Serum Tyrosinase in Malignant Disease, Its Activity, and the Electrophoretic Patterns of the Enzyme as Carried by lmmunoglobulins1

needed to bring about 60% saturation must be accurately calculated. The serum protein fraction salted out at 50 to 60% saturation with the salt was dissolved in deionized water and diluted with large volume of 1 m@phosphate buffer (monobasic and dibasic sodium phosphates; Mallinckrodt Chemical Works, St. Louis, Mo.) pH 6.8; desalted against the same phosphate buffer to remove ammonium sulfate; and concentrated by ultra filtration (Amicon, PM 10; 0—4°). The ammonium sulfate-free serum fraction thus obtained was especially useful for assay of tyrosinase activity. However, the serum fraction which was not ammonium sulfate free could be used as such for electropho resis. The serum fractions with and without ammonium sulfate were frozen until use. The tyrosinase activity in the serum tyrosinase fraction was determined by formation of [14C]melanin from L-j14C]tyrosine (4, 5). The electrophoretic patterns of serum tyrosinase were obtained by acrylamide disc gel electrophoresis of the serum tyrosinase fraction, followed by incubation of the gel sample with L-dopa (1 60 @ig/ml; Nutritional Biochemical Corp. , Cleve land, Ohio) at room temperature overnight (7, 9). In the previous studies (7, 9), 5% sucrose (w/v) was added to a portion of serum tyrosinase fraction prior to electrophoresis in order to eliminate enzyme diffusion during electrophoresis. However, 7.5% sucrose (w/v) was used in the present study. The lipase (porcine pancrease; Schwarz/Mann, Orangeburg, N. V.; spe cific activity, 66.9 units/mg) digestion of the serum enzyme preparation to free tyrosinase followed procedures previously described (1, 7, 9). Protein localization of the gel sample after electrophoresis was performed by staining for 1 hr with 0.25% Coomassie Brilliant Blue (16) and destaining in 7% acetic acid (1 1) until clear protein bands appeared. Both enzymic and protein bands were preserved in 7.5% acetic acid (1 1) and scanned at 540 nm in the same solution using a densitometer (Gilford Instrument Model 2400 5 spectrophotometer) to reveal corresponding peaks. Bromphenol blue was used for dye front during electrophoresis. With known information of gel length and dye front at the end of electrophoresis and of gel length and band position after incubation or staining, the RF's of the enzymic and protein bands (or peaks) were calculated. Since peroxidase may also play a role in melanogenesis (15), it is necessary to differentiate tyrosinase activity from peroxidase enzymic activity or vice versa. In the present study, the serum enzymic activity in melanin formation has been experimentally shown to be controlled by tyrosinase. The evi dence has been presented in †̃ †̃ Results and Discussion.― After separation of the serum tyrosinase fraction from the ammonium sulfate fraction, a supernatant was obtained. The presence of tyrosinase inhibitors in the supernatant was dem onstrated by ultrafiltration of the supernatant into different fractions with different molecular weight ranges and by testing their inhibitory effect separately on mushroom tyrosinase (Nu tritional Biochemical Corp.). The advantages of using mush