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Hej Dustin,
<p>try this, relatively cheap and non radioactive, ...works.
<p>good luck
<br>
<p>Regulation of phosphotransferase activity of hexokinase 2 from Saccharomyces
cerevisiae by modification at serine-14
<br>Golbik R, Naumann M, Otto A, Muller EC, Behlke J, Reuter R, Hubner
G, Kriegel TM
<br>BIOCHEMISTRY : 40 (4): 1083-1090 JAN 30 2001
<p>Abstract:
<br>Isoenzyme 2 of hexokinase functions in sugar sensing and glucose repression
in Saccharomyces cerevisiae, The degree of in
<br>vivo phosphorylation of hexokinase 2 at serine-14 is inversely related
to the extracellular glucose concentration [Vojtek, A. B.,
<br>and Fraenkel, D. G. (1990) fur. J, Biochem. 190, 371-375]; however,
a physiological role of the modification causing the
<br>dissociation of the dimeric enzyme in vitro [as effected by a serine-glutamate
exchange at position 14; Behlke et al. (1998)
<br>Biochemistry 37, 11989-11995] is unclear. This paper describes a comparative
stopped-flow kinetic and sedimentation
<br>equilibrium analysis performed with native unphosphorylated hexokinase
2 and a permanently pseudophosphorylated
<br>glutamate-14 mutant enzyme to determine the functional consequences
of phosphorylation-induced enzyme dissociation, The
<br>use of a dye-linked hexokinase assay monitoring proton generation allowed
the investigation of the kinetics of glucose
<br>phosphorylation over a wide range of enzyme concentrations. The kinetic
data indicated that monomeric hexokinase represents
<br>the high-affinity form of isoenzyme 2 for both glycolytic substrates.
Inhibition of glucose phosphorylation by ATP [Moreno et
<br>al, (1986) Eur, J, Biochem. 161, 565-569] was only observed at a low
enzyme concentration, whereas no inhibition was
<br>detected at the high concentration of hexokinase 2 presumed to occur
in the cell. Pseudophosphorylation by glutamate
<br>substitution for serine-14 increased substrate affinity at high enzyme
concentration and stimulated the autophosphorylation of
<br>isoenzyme 2, The possible role of hexokinase 2 in vivo phosphorylation
at serine-14 in glucose signaling is discussed.
<p>dustjohn@hotmail.com wrote:
<blockquote TYPE=CITE>I am in need of a decent protocol to measure ATP
hydrolysis in solution. One way to do it is to hydrolyze radioactive
ATP (cleave off the gamma-32P), use charcoal and TCA to remove protein/ATP/ADP
and then scintillation count an aliquot of the supernatant. However
this method is not the greatest because there are high background levels
of 32PPi in the supernatant if the ATP solution is not pure. Does
anybody have any suggestions as to what I should do? Are there any
relatively cheap non-radioactive ATPase assays? Any ideas of a good
positive control (I'm working with a Ser/Thr protein kinase so any enzyme
along this line would be fantastic).
<p>Thanks for your help!
<p>Dustin
<p><a href="http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0">http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0</a></blockquote>
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