Protocol for ATPase Assay? i.e. radioactive vs non-radioactive
Gregor.Madej at mpibp-frankfurt.mpg.de
Wed Nov 13 13:39:35 EST 2002
try this, relatively cheap and non radioactive, ...works.
Regulation of phosphotransferase activity of hexokinase 2 from Saccharomyces cerevisiae by modification at serine-14
Golbik R, Naumann M, Otto A, Muller EC, Behlke J, Reuter R, Hubner G, Kriegel TM
BIOCHEMISTRY : 40 (4): 1083-1090 JAN 30 2001
Isoenzyme 2 of hexokinase functions in sugar sensing and glucose repression in Saccharomyces cerevisiae, The degree of in
vivo phosphorylation of hexokinase 2 at serine-14 is inversely related to the extracellular glucose concentration [Vojtek, A. B.,
and Fraenkel, D. G. (1990) fur. J, Biochem. 190, 371-375]; however, a physiological role of the modification causing the
dissociation of the dimeric enzyme in vitro [as effected by a serine-glutamate exchange at position 14; Behlke et al. (1998)
Biochemistry 37, 11989-11995] is unclear. This paper describes a comparative stopped-flow kinetic and sedimentation
equilibrium analysis performed with native unphosphorylated hexokinase 2 and a permanently pseudophosphorylated
glutamate-14 mutant enzyme to determine the functional consequences of phosphorylation-induced enzyme dissociation, The
use of a dye-linked hexokinase assay monitoring proton generation allowed the investigation of the kinetics of glucose
phosphorylation over a wide range of enzyme concentrations. The kinetic data indicated that monomeric hexokinase represents
the high-affinity form of isoenzyme 2 for both glycolytic substrates. Inhibition of glucose phosphorylation by ATP [Moreno et
al, (1986) Eur, J, Biochem. 161, 565-569] was only observed at a low enzyme concentration, whereas no inhibition was
detected at the high concentration of hexokinase 2 presumed to occur in the cell. Pseudophosphorylation by glutamate
substitution for serine-14 increased substrate affinity at high enzyme concentration and stimulated the autophosphorylation of
isoenzyme 2, The possible role of hexokinase 2 in vivo phosphorylation at serine-14 in glucose signaling is discussed.
dustjohn at hotmail.com wrote:
> 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).
> Thanks for your help!
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