Abstract
A microchemical technique has been adapted to the evaluation of the membrane‐bound enzyme alkaline phosphatase within human malignant and benign breast lesions. Frozen tissue sections were either stained or lyophilized in alternating sequence in order that samples for enzyme analysis could be chosen from well‐defined regions of epithelial proliferation. Microgram quantities of human tumors could then be examined with multiple replications to elucidate patterns of overall enzyme activity, the activity of tissue fractions, and the isoenzyme form present within individual tumors. A significant difference (p<0.001) was found to exist between the specific activity of alkaline phosphatase from human infiltrating ductal carcinomas and human fibroadenomas. The activity value determined for the malignant group of tumors was 2.12±1.80, while that for benign tumors was 7.52±4.31 mmoles/min/kg dry weight at 37° C. The percentage of alkaline phosphatase activity found within 29,000 g pellets of malignant tumors was variable and constituted approximately 60% of the total activity found in all fractions, while that for benign tumors was more constant and amounted to approximately 90% of the total found. The malignant and benign tumor alkaline phosphatase of both supernatant and pellet fractions was shown to be 90% labile to incubation at 65° C for 5 min and to be 50% inhibited by 20 mM 1‐phenylalanine. Polyacrylamide gel electrophoresis revealed a single enzyme band of identical Rf for each of these fractions from the two tumor types. When varying proportions of malignant and benign tissue samples were co‐incubated, no influence of tumor type on the total activity or the percentage membrane‐bound activity was found. However, a change in the proportion of enzyme activity pelleted at 29,000 g could be shown when the cellularity of the tissue sample was varied. While homogenization of the total sample or of the insoluble material could release some of this activity, greater enzyme activities were always found within insoluble, 480 g pellets and 29,000 g pellets taken from benign tumors. An entrapment of membrane proteins could account for these results and should be considered when tissues are analyzed in the manner described.