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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 –70 C. A 50 L 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 L) in duplicate tubes. Each tube contained 1000L 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), 500L substrate solution (100mM L-glutamine), 470L H2O, and 30L enzyme. The addition of enzyme initiates the reaction. The tubes are then incubated at 37C 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 0C wherever possible (separation techniques carried out at Tate, et al 1972). First, a 100L 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. 20L 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 –70 C. A 50 L 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 - 1000L 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) + 500L L-glutamine substrate solution + 450L H2O
Tube 2 – 1000L 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 50L enzyme and the tubes were incubated for 15 minutes at 37C. 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 500L 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 100/400L. 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 100g. 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 0.142moles/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, G. (1972) Chinese Hamster Liver
Glutamine Synthetase
. J. Biol. Chem. 247: 2272-2277.


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