Related Experiment Video
Updated: Aug 9, 2026
Identification of protein complexes with quantitative proteomics in S. cerevisiae
Published on: March 4, 2009
Purification and characterization of cAMP dependent protein kinase from Microsporum gypseum
1Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Abstract:
A cyclic AMP dependent protein kinase (PKA), its regulatory (R) and catalytic (C) subunits were purified to homogeneity from soluble extract of Microsporum gypseum. Purified enzyme showed a final specific activity of 277.9 nmol phosphate transferred min(-1) mg protein(-1) with kemptide as substrate. The enzyme preparation showed two bands with molecular masses of 76 kDa and 45 kDa on sodium dodecyl polyacrylamide gel electrophoresis. The 76 kDa subunit was found to be the regulatory (R) subunit of PKA holoenzyme as determined by its immunoreactivity and the isoelectric point of this subunit was 3.98. The 45 kDa subunit was found to be the catalytic (C) subunit by its immunoreactivity and phosphotransferase activity. Gel filtration using Sepharose CL-6B revealed the molecular mass of PKA holoenzyme to be 240 kDa, compatible with its tetrameric structure, consisting of two regulatory subunits (76 kDa) and two catalytic subunits (45 kDa). The specificity of enzyme towards protein acceptors in decreasing order of phosphorylation was found to be kemptide, casein, syntide and histone IIs. Purified enzyme had apparent K(m) values of 71 microM and 25 microM for ATP and kemptide, respectively. Phosphorylation was strongly inhibited by mammalian PKA inhibitor (PKI) but not by inhibitors of other protein kinases. The PKA showed maximum activity at pH 7.0 and enzyme activity was inhibited in the presence of N-ethylmaleimide (NEM) which shows the involvement of sulfhydryl groups for the activity of PKA. PKA phosphorylated a number of endogenous proteins suggesting the multifunctional role of cAMP dependent protein kinase in M. gypseum. Further work is under progress to identify the natural substrates of this enzyme through which it may regulate the enzymes involved in phospholipid metabolism.
Insights
Cyclic AMP dependent protein kinase (PKA) was purified from Microsporum gypseum, revealing its regulatory and catalytic subunits. This enzyme plays a multifunctional role in the fungus, with further research ongoing to identify its natural substrates.
Area of Science:
- Biochemistry
- Molecular Biology
- Mycology
Background:
- Cyclic AMP dependent protein kinase (PKA) is a crucial enzyme in cellular signaling pathways across various organisms.
- Understanding PKA function in fungi like Microsporum gypseum is essential for elucidating fungal physiology and potential therapeutic targets.
Purpose of the Study:
- To purify and characterize cyclic AMP dependent protein kinase (PKA) from the fungus Microsporum gypseum.
- To determine the molecular properties, substrate specificity, and kinetic parameters of the purified PKA.
- To investigate the potential role of PKA in regulating endogenous protein phosphorylation and metabolic pathways in M. gypseum.
Main Methods:
- Purification of PKA holoenzyme and its subunits (regulatory and catalytic) from soluble extracts of M. gypseum.
- Analysis of subunit molecular masses using SDS-PAGE and holoenzyme mass via gel filtration.
- Determination of enzyme kinetics, substrate specificity, and inhibition by specific PKA inhibitors.
Main Results:
- Purified PKA exhibited a specific activity of 277.9 nmol phosphate transferred min(-1) mg protein(-1) with kemptide.
- The enzyme preparation contained regulatory (76 kDa) and catalytic (45 kDa) subunits, forming a tetrameric holoenzyme (240 kDa).
- PKA demonstrated specificity for kemptide, casein, syntide, and histone IIs, with apparent K(m) values of 71 µM for ATP and 25 µM for kemptide. Activity was optimal at pH 7.0 and dependent on sulfhydryl groups, inhibited by N-ethylmaleimide (NEM).
Conclusions:
- The study successfully purified and characterized PKA from M. gypseum, defining its subunit composition and enzymatic properties.
- The phosphorylation of endogenous proteins by M. gypseum PKA suggests a significant and multifunctional role in fungal cellular processes.
- Further investigation into PKA's natural substrates is warranted to fully understand its regulatory functions, particularly in phospholipid metabolism.
