质性增加,也显著改变了土壤系统中碳矿化的生物学过程。土壤有机碳的保持可以促进团聚体的形成,使土壤物理稳定性增加,减缓风蚀。对易风蚀土地进行退耕还林还草、实行保护性耕作等措施可以有效增加土壤碳的固存。     

Terrestrial organic carbon storage in a British moorland

Accurate estimates for the size of terrestrial organic carbon (C) stores are needed to determine their importance in regulating atmospheric CO₂ concentrations. The C stored in vegetation and soil components of a British moorland was evaluated in order to: (i) investigate the importance of these ecosystems for C storage and (ii) test the accuracy of the United Kingdom's terrestrial C inventory. The area of vegetation and soil types was determined using existing digitized maps and a Geographical Information System (GIS). The importance of evaluating C storage using 2D area projections, as opposed to true surface areas, was investigated and found to be largely insignificant. Vegetation C storage was estimated from published results of productivity studies at the site supplemented by field sampling to evaluate soil C storage. Vegetation was found to be much less important for C storage than soil, with peat soils, particularly Blanket bog, containing the greatest amounts of C. Whilst the total amount of C in vegetation was similar to the UK national C inventory's estimate for the same area, the national inventory estimate for soil C was over three times higher than the value derived in the current study. Because the UK's C inventory can be considered relatively accurate compared to many others, the results imply that current estimates for soil C storage, at national and global scales, should be treated with caution.     

Insect herbivory, organic matter deposition and effects on belowground organic matter fluxes in a central European oak forest

Apart from the forest floor, the canopy of forested ecosystems functions as the second most important source for dissolved and particulate fractions of organic and inorganic C and N compounds. However, under mass outbreak situations of insect herbivores this flux path of organic matter is considerably intensified clearly exceeding C and N fluxes from the forest floor. In this paper we report on herbivore-altered C and N fluxes from the canopy to the forest floor and effects on forest floor nutrient fluxes during severe defoliating herbivory of the winter moth (Operophtera brumata) and the mottled umber moth (Eranis defoliaria) in an oak forest in Germany. Over the course of 6.5 months we followed the C and N fluxes with bulk deposition, throughfall solution, insect frass deposits (green-fall together with insect faeces) and with forest floor solution in an 117-yr-old oak (Quercus petraea) forest. Compared to the control, herbivore defoliation significantly enhanced throughfall inputs of total and dissolved organic carbon and nitrogen by a factor of 3 and 2.5 (for TOC and DOC), and by 1.4 and 1.3 times (for TNb and DNb), respectively. Frass plus green-fall C and N fluxes peaked in May with 592 kg C?ha^?1 and 33.5 kg N?ha^?1 representing 79.6% (for C) and 78.3% (for N) of the total C and N input over 2.5 months. The quantitative and qualitative C and N input via faeces and litter deposition significantly differ between the insect affected and non-affected site. However, the C and N fluxes with throughfall did not significantly correlate with forest floor leachates. In this context, forest floor fluxes of TOC, DOC and NO₃-N were significantly lower at the infested site compared to the control, whereas fluxes of NH₄-N together with DON were significantly higher. The study demonstrates the importance of linking the population and associated frass dynamics of herbivorous insects with the cycling of nutrients and organic matter in forest ecosystems, highlighting the remarkable alterations in the timing, amounts and nature of organic matter dynamics on the ecosystem level. Consequently, the ecology of phytophagous insects allows partly to explain temporal-spatial alterations in nutrient cycling and thus ecosystem functioning.     

Carbon,nitrogen and phosphorus net mineralization in organic horizons of temperate forests: stoichiometry and relations to organic matter quality

The rates of mineralization processes influence C sequestration and soil fertility, but despite their importance for ecosystem functioning, C, N and P net mineralization rates are seldom investigated together. Hence, we studied the relationships between net mineralization rates and organic matter stoichiometry in an 8-week incubation experiment with Oi, Oe and Oa horizon material of six beech, one spruce and one pine site. We determined C, N and P net mineralization rates, organic C quality and C:N:P stoichiometry. Net N mineralization only occurred below molar organic matter C:N ratios of 40 (Oi) or 28 (Oa) and N:P ratios of 42 (Oi) or 60 (Oa), and increased with decreasing C:N and N:P ratios. Net P mineralization only occurred below C:P ratios of 1400 (Oi) and N:P ratios of 40 (Oi), and increased with decreasing C:P and N:P ratios. Net N and P mineralization were strongly positively correlated with each other (r = 0.64, p < 0.001), whereas correlations of both net N and net P mineralization with C mineralization were weak. The average C:N:P stoichiometry of net mineralization was 620:4:1 (beech, Oi), 15,350:5:1 (coniferous, Oi), 1520:8:1 (Oe) and 2160:36:1 (Oa). On average, ratios of C:N net mineralization were higher, and ratios of N:P net mineralization lower than organic matter C:N and N:P ratios. This difference contributed to the decrease of C:N ratios and increase of N:P ratios from the Oi to the Oa horizons. In conclusion, the study shows that C, N and P net mineralization rates were closely correlated with the organic matter stoichiometry and that these correlations were modified by the degree of decomposition of the organic matter.     

Biochar stimulates the decomposition of simple organic matter and suppresses the decomposition of complex organic matter in a sandy loam soil

Incorporating crop residues and biochar has received increasing attention as tools to mitigate atmospheric carbon dioxide (CO₂) emissions and promote soil carbon (C) sequestration. However, direct comparisons between biochar, torrefied biomass, and straw on both labile and recalcitrant soil organic matter (SOM) remain poorly understood. In this study, we explored the impact of biochars produced at different temperatures and torrefied biomass on the simple C substrates (glucose, amino acids), plant residues (Lolium perenne L.), and native SOM breakdown in soil using a ¹⁴C labeling approach. Torrefied biomass and biochars produced from wheat straw at four contrasting pyrolysis temperatures (250, 350, 450, and 550 °C) were incorporated into a sandy loam soil and their impact on C turnover compared to an unamended soil or one amended with unprocessed straw. Biochar, torrefied biomass, and straw application induced a shift in the soil microbial community size, activity, and structure with the greatest effects in the straw‐amended soil. In addition, they also resulted in changes in microbial carbon use efficiency (CUE) leading to more substrate C being partitioned into catabolic processes. While overall the biochar, torrefied biomass, and straw addition increased soil respiration, it reduced the turnover rate of the simple C substrates, plant residues, and native SOM and had no appreciable effect on the turnover rate of the microbial biomass. The negative SOM priming was positively correlated with biochar production temperature. We therefore ascribe the increase in soil CO₂ efflux to biochar‐derived C rather than that originating from SOM. In conclusion, the SOM priming magnitude is strongly influenced by both the soil organic C quality and the biochar properties. In comparison with straw, biochar has the greatest potential to promote soil C storage. However, straw and torrefied biomass may have other cobenefits which may make them more suitable as a CO₂ abatement strategy.     

Soil organic carbon fractions differ in two contrasting tall fescue systems

The value of tall fescue (Festuca arundinacea Schreb.) for C sequestration in addition to forage production and soil conservation is of current interest. However, studies relating to the impacts of endophyte infected (E+) and endophyte free (E?) tall fescue on soil organic matter fractions are few. This study examined how E+ and E? growth affected soil C fractions 4 years after establishment. The study site was at the University of Kentucky research farm, Lexington, Kentucky, USA. From soil cores in replicated fields we measured total C, particulate organic matter C (POM C), microbial biomass C (MBC), mineralizable C (Min C), C in aggregates, and aggregate distribution at depths of 0 to 15 and 15 to 30 cm. Significant effects between E+ and E- fescue were sometimes observed for MBC, Min C, C in micro-aggregates, and aggregate distribution, but not for total C, and POM C. At 0?C15 cm MBC (E+ 26% greater than E?), Min C (E+ 43% lower than E?), C associated with micro-aggregates (E+ 15% lower than E?), and micro-aggregates (46% more micro-aggregates in E+ than E?), were affected by endophyte infection, confirming hypotheses that early changes in soil properties were reflected in labile C fractions and soil structure. Endophyte infection status in tall fescue has quantifiable effects on C sequestration and soil structure, achievable in a relatively short period that can be used to monitor conservation efforts and the consequences of pasture renovation strategies.     

Primary organic production in the Atlantic Ocean

Summary The determination of 24-h primary organic production of phytoplankton photosynthesis under 1 m² in the vast areas of open parts of the Atlantic ocean was made by using isopic technique. It was stated that the production is maximal in coastal waters of Europe (0,8–1,3 g C/m²) and especially of the west coast of Africa (to 3,4 g C/m²). Middle rate of production was observed in the North Atlantic Current, and the Equatorial Current (0,2–0,6 g C/m²). Relatively low organic production was found in the tropical waters of the Equatorial Current (about 0,1 g C/m²). Production in the transparent waters of the Sargasso sea was very low (0,03–0,05 g C/m²). Upper data corresponds well with that which had been collected by the Galathea expedition in the same regions of the Ocean. Definite correlation between the rate of primary production and standing crop of crustacean zooplankton was observed in tropical waters.     

Soil organic matter turnover is governed by accessibility not recalcitrance

Mechanisms to mitigate global climate change by sequestering carbon (C) in different ‘sinks' have been proposed as at least temporary measures. Of the major global C pools, terrestrial ecosystems hold the potential to capture and store substantially increased volumes of C in soil organic matter (SOM) through changes in management that are also of benefit to the multitude of ecosystem services that soils provide. This potential can only be realized by determining the amount of SOM stored in soils now, with subsequent quantification of how this is affected by management strategies intended to increase SOM concentrations, and used in soil C models for the prediction of the roles of soils in future climate change. An apparently obvious method to increase C stocks in soils is to augment the soil C pools with the longest mean residence times (MRT). Computer simulation models of soil C dynamics, e.g. RothC and Century, partition these refractory constituents into slow and passive pools with MRTs of centuries to millennia. This partitioning is assumed to reflect: (i) the average biomolecular properties of SOM in the pools with reference to their source in plant litter, (ii) the accessibility of the SOM to decomposer organisms or catalytic enzymes, or (iii) constraints imposed on decomposition by environmental conditions, including soil moisture and temperature. However, contemporary analytical approaches suggest that the chemical composition of these pools is not necessarily predictable because, despite considerable progress with understanding decomposition processes and the role of decomposer organisms, along with refinements in simulation models, little progress has been made in reconciling biochemical properties with the kinetically defined pools. In this review, we will explore how advances in quantitative analytical techniques have redefined the new understanding of SOM dynamics and how this is affecting the development and application of new modelling approaches to soil C.     

The retention of organic matter in soils

The turnover of C in soils is controlled mainly by water regimes and temperature, but is modified by factors such as size and physicochemical properties of C additions in litter or root systems, distribution of C throughout the soil as root systems, or addition as litter, distribution of C within the soil matrix and its interaction with clay surfaces.Soil factors which retard mineralization of C in soils are identified from correlations of C contents of soils with other properties such as clay content and base status. The rate and extent of C mineralization depends on the chemistry of the added organic matter and interaction with clays of the microbial biomass and metabolites.The organomineral interactions are shown to depend on cation bridges involving mainly Ca in neutral to alkaline soils, Al in acid soils and adsorption of organic materials on iron oxide surfaces. The various organomineral interactions lead to aggregations of clay particles and organic materials, which stabilizes both soil structure and the carbon compounds within the aggregates.     

Improving estimates of maximal organic carbon stabilization by fine soil particles

Organic carbon (C) associated with fine soil particles (<20 μm) is relatively stable and accounts for a large proportion of total soil organic C (SOC). The soil C saturation concept proposes a maximal amount of SOC that can be stabilized in the fine soil fraction, and the soil C saturation deficit (i.e., the difference between current SOC and the maximal amount) is presumed to affect the capacity, magnitude, and rate of SOC storage. In this study, we argue that predictions using current models underestimate maximal organic C stabilization of fine soil particles due to fundamental limitations of using least-squares linear regression. The objective was to improve predictions of maximal organic C stabilization by using two alternative approaches; one mechanistic, based on organic C loadings, and one statistical, based on boundary line analysis. We collected 342 data points on the organic C content of fine soil particles, fine particle mass proportions in bulk soil, dominant soil mineral types, and land use types from 32 studies. Predictions of maximal organic C stabilization using linear regression models are questionable because of the use of data from soils that may not be saturated in SOC and because of the nature of regression itself, resulting in a high proportion of presumed over-saturated samples. Predictions of maximal organic C stabilization using the organic C loading approach fit the data for soils dominated by 2:1 minerals well, but not soils dominated by 1:1 minerals; suggesting that the use of a single value for specific surface area, and therefore a single organic C loading, to represent a large dataset is problematic. In boundary line analysis, only data representing soils having reached the maximal amount (upper tenth percentile) were used. The boundary line analysis estimate of maximal organic C stabilization (78 ± 4 g C kg^?1 fraction) was more than double the estimate by the linear regression approach (33 ± 1 g C kg^?1 fraction). These results show that linear regression models do not adequately predict maximal organic C stabilization. Soil properties associated with soil mineralogy, such as specific surface area and organic C loading, should be incorporated to generate more mechanistic models for predicting soil C saturation, but in their absence, statistical models should represent the upper envelope rather than the average value.     

Assay of penicillin acylase in organic media

Summary The synthetic substrate 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB) is an appropriate substrate for assaying penicillin acylase activity in reversed micellar systems of Aerosol - OT in isooctane. Accumulation of 6-nitro-3-aminobenzoic acid (NABA) produced by the enzymatic hydrolysis of NIPAB, followed by the increase in absorbance at 405 nm, was linear at 4 to 20 mM for up to 30 minutes and 15 °C to 40 °C.Abbreviations PA penicillin acylase (penicillin amidohydrolase EC 3.5.1.11) - AOT Aerosol OT (sodium bis- (2-ethylhexyl) sulfosuccinate) - NIPAB 6-nitro-3-(phenylacetamido)-benzoic acid - NABA 6-nitro-3-aminobenzoic acid - BSA bovine serum albumin     

Dynamics of organic matter in soils

Summary Dynamics of C, N, S, and to some extent P are expressed by a knowledge of the size and turnover rates of plant constituents such as soluble C and N components, cellulose and hemicellulose, and lignin. Soil organic matter constituents include: the microbial biomass as determined chemically or microscopically, non-biomass active components determined by isotopic dilution, stabilized N constituents for which good techniques are not yet available, and resistant or old C and associated N determined by carbon dating. The processes involved in the nutrient transformations and transfers are reasonably well understood. The control mechanisms require further elucidation to be able to extrapolate from the laboratory to the field, and between field sites. Major control mechanisms requiring further insight include the effects of C availability on transformations of C and N. The other control for which every little is known is that of spatial compartmentalization. Compartmentalization ranges from landscape or management sequences to pedogenic layers, rhizosphere-mycorrhizal effects, clay-sesquioxide surfaces, aggregation, localized enzymes, and microbial effects such as membrane boundaries. Control mechanisms for concurrent mineralization-immobilization, the stabilization of microbial products, and the relative role of the biomass as a catalyst rather than as a source-sink for nutrients, must be understood. There is potential for combining a knowledge of microbial production and turnover with that of the roles of the soil organic active fraction as a temporary storehouse for nutrients. This, in conjunction with management techniques such as zero tillage and crop rotation, should make it possible to better utilize soil and fertilizer N, especially in areas of the world where the cost of nutrients is high relative to the value of the crop grown.Introductory lecture     

火烧对森林土壤有机碳的影响研究进展

对国内外火烧影响森林土壤有机碳动态的研究成果进行了综合述评。较多研究表明低强度火烧不会造成土壤有机碳贮量的明显变化,但火烧非常强烈而彻底,土壤有机碳明显减少。有限研究表明火烧对森林土壤呼吸的影响结果有增加、降低或无影响,因火烧强度、火后观测时间、森林类型、火烧迹地上植被恢复进程和气候条件等而异。同时,火烧对土壤有机碳组分(活性有机碳和黑碳)也具有不同程度的影响。随着全球变化研究的深入,火烧作为森林主要管理措施对大气CO2浓度影响亦愈来愈受重视,今后应着重开展以下几方面研究:(1)扩大气候和经营管理的变化对森林土壤有机碳贮量时空动态影响研究;(2)深入探讨火烧影响土壤CO2释放的过程及机理;(3)加强火烧历史和频率对黑碳影响的研究;(4)从广度和深度上加强火烧等经营措施对亚热带森林土壤碳动态影响的研究。     

Spectroscopic studies of bacteriorhodopsin fragments dissolved in organic solution.

Fourier transform infrared and UV fourth-derivative spectroscopies were used to study the secondary structure of bacteriorhodopsin and its chymotryptic and one of the sodium borohydride fragments dissolved in chloroform-methanol (1:1, v/v), 0.1 M LiClO4. The C1 fragment (helices C, D, E, F, and G) showed an alpha-helical content of about 53%, whereas C2 (helices A and B) had about 60%, and B2 (helices F and G) about 65% alpha-helix. The infrared main band indicated differences in alpha-helical properties between these fragments. These techniques were also used to obtain information on the interactions among helices. According to the results obtained from the hydrogen/deuterium exchange kinetics, about 40% of the amide protons of C2 are particularly protected against exchange, whereas for the C1 fragment this process is unexpectedly fast. UV fourth-derivative spectra of these samples were used to obtain information about the environment of Trp side chains. The results showed that the Trp residues of C2 are more shielded from the solvent than those of C1 or B2. The results of this work indicate that the specific interactions existing between the transmembrane segments induce different types of helical conformations in native bacteriorhodopsin.     

Intake of dissolved organic matter from deep seawater inhibits atherosclerosis progression

Dissolved organic matter (DOM) in seawater can be defined as the fraction of organic matter that passes through a filter of sub micron pore size. In this study, we have examined the effect of DOM of deep seawater (DSW) from Pacific Ocean on platelet aggregation and atherosclerosis progression. DSW was passed through a series of filters and then through an Octadecyl C18 filter; the retained substance in ethanol was designated as C18 extractable DOM (C18-DOM). Our studies showed that C18-DOM treatment inhibited platelet aggregation, P-selectin expression and activity of COX-1 significantly. C18-DOM increased the expression of anti-atherogenic molecule namely heme oxygenase-1 in endothelial cells and all these data showed that C18-DOM is exhibiting aspirin-like effects. Moreover our in vivo studies showed that C18-DOM feeding slowed remarkably the progression of atherosclerosis. Our study demonstrated a novel biological effect of oceanic DOM, which has several important implications, including a possible therapeutic strategy for atherosclerosis.     

Functional significance of conserved cysteines in the human organic cation transporter 2

The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C63, C89, C103, and C143), and the C89 and C103 mutants had reduced Michaelis constants (K(t)) for MPP. The loop cysteines were refractory to interaction with thiol-reactive biotinylation reagents, except after pretreatment of intact cells with dithiothreitol or following cell membrane solubilization. Reduction of disulfide bridge(s) did not affect transport, but labeling the resulting free thiols with maleimide-PEO(2)-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K(t) value for MPP. In contrast, the K(t) value associated with TEA transport was reduced sevenfold in the C474A mutant, and the C474F mutant failed to transport TEA. This study shows that some but not all of the six extracellular loop cysteines exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.