Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization

Abstract

Key words: Proteolysis; Calcium; Calpain; activity and thus it can be considered as an active species of Activation process calpain. Materials and methods I. Introduction Purification of human erythrocyte calpainThe Ca2+-dependent proteinase, calpain, is normally locaHuman erythrocyte calpain was purified as previously described lised in the cytosol of the cells [1][2][3][4][5], together with its natural[15], modified as follows: packed red cells 50 ml were lysed in inhibitor calpastatin. In this cell localisation, calpain is pos-5 vols. of water containing 1 mM EGTA; the membranes were discarded by centrifugation at 25 000 ×g for 10 min and the supernatant tulated to be inactive, due to the high requirement for Ca 2+ was treated with 125 g (wet powder) of DE 32 previously extensively [6][7][8], in the absence of which the enzyme retains a native washed with 50 mM sodium acetate, pH 6.7, containing 0.1 mM conformation with an inaccessible active site. The transition EGTA (buffer A) and stirred for 10 min at 5°C. The resin was colfrom a high to low calcium requiring form results from aulected on a Buckner funnel, washed with 1 1 of buffer A and transferred on a glass column (2.5x 15 cm). The absorbed proteins were toproteolysis, which causes the removal of fragments at the Neluted in a single step with 0.2 M NaC1 dissolved in buffer A. The terminal region of both 80 kDa catalytic and 30 kDa subunits fractions containing calpain activity were collected, precipitated in [9][10][11][12]. The native catalytic subunit is then converted into a 75 50% saturated ammonium sulfate and centrifuged at 25000×g for kDa species, and the small subunit into a 18 kDa fragment.10 min. The pellet was suspended in buffer A and dialyzed for 4 h This new enzyme form expresses full catalytic activity at conin the same buffer solution. The dialyzed material was then loaded onto a column (2.5×3 cm) of Source 15Q (Pharmacia) previously centrations of calcium 50-100-times lower than that required equilibrated in buffer A. The proteins were eluted with a linear graby the native form, and has also been identified in red cells dient of 0~).3 M sodium chloride. The fractions containing calpain enriched with Ca 2+ following exposure of cells to a ionophore, activity were collected, dialyzed for 4 h against sodium borate (50 It seems therefore that autoproteolysis is an essential step that mM, pH 7.5) containing 0.1 mM EGTA (buffer B). To the dialyzed material was added sodium chloride at a final concentration of 0.3 M and the solution loaded onto a column (1 × 10 cm) containing PhenylSepharose CL 4B (Pharmacia) equilibrated with buffer B, containing *Corresponding author. Fax: (39) (10) 354415.0.3 M NaC1. The resin was washed and the bound proteins were 0014-57931961512.00

Spatial Localization of m-Calpain to the Plasma Membrane by Phosphoinositide Biphosphate Binding during Epidermal Growth Factor Receptor-Mediated Activation

“…Calpains are predominantly found in the cytosol, presumably in an inactive state (16). When -calpain is exposed to Ca 2ϩ , it translocates to membranes (42,52), where it then undergoes autolysis (40). The 80-kDa subunit of -calpain has been shown to associate with membranes in the presence of Ca 2ϩ , followed by autolysis to an intermediate, active 78-kDa form found at the membrane, which then autolyzes to the 75-kDa form found only in the cytosol with subsequent proteolytic degradation (40).…”

Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells

“…Mpp5 (21,22), drebrin-like protein isoform 3 (Dbn1) (23)(24)(25), and LIM and SH3 domain protein 1 (Lasp1) (26) are actinbinding adaptor proteins. Calpain-1 catalytic subunit (Capn1) is a protease (27,28). Serine/threonine-protein kinase MRCK β (Cdc42bpb) (29) and Rho guanine nucleotide exchange factor 2 (Arhgef2) (30) are actin-regulatory proteins.…”

Activity profile of calpains I and II in chronically infarcted rat myocardium – influence of the calpain inhibitor CAL 9961

“…A dissociation of the large and small subunit following separation of the calpains has been described previously by Elce et al (1997a). In addition, the use of Ca 2+ -free media in all steps of the preparation prevented the autolysis of the large 80 kDa subunit of calpains I and II into inactive fragments (Michetti et al, 1996). In the work reported here, the Ca 2+ -dependent proteolytic activity of the 80 kDa subunits was measured using the¯uorimetric enzyme assay in fractions following DEAE-Sepharose chromatography in the presence of 5 mM Ca 2+ , suggesting that the large catalytic subunits of both calpain isoforms were intact and active in the presence of Ca 2+ .…”