Related Experiment Videos
Isolation, purification and characterization of intracellular calmodulin like protein (CALP) from Mycobacterium phlei
P V Sarma1, P U Sarma, P S Murthy
1Department of Biochemical Technology, Sri Venkateswara College, New Delhi, India.
Abstract:
A monomeric acidic protein of 14,000 Da with an isoelectric point of 4.5 was isolated from Mycobacterium phlei, which stained poorly with Coomassie brilliant blue. This protein showed retardation in mobility in SDS-PAGE upon treatment with calcium, similar to eukaryotic calmodulin proteins. Activation of cAMP phosphodiesterase and NAD kinase by this protein was observed. The CD spectral analysis indicated that the CALP has 52% of beta-conformation. The regular beta-conformation of the calmodulin like protein was shifted to 46% alpha-helical structure when calcium ions reacted with the protein, however, 42% of the CALP still retained its original beta-conformation. These observations indicated homology of this calcium binding protein with that of eukaryotic calmodulins in few structural and functional properties.
Insights
Researchers discovered a novel calcium-binding protein in Mycobacterium phlei. This protein shares structural and functional similarities with eukaryotic calmodulin, suggesting conserved calcium-binding mechanisms across species.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Mycobacterium phlei possesses unique proteins.
- Calcium-binding proteins play crucial roles in cellular signaling.
- Eukaryotic calmodulin is a key calcium sensor.
Purpose of the Study:
- To isolate and characterize a novel calcium-binding protein from Mycobacterium phlei.
- To investigate the structural and functional properties of the isolated protein.
- To determine potential homologies with eukaryotic calmodulin.
Main Methods:
- Protein isolation and purification from Mycobacterium phlei.
- SDS-PAGE analysis to assess protein mobility changes with calcium.
- Enzyme activity assays to evaluate protein function (cAMP phosphodiesterase, NAD kinase activation).
- Circular Dichroism (CD) spectral analysis for conformational studies.
Main Results:
- A monomeric acidic protein (14,000 Da, pI 4.5) was isolated.
- The protein exhibited calcium-dependent mobility shifts in SDS-PAGE.
- The protein activated cAMP phosphodiesterase and NAD kinase.
- CD spectral analysis revealed significant beta-conformation (52%) that shifted towards alpha-helical structure upon calcium binding, while retaining beta-conformation.
Conclusions:
- The isolated protein from Mycobacterium phlei displays functional and structural similarities to eukaryotic calmodulin.
- This suggests a conserved calcium-binding protein domain or mechanism in prokaryotes and eukaryotes.
- Further research into this calcium-binding protein could elucidate fundamental cellular processes.