STRUCTURAL STABILITY OF CHLOROPLAST COUPLING FACTOR-1 AS DETERMINED BY DIFFERENTIAL SCANNING CALORIMETRY AND COLD INACTIVATION

At least part of the gamma subunit of the catalytic portion of the chl oroplast ATP synthase (CF1) is present in the middle of the alpha(3) b eta(3) heterohexamer, Interactions of the alpha/beta subunits with the gamma subunit stabilize the hexameric structure. Surprisingly, neithe r reduction of the gamma disulfide nor selective proteolysis of alpha, beta, and gamma affects the thermal stability of EDTA-treated CF1 pre parations, as determined by differential scanning calorimetry. Dissoci ation of the enzyme in the cold may be monitored by loss of the ATPase activity of CF1 depleted of its epsilon subunit [CF1(-epsilon)]. The rate of cold inactivation of ATPase activity of reduced and alkylated CF1(-epsilon) treated with trypsin in solution was much faster than th at of CF1(-epsilon) (8.1 versus 38.7 min, respectively, for 50% loss o f activity). The increased cold lability of the trypsin-treated enzyme was not a consequence of the cleavage of the gamma. CF1 incubated wit h trypsin under conditions in which gamma is not cleaved was as cold l abile as CF1 with cleaved gamma. Instead, loss of the delta subunit an d a few residues from the C-termini of the beta subunits were responsi ble for the increased cold lability of the enzyme.