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martys_data_sheet.xlsx
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Sheet1

Effect of pH on alcohol dehydrogenase activity:
activity (NADH/min)
pH mean standard deviation
1 0.0 0.0 Activity was measured as NADH produced per minute (measured using a spectrophotometer at 340 nm)
2 1.2 0.3
3 3.5 1.2 The reaction buffer was 100 mM HEPES, 2 mM EDTA, 2 mM MgCl2, 500 mM NAD+, 500 mM ethanol
4 8.5 2.3
5 12.2 3.2 For pH studies, the HEPES buffer was changed from 1-14 at 30°C
6 26.9 3.5
7 51.2 6.3 For temperature studies, the pH was 7 and temperature changed from 5-70°C
8 35.7 4.6
9 22.2 3.5
10 13.3 2.4 For seqeuncing I took 1 g of mouse liver, ground the tissue with a mortar and pestle, and extracted genomic DNA using the
11 4.3 1.2 "Quick DNA" kit from Bio-Rad. I used PCR to amplify the amylase gene using these primers:
12 0.9 0.2
13 0.5 0.3 ALD-F 5'-ATGGCTGGCCTTGCTACGGGC-3'
14 0.0 0.0 ALD-R 5'-AATCTACCATGTTTGGTTGAC-3'
I then used 22 uL of "Quick PCR Reagent" (Bio-Rad) with 1 uL of genomic DNA and 1 uL of each primer (above).
Effect of temperature on alcohol dehydrogenase activity: Thermocycler conditions were: 94°C for 10 min, 35 cycles of [94°C for 30 s, 55°C for 30 s, 72°C for 1 min], followed by
72°C for 10 min
activity (NADH/min)
Temp °C mean standard deviation I sent the amplified product for sequencing to TCAG Laboratories and processed the sequence using the FinchTV program
5 3.5 1.0
10 5.7 1.2
15 10.5 1.3 For enzyme analyses, I extracted protein from 1 g of frozen mouse liver (with mortar and pestle) using a buffer consisting of 50 mM HEPES (pH 7.0),
20 50.3 5.2 2 mM EDTA, 2 mM MgCl2. I filtered the extract through cheesecloth and then centrifuged the suspension at 13,000 rpm for 10 min at 4°C, and then collected the supernatant
25 100.6 12.5 (containing the enzymes) for the activity analyses.
30 181.0 20.5
35 225.4 19.9
40 180.3 17.9
45 65.5 8.5
50 12.2 3.2
55 5.1 1.2
60 1.0 0.2
65 0.0 0.0
70 0.0 0.0

Sheet2

Sheet3