A procedure was developed for the isolation of reticuloplasm, the luminal material of the endoplasmic reticulum (ER). A reticuloplasm-rich extract was prepared from a murine plasmacytoma cell line that contains large amounts of ER, by first extracting the cytoplasmic contents using hypotonic lysis to yield ER-rich ‘shells’ followed by mechanical lysis to release the ER contents. The extract contains five major proteins with apparent molecular weights of 100, 75, 60, 58 and 55 (×10³)M_r by SDS-polyacrylamide gel electrophoresis. The 100, 75 and 58 (×10³)M_r species were identified as the known ER proteins endoplasmin, BiP and PD1, respectively. The ER association of the 60 and 55 (×10³) M_r proteins was confirmed by confocal fluorescence microscopy with affinity-purified antibodies.

Equilibrium dialysis with isolated reticuloplasm gave a calcium-binding capacity of 300 nmoles calcium per mg protein with half-maximal binding at 3 mM-Ca²⁺. Purified endoplasmin bound 280 nmoles calcium per mg protein at a calcium concentration of 5 mM-Ca²⁺.

A calcium overlay test revealed that, in addition to endoplasmin, reticuloplasm contained at least three other calcium-binding proteins: i.e. BiP, PDI and the 55×10³M_r protein, respectively, with endoplasmin and the 55×10³M_r protein (CRP55) accounting for the major proportion of the calcium-binding activity.

Treatment of cells with calcium ionophore led to the specific over-expression of the major calcium-binding reticuloplasmins endoplasmin, BiP and CRP55.

These studies show that the lumen of the ER contains a family of proteins with the capacity to bind significant amounts of calcium in the millimolar range and thereby to confer upon the ER the ability to perform a calcium storage function analogous to that of the sarcoplasmic reticulum in muscle cells.