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The re-use of scientific data has the potential to greatly increase communication, collaboration and synthesis within and among disciplines, and thus is fostered, supported and encouraged. Permission to use this dataset is granted to the Data User free of charge subject to the following terms:

1) Acceptable use. Use of the dataset will be restricted to academic, research, educational, government, recreational, or other not-for-profit professional purposes. The Data User is permitted to produce and distribute derived works from this dataset provided that they are released under the same license terms as those accompanying this Data Set. Any other uses for the Data Set or its derived products will require explicit permission from the dataset owner.
2 ) Redistribution. The data are provided for use by the Data User. The metadata and this license must accompany all copies made and be available to all users of this Data Set. The Data User will not redistribute the original Data Set beyond this collaboration sphere.
3 ) Citation. It is considered a matter of professional ethics to acknowledge the work of other scientists. Thus, the Data User will properly cite the Data Set in any publications or in the metadata of any derived data products that were produced using the Data Set. Citation should take the following general form: Creator, Year of Data Publication, Title of Dataset, Publisher, Dataset identifier. For example:

Shaver, G. 1989. Above ground biomass in acidic tussock tundra experimental site, 1989, Arctic LTER, Toolik, Alaska. Arctic LTER, Marine Biological Lab, Woods Hole, Ma 02543. 1989gsttbm http://ecosystems.mbl.edu/arc/terrest/biomass/index.shtml 

4 ) Acknowledgement. The Data User should acknowledge any institutional support or specific funding awards referenced in the metadata accompanying this dataset in any publications where the Data Set contributed significantly to its content. Acknowledgements should identify the supporting party, the party that received the support, and any identifying information such as grant numbers. For example:

Data sets were provided by the Arctic LTER. This material is based upon work supported by the National Science Foundation under Grants #DEB-981022, 9211775, 8702328; #OPP-9911278, 9911681, 9732281, 9615411, 9615563, 9615942, 9615949, 9400722, 9415411, 9318529; #BSR 9019055, 8806635, 8507493.

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Dataset URLs:METADATA: HTML, Rich Text, XML(EML compliant)
DATA: Comma Delimited, Excel file with Metadata and data, Dataset via LTER Data Poral
Dataset ID:2008Toolik_ProteolyticActivity.01
Dataset Title:Proteolytic enzyme activity of organic and mineral soil core samples collected near Toolik Lake field station, Alaska, July 2001
Investigator 1: 
First Name:Julia
Last Name:Reiskind
Organization:University of Florida
Address line 2:Department of Biology
Address line 3:P.O. Box 118525
City:Gainesville
State:FL
Zip Code:32611
Country:USA
Investigator 2: 
First Name:Michelle
Last Name:Mack
Organization:University of Florida
Address line 2:Department of Biology
Address line 3:P.O. Box 118525
City:Gainesville
State:FL
Zip Code:32611
Country:USA
Investigator 3: 
First Name:Martin
Last Name:Lavoie
Organization:St. Francis Xavier University
Address line 2:Department of Earth Sciences
Address line 3:5009 Chapel Square
City:Antigonish
State:Nova Scotia
Zip Code:B2G 2W5
Country:Canada
Associate Investigators:
Keywords:arctic, mineral layer, organic layer, enzyme activity, tundra, moist acidic tundra, soil
Abstract: The original focus of this study was an analysis of proteolytic enzyme activity of Alaskan arctic tundra soils, however initial results raised questions regarding the method (Watanabe and Hayano, 1995). Thus, the goals of the study changed to 1) an investigation of the method, and 2) a comparison of enzyme activities of two different soil layers from the arctic tundra. Methodological examination included the impact of toluene, used to prevent immobilization of the product, and blank correction of enzyme activity, and a search for a true 6-h linear rate of activity during a 48-hour incubation. We measured native and potential, using casein as an artificial substrate, activities as net amino acid production in mineral and organic soil layer samples. Varying toluene concentration had no clear effect on activity; omitting toluene resulted in zero native activity and reduced potential for the organic samples, but not for the mineral. Comparison of activities with and without blank correction indicated, particularly for potential activity of samples with low native rates, that correction was required for accuracy. Native and potential activity of the organic samples, and native of the mineral were linear for the first 6 h of incubation; linearity was observed during the 6 to 24 h incubation for potential activity of the mineral. Soil layer activity data indicated that native activity was higher in organic soils as compared with mineral. The organic layer potential activity was ten-fold greater than the native, suggesting substrate limitation; potential and native activities did not differ in the mineral layer, indicating substrate sufficiency. Casein addition changed the kinetic pattern for both layers from hyperbolic to sigmoidal for the mineral and linear for the organic, implying different enzyme pools or behavioral changes of existing pools. Native activity based on total soluble protein was higher for the mineral samples relative to the organic, reiterating substrate capacity differences and variations in enzyme/substrate interactions.
For questions about the Metadata and data contact the Investigators.
For information about this web site contact:
Arctic LTER Information Manager
The Ecosystems Center
Marine Biological Lab
7 MBL St
Woods Hole, MA 02543
Phone (508) 289 7496
Email: arc_im@mbl.edu
Online URL: http://ecosystems.mbl.edu/ARC/
DATA FILE INFORMATION:
Data File URL http://metacat.lternet.edu/das/dataAccessServlet?docid=knb-lter-arc.10528&urlTail=terrest/bulk/data/2008Toolik_ProteolyticActivity.csv
Data File Name 2008Toolik_ProteolyticActivity.01
Beginning Date 1/1/2008
End Date 3/30/2008
Number of Data Records 162
Other Files to Reference 0
Availability Status Type 1
Quality Control Information
Maintenance Description
Log of Changes: Updated Metadata sheet
 
RESEARCH LOCATION:                  
Location Name LTER Moist Acidic Tussock Tundra Select Site or enter New One Select Site or enter New One Select Site or enter New One Select Site or enter New One Select Site or enter New One Select Site or enter New One Select Site or enter New One  
Geographic Description west of Toolik Lake Enter Description Enter Description Enter Description Enter Description Enter Description Enter Description Enter Description  
Location Bounding Box                  
West Bounding Coordinate                  
East Bounding Coordinate                  
North Bounding Coordinate                  
South Bounding Coordinate                  
OR if single point location                  
Latitude 68.624411 In Decimal Degrees In Decimal Degrees In Decimal Degrees In Decimal Degrees In Decimal Degrees In Decimal Degrees In Decimal Degrees  
Longitude -149.609589 In Decimal Degrees In Decimal Degrees In Decimal Degrees In Decimal Degrees In Decimal Degrees In Decimal Degrees In Decimal Degrees  
Elevation 750 In Meters In Meters In Meters In Meters In Meters In Meters In Meters  
Link to Google Map View on Google Map                
                   
 
TAXONOMIC COVERAGE:
Organisms studied Betula nana,Salix sp., Vaccinium vitis-idaea, Hylocomium splendens, Dicranum elongatum
 
Methods: Samples. Six moist acidic intertussock tundra soil cores, three organic (depth 0 to 15 cm) and three mineral (depth 0 to 10 cm) (interface between the layers was the 0 cm of the mineral core), were collected in late July 2001. Duplicate cores were randomly selected within a 100 m2 area with homogeneous vegetation cover. The cores were frozen at -20 C and shipped to the University of Florida for storage as above. Subsamples were randomly selected from each thawed core and homogenized. These were used for determination of %C and %N, pH, organic and inorganic N, total soluble protein (TSP), total free amino acids (TFAA) and proteolytic enzyme activity. Bulk density was determined (Hobbie SE, Gough L, 2002, Foliar and soil nutrients in tundra on glacial landscapes of contrasting ages in northern Alaska, Oecologia 131:453-462).
Determination of soil chemical characteristics. Total C and N were determined using a Costech ECS 4010 Elemental Analyzer (Valencia, CA). The pH was measured for each soil core using a soil:water ratio of 1:2.5 for the mineral samples and 1:10 for the organic. Inorganic N was extracted with 0.5 M K2SO4 (1:5:w:v) and determined colorimetrically using an autoanalyzer (Astoria-Pacific International, Clackamas, OR). Persulfate oxidation digestion was used to extract total dissolved nitrogen from K2SO4 extracts (Sollins P, Glassman C, Paul EA, Swanston C, Lajtha K, Heil JW, Elliott ET, 1999, Soil Carbon and Nitrogen: Pools and Fractions. Oxford University Press, Inc., New York, USA). The samples were digested at 80 C overnight; nitrate was determined colorimetrically as above. Check solutions of N-nitrate and N-glycine were included in the assay. Total dissolved organic nitrogen (DON) was calculated from this value minus the total inorganic value (DIN). Extraction of TSP was done with 0.1 M NaHCO3 (1:5:w:v) (Ladd JN, Paul EA, 1973, Changes in enzymic activity and distribution of acid-soluble, amino acid-nitrogen in soil during nitrogen immobilization and mineralization. Soil Biology and Biochemistry 5:825-840; Weintraub MN, Schimel JP, 2005, Seasonal protein dynamics in Alaskan arctic tundra soils. Soil Biology and Biochemistry 37:1469-1475). After vacuum filtration the samples were spun 5 min at 14000 rev min-1 (Eppendorf Micro Centrifuge, Model 5415C, Brinkmann Instruments, Inc., Westbury NY) and the supernatants frozen at -20 C prior to analyses. Protein was measured against ?-globulin (0 to 500 g ml-1) (Bradford MM, 1976, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye-binding. Analytical Biochemistry 72:248-254) adapted for absorbance readings using a microplate reader (BioTek Instruments, Inc., Winooski VT, Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, CA). Distilled water was used to extract TFAA (1:5:w:v) (Kielland K, 1995, Landscape patterns of free amino acids in arctic tundra soils, Biogeochemistry 31:85-98). Amino acid concentration was determined as N using leucine as the standard (0 to 2.8 g amino acid-N) and ninhydrin, which reacts with primary and secondary amines, and ammonia, as the detection agent (Lipson DA, Monson RK, 1998, Plant-microbe competition for soil and amino acids in the alpine tundra: effects of freeze-thaw and dry re-wet events, Oecologia 113:406-414). Effect of toluene concentration on proteolytic enzyme activity. Core subsamples were weighed and divided into one or 2 gram wet weight aliquots, depending on the subsequent toluene concentration; there were three replicas per concentration. The samples were placed in specimen cups at 15 C overnight; incubation buffer (see below) was added the next morning. Three toluene concentrations were tested: 1) 2 g wet soil, 0.16 ml toluene, with a final volume of 16 ml (1.0% v:v) (modified from Weintraub JN and Schimel JP 2005); 2) 1 g wet soil, 0.4 ml toluene, with a final volume of 12 ml (3.33% v:v) (Watanabe K, Hayano K, 1995, Seasonal variation of soil protease activities and their relation to proteolytic bacteria and bacillus spp in paddy field soil, Soil Biology and Biochemistry 27:197-203); and 3) 2 g wet soil, 0.8 ml toluene, with a final volume of 16 ml (5.0% v:v). Samples without toluene were 1 g wet soil and 12 ml incubation buffer. A formula ((0 or 3.33% activity [g amino acid-N]/12 ml incubation buffer) x (16 ml incubation buffer/2 g])) was used to correct the variation in mass/volume vs toluene concentration between the two different sample sets with the assumption that the effect is linear.
Soil incubation and measurement of proteolytic enzyme activity. Soil slurries were prepared by adding incubation buffer (50 mM sodium citrate, pH adjusted to within 0.1 unit of the respective soil sample) acclimated to 15 C and the respective quantity of toluene (Watanabe and Hayano, 1995; Lipson DA, Schmidt SK, Monson RK, 1999, Links between microbial population dynamics and nitrogen availability in an alpine ecosystem, Ecology 80:1623-1631; Weintraub and Schimel, 2005). Native proteolytic enzyme activity was determined on these samples; 0.3% (final concentration) casein was added to a replicate set for determination of potential proteolytic activity. All samples were incubated at 15 C for a total of 48 h. Subsamples were taken at 0, 3, 6, 12, 24 and 48 h. Enzyme activity was stopped by adding trichloroacetate (TCA) buffer (1:1:v:v) followed by freezing at -20 C (Lipson et al., 1999). Buffer controls in the absence of soil were treated exactly the same as the soil samples and will be referred to as blank corrected. Proteolytic enzyme activity was determined with modifications and adaptations for absorbance measurements using a microplate reader (Watanabe and Hayano 1995; Lipson and Monson 1998; Weintraub and Schimel, 2005). Thawed samples were spun 5 min at 14000 rev min-1, and the supernatant was used for activity determinations. Activity was measured as net amino acid production against leucine in 50% TCA buffer, which corresponded with the sample matrix and was found to have no effect on leucine concentration. Two standard curves (0 2.8 g amino acid-N and 0 7 g amino acid-N) were used depending on the activity of the samples; the lower curve was sensitive enough to detect quantities as low as 0.01 g amino acid-N. Three activity measurements were taken per sample, and activity was determined on soil dry weight or total soluble protein bases. The effect of toluene on leucine measurements was also assessed and no effect was found. Reactive NH4+ was not accounted for (see Weintraub and Schimel, 2005). Control values were subtracted from the sample values throughout the 48-h time course. Progress curves, which measure product increase over time, were used to examine reaction kinetics (Duggleby RG, 1995, Analysis of enzyme progress curves by nonlinear regression, Methods in Enzymology 249:61-90). These curves reflect the impact of changing substrate concentrations and product accumulation, providing a clearer idea of enzyme kinetics during long-term incubations. Sigma Plot (Systat Software, Inc., Richmond CA) was used to select the best curve fit.
Statistical analysis. For each soil layer the effects of toluene concentration, casein, and interaction between the two on proteolytic enzyme activity, as determined by net amino acid accumulation, between zero and 6 h, and zero and 48 h were assessed with a two-way mixed analysis of variance (ANOVA) model. Time zero and all negative values were treated as all the others. F-values were based on type III sums of square (Sokal R, Rohlf FJ,1994, Biometry. 3rd Edition, WH Freeman and Company, NY, USA). The degrees of freedom associated with appropriate F-values were computed using Satterwaites approximation (Littell RC, Milliken GA, Stroup WW, Wolfinger RD, Schabenberger O, 2006, SAS for mixed models, 2nd Edition, Cary, NC: SAS Institute Inc.). Significance was determined using a Bonferroni test at the level of p<0.05. A residual analysis was performed to check normality and homogeneity of variance (Levenes and Welchs tests), and logarithmic transformation was used to achieve normality when necessary. Repeated measures ANOVA were used to test the effects of toluene concentration (excluding the 0%) and time, and their interaction on enzyme activity for the observed linear portion of the time course curves. All statistical analyses were computed using SAS 9.1.
Note. The data collected in this study are published in Soil Biology and Biochemistry 43:70 (2011). A corrigendum was sent in January 2013 indicating that all enzyme activities, accumulation values and the total free amino acid content should be reduced by a factor of five. The data presented reflect this change as do the standard curve values described in the methods section.

Data Table

Variable Name Variable Description Data Type Units DateTime Format Code Information Missing Value Code
Soil Layer Organic layer, mineral layer text       ""=BLANKS (NO DATA)
% C percentage carbon per soil mass number percent     ""=BLANKS (NO DATA)
% N percentage nitrogen per soil mass number percent     ""=BLANKS (NO DATA)
C/N ratio percentage carbon to percentage nitrogen number number     ""=BLANKS (NO DATA)
DON dissolved organic nitrogen number microgramPerGram     ""=BLANKS (NO DATA)
TSP total soluble protein number milligramPerGram     ""=BLANKS (NO DATA)
TFAA total free amino acids number microgramPerGram     ""=BLANKS (NO DATA)
DIN dissolved inorganic nitrogen number microgramPerGram     ""=BLANKS (NO DATA)
pH acidity or alkalinity determination number number     ""=BLANKS (NO DATA)
BD bulk density number gramPerCentimeterCubed     ""=BLANKS (NO DATA)
MC moisture content number percent     ""=BLANKS (NO DATA)
Subsampling Time time subsamples taken during a time course number hour     ""=BLANKS (NO DATA)
[Toluene] toluene concentration to reduce immobilization number percent     ""=BLANKS (NO DATA)
NPEA native proteolytic enzyme activity (amino acid N/gDW) number microgramPerGram     ""=BLANKS (NO DATA)
PPEA potential proteolytic enzyme activity (amino acid N/gDW) number microgramPerGram     ""=BLANKS (NO DATA)
Matrix (Buffer) blank values (amino acid N) (blank values = control values) Note: 0 values are real (data not missing) number microgram     ""=BLANKS (NO DATA)
Matrix (Buffer + Casein) blank values (amino acid N) (blank values = control values) number microgram     ""=BLANKS (NO DATA)

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