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Cardiac high molecular weight calmodulin binding protein contains calpastatin activity
R Kakkar1, R V Raju, R L Mellgren
1Department of Pathology, College of Medicine, University of Saskatchewan, Royal University Hospital, Saskatoon, SK, S7N 0W0, Canada.
Insights
A high molecular weight calmodulin binding protein (HMWCaMBP) from bovine heart is homologous to calpastatin, a calpain inhibitor. This protein may represent a calmodulin-binding form of calpastatin, impacting cellular processes.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- A high molecular weight calmodulin binding protein (HMWCaMBP) was previously isolated from bovine heart cytosol.
- The biological function of HMWCaMBP remained largely uncharacterized.
Purpose of the Study:
- To elucidate the biological function of HMWCaMBP.
- To determine the relationship between HMWCaMBP and known cellular proteins.
Main Methods:
- Peptide mapping and sequencing of HMWCaMBP.
- Sequence homology analysis against protein databases.
- Western blot analysis using antibodies against HMWCaMBP and calpastatin.
- In vitro assays to assess calpain inhibition activity.
Main Results:
- Two of three sequenced peptides from HMWCaMBP showed high homology to calpastatin.
- HMWCaMBP exhibited immunoreactivity with antibodies against calpastatin.
- HMWCaMBP dose-dependently inhibited the activity of calpain I and calpain II.
Conclusions:
- HMWCaMBP is homologous to calpastatin, a known calpain inhibitor.
- The findings suggest HMWCaMBP may be a calmodulin-binding variant of calpastatin.
- This discovery offers insights into the regulation of calpain activity via calmodulin-dependent mechanisms.
Abstract:
A high molecular weight calmodulin binding protein (HMWCaMBP) was previously identified and purified from bovine heart cytosolic fraction [Sharma, R.K. (1990) J. Biol. Chem. 265, 1152-1157]. In this study, we report the biological function of this protein. HMWCaMBP was subjected to peptide mapping and three peptides were sequenced. Two of the three peptide sequences were shown to be highly homologous to the calpain inhibitor, calpastatin. However, the third peptide did not show homology to any known proteins. The Western blot analysis of HMWCaMBP and purified calpastatin from bovine cardiac muscle showed immunoreactivity with polyclonal antibody raised against HMWCaMBP. Furthermore, HMWCaMBP inhibited calpain II and calpain I activities in a dose dependent fashion. Our data based on sequence homology, amino acid analysis, antibody reactivity and calpain inhibition suggests that HMWCaMBP is homologous to calpastatin and may be a CaM-binding form of calpastatin.