Activation of Enzymes, HIV, and HSPs
NSF Grant # DUE0653234
David Coreas
Molecular Biology
dvdcoreas@yahoo.com
Elena Betancourt
Physiology and Neuroscience
ebetancourt@ucsd.edu
Melody Sycks
Biochemistry
msycks@ucsd.edu
Activation of Enzymes
The goal of Moores UCSD Cancer Center's Continuing
Umbrella of Research Experiences program in Cancer Cells, is
to understand the glucose metabolism in cancer tissues by
measuring the activity of enzymes: 1phosphofructokinase
(PFK-1), Muscle 1phosphofructokinase (mPFK1), Liver
1phosphofructokinase (lPFK1) heart 1phosphofructokinase
(hPFK1), lactate dehydrogenase (mLDH). The role/effect that
Vitamin C has in glycolysis.
PFK-1 
Hsp’s
The aggregation (clumping together of denatured proteins) of
proteins is harmful to cells, and are indicatory of diseases such
as Alzheimer's, Parkinson’s, viral disease, and fever. Hep G2
(Hepatocellular Carcinoma), produce different heat shock
proteins (HSP’s), as a defense mechanism against heat stress.
Hsp70 prevents protein aggregation; Hsp90 is responsible for
the creation, destruction, and maintenance of proteins; both
HSP70 and HSP90 assist in the folding of proteins; HSP27 acts
as a chaperone to preventing denatured proteins from
aggregating and plays an important role in stabilizing them.
Method
Results
Activation of Enzymes
To measure the activity of the enzymes, different methods were
used: Dimensional analysis (mathematical calculation) to weigh
chemicals and prepare dilutions, spectrophotometers used to
measure inhibition/purification/activity/stabilization of muscle
enzymes (phosphofructokinase-1, PFK-1; aldolase; and lactate
dehydrogenase ,mLDH)
Hsp’s
The experiment being conducted on Hsp70, 90, and 27,
involves locating and identifying Hsp’s. Data is then analyzed
to determine their relative concentrations after predetermined
stress conditioning. The induction of Hsp’s in HepG2 is
measured through the use of Protein Concentration Assay,
LDS-PAGE, PCR, and Western Blot techniques.
HIV
Three decades of Human Immunodeficiency Virus (HIV)
research has resulted in various antiviral drugs which target
viral proteins such as Protease (PR), Reverse Transcriptase,
Integrase, or the cellular receptors involved in viral entry.
Roughly 32 inhibitors, 11 of which target protease, have been
approved by the FDA since the first Protease Inhibitor (PI) was
launched in 1995. With the use of Green Fluorescent Proteins
(GFP), acting as a downstream reporter, assays can be
developed that show whether or not Protease has been
inhibited.
VOL (mL)
150
Tvol
150
eu/mL A
0.2835
Teu A
42.525
eu/mL P
0.2628
Teu P
39.42
eu/mL L
0.2349
Teu L
35.235
MTP8
0
MTP8 *40
0
10
160
0.3495
3.495
0.2572
2.572
0.3354
3.354
0.04
0.2
10
170
0.3197
3.197
0.2194
2.194
0.0480
0.48
0.04
0.2
10
180
0.2038
2.038
0.1921
1.921
0.0892
0.892
0.04
0.2
10
190
0.1757
1.757
0.2177
2.177
0.1156
1.156
0.60
3
10
200
0.1246
1.246
0.2182
2.182
0.0567
0.567
0.60
3
10
210
0.0597
0.597
0.1828
1.828
0.0429
0.429
0.6
3
10
220
0.0352
0.352
0.1842
1.842
0.0312
0.312
0.60
3
Eluted
40TP8E/DT
T
40TP8E/DT
T
40TP8E/DT
T
600TP8E/DT
T
600TP8E/DT
T
600TP8E/DT
T
600TP8E/DT
T
Table 1: 50 grams of rabbit muscle measured in different
concentrations and environments at volumes from 150mls to
220mls.
7/11/11 RM PFK-1 1st DEAE Sephacel
Hsp’s
Protein concentration assay, LDS-PAGE, PCR and Western
Blot techniques were used to locate and identify heat shock
proteins. Hsp70 and Hsp27 were induced by heat shock,
whereas the presence of Hsp90 was independent of heat shock.
This means that Hsp70 and Hsp27 production in a cell
increases when exposed to heat stress. Heat stress is not a
factor in Hsp90 production.
Tritons
Interpretation:
-Hsp24 production progressively increased with resting time
after stress.
5
4.5
4
3.5
Teu A
3
Teu P
2.5
Teu L
2
MTP8 *40
1.5
1
0.5
0
150
160
170
180
190
200
210
220
230
(30ug/µl)
Volume (ml)
(10 ug/µl)
Graph 1: Represents an equilibrium of enzymes from 200mls to
220mls.
HIV
HIV
Research is ongoing.
Illustrates the
process of DNA
amplification
HSP90 
Conclusion
Activation of Enzymes
Teu A, Teu P, Teu L, MTP8*40
Introduction
HIV
The premise of the assay is to monitor the effectiveness of a
protease inhibitor by the use of five major components: Protease
(viral-enzyme), Protease Inhibitor (peptide library), Upstream
Activation Sequence (‘UAS’/promoter sight), GFP (Downstream
Reporter), and Gal4 (2-domain structure that binds to the UAS; if
un-cleaved by viral-enzymes).
By expressing a random peptide library within the cytoplasm of a
cell, it is possible to interfere with the proteolytic activity of
protease and keep it from cleaving the long precursor protein of
HIV into the functional HIV proteins.
Research on peptide libraries acting as Protease Inhibitors is
ongoing novel research , and any conclusions drawn at the
moment are speculative.
Hsp’s
Western Blot Results Hsp90
Low
Concentration
----(10ug/20µl)
High concentration
(30 ug/20µl )---->
Hep G cells were exposed to heat stress and then rested for different periods of time. Four
different examples are shown: Standard “C,” No rest time “HS0,” four hours rest “Hs4,” and
twenty-four hours rest “Hs24.”
Interpretation:
-Pictured above: results from Hsp90 show no change in concentration.
-Picture blow: results from Hsp70 show progressive increase in concentration over resting
times.
Western Blot Results Hsp70
HIV Virus 
Assay developed to
monitor proteolytic
activity by expression
of green-florescent
protein.
High
concentration
(30 ug/20µl)
Low
Concentration
(10ug/20µl)
(HS0, HS24, HS4, C)
Activation of Enzymes
From 50 grams of rabbit muscle, there were 40.3 total enzymes
units, a 4.26% yield of enzymes, a specific activity of
0.61 eu/mg, and a 5.2 fold purification.
Acknowledgements
Special thanks to: Moores Cancer Center, National Science
Foundation, LIPP Family Foundation, M.E.S.A. Program ,
Wolkowicz Laboratory, De Maio Laboratory, Percy Russell
Laboratory, Dr. Lawrence Alfred, Dr. Raga Bakhiet, Dr. Nouna
Bakhiet, Dr. Antonio De Maio, Dr. David Cauvi, Dennis
Hawisher B.S., Dr. Percy Russell, Dr. Roland Wolkowicz,
Aleksandr Stotland PhD Candidate, Dean Kathy Tyner, and
Dr. Percy Russell