Multiple forms of cathepsin D from bovine uterus.

Preparations of cathepsin D from the bovine uterus contain at least 12 distinct forms of enzyme which digest hemoglobin at pH 3.2. Ten of these forms (2 through 11) have been purified by DEAE-Sephadex chromatography and disc electrophoresis. Six major forms (4, 5, 6, 7, 8, and 12) account for 94% of the total cathepsin D activity of the uterus. These six forms have been unequivocally identified as cathepsin D on the basis of their specificity in cleaving the B chain of insulin. Forms 2 and 3 show pH optima at pH 3.2 and are 50 % inhibited by 0.001 M dithiothreitol in agreement with the properties of cathepsin D. Forms 1, 9, 10, and 11 are present only in trace amounts; they have approximately the same molecular weight as cathepsin D and digest hemoglobin at acid pH. Forms 2 through 8 have also been resolved by isoelectric focusing; their isoelectric points range from 5.8 to 7.2. Disc electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol gave a single band of molecular weight 40,000 to 42,000 for Forms 2,3, and 4; Forms 5 through 9 gave two distinct bands corresponding to weights of 13,000 to 14,000 and 25,000 to 28,000. Equilibrium ultracentrifugation gave a weight average molecular weight of 40,000 for Form 4 and slightly lower weights for Forms 5 and 6. Form 4 was more stable in acid and urea than Form 5. Forms 4 through 7 all had the same V,,, and K, when hemoglobin was used as substrate. This evidence suggests that Form 4 is the primary form of cathepsin D, that it consists of a single polypeptide chain of molecular weight 40,000, and that this chain is cleaved in viuo without loss of activity into fragments of + and % length to produce higher numbered forms.