written 4/24/00 (figures omitted)
Abstract
Rat
hepatocytes were fractionated via differential centrifugation. The fractions
were assayed for enzyme activity and subsequently separated using ammonium sulfate
fractionation, desalting by gel filtration, and ion-exchange chromatography.
Each of these fractions were assayed for enzyme activity. The Km and Vmax of
the enzyme were determined experimentally, and the protein distribution was
assayed via the Bradford technique. Finally, sodium dodecyl-sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) was used to determine which fractions contained
the enzyme.
Introduction
Glutamine
synthetase performs key functions in nitrogen metabolism, specifically that
of ammonia assimilation and glutamine biosynthesis. Glutamine synthetase metabolizes
nitrogen by an ATP-driven pathway, condensing ammonia with glutamate to form
glutamine. Glutamine then acts as a source of nitrogen in the biosynthetic reactions
of various nitrous metabolites, such as amino acids, nucleotides, and amino-sugars.
(Pfluegl/Eisenberg 1995)
Glutamine synthetase can be found in the liver
and most likely functions in the general amino acid and nitrogen metabolism
of mammalian organisms, as glutamine is utilized in the synthetic reactions
of histidine, purines, pyridine nucleotides, carbamyl phosphate and the transamination
synthetic reactions of alanine and glycine. (Tiemeier et al 1972) The large
size (Mw ~620 KDa) and the complex regulation patterns of glutamine synthetase
stem from its central role in cellular nitrogen metabolism. (Liaw, Pfluegl,
and Eisenberg 1999)
It was found in 1993 that glutamine synthetase
activity was decreased in human hepatocellular carcinomas. It was also found
that glutamate was oxidized via transamination and deamination pathways in the
liver and cirrhotic liver mitochondria. However in tumor mitochondria, glutamate
oxidation was preferentially initiated by the transamination route. (Matsuno/Goto
1993)
Materials and Methods
A
series of 2mL L-gamma-glutamylhydroxamate dilutions were made at concentrations
of 2, 1, 0.5, 0.2, and 0.1 mM, starting with the stock of 10mM L-gamma-glutamylhydroxamate.
50 mM Imidazole/HCl buffer (pH 7.2) was used for the dilutions. 2mL FeCl3 was
added to each tube and the optical density at 535nm was taken against a blank
of 2mL imidazole buffer to which 2mL FeCl3 had been added. A graph was then
constructed of these results. We then used the 1mM solution against the blank
to plot a graph of the OD at varying wavelengths by adjusting the wavelength
on the spectrophotometer to the minimum (zero absorbance) in 20nm increments
until zero absorbance can no longer be obtained.
To obtain a liver specimen, a medium sized rat
is sacrificed by a rapid method (etherization, decapitation, cervical dislocation)
and the liver removed, weighed, and placed in a 100mL beaker (on ice) containing
50mL homogenization buffer (0.25 M sucrose, 2 mM 2-mercaptoethanol, 0.2 mM EDTA,
10 mM imidazole/HCl, pH 7.5).
The tissue is then divided into several pieces
and the solution allowed to perfuse the tissue to remove blood cells. This procedure
is repeated until the RBCs are removed. The liver is then cut up in small (0.5cm)
pieces and placed in a 50mL beaker containing 10-15mL ice cold buffer. The cut
tissue is added to 4X buffer (by weight) and the cells disrupted in a Polytron
tissue disrupter or Waring blender for 2x15 seconds at medium setting.
The homogenate was then filtered through 3-4 layers
or cheesecloth, the volume measured, and all containers and cheesecloth washed
with an equal volume of buffer. The two fractions were combined and two 1mL
samples removed and saved on ice. The remaining homogenate was separated into
50mL centrifuge tubes and spun at 1000, 5000, and 20000g for 10 minutes each.
After each centrifugation the supernatants were removed and recombined and the
pellets resuspended in 20mL (total) buffer. Two 1mL samples were removed from
each fraction and stored at –70º C. A 50 µL from each tube
was removed and suspended in an equal volume of solubilization buffer, then
placed in a boiling water bath for 3 minutes and stored as above. (differential
centrifugation performed as Tate, et al. 1972)
Each fraction was then assayed at a single concentration (30 µL) in duplicate
tubes. Each tube contained 1000µL 2X reaction buffer (100mM imidazole/HCl
buffer at pH 7.2, 10mM MnCl2, 50mM Na/AsO4 buffer at pH 7.2, 0.2mM ADP, and
200mM hydroxylamine), 500µL substrate solution (100mM L-glutamine), 470µL
H2O, and 30µL enzyme. The addition of enzyme initiates the reaction. The
tubes are then incubated at 37ºC for 15 minutes and the reaction stopped
upon the addition of 2mL FeCl3. The tube is then centrifuged and the OD taken
at 535nm. (performed as Rowe et al 1972)
The following fractionation procedures were carried
out at 0ºC wherever possible (separation techniques carried out at Tate,
et al 1972). First, a 100µL sample of crude cytosol was removed to later
be used to correct our figures. The volume of crude cytosol was then measured
and saturated (NH4)2SO4 solution added to it to bring it to 30% saturation.
The solution was mixed, the volume measured, and the solution allowed to stand
for 15 minutes. It was then spun at 5000rpm for 15 minutes and the supernatant
collected and measured. To the supernatant was added enough (NH4)2SO4 to bring
the saturation to 55%. This was then mixed, measured, and allowed to stand for
15 minutes, then spun again at 5000rpm for 15 minutes. The supernatant was then
removed and the buffers of each pellet suspended in 6mL and 20mL (total) buffer,
respectively. Buffer contained 50mM KCl, 20mM MgCl2, 2mM 2-mercaptoethanol,
0.1mM EDTA, and 10mM imidazole/HCl at pH 7.5.
All three above fractions were desalted by gel
filtration using 20mL and 60mL syringes, glass wool, and 100mL of Sephadex G-25.
Glass wool was placed on the bottom of the 20mL syringe and it was packed to
the 18mL mark with Sephadex G-25 (equilibrated with the buffer). The column
was washed with 18mL buffer and allow it to run dry. The <30% sample (6mL)
was run through the column, eluted with 6mL buffer, and the fraction was saved
in a test tube. The column was then washed with 25mL buffer and used again for
the >55% sample.
Using the 60mL syringe for the 30-55% fraction,
the syringe was packed with glass wool and Sephadex G-25 to the 60mL mark and
washed with 60mL buffer. The 30-55% fraction (20mL) was run through the syringe,
eluted with 20mL buffer and the fraction was saved in a test tube. The column
was then washed with 80mL buffer.
For ion-exchange chromatography, glass wool was
placed in the bottom of a 5mL syringe which was then filled with 2mL of packed
Dowex-1. The column was washed with 10mL buffer and 5mL of the desalted 30-55%
fraction was run through the column, eluted three times with 5mL buffer. The
four fractions were collected and stored in test tubes. 20µL of each of
the above fractions were then assayed for glutamine synthetase activity as described
in the procedure above. Two 1mL samples were removed from each fraction and
stored at –70º C. A 50 µL from each tube was removed and suspended
in an equal volume of solubilization buffer, then placed in a boiling water
bath for 3 minutes and stored as above. The 30-55% fraction was saved on ice
(not frozen) for the following enzyme kinetics experiment.
To measure transferase and synthetase enzyme activity,
we prepared duplicate tubes of each of the following:
Tube 1 - 1000µL 2x transferase reaction buffer (100mM imidazole/HCl buffer
at pH 7.2, 10mM MnCl2, 50mM Na/AsO4 buffer at pH 7.2, 0.2mM ADP, and 200mM hydroxylamine)
+ 500µL L-glutamine substrate solution + 450µL H2O
Tube 2 – 1000µL 2x synthetase reaction buffer (100mM imidazole/HCl
buffer at pH 7.2, 20mM MnCl2, 25mM 2-mercaptoethanol, 200mM hydroxylamine, and
10mM ATP) + 500 L-glutamate substrate solution.
The transferase reaction was initiated upon addition
of 50µL enzyme and the tubes were incubated for 15 minutes at 37ºC.
The reaction was stopped by the addition of 2mL FeCl3 solution, the samples
were centrifuged and the ODs taken. The synthetase procedure is the same except
500µL were necessary to initiate the reaction. Five sets of duplicate
assay tubes (and one blank using water in place of enzyme) were prepared and
incubated for 2, 5, 10, 20, and 30 minutes. After the reactions were terminated
the ODs were taken. We then performed another experiment where the amount of
enzyme was varied and the final volume was kept at 2mL by adjusting with water.
Four duplicate sets of assay tubes were prepared with the enzyme/water amounts
as follows: 10/490, 25/475, 40/450, and 100/400µL. The tubes were incubated
for 15 minutes and the ODs taken.
We then performed a similar experiment where we
varied substrate concentration as shown in the Transferase and Synthetase M-M/E-H
kinetics graphs. The tubes were incubated for 15 minutes and the ODs taken.
To determine protein distribution (as Bradford
1976), we first had to construct a standard curve consisting of 10, 20, 40,
60, 80, and 100µg. The appropriate volume of 1mg/mL BSA and 0.05N NaOH
was added to duplicate test tubes to bring the sample volume to 0.1mL. Known
volumes of our samples (diluted 1:10 with NaOH) were added to other test tubes
and their volume was adjusted to 0.1mL. 5mL of Bradford Reagent (Brilliant Blue
G-250 in ethanol/phosphoric acid/water) was added to each of the tubes and thoroughly
vortexed. The tubes were incubated for 5 minutes and their ODs taken at 595nm.
Finally, all stored fractions from previous fractionations
were assayed by means of sodium dodecyl-sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). (as Laemmli 1970)
Results
The
standard curve for the concentration of L-gamma-glutamylhydroxamate was found
to have a slope of 0.52. It was also found that the optimal wavelength for the
L-gamma-glutamylhydroxamate/FeCl3 complex was around 500nm.
The hepatocytes were fractionated by means of
differential centrifugation and were separated into several fractions as shown
in Figure 5. Each fraction was then assayed for enzyme activity. These results
are also shown on Figure 5.
Ammonium sulfate fractionation was then used to
separate the crude cytosol into three fractions: <30% (NH4)2SO4, 30-55% (NH4)2SO4,
and >55% (NH4)2SO4. These fractions were then desalted by gel filtration.
The 30-55% fraction was then used to make 4 Dowex fractions by Ion-Exchange
Chromatography. Samples were removed from each of the above fractions and assayed
for enzyme activity by the same means as before. These results are also shown
in Figure 5, bottom table.
A standard curve of BSA was obtained (see Figure
7) as a reference for the enzyme kinetics and protein content experiments. The
enzyme was then assayed to determine the relationship between product and incubation
time (results in Figure 8). The effect of varying enzyme concentration on enzyme
activity was tested and a specific enzyme activity of 0.142µmoles/hour/mg
found. The effects of varying substrate concentration were tested and Michaelis-Menten
and Eadie-Hofstee graphs were constructed of each result (see Figures 10-13).
Vmax for Transferase was determined to be around 0.35-0.46, Km was uncertain
for reasons I will discuss in the next section. Vmax for Synthetase was found
to be around 3.4-3.88 and Km was also uncertain.
Each of the fractions were assayed for protein
content by the Bradford technique. These results may be viewed in Figure 6.
SDS-PAGE was then used to assay each of the fractions and to locate the band
for glutamine synthetase. Using the PAGE standard curve I constructed based
on the standards (Figure 14), I estimated where on the gel glutamine synthetase
would display a band. On Figure 15 I labeled the bands on the gel according
to where each protein showed up.
Discussion
The
slope of the standard curve of L-gamma-glutamylhydroxamate was determined graphically
to be 0.52. This is in accordance with McIlroy’s proposed slope. From
the spectrophotometry measurements, our optimal wavelength was experimentally
determined to be about 500nm, which falls short of the 535 optimal wavelength
used for the experiments. This must be due to either improper mixing of reagents
or a faulty spectrophotometer. I am inclined to believe that the spectrophotometer
was off, which means that data in all subsequent experiments would exhibit this
significant error.
Enzyme activity assays performed after the fractionation
procedure determined that the enzyme was distributed mainly in the crude cytosol
and the 30-55% (NH4)2SO4 fractions. The following enzyme kinetics experiments
determined that the enzymes did in fact display Michaelis-Menten enzyme kinetic
properties. As I explained in the previous section, the Vmax values obtained
for each graph were relatively consistent, however the Km values showed an enormous
discrepancy. I am not sure how to account for this, because the Eadie-Hofstee
equation is the same as the Michaelis-Menten equation, just rearranged. Slope
calculations for the Eadie-Hofstee graphs were calculated by computer using
Microsoft Excel. Looking at the equations, the y-intercept seems to be correct
but the slope appears to be off. Again, I don’t know how to account for
this.
Using the standard curve obtained from the standards
column of the SDS-PAGE gel, an Rf value was calculated for glutamine synthetase
and it’s relative location on the gel was then ascertained (see Figures
14 and 15). The relative locations of each protein band were then marked on
the gel and a band could then be chosen to represent glutamine synthetase. Virtually
every well showed a glutamine synthetase band except the standards and >55%
lanes.
References
Bradford, M.M. (1976) A
Rapid and Sensitive Method for the
Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye
Binding. Analytical Biochem. 72, 248-254.
Laemmli, U.K. (1970) Cleavage
of Structural Proteins During
the Assembly of the Head of Bacteriophage T4. Nature 227: 680-685.
Liaw, Pfluegl, and Eisenberg.
(1999) Structural Models for
Reaction Mechanism and Regulation of Glutamine Synthetase.
http://www.doe-mbi.ucla.edu/People/Eisenberg/Gallery/GS.html
Matsuno, T. and I. Goto. (1993)
Glutamine Synthetase and Glutaminase
Activities In Human Cirrhotic Liver and Hepatocellular
Carcinoma. http://www.cancerprev.org/Meetings/1993/abs/abs33-40/ses35/93c0096.htm
McIlroy, P.J. (2000) A
Laboratory Manual For General
Physiology.
Pfluegl, G.M.U. and D. Eisenberg
(1995) Towards the Three-
Dimensional Structure of Type II (Eucaryotic) Glutamine Synthetase From Human.
http://www.doe-mbi.ucla.edu/people/gaston/gs/hgs/hgsabs.html
Rowe, W.B., Ronzio, R.A.,
Wellner, V.P., and Meister, A.
(1972) Glutamine Synthetase (Sheep Brain). Meth. Enz. 17:900-910.
Tate,S.S., Leu, F-Y, and Meister,
A. (1972) Rat Liver
Glutamine Synthetase. J. Biol. Chem. 247: 5312-5321.
Tiemeier, T.F., and Milman,
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