Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum.
Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.
Samanta
Krishna
K
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
Kar
Pulak
P
Ghosh
Biswarup
B
Chakraborti
Tapati
T
Chakraborti
Sajal
S
D000818:Animals; D002135:Calcium-Binding Proteins / Q000032:analysis*; D002154:Calpain / Q000032:analysis* / Q000037:antagonists & inhibitors; D002254:Carbonates / Q000494:pharmacology; D002364:Caseins; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D009131:Muscle, Smooth, Vascular / Q000737:chemistry* / Q000648:ultrastructure; D011651:Pulmonary Artery / Q000737:chemistry / Q000648:ultrastructure
D002135:Calcium-Binding Proteins; D002254:Carbonates; D002364:Caseins; C005686:sodium carbonate; C033668:calpastatin; D002154:Calpain; C449000:m-calpain