Factors affecting the mutual interaction between the catalytic core [octamer of large subunit (A)] and the small subunit (B) comprising ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from the superhalophilic cyanobacterium, Aphanothece halophytica, were investigated. The enzyme molecule dissociated into the catalytic core highly depleted of subunit B and the monomeric form of subunit B during density gradient centrifugation (15 h, 4 degrees C) in a sucrose solution of low ionic strength ([I] less than or equal to 50 mM), whereas dissociation was effectively prevented in the presence of 0.3 M KCl. Under the latter condition, dissociation of the enzyme molecule was almost completely prevented by raising the temperature to 20 degrees C, suggesting hydrophobic interaction between catalytic core and subunit B. The addition of RuBP to the sucrose gradient was shown to effectively reduce the molecular dissociation, suggesting a close interaction between the catalytic site and the binding site of subunit B with the catalytic core directly or indirectly. The dissociation was accelerated at alkaline pH higher than 8.5. Reconstitution of the enzymatically active molecular form from the separated components, catalytic core highly depleted of subunit B and B1, was done under various conditions. Both carboxylase and oxygenase activities increased proportionately with the amount of subunit B and then became saturated. From the reconstitution kinetics of RuBP carboxylase, the binding constant of subunit B (KD) was estimated to be about 30 nM in the presence of bovine serum albumin under the usual assay conditions at pH 7.5 and 25 degrees C, but decreased to about 1 nM by the further addition of 0.3 M KCl. Alkaline pH (8.5 or 9) could increase KD by one order of magnitude. High KD was also observed as a result of lowering the temperature; however, the presence of 0.3 M KCl or 0.4 M sucrose or glycerol could effectively decrease the KD at low temperature from 900 nM to less than 50 nM. All these data indicate that the enzyme dissociation at low temperature can be prevented in vivo by cellular components such as salts, polyols, and substrate RuBP besides a factor of enzyme concentration.