Purification and characterization of a factor catalyzing the conversion of the multiple forms of tyrosine aminotransferase from rat liver.

Using glycerol as a stabilizing agent, a lysosomal factor (termed “convertase”) which converts the multiple forms of tyrosine aminotransferase has been purified about 16,000-fold with a 6% yield from rat liver. The purification method involves differential centrifugation, salt extraction of the mitochondrial-lysosomal fraction, acetone fractionation, acid precipitation, and chromatography on CM-cellulose, hydroxylapatite, Sephadex G-75, and DEAE-cellulose. The purified convertase was apparently homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate and was a monomeric protein with a molecular weight of 33,500 to 35,000 as determined by Sephadex G-75 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and was heatand alkaline-labile even in the presence of glycerol. Incubation of purified Form I of tyrosine aminotransferase with the purified convertase resulted in a prompt conversion of this form to Form Il and subsequently to Form UI. Concomitant studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the same preparations demonstrated a rapid reduction of the 52,000-Mr tyrosine aminotransferase subunit with a concomitant increase in a 48,000-Mr subunit such that the quantitative changes in the forms measured by enzyme activity were essentially identical with those seen on electrophoresis assuming a subunit structure of: Form I = two 52,000-Mr subunits; Form 11 = one 52,000 subunit + one 48,000-Mr subunit; and Form 111 = two 48,000-M, subunits. The purified convertase showed a potent azocaseinolytic activity. These findings demonstrate the proteolytic nature of the convertase. The distinction of this converting proteinase (“convertase”) from the cathepsins previously reported to occur in rat liver lysosomes is also discussed.