[KINETICS OF S-NITROSO-L-CYSTEINE UPTAKE IN RAW 264.7
MACROPHAGES]{.underline}, [Meagan M. Kochel]{.underline}, Ripon College,
Chemistry Department, Ripon, WI 54971, Dr. Neil Hogg\*, Medical College
of Wisconsin, Department of Biophysics, Milwaukee, WI 53226,
nhogg@mcw.edu

Michaelis-Menten constants (K~m~'s) represent the dissociation constants
of specific enzymes. Due to the nature of enzymes and the fact that they
are almost always less concentrated than their specified substrates, an
enzymatic reaction will reach a maximum rate or velocity when all enzyme
particles are complexed with substrate. At that point, the reaction can
only proceed as fast as the product particles can detach. The idea of
V~max~ describes the maximum speed a certain reaction can possibly go at
a given concentration, when all reaction sites are in use, and the K~m~
is the substrate concentration at half the V~max~. When an enzymatic
reaction follows Michaelis-Menten kinetics, the rate increases steeply
at lower concentrations but begins to level out and become constant as
the enzyme becomes saturated; a graph of concentration versus rate would
take the form of a rectangular hyperbola.

L-Cysteine is an amino acid with a thiol group that can be modified to
form S-nitroso-L-cysteine, or Cys-NO. Cys-NO stops respiration in cells
and turns critical intracellular thiols into intracellular -SNO's
(S-nitrosothiols), and at concentrations higher than 500µM, eventually
kills the cells. It is known to break down in light, but was also found
to break down even more quickly when left at room temperature or with
exposure to any amount of metal ions. For this reason, all reactions
with Cys-NO need to be done in the presence of DTPA (a metal chelator)
and any samples have to be kept on ice. This compound enters cells
through an L-amino acid transport (L-AT) protein, however, the K~m~ and
V~max~ are not known. The objective of this research was to find them,
which would also prove the enzymatic transport mechanism. By measuring
the rate of uptake over a given time period at varying concentrations of
Cys-NO and then plotting the rates versus the starting concentrations,
the K~m~ and V~max~ can be extrapolated. To measure the rate, the
extracellular concentration of Cys-NO can be measured at consecutive
intervals and plotted versus time. The concentration of Cys-NO can be
tested in several ways. The first is the Griess-Saville reaction which
measures the total nitrite level and then the total --SNO level, with
the difference between the two being the Cys-NO concentration; however,
this proved relatively inaccurate. The second and presumably the best
method of measuring Cys-NO concentration is to measure the absorbance at
334nm with a spectrophotometer. As the extinction coefficient is 0.9 for
a 1mM solution, the concentration can quite easily be calculated using
Beer's law (A=εbc). This method is clear and accurate until about 25µM,
wherein the data starts to get rather noisy and the instrument reaches a
sensitivity threshold. The final method, which would be useful for
concentrations under what the spectrophotometer was capable of
measuring, was the use of chemiluminescence.

To get an idea for the time frame of this reaction, the uptake was first
tested in twenty minute intervals over an eighty minute time frame, over
RAW cells. Samples were taken in triplicate and put directly onto ice.
The uptake leveled out after about forty minutes, so the time frame for
testing was adjusted, with samples taken every five minutes for forty
minutes. Concentrations were tested both above and below 50µM to
determine relative placement on the curve, and from these it was
determined that the rate was still climbing. Assays were done at
concentrations up to 1mM, however, any rate taken above 500µM was
unreliable due to the number of cells that were dying, which happened
sooner and sooner as the concentration was increased. Even with
adjustments to the time frame, it soon became close to impossible to
obtain a confident rate at all.

When the rates that were reliable were graphed against their starting
concentrations, a best-fit Km and V~max~ were found, however, it is
quite possible that an entirely correct Km and V~max~ cannot be found
because of the detrimental effects of Cys-NO on a cell's ability to
survive. Though there is plenty of data to support the lower end of the
curve, the higher saturation points are not well represented in the
data, and so the more influential part of the curve might be different
than what is proposed by these data.