Activation and inhibition of human cancer cell hyaluronidase by proteins

Results regarding hyaluronidase activity in tumor extracts or cell lines ar e subject to variations according to the method used for the assay and, som etimes, within an assay. Hyaluronidase was assayed at pH 3.8 in the culture medium of the human cancer-derived cell lines SA87 and H460M by several te chniques: HPLC, Reissig technique, ELSA technique, and zymographic analysis . The optimal pH was between 3.3 and 4 in solutions at constant 150 mM sodi um concentration. The enzyme was reversibly inhibited by sodium concentrati on over 200 mM. The activity of purified hyaluronidase increased in the pre sence of low concentrations of the specific HA-binding glycoprotein hyaluro nectin, or of bovine serum albumin or immunoglobulins, or of human albumin, transferrin, or hemoglobin, showing that proteins cooperate in enzyme acti vity. The ELSA technique showed that optimal pH was slightly lower in the p resence of HN than that with BSA. The optimal BSA concentration was determi ned with the ELSA technique at 0.1 g/liter, and excess of either protein in hibited hyaluronidase. When measured with the Reissig technique, the activi ty of purified enzyme in the presence of 0.1 g/liter BSA was up to fourfold that without BSA. The cooperative effect of BSA was visualized by zymograp hy. We conclude that the total protein content of hyaluronidase solutions m ust be considered to correctly interpret quantitation of the enzyme in sera or tissue extracts because protein concentrations above 200 mu g/liter lea d to underestimation of the enzyme. (C) 1999 Academic Press.