Activity of TbTryS measured by HPLC

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Asset Title	Activity of TbTryS measured by HPLC			The name of the data file
Uploader	Alejandro Leroux			The person submitting the asset to SEEK
Uploader SEEK ID	346			If you add your own SEEK ID, this will help us link this asset with your profile
Project	SilicoTryp			The project that the asset belongs to

ASSAY
Assay SEEK ID	194			If referring to an exisitng Assay, you can link to it via the Assay SEEK ID.
Assay Title	Trypanothione synthetase Gsp and T(SH)2 production measured by HPLC			The title of an exisitng assay
Assay_type	discontinuousEnzymatic			The assay_type describes the type of experiment you are performing
Technology_type	enzymatic_activity_measurements			Describes the type of instruments and/or equipment used for the experiment
Description	This assay is designed to obtain the in vitro kinetic data of T. brucei brucei trypanothione synthetase (TbTryS). The enzyme catalyzes the ATP-dependent ligation of spermidine (Spd) and GSH to generate glutathionylspermidine (Gsp) and also of Gsp and GSH to finally produce trypanothione [T(SH)2]. The data was obtained by taking different samples of the assay and analyzing them with an HPLC-linked method.			A brief, human readable description.
Experimentalist	Alejandro Leroux			The names of the people who carried out the experiments. These can either be SEEK members or external scientists
Date	2013-01-30T0:0:0+0000			The start date for the experiment if different from the upload date
SOP	https://seek.sysmo-db.org/sops/180	https://seek.sysmo-db.org/sops/181	https://seek.sysmo-db.org/sops/189	Links to SOPs and protocols used to carry out the experiment. If they are already in SEEK, you can refer to them by their SEEK ID
Publication (optional)	PMID: 23814051			If this data appears in a publication, you can link it directly, or via the assay or study. If it is already registered in SEEK, you can use the PubMed ID or DOI as a reference.

Experimental_conditions
Item	pH	buffer	temperature	The name of the experimental condition you are fixing in your experiment (e.g. temperature, concentration, pH etc). If there is more than 1, please list them in columns across the spreadsheet
Compound (if concentration)				The compound name is only required if the item is concentration.
Unit				The SI units of the experimental conditions measurements.
Start_value (optional)				This field is used for recording changes throughout the experiment to measure different conditions (e.g. pH or dilutions)
End_value (optional)				This field is used for recording changes throughout the experiment to measure different conditions (e.g. pH or dilutions)
Comments	TbTryS activity was measured at 37°C in the in vivo-like buffer. All substrate stock solutions were prepared in the in vivo-like buffer and the pH was adjusted to 7.0. The assays were performed in a final volume of 2 ml and contained 0.2 mM NADH, 1 mM phosphoenolpyruvate, 4 units pyruvate kinase, 4 units L-lactate dehydrogenase, 0.17 µM TbTryS, 2.1 mM ATP, and varying amounts of GSH and spermidine.			Additional information that would be useful for people reading this data file

Culture growth	CultureGrowth

FACTORS_STUDIED
Item	enzymeActivity	enzymeActivity	enzymeActivity	Factors Studied are the factors that you deliberately change during the course of your experiment to see the effects. For example, if you measure the concentration of compounds at pH 5, 6 & 7, the factor studied is the pH (i.e. it is about the treatment you apply rather than the measurements you make).
Compound (if concentration)				The compound name is only required if the Factors Studied item is concentration.
Unit				The SI units of the factors studied measurements
Start_value (optional)				This field is used for recording changes throughout the experiment for one particular factor studied (e.g. pH in crease from 4 - 7 or dilution rate doubling)
End_value (optional)				This field is used for recording changes throughout the experiment for one particular factor studied (e.g. pH in crease from 4 - 7 or dilution rate doubling)
SD (optional)				The standard deviation for the difference in factors studied measurements (optional)
Comments				Additional information that would be useful for people reading this data file





TbTryS activity was measured at 37°C in the in vivo-like buffer. All substrate stock solutions were prepared in the in vivo-like buffer and the pH was adjusted to 7.0. The assays were performed in a final volume of 2 ml and contained 0.2 mM NADH, 1 mM phosphoenolpyruvate, 4 units pyruvate kinase, 4 units L-lactate dehydrogenase, 0.17 µM TbTryS, 2.1 mM ATP and varying amounts of GSH, and Spd.



Experiment A: 0.33 mM GSH and 8 mM Spd
Experiment B: 0.33 mM GSH and 1.6 mM Spd
Experiment C: 2 mM GSH and 8 mM Spd

SOP	https://seek.sysmo-db.org/sops/180	https://seek.sysmo-db.org/sops/181

Abreviation	Full name	ChEBI ID	Uniprot ID
Spd	Spermidine	CHEBI:16610
Gsp	N-glutathionylspermidine	CHEBI:16613
ATP	Adenosine-5'-triphosphate	CHEBI:15422
GSH	Glutathione	CHEBI:16856
T(SH)2	Trypanothione	CHEBI:17842
TbTryS	Trypanosoma brucei trypanothione synthetase		Q8IEX1

	Assay						[Gsp] production		[T(SH)2] production
	Total Assay Vol.	[Spd]	[ATP]	[GSH]	[TbTryS]	Assay Time	Mean	SD	Mean	SD
Unit	µl	µM	µM	µM	µM	min	µM	µM	µM	µM
	2000	8000	2100	330	0.17278065010960522	0	0	0	0	0
	2000	8000	2100	330	0.17278065010960522	0.3	7.050000000000001	0.4949747468305834	0.13333333333333333	0.23094010767585033
	2000	8000	2100	330	0.17278065010960522	0.7	11.333333333333334	0.6806859285554041	0.6333333333333334	0.3055050463303894
	2000	8000	2100	330	0.17278065010960522	1	15.033333333333331	2.402776172125354	1.0999999999999999	1.0148891565092222
	2000	8000	2100	330	0.17278065010960522	1.3	19.233333333333334	4.73849483837781	2.2333333333333334	1.4011899704655804
	2000	8000	2100	330	0.17278065010960522	1.7	22.733333333333334	2.5579940057266217	4.033333333333333	1.050396750439248
	2000	8000	2100	330	0.17278065010960522	2	24.3	2.951270912674741	4.833333333333333	1.2858201014657256
	2000	8000	2100	330	0.17278065010960522	4	40.699999999999996	3.517101079013795	16.333333333333332	2.7537852736430475
	2000	8000	2100	330	0.17278065010960522	8	53.86666666666667	4.654388610046793	44.1	6.999999999999967
	2000	8000	2100	330	0.17278065010960522	15	64.93333333333334	4.2594991880892925	99.66666666666667	14.483208668431606
	2000	8000	2100	330	0.17278065010960522	24	76.69999999999999	10.4651803615609	156	15.697770542341347
	2000	1600	2100	330	0.17278065010960522	0	0	0	0	0
	2000	1600	2100	330	0.17278065010960522	0.3	1.3		0.15000000000000002	0.07071067811865474
	2000	1600	2100	330	0.17278065010960522	0.7	4.8999999999999995	0.529150262212918	0.7666666666666666	0.20816659994661382
	2000	1600	2100	330	0.17278065010960522	1	6.633333333333333	0.3055050463303896	1.5999999999999999	0.6082762530298214
	2000	1600	2100	330	0.17278065010960522	1.3	8.033333333333333	0.5686240703077323	2.8666666666666667	0.98657657246325
	2000	1600	2100	330	0.17278065010960522	1.7	9.166666666666666	0.907377172587747	4.2	1.345362404707368
	2000	1600	2100	330	0.17278065010960522	2	9.8	0.9899494936611668	5.75	1.3435028842544428
	2000	1600	2100	330	0.17278065010960522	4	12.366666666666667	1.40118997046558	14.633333333333333	4.314317249963578
	2000	1600	2100	330	0.17278065010960522	8	15.533333333333331	8.483120495037983	34.23333333333333	6.900966115938688
	2000	1600	2100	330	0.17278065010960522	15	17	3.5552777669262334	93.5	27.272146963523067
	2000	1600	2100	330	0.17278065010960522	24	15.3	0.14142135623731025	123.8	17.253405460951765
	2000	8000	2100	2000	0.17278065010960522	0	0	0	0	0
	2000	8000	2100	2000	0.17278065010960522	0.3	3.3	0.42426406871192857	0	0
	2000	8000	2100	2000	0.17278065010960522	0.7	5.533333333333334	0.6350852961085887	0.06666666666666667	0.05773502691896258
	2000	8000	2100	2000	0.17278065010960522	1	7.5	1.5588457268119917	0.13333333333333333	0.11547005383792516
	2000	8000	2100	2000	0.17278065010960522	1.3	9.8	3.394112549695417	0.35	0.3535533905932738
	2000	8000	2100	2000	0.17278065010960522	1.7	11.800000000000002	1.7999999999999867	0.6	0.2000000000000001
	2000	8000	2100	2000	0.17278065010960522	2	13.45	2.4748737341529163	0.9	0.2828427124746191
	2000	8000	2100	2000	0.17278065010960522	4	21.766666666666666	1.8876793513023689	3.3666666666666667	0.9451631252505223
	2000	8000	2100	2000	0.17278065010960522	8	31.900000000000002	6.148983655857279	7.833333333333333	0.945163125250522
	2000	8000	2100	2000	0.17278065010960522	15	43.56666666666666	2.821937868439581	27.266666666666666	4.754296302643877
	2000	8000	2100	2000	0.17278065010960522	24	52.4		55.6