33P translocation in the thallus of the mat-forming lichen Cladonia portentosa

The extent of vertical migration of ³³P in thalli of the heathland lichen Cladonia portentosa was investigated under field conditions. ³³P-labelled orthophosphate was introduced into the bottom 25 mm of podetia cut to a length of 50 mm from the apices. The distribution of label was scanned using a molecular imager immediately after incubation, and after growing for 8 wk and 6 months. Differences in the relative distribution of label between podetia harvested at the beginning and the end of the experiment showed that there had been a significant migration of ³³P upwards out of the labelled 25 mm stratum towards the apex. This was confirmed by statistically significant changes in the median (md) and the 90 percentile of total relative distribution of ³³P label. In a control treatment in which label was introduced into the apical 25 mm of podetia, which were then grown inverted (top down), no upward movement of label was detected. By contrast, a statistically significant reduction in the md of the distribution indicated migration downwards towards the thallus apex. The results are consistent with the hypothesis that P is recycled within podetia of mat-forming lichens, migrating from degrading basal regions upwards to the growing apices following a source–sink relationship.     

Growth and nitrogen relations in the mat-forming lichens Stereocaulon paschale and Cladonia stellaris

BACKGROUND AND AIMS: Mat-forming lichens in the genera Stereocaulon and Cladonia have ecosystem-level effects in northern boreal forests. Yet the factors affecting the productivity of mat-forming lichens are not known. The aim of the presented work was to investigate whether mat-forming lichens adapted to low N availability employ N-conserving mechanisms similar to those of vascular plants in nutrient-poor ecosystems. Specifically, the following questions were asked: (a) Do lichens translocate N from basal areas to apical growth areas? (b) Are the quantities of N translocated of ecological significance. (c) Is lichen growth dependent on tissue N concentration [N]. METHODS: Two different, but complementary, field experiments were conducted using the mat-forming N2-fixing Stereocaulon paschale and non-fixing Cladonia stellaris as model species. First, N translocation was investigated by feeding lichens with Na(15)NO3 either directly to the apex (theoretical sink) or to the basal part (theoretical source) and observing the redistribution of (15)N after a growth period. Secondly, growth and variation in [N] in thalli of different lengths was measured after a growth period. KEY RESULTS: (15)N fed to lower parts of lichen was translocated towards the growing top, but not vice versa, indicating physiologically dependent translocation that follows a sink-source relationship. In the growth experiment where thalli were cut to different lengths, the significant decrease in [N] in apices of short vs. longer thalli after a growth period is consistent with internal relocation as an ecologically important source of N. CONCLUSIONS: The presented results demonstrate that internal recycling of N occurs in both species investigated and may be ecologically important in these mat-forming lichens under field conditions. The higher nitrogen use efficiency and relative growth rate in C. stellaris in comparison with S. paschale probably enable C. stellaris to dominate the ground cover vegetation in dry boreal coniferous forests under undisturbed conditions.     

Translocation,remineralization, and turnover of nitrogen in the roots and rhizomes of Spartina alterniflora (Gramineae)

We used ¹⁵N to quantify rates of N translocation from aerial to belowground tissues, foliar leaching, and turnover and production of root and rhizome biomass in the plant-sediment system of short Spartina alterniflora areas of Great Sippewissett Marsh, Massachusetts. Decay of belowground tissues in litterbag incubations at 1- and 10-cm depths resulted in 80% remineralization of the original plant (¹⁵N-labeled) N and 20% burial after 3 years. Translocation of ¹⁵N from plant shoots in hydrologically controlled laboratory lysimeters maintained under field conditions was 38% of the aboveground pool while leaching of N was 10% from June to October. Most of the translocated N was not retranslocated to new aboveground growth in December but appeared to be either remineralized or buried in the sediment. Injection of ¹⁵N into field stands of grass showed initially high incorporation into plants followed by a continuous decline over the next 7 years yielding a gross tumover time of 1.5–1.6yr. Correcting the gross N turnover for recycling of label via translocation and uptake of remineralized label during this period, a net root and rhizome turnover time of 1.0–1.1 yr was obtained. Combining the turnover time with independent estimates of seasonal belowground biomass yielded an estimate of belowground production of 929–1,022 g C m⁻² yr⁻¹, similar to measurements by traditional biomass harvest, CO² based budgets and models for comparable areas of this marsh. Integration of the production and nitrogen balance estimates for short Spartina marsh yielded translocation, 1.4 g N m⁻² yr⁻¹, leaching, 0.4 g N m⁻² yr⁻¹, remineralization, 14.9–16.3 g N m⁻² yr⁻¹, and burial, 3.7–4.1 g N m⁻² yr⁻¹.     

Lobe formation and division in the foliose lichen <Emphasis Type="Italic">xanthoparmelia conspersa</Emphasis>

New lobe development and lobe division was studied in the foliose lichen Xanthoparmelia conspersa (Ehrh. ex. Ach.) Hale. In thalli with either the centre or margin removed, the inside edge of the perimeter, the outer edge of the reproductive centre, and fragments derived from the thallus perimeter all regenerated growing points (‘lobe primordia’) within a year. Thalli possessing isidia had the greatest ability to regenerate growing points. In reproductive thalli, there was a positive correlation between the density of new growing points and thallus size. When fragments were cut from the perimeters of mature X. conspersa thalli and glued to pieces of slate, the ratio of growing points to mature lobes increased over 54 months. Lobes within a thallus exhibited different degrees of bifurcation. In some bifurcating lobes, the point of origin of the bifurcation advanced at the same rate as the lobe tips over 4 months but in most lobes, the bifurcation point either advanced less rapidly than the lobe tips or retreated from its original location. Removing adjacent lobes had no significant effect on the radial growth of a lobe over 4 months or on the location of the bifurcation point but it increased the number of growing points. These results suggest that for X. conspersa: 1) all portions of of thalli can regenerate growing points, 2) few growing points actually develop into mature lobes, 3) individual lobes within a thallus grow and divide differently, and 4) adjacent lobes inhibit the development of growing points on their neighbours.     

Uptake of 15N by Kiwifruit Vines from Applications of Nitrogen Fertilizer Prior to Budbreak

Mature field-grown kiwifruit vines (Actinidia deliciosa var.deliciosa cv. Hayward) were fertilized with ¹⁵N-labelled fertilizer(ammonium sulphate, 10 atom % ¹⁵N, 50 kgN ha^-1) to investigatethe timing of uptake of fertilizer nitrogen (N) and its availabilityfor new season's growth. Treatments were applied on four occasions,representing 2, 6, 10 and 14 weeks prior to budbreak. Samplesof root, stem, cordon, fruiting cane, vacuum-extracted xylemsap, and new season's growth were collected at fortnightly intervalfrom early winter until 2 months after budbreak. Two weeks after application of each treatment, ¹⁵N equivalentto an average of 7% of the applied label was recovered in rootmaterial. Although label was taken up by roots, there was nomovement of ¹⁵N within the plant until about 1 month prior tobudbreak when it was measured in the stem and cordon. Fertilizernitrogen was not detected at the distal end of fruiting canes,and in new season's growth until 3-4 weeks after budbreak. Beforebudbreak, all nitrogen in the xylem sap was in amino forms.Nitrate appeared 4 weeks after budbreak, and although more enrichedwith ¹⁵N than the amino nitrogen, accounted for only 19% ofthe label. Eight weeks after budbreak, nitrate nitrogen accountedfor 57% of the label. There were no major treatment effects of ¹⁵N on vines in eitherspring or at harvest, although enrichments in fruit and leavesfrom the earliest treatment tended to be less at the end ofthe season than those from the later applications.Copyright1993, 1999 Academic Press Actinidia deliciosa, kiwifruit, nitrogen, ¹⁵N, nutrient uptake     

Use of stable isotopes to quantify nitrogen, potassium and magnesium dynamics in young Scots pine (Pinus sylvestris)

Two-yr-old Scots pine ( Pinus sylvestris ) seedlings were grown in sand culture for 1 yr with a generous supply of a balanced nutrient solution. Trees were repotted into clean sand in February 1998 and given either a reduced or adequate nutrient supply containing enriched ¹⁵N, ⁴¹K and ²⁶Mg to label nutrient uptake during spring 1998. Trees doubled their biomass during the experiment. Whole-tree net photosynthesis was reduced by 43% after 95 d in trees that received the lower nutrient supply ( P < 0.001), although differences in biomass between the two treatments were less pronounced. Remobilization contributed 83, 82 and 52% of the N, K and Mg, respectively, used to support growth of new tissues in trees that received reduced nutrient supply. Those receiving the higher nutrient supply still obtained 44–59% of nutrients used for spring growth of new tissues from remobilization. Current nutrient supply had no significant effect on the amount of N or Mg remobilized to new tissues but K remobilization was less in trees that received the lower nutrient supply ( P = 0.025). The importance of remobilization in young trees and problems associated with quantifying internal cycling of nutrients are discussed.     

Structure and unusual characteristics of a new family of transposable elements in the sea urchin Strongylocentrotus purpuratus.

The transposable element family TU of the sea urchin Strongylocentrotus purpuratus, a higher eucaryote, has recently been described (D. Liebermann, B. Hoffman-Liebermann, J. Weinthal, G. Childs, R. Maxson, A. Mauron, S.N. Cohen, and L. Kedes, Nature [London] 306:342-347, 1983). A member of this family, TU4, has an insertion, called ISTU4, of non-TU DNA. ISTU4 is a member of a family of repetitive sequences, which are present in some 1,000 copies per haploid S. purpuratus genome (B. Hoffman-Liebermann, D. Liebermann, L.H. Kedes, and S.N. Cohen, Mol. Cell. Biol. 5:991-1001, 1985). We analyzed this insertion to determine whether it is itself a transposable element. The nucleotide sequence of ISTU4 was determined and showed an unusual structure. There are four, approximately 150 nucleotides long, imperfect direct repeats followed by a single truncated version of these repeats. This region is bounded at either side by approximately 100-nucleotide-long sequences that are not related to each other or to the repeats. Nucleotide sequences at the boundaries of ISTU4-homologous and flanking regions in five genomic clones show that ISTU4 represents a family of sequences with discrete ends, which we call Tsp elements. We showed that the genomic locus that carries a Tsp element in one individual was empty in other individuals and conclude that Tsp elements are a new and different type of transposable element. Tsp elements lack two features common to most other transposable elements: Tsp integration does not result in the duplication of host DNA, and there are no inverted repeats at their termini, although short inverted repeats are present at a distance from the termini.     

Symbiosis constraints: Strong mycobiont control limits nutrient response in lichens

Symbioses such as lichens are potentially threatened by drastic environmental changes. We used the lichen Peltigera aphthosa—a symbiosis between a fungus (mycobiont), a green alga (Coccomyxa sp.), and N₂‐fixing cyanobacteria (Nostoc sp.)—as a model organism to assess the effects of environmental perturbations in nitrogen (N) or phosphorus (P). Growth, carbon (C) and N stable isotopes, CNP concentrations, and specific markers were analyzed in whole thalli and the partners after 4 months of daily nutrient additions in the field. Thallus N was 40% higher in N‐fertilized thalli, amino acid concentrations were twice as high, while fungal chitin but not ergosterol was lower. Nitrogen also resulted in a thicker algal layer and density, and a higher δ¹³C abundance in all three partners. Photosynthesis was not affected by either N or P. Thallus growth increased with light dose independent of fertilization regime. We conclude that faster algal growth compared to fungal lead to increased competition for light and CO₂ among the Coccomyxa cells, and for C between alga and fungus, resulting in neither photosynthesis nor thallus growth responded to N fertilization. This suggests that the symbiotic lifestyle of lichens may prevent them from utilizing nutrient abundance to increase C assimilation and growth.     

Growth,Net Production,Litter Decomposition,and Net Nitrogen Accumulation by Epiphytic Bryophytes in a Tropical Montane Forest1

To understand the ecological roles of epiphytic bryophytes in the carbon (C) and nitrogen (N) cycles of a tropical montane forest, we used samples in enclosures to estimate rates of growth, net production, and N accumulation by shoots in the canopy, and litterbags, to estimate rates of decomposition and N dynamics of epiphytic bryophyte litter in the canopy and on the forest floor in Monteverde, Costa Rica. Growth of epiphytic bryophytes was estimated at 30.0–49.9 percent/yr, net production at 122–203 g/m²/yr, and N accumulation at 1.8–3.0 g N/m²/yr. Cumulative mass loss from litterbags after one and two years in the canopy was 17 ± 2 and 19 ± 2 percent (mean ± 1 SE) of initial sample mass, respectively, and mass loss from litter and green shoots in litterbags after one year on the forest floor was 29 ± 2 and 45 ± 3 percent, respectively. Approximately 30 percent of the initial N mass was released rapidly from litter in both locations. Nitrogen loss from green shoots on the forest floor was greater; about 47 percent of the initial N mass was lost within the first three months. There was no evidence for net N immobilization by litter or green shoots, but the remaining N in litter was apparently recalcitrant. Annual net accumulation of C and N by epiphytic bryophytes was estimated at 37–64 g C/m²/yr and 0.8–1.3 g N/m²/yr, respectively. Previous research at this site indicated that epiphytic bryophytes retain inorganic N from atmospheric deposition to the canopy. Therefore, they play a major role in transforming N from mobile to highly recalcitrant forms in this ecosystem.     

General introduction to the lists of threatened biotopes,flora and fauna of the trilateral Wadden Sea Area (red data book)

The effect of the acclimation temperature on the temperature tolerance ofPorphyra leucosticta, and on the temperature requirements for growth and survival ofEnteromorpha linza was determined under laboratory conditions. Thalli ofP. leucosticta (blade or Conchocelis phases), acclimated to twenty-five degrees, survived up to 30°C, i.e. 2°C more than those acclimated to 15°C which survived up to 28°C. Lower temperature tolerance of bothPorphyra phases that were acclimated to 15°C was −1°C after an 8-week exposure time at the experimental temperatures. The upper temperature tolerance ofE. linza also increased by 2°C, i.e. from 31 to 33°C, when it was acclimated to 30°C instead of 15°C. The lower temperature tolerance increased from 1 to −1°C, when it was acclimated to 5°C instead of 15°C.E. linza thalli acclimated for 4 weeks to 5 or 10°C reached their maximum growth at 15°C, i.e. at a 5°C lower temperature than those acclimated to 15 or 30°C. These thalli achieved higher growth rates in percent of maximal growth at low temperatures than those acclimated to 15 or 30°C. Thalli acclimated for 1 week to 5°C reached their maximum growth rate at 20°C and achieved growth rates at low temperatures similar to those recorded for thalli acclimated to 15°C. Thalli ofE. linza acclimated for 4 weeks to 5°C lost this acclimation after being post-cultivated for the same period at 15°C. That was not the case with thalli acclimated for 8 weeks to 5°C and post-acclimated for 4 weeks to 15°C. These thalli displayed similar growth patterns at 10–25°C, while a decline of growth rate was observed at 5 or 30°C. The significance of the acclimation potential ofE. linza with regard to its seasonality in the Gulf of Thessaloniki, and its distribution in the N Atlantic, is also discussed.     

Heterogeneity of Chlorophyll Fluorescence over Thalli of Several Foliose Macrolichens Exposed to Adverse Environmental Factors: Interspecific Differences as Related to Thallus Hydration and High Irradiance

Spatial heterogeneity of chlorophyll (Chl) fluorescence over thalli of three foliose lichen species was studied using Chl fluorescence imaging (CFI) and slow Chl fluorescence kinetics supplemented with quenching analysis. CFI values indicated species-specific differences in location of the most physiologically active zones within fully hydrated thalli: marginal thallus parts (Hypogymnia physodes), central part and close-to-umbilicus spots (Lasallia pustulata), and irregulary-distributed zones within thallus (Umbilicaria hirsuta). During gradual desiccation of lichen thalli, decrease in Chl fluorescence parameters (F_O - minimum Chl fluorescence at point O, F_P - maximum Chl fluorescence at P point, ₂ - effective quantum yield of photochemical energy conversion in photosystem 2) was observed. Under severe desiccation (>85 % of water saturation deficit), substantial thalli parts lost their apparent physiological activity and the resting parts exhibited only a small Chl fluorescence. Distribution of these active patches was identical with the most active areas found under full hydration. Thus spatial heterogeneity of Chl fluorescence in foliose lichens may reflect location of growth zones (pseudomeristems) within thalli and adjacent newly produced biomass. When exposed to high irradiance, fully-hydrated thalli of L. pustulata and U. hirsuta showed either an increase or no change in F_O, and a decrease in F_P. Distribution of Chl fluorescence after the high irradiance treatment, however, remained the same as before the treatment. After 60 min of recovery in the dark, F_O and F_P did not recover to initial values, which may indicate that the lichen used underwent a photoinhibition. The CFI method is an effective tool in assessing spatial heterogeneity of physiological activity over lichen thalli exposed to a variety of environmental factors. It may be also used to select a representative area at a lichen thallus before application of single-spot fluorometric techniques in lichens.     

Effects of spaceflight and thyroid deficiency on hindlimb development. I. Muscle mass and IGF-I expression.

Thyroid deficiency (TD) in neonatal rats causes reduced growth of skeletal muscle that is disproportionately greater than that for other tissues (G. R. Adams, S. A. McCue, M. Zeng, and K. M. Baldwin. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 276: R954-R961, 1999). TD depresses plasma insulin-like growth factor I (IGF-I) levels, suggesting a mechanism for this effect. We hypothesized that TD and exposure to spaceflight (SF) would interact to reduce skeletal muscle growth via a reduction in IGF-I levels. Neonatal rats were flown in space for 16 days. There was a similar, nonadditive reduction in the growth of the body ( approximately 50%) and muscle weight (fast muscles, approximately 60%) with either TD or SF. In the soleus muscle, either SF or TD alone resulted in growth reductions that were augmented by SF-TD interactions. There were strong correlations between 1) muscle mass and muscle IGF-I levels and 2) circulating IGF-I and body weight. These results indicate that either hypothyroidism or exposure to SF will limit the somatic and muscle-specific growth of neonatal rats. The impact of these perturbations on skeletal muscle growth is relatively greater than the effect on somatic growth. The mechanisms by which either TD or SF impact growth appear to have a common pathway involving the control of plasma and muscle IGF-I concentrations.     

Specific Incorporation of Precursors into DNA by Feeding Labeled Bacteria to Paramecium aurelia

SYNOPSIS. Studies were carried out on the introduction of labeled precursors into the DNA of Paramecium aurelia (syngen 4, stock 51) by way of the bacteria that are used for food. A thymine-requiring strain of Escherichia coli (15 T⁻) was labeled by growth in either H³-methyl thymidine or 2-C¹⁴ bromouracil, washed free of the exogenous label, and fed to the paramecia. The tritium label from the bacteria was incorporated almost exclusively into the DNA of the paramecia, whereas it was much less specifically incorporated when introduced directly from the medium. The Cu label from bromouracil was also incorporated mainly into the DNA of the paramecia although a small amount appeared in RNA. The formation of labeled food vacuoles was followed. Food vacuoles were formed at a nearly constant rate, with the total number of vacuoles increasing throughout the cycle. The lifetime of the vacuoles was about 2.5 hours. Incorporation of the label into the DXA of the paramecia begins within a few minutes of the formation of the first labeled vacuole. DNA synthesis begins about 1.5 hr after the previous fission (total cell cycle about 5.8 hr) and progresses at a nearly constant rate throughout the remainder of the cycle.     

Translocation of Amyloid Precursor Protein C-terminal Fragment(s) to the Nucleus Precedes Neuronal Death due to Thiamine Deficiency-induced Mild Impairment of Oxidative Metabolism

Thiamine deficiency (TD) is a model of neurodegeneration induced by mild impairment of oxidative metabolism. TD produces time-dependent glial activation, inflammation, oxidative stress, altered metabolism of amyloid precursor protein (APP), exacerbation of plaque formation from APP, and finally, selective neuron death in specific brain regions. The sub-medial thalamic nucleus (SmTN) is the most sensitive region to TD. Alteration in APP metabolism and nuclear translocation of carboxy-terminal fragments (CTF) of APP has been implicated in neuron death in other models of neurodegeneration. These experiments tested whether TD causes translocation of CTF into the nucleus of neurons in the SmTN that are destined to die after 9 days of TD by examining overlapping immunoreactivity (IR) of antibody APP 369 with either Alz90, 6E10 or 4G8 epitopes in the nuclei of the neurons in the SmTN. TD caused the accumulation of the CTF of APP in nuclei of SmTN neurons within 3 days of TD. These changes did not occur in the cortex which is spared in TD. Western blot analysis of nuclear fractions revealed a significant (61%; P < 0.026) increase in CTF 12 levels in TD SmTN (2.08 +/- 0.56) compared to control SmTN (1.29 +/- 0.41). Although TD increased CTF 15 levels in TD SmTN (1.95 +/- 0.73) compared to control SmTN (0.62 +/- 0.52) by 214%; P < 0.665 and decreased the full-length holo-APP levels in TD SmTN (0.32 +/- 0.30) compared to control SmTN (0.47 +/- 0.18) by 34%; P < 0.753, the differences were statistically insignificant. TD did not alter CTF 15 or CTF 12 levels in cortex. These findings demonstrate that changes in APP metabolism occur in early stages of TD, and they may play an important role in TD-induced selective neuronal loss.     

Distribution of nitrogen-13 from labeled nitrate and nitrite in germfree and conventional-flora rats

The in vivo distribution of physiological concentrations of NO3- and NO2- labeled with 13N was studied in germfree and conventional-flora Sprague-Dawley rats after gastric intubation (gavage), intravenous (cardiac or tail vein), or intraluminal (intestinal) injection. Some in vitro studies were performed to determine the influence of the bacterial flora on ion distribution. After gavage with 13NO3-, essentially all of the label passed into the upper small intestine, where most was absorbed; however, up to 24% of the 13N could reach the ileum within 1 h. Gavage with 13NO2- resulted in some gastric absorption of the label, but most seemed to exit the stomach via passage into the duodenum. The exit of 13NO2- from the stomach was slower, and less 13N appeared to be absorbed from the small intestine than with 13NO3-. Movement of label through the gastrointestinal tract could be enhanced by inducing diarrhea. Absorbed 13N was either excreted in the urine, reentered the gastrointestinal tract at various points, or was temporarily stored in the eviscerated carcass. The bacterial flora, either by incorporation or chemical alteration, appeared to have some influence on the distribution of 13N from 13NO3- or 13NO2-.     

Distribution of nitrogen-13 from labeled nitrate and nitrite in germfree and conventional-flora rats.

The in vivo distribution of physiological concentrations of NO3- and NO2- labeled with 13N was studied in germfree and conventional-flora Sprague-Dawley rats after gastric intubation (gavage), intravenous (cardiac or tail vein), or intraluminal (intestinal) injection. Some in vitro studies were performed to determine the influence of the bacterial flora on ion distribution. After gavage with 13NO3-, essentially all of the label passed into the upper small intestine, where most was absorbed; however, up to 24% of the 13N could reach the ileum within 1 h. Gavage with 13NO2- resulted in some gastric absorption of the label, but most seemed to exit the stomach via passage into the duodenum. The exit of 13NO2- from the stomach was slower, and less 13N appeared to be absorbed from the small intestine than with 13NO3-. Movement of label through the gastrointestinal tract could be enhanced by inducing diarrhea. Absorbed 13N was either excreted in the urine, reentered the gastrointestinal tract at various points, or was temporarily stored in the eviscerated carcass. The bacterial flora, either by incorporation or chemical alteration, appeared to have some influence on the distribution of 13N from 13NO3- or 13NO2-.     

Effects of leaf nitrogen availability and leaf position on nitrogen allocation patterns in Vaccinium vitis-idaea and Vaccinium uliginosum

Summary Nitrogen allocation patterns from leaves of Vaccinium vitis-idaea (evergreen) and Vaccinium uliginosum (deciduous) were assessed using a foliar application of ¹⁵N labeled ammonium sulfate. These are wild perennial shrubs inhabiting arctic and subarctic regions. More label was transported from labeled leaves of Vaccinium uliginosum then Vaccinium vitis-idaea. In Vaccinium uliginosum, the amount of label transported from the labeled leaf increased as the concentration of nitrogen in the label increased. Current growth in Vaccinium uliginosum was a strong sink for nitrogen because most of the ¹⁵N transported from the labeled leaf was contained in this region. In addition, when greater quantities of nitrogen were applied, larger quantities were retained in current growth. Current growth of Vaccinium vitis-idaea, on the other hand, was not as strong a sink because regardless of the nitrogen available thru various label concentrations, the enrichment of current growth was not affected and was not significantly different from older stems or leaves. Yet, in both species, nitrogen was transported freely from leaves of all positions along the stem to all parts of the plant including roots and rhizomes. The position of the leaf along the stem had no effect on the patterns of allocation to other organs.     

Differential behavior of the two free sulfhydryl groups of human plasma fibronectin: effects of environmental factors.

We report here a novel approach to label specifically one of the two cryptic, free sulfhydryl groups per subunit of human plasma fibronectin with either an 15N,2H-maleimide spin label or a coumarinylphenyl maleimide fluorescent label. This permits the use of electron spin resonance (ESR) or fluorescence techniques to study molecular dynamics of fibronectin with the label attached to a single site per chain on the protein molecule. The method is based on our observation that upon adsorption of fibronectin to a gelatin-coated surface, the SH1 site, located between the DNA-binding and the cell-binding domains, is partially exposed, while the SH2 site, located within the carboxyl-terminal fibrin-binding domain, remains buried and unreactive. The procedures for the preparation of the selectively labeled fibronectins are described in detail. The physicochemical properties of these single-site labeled fibronectins, particularly as affected by high salt, heparin, surface binding, and temperature, were characterized by ESR spin-label and steady-state fluorescence techniques. The steady-state fluorescence measurement indicates that both local environments of SH1 and SH2 sites are relatively hydrophobic, and that the SH2 site is more hydrophobic than the SH1 site. The ESR results show that heparin or high salt induces an increase in the domainal flexibility in both SH1 and SH2 regions, perhaps through the disruption of domain-domain interactions in the fibronectin molecule, and that the former is more effective than the latter in producing such an effect. The observed heparin effect is reversible by addition of calcium ions in the SH2 regions but not in the SH1 regions. In addition, at temperatures above 44 degrees C, both type III homologous regions containing the free sulfhydryl groups are shown to undergo denaturation and aggregation processes. The data presented here suggest that the newly developed method for differential labeling of the free sulfhydryl groups in fibronectin should be useful for mapping the spatial arrangement of structural domains in the protein molecule using spin-label-spin-probe and fluorescence energy transfer techniques.