Purification of a Synaptic Membrane Na⁺/Ca²⁺ Antiporter and Immunoextraction with Antibodies to a 36‐kDa Protein

Abstract

The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na+/Ca2+ exchange activity were examined and a series of chromatographic procedures were used for the isolation of a protein involved in this transport activity. The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of 20% (vol/vol) glycerol led to optimal solubilization, and soybean phospholipids in low-pH medium were found to produce optimal reconstitution of activity after dialysis to remove the detergent. Sequential chromatography steps involving the use of gel filtration on Sephacryl S-400 HR, ion exchange on diethylaminoethyl-Sephacel, and metal chelate chromatography on tris-(carboxymethyl)ethylenediamine loaded with LaCl3 led to the isolation of a fraction highly enriched in both Na+/Ca2+ exchange activity and two protein bands identified by denaturing electrophoresis. The estimated molecular masses of the two proteins were 50 and 36 kDa. Development of polyclonal antibodies to the 36-kDa protein permitted immunoextraction of greater than 95% of the antiporter activity from solubilized synaptic plasma membranes. These antibodies cross-reacted with the electroeluted 50-kDa protein on enzyme-linked immunosorbent assays, suggesting a close relationship between the two proteins. These results indicate that the 36-kDa protein is at least a component of the brain membrane Na+/Ca2+ antiporter.

The properties, distribution and function of Na+–Ca2+ exchanger isoforms in rat cutaneous sensory neurons

“…To begin to explore the basis for the apparent difference between these previous results and those of the present study, we probed protein from DRG, sciatic nerve and skin with the anti-NCX2 antibody used in this previous study. However, we were only able to detect a band at 36 kDa (not shown), suggestive of an NCX ancillary protein (Michaelis et al 1992) as the estimated size of NCX2 protein is 102 kDa. Nonetheless, the present results with an antibody obtained from Dr Philipson at UCLA were consistent with previous results in brain tissue (Thurneysen et al 2002a;Papa et al 2003).…”

“…However, we were only able to detect a band at 36 kDa (not shown), suggestive of an NCX ancillary protein (Michaelis et al . ) as the estimated size of NCX2 protein is 102 kDa. Nonetheless, the present results with an antibody obtained from Dr Philipson at UCLA were consistent with previous results in brain tissue (Thurneysen et al .…”

Mechanisms of Injury‐Induced Calcium Entry into Peripheral Nerve Myelinated Axons: Role of Reverse Sodium‐Calcium Exchange

“…0 Na, zero-Na"/Li~substitution; 0 Ca, zero-Ca 2 '/EGTA. sitive to Bep blockade than that of other nonneuronal and neuronal tissues (Michaelis et al, 1992(Michaelis et al, , 1994Marlier et a!., 1993). Such a possibility might explain the partial effectiveness of Bep.…”

Amyloid beta-peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca2+ homeostasis and cell death

“…ATPase activity assay. Membrane ATPase activities were assayed by a method adapted from Rohn et al (1993 Michaelis et al (1992). Following treatment, cells were washed with PBS then scraped in a sodium phosphate (150 mM) solution, transferred to 15 ml conical tubes and stored at 4°C for 12-16 hr.…”