Case 19 Purification of Rat Kidney Sphingosine Kinase Focus concept The purification and kinetic analysis of an enzyme that produces a product important in cell survival is the focus of this study. Prerequisites Protein purification techniques and protein analytical methods. Basic enzyme kinetics. Background Sphingolipid metabolites have recently been shown to be involved in cell viability. For example, ceramide has been shown to stimulate apoptosis, or programmed cell death, whereas sphingosine-1- phosphate (SPP) promotes cell survival. A number of different factors influence the levels of ceramide and SPP in the cell, as shown in Figure 19.1. Clearly, the balance of ceramide and SPP will be one of the many factors that influences whether a cell lives or dies. An understanding of the biochemical processes involved in regulating these sphingolipids has important applications in the treatment of cancer. We will concentrate on sphingosine- 1-phosphate (SPP). SPP is synthesized in the cell from sphingosine and ATP as shown in Figure 19.2. The enzyme that catalyzes the reaction is sphingosine kinase. In this case, we will study the purification of the sphingosine kinase enzyme. 1
Radiation Anticancer drugs TNF- Platelet-derived growth factor Other growth factors Ceramide Spingosine-1-phosphate (SPP) Programmed Cell Death Cell Survival Figure 19.1: Factors (Apoptosis) that influence cellular levels of ceramide and spingosine-1-phosphate. O - O P O - HO OH O OH 2 NH 3 + + ATP sphingosine kinase 2 NH 3 + + ADP ( 2 ) 12 ( 2 ) 12 3 Figure 19.2: Synthesis of sphingosine-1-phosphate from sphingosine and ATP. 3 Questions 1. The investigators purified the enzyme sphingosine kinase using rat kidneys as a source of enzyme since it had been shown previously that kidneys contain a high concentration of SPP. Kidneys were homogenized in buffer, filtered, and then fractionated by ammonium 2 2 OH H 2 N C 2 OH 2 OH Figure 19.4: Structure of Tris conjugate base.
Kinase activity, units Protein, mg sulfate precipitation. The ammonium sulfate precipitate was dissolved in buffer and then applied to a DEAE-cellulose (anion exchange) column. The investigators eluted the protein using Tris buffer at ph = 7.4 with increasing concentrations of sodium chloride (from 0 to 0.50 M). Fractions were collected and assayed for sphingosine kinase in a procedure that involved the addition of sphingosine to radioactively labeled (γ- 32 P) ATP and the subsequent quantitation of 32 P in the SPP product. The results are shown in Figure 19.3. Compared to the other proteins in the rat kidney, what statements can you make about the sphingosine kinase enzyme, based on its elution behavior from the DEAE column? 2. The investigators reported that the sphingosine kinase enzyme bound to the DEAE column when Tris was used as a buffer, but not when a phosphate buffer was used. The structure of the conjugate base of Tris buffer is shown in Figure 19.4. (Both buffers were adjusted to an identical ph of 7.4.) Explain why. 20000 0.5 M NaC 25 15000 10000 20 15 10 5000 0 M NaC 5 3. The 0 0 50 100 150 200 Fraction number 0 Kinase activity, units Figure 19.3: Elution profile of rat kidney homogenate on DEAE-cellulose (based on Olivera, et al., 1998). investigators carried out several additional chromatographic steps and finally obtained a fraction that contained pure sphingosine kinase, as determined by SDS-PAGE. The molecular weight was determined by comparing the migration of the enzyme with the migration of standard molecular weight markers in Lane 1. The investigators also carried out an additional experiment in which one 3 Protein, mg
batch of the enzyme was treated with the reducing agent β-mercaptoethanol (Lane 2). A second batch was not treated (Lane 3). The results are shown in Figure 19.5. a. Estimate the molecular weight of sphingosine kinase. b. Interpret the results of the experiment described above. What can you say about the structure of sphingosine kinase? 4. The investigators next carried out simple kinetic analyses using sphingosine and ATP as substrates in separate experiments. Michaelis-Menten plots and the corresponding Lineweaver-Burk plots are shown in Figures 19.6 and 19.7. a. Calculate the K M and v max values for both substrates. b. The activity of sphingosine kinase was measured in the presence of threo-dihydrosphingosine, a stereoisomer of sphingosine. The Lineweaver-Burk plot obtained from a kinetic analysis of sphingosine kinase activity carried out in the presence of this inhibitor is shown in Figure 19.6. Calculate the K M and v max values in the presence of the inhibitor. What kind of an inhibitor is threo-dihydrosphingosine? Explain your answer completely. 4
(Kinase activity) -1, mg/min/ mol) Mwt markers (Kd) 1 2 3 102 Figure 19.5: SDS-PAGE gel of the sphingosine kinase purification (based on Olivera, et al., 1998). 78 50 34.2 28.3 0.12 0.1 y = 0.271x + 0.011 0.08 0.06 0.04 y = 0.0580x + 0.0081 0.02 0 Figure 19.5: SDS-PAGE gel of purified -0.2 sphingosine -0.1 kinase. 0 0.1 0.2 0.3 0.4 [Sphingosine] -1, M -1 No inhibitor With threo-dihydrosphingosine Linear (With threo-dihydrosphingosine Figure 19.6: Lineweaver-Burk analysis of sphingosine kinase activity using varying amounts of sphingosine substrate, in the presence and in the absence of the inhibitor threodihydrosphingosine (based on Olivera, et al., 1998). 5
(Kinase activity) -1, mg/min/ mol) 0.014 0.012 0.010 y = 0.00047x + 0.00781 0.008 0.006 0.004 0.002 0.000-18 -16-14 -12-10 -8-6 -4-2 0 2 4 6 8 10 [ATP] -1, mm -1 Figure 19.7: Lineweaver-Burk analysis of sphingosine kinase activity using varying amounts of ATP substrate (based on Olivera, et al., 1998) Reference Olivera, A., Kohama, T., Tu, Z., Milstein, S., and Spiegel, S. (1998) J. Biol. Chem. 273, 12576-12583. 6