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Calcium-binding properties of human erythrocyte calpain.

ABSTRACT: The results presented provide more information on the sequential mechanism that promotes the Ca2+-induced activation of human erythrocyte mu-calpain under physiological conditions. The primary event in this process corresponds to the binding of Ca2+ to eight interacting sites, of which there are four in each of the two calpain subunits. Progressive binding of this metal ion is linearly correlated with the dissociation of the proteinase, which reaches completion when all eight binding sites are occupied. The affinity for Ca2+ in the native heterodimeric calpain is increased 2-fold in the isolated 80 kDa catalytic subunit, but it reaches a Kd consistent with the physiological concentration of Ca2+ only in the active autoproteolytically derived 75 kDa form. Binding of Ca2+ in physiological conditions, and thus the formation of the 75 kDa subunit, can occur only in the presence of positive modulators. These are represented by the natural activator protein, found to be a Ca2+-binding protein, and by highly digestible substrates. The former produces a very large increase in the affinity of calpain for Ca2+, and the latter a smaller but still consistent decrease in the Kd of the proteinase for the metal ion. As a result, both dissociation into the constituent subunits and the autoproteolytic conversion of the native 80 kDa subunit into the active 75 kDa form can occur within the physiological fluctuations in Ca2+ concentration. The delay in the expression of the proteolytic activity with respect to Ca2+ binding to native calpain, no longer detectable in the 75 kDa form, can be attributed to a Ca2+-induced functional conformational change, which is correlated with the accessibility of the active site of the enzyme.

SUBMITTER: Michetti M

PROVIDER: S-EPMC1218616 | biostudies-other | 1997 Aug

REPOSITORIES: biostudies-other

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Calcium binding by human erythrocyte membranes.

Project description:1. The characteristics of Ca(2+) binding to haemoglobin-free human erythrocyte membranes were investigated by using (45)Ca and centrifugation partition of ;ghosts' from their external incubation medium. Equilibrium of ;ghosts' with external Ca(2+) required less than 15min. 2. The binding did not vary with temperature in the range 0-37 degrees C. 3. At pH7.4 ;ghosts' bound a maximum of 283mumol of Ca(2+)/g of ;ghost' protein, equivalent to 6.85x10(7) Ca(2+) ions per cell. 4. Increasing the ionic strength from 0.01 to 0.46 diminished Ca(2+) binding, as did ATP in concentrations ranging from 0 to 15mm in the incubation medium. 5. An increase of the pH from 3.0 to 9.3 caused a marked increase in the amount of Ca(2+) bound. 6. Extraction of (45)Ca-labelled ;ghosts' with chloroform-methanol showed that the distribution of Ca(2+) was: 79% protein-bound, 16% lipid-bound, 5% in the aqueous phase, presumably non-bound. Most of the lipid-bound Ca(2+) (about 80%) was associated with a phospholipid fraction containing phosphatidylserine, phosphoinositides and phosphatidylethanolamine, giving a molar Ca(2+): phosphorus ratio of about 1:2.

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Discrete proteolysis of neuronal calcium sensor-1 (NCS-1) by mu-calpain disrupts calcium binding.

Project description:Neuronal calcium sensor-1 (NCS-1) is a high-affinity, low-capacity Ca(2+)-binding protein expressed in many cell types. We previously showed that NCS-1 interacts with inositol 1,4,5-trisphosphate receptor (InsP(3)R) and modulates Ca(2+)-signaling by enhancing InsP3-dependent InsP(3)R channel activity and intracellular Ca(2+) transients. Recently we reported that the chemotherapeutic agent, paclitaxel (taxol) triggers mu-calpain dependent proteolysis of NCS-1, leading to reduced Ca(2+)-signaling within the cell. Degradation of NCS-1 may be critical in the induction of peripheral neuropathy associated with taxol treatment for breast and ovarian cancer. To begin to design strategies to protect NCS-1, we treated NCS-1 with mu-calpain in vitro and identified the cleavage site by N-terminal sequencing and MALDI mass spectroscopy. mu-Calpain cleavage of NCS-1 occurs within an N-terminal pseudoEF-hand domain, which by sequence analysis appears to be unable to bind Ca(2+). Our results suggest a role for this pseudoEF-hand in stabilizing the three functional EF-hands within NCS-1. Using isothermal titration calorimetry (ITC) we found that loss of the pseudoEF-hand markedly decreased NCS-1's affinity for Ca(2+). Physiologically, this significant decrease in Ca(2+) affinity may render NCS-1 incapable of responding to changes in Ca(2+) levels in vivo. The reduced ability of mu-calpain treated NCS-1 to bind Ca(2+) may explain the altered Ca(2+) signaling in the presence of taxol and suggests a strategy for therapeutic intervention of peripheral neuropathy in cancer patients undergoing taxol treatment.

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Elimination of Teratogenic Human Induced Pluripotent Stem Cells by Bee Venom via calcium-calpain pathway

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Oligomeric state, hydrodynamic properties and target recognition of human Calcium and Integrin Binding protein 2 (CIB2).

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Elimination of Teratogenic Human Induced Pluripotent Stem Cells by Bee Venom via Calcium-Calpain Pathway.

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