Biochemistry 1982, 21, 875-879 and this ancillary activity was eluted just before the acetyltransferase peak in the Sephacryl profile. The stabilities of this and other aminoglycoside-modifying enzymes (R. G. Coombe and AM George, unpublished results) to gel filtration on Sephadex columns in which there is substantial loss of enzyme activity have been reported (Williams & Northrop, 1976; Umezawa et al., 1973). In these experiments, the criteria for homogeneity of the enzyme were (a) elution of the purified enzyme from Sephacryl and DEAE-Sephacel columns in superimposed activity-protein peaks and (b) a single protein band coincident with acetyltransferase activity in acrylamide gel electrophoresis. Stability studies and definition of buffer and ion require-ments established limits for the successful purification of AAC (3)-V with maximal retention of activity, and for its subsequent employmentin kinetic, pH, and substratestudies.