EnzymeKineticsassayoftheWT

To assay 17 b-HSD activity in lysates, cells were harvested 48h after transfection using PBS Enzyme Free Cell Dissociation Solution ( specialty Media Inc., Lavellette, NJ), frozen on dry ice and stored at -80 C. 

Cell pellets were thawed on ice, resuspended in : 

* 10mM Tris-HCl(pH 7.4)/150mM KCL/1 mM EDTA/2mM Dithiothreitol (DTT) at protein concentration of 3-6 mg/ml, and snap-frozen on dry ice . After thawing at 37?C, the extracts were chilled on ice and sonicated for 30sec.

A typical assay for establish the apparent Vmax and Km of the Substrate ANDROST-4-ENE-3,17-DIONE of the normal & mutant HSD17B3 enzyme used: 

1. *

 20-30 microgram 

2. of total protein in

 0.2 mL of 100mM Tris - citrate(pH6)/2mM DTT.

1. * Steroid substrate was added in different concent.

(0.1-8 microM)

2. , and NADPH was added to a final cocent. of 2mM.

1. * Reactions were initiated by the addition of enzyme and were carried out for 25? 30?min at 37 ?C .

A typical assay to establish the apparent pH of the normal & mutant HSD17B3 enzyme used: 

1. * Serial variation of pH (range4.5pH -8.5pH), for the reaction Buffer :

 100mM Tris -citrate/2mM DTT

2. .

1. * Steroid substrate was added at final concent. of 5 mM ( where 1 mM were 14C- ANDROST-4-ENE-3,17-DIONE and 4mM were cold substrate).

Transfection of DNA into 293T cells by using LIPOFECTAMINE PLUS Reagent package (LIFE TECHNOLOGIES Cat.No.10964-013): 
Protocol 

1. * The day before transfection, split and plating the cells, so the that they are 50%- 60% confluent the day of transfection. Avoid antibiotics at the time of transfection and during.

1. * Culture vessel:

100mm

1. * plasmid DNA ;

 4 microg


2. * plasmid b-gal ;

 1 microg

1. * PLUS reagent;

 20 microliter

1. * LIPOFECTAMINE PLUS Reagent;

 30 microliter

1. * Incubate at 37癈 at 5% CO2 for 3h

 (see LIFE TECHNOLOGIES protocol.)

1. * After 3h inc., increase volume of medium to normal volume

 (see LIFE TECHNOLOGIES protocol.)

1. * The cell extract were assayed for reporter gene activity 48h after the start of transfection.