Development of myofibrillar ATPase assay system on pH stat

The ATPase assay system on pH stat apparatus was developed. For the ATPase activity measurement, hydrogen ion (H+) concentration delivered from inorganic phosphate (Pi) as a hydrolysis production of ATP was estimated by titrating with 20 mM NaOH solution instead of colorimetric measurement of Pi. In...

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Bibliographic Details
Published in:Fisheries science 2005-04, Vol.71 (2), p.380-387
Main Authors: Koseki, S. (Hokkaido Univ., Sapporo (Japan)), Nomura, K, Konno, K
Format: Article
Language:English
Subjects:
ATP
ATPASE
denaturation
FISH
gelation
Hydrogen ions
Hydrolysis
MEASUREMENT
MUSCLE FIBRES
Muscular system
myofibrils
pH stat
Salts
Sodium hydroxide
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Summary:The ATPase assay system on pH stat apparatus was developed. For the ATPase activity measurement, hydrogen ion (H+) concentration delivered from inorganic phosphate (Pi) as a hydrolysis production of ATP was estimated by titrating with 20 mM NaOH solution instead of colorimetric measurement of Pi. Inclusion of 0.5 M KCI in the ATP stock solution and 2 mM Tris-maleate (pH 7.0) buffer in the reaction medium reduced the extent of pH change upon addition of ATP to initiate the ATPase reaction. The amount of H+ liberated from Pi was strongly affected by the set pH for the ATPase assay with a promotion at alkaline pH. Thus, it was required to estimate the coefficient to convert H+ to a Pi concentration at a specific pH. The specified coefficient at pH 7.0 was 1.248. ATPase assay on pH stat allowed us to follow the ATP hydrolysis by myofibrils continuously showing a curvature profile at low salt medium (=0.3 M KCI).
ISSN:0919-9268
1444-2906
DOI:10.1111/j.1444-2906.2005.00975.x