Question: The investigators purified the enzyme sphingosine kinase using rat kidneys as a source of enzyme …

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 sphingosine-1-phosphate (SPP). Kidneys were homogenized in
buffer, filtered, and then fractionated by ammonium 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). The results are shown in the following figure.
Fractions were collected and assayed for sphingosine kinase in a
procedure that involved the addition of sphingosine to radioactively labeled (?-32P) ATP
and the subsequent quantitation of 32P in the SPP
product.

a. At what concentration range of NaCl
does most of this enzyme elute from the DEAE column?

b. 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.

c. The investigators next carried out
simple kinetic analyses using sphingosine and ATP as substrates in
separate experiments. 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 KM and Vmax values in the absence and presence of the
inhibitor. What kind of an inhibitor is
threo-dihydrosphingosine? Explain your answer
completely.