Ex vivo measurement of calpain activation in human peripheral blood lymphocytes by detection of immunoreactive products of calpastatin degradation.
Limited proteolysis of multiple intracellular proteins by endogenous Ca-dependent cysteine proteases--calpains--is an important regulatory mechanism for cell proliferation, apoptosis etc. Its importance for cellular functions is stressed by existence of endogenous calpain inhibitors--calpastatins. The calpain-calpastatin system within living cells is in a fragile balance, which depends on both partners. The interdependence of calpain--a protease--and calpastatin--an endogenous inhibitor and at the same time a substrate for this enzyme makes any assessment of actual activity of this enzyme in the cells very difficult. In this work we made an attempt to estimate and compare the activity of calpain in human peripheral blood lymphocytes by assessing the levels of limited proteolysis of calpastatin in these cells by western blot, while at the same time the levels of calpain protein inside these cells was measured by flow cytometry. Our results indicate that it is possible to compare (semi-quantitatively) the activities of calpain in peripheral blood CD4+ and CD19+ lymphocytes from various donors that way. Preliminary results showed that calpain activity is increased in the CD4+ T cells isolated from peripheral blood of rheumatoid arthritis patients as compared to control lymphocytes. Extremely high intrinsic activity of calpain was detected in chronic lymphocytic leukemia (CD19+) cells. All this confirms the detection of immunoreactive products of calpastatin as a good maker of endogenous calpain activity.
Mikosik
Anna
A
Department of Patophysiology, Medical University of Gdańsk, Poland.
Zaremba
Anna
A
Puchalska
Zofia
Z
Daca
Agnieszka
A
Smolenska
Zaneta
Z
Lopatniuk
Paulina
P
Mital
Andrzej
A
Hellman
Andrzej
A
Bryl
Ewa
E
Witkowski
Jacek M
JM
D018941:Antigens, CD19 / Q000378:metabolism; D015153:Blotting, Western; D015704:CD4 Antigens / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000276:immunology*; D002154:Calpain / Q000378:metabolism*; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D008214:Lymphocytes / Q000097:blood* / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D018941:Antigens, CD19; D015704:CD4 Antigens; D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; C033668:calpastatin; D002154:Calpain