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Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Rabbit liver phosphofructokinase: rapid purification and phosphorylation site identification
Z Zhao1, R W Pascalar, D A Malencik
1Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331, USA.
Biochemical and Biophysical Research Communications
|May 15, 1996
Summary
Researchers purified liver phosphofructokinase using protamine sulfate precipitation. This method yields highly active, homogeneous enzyme efficiently, aiding further biochemical studies.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Liver phosphofructokinase (PFK) is a key glycolytic enzyme.
- Efficient purification methods are crucial for biochemical and structural studies.
Purpose of the Study:
- To develop an efficient purification protocol for liver phosphofructokinase.
- To characterize the purified enzyme and a phosphorylated fragment.
Main Methods:
- Selective precipitation of PFK using protamine sulfate from a heat-treated crude extract.
- Incorporation of protamine sulfate precipitation into a five-step purification procedure.
- In vitro phosphorylation of purified PFK using cAMP-dependent protein kinase and subsequent CNBr digestion.
Main Results:
- A 67-fold increase in specific activity and nearly full recovery of catalytic activity.
- Electrophoretically homogeneous liver PFK obtained with 47% recovery in a single day.
- Isolation and sequencing of a radio-labeled fragment (AEYVSGELEHVTRRSLS) from a CNBr digest of phosphorylated PFK.
Conclusions:
- Protamine sulfate precipitation is an effective method for liver PFK purification.
- The developed protocol provides a rapid and efficient means to obtain highly pure, active enzyme.
- Characterization of the phosphorylated fragment provides insights into enzyme regulation.
