Activation of r-[D⁶⁴⁶A]E¹-HPg by SK-HPm and Sak-HPm. Lane 1, wtr-E¹-HPg. Lane 2, wtr-E¹-HPg (200 ng) + SK-HPm (10 ng). Lane 3, wtr-E¹-HPg (200 ng) + Sak-HPm (10 ng). Lane 4, r-[D⁶⁴⁶A]E¹-HPg. Lane 5, r-[D⁶⁴⁶A]E¹-HPg (200 ng) + SK-HPm (10 ng). Lane 6, r-[D⁶⁴⁶A]E¹-HPg (200 ng) + Sak-HPm (10 ng). Lane 7, molecular-weight markers (phosphorylase b, 94,000; bovine serum albumin, 66,200; rabbit muscle aldolase, 39,200; and triose phosphate isomerase, 26,600). The slightly lower molecular weight of the HPm heavy chain in lane 5 compared with lane 6 is due to a small amount of free HPm in the SK-HPm mixture that was released consequent to degradation of the SK within the complex, and which would then catalyze conversion of the E¹-HPm heavy chain to the K⁷⁸-HPm heavy chain. The buffer for the activations was 10 mmol/L HEPES-NaOH/150 mmol/L NaOAc, pH 7.4, at 37°C.