Activation and inhibition of human cancer cell hyaluronidase by proteins.
Results regarding hyaluronidase activity in tumor extracts or cell lines are subject to variations according to the method used for the assay and, sometimes, 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 techniques: HPLC, Reissig technique, ELSA technique, and zymographic analysis. The optimal pH was between 3.3 and 4 in solutions at constant 150 mM sodium concentration. The enzyme was reversibly inhibited by sodium concentration over 200 mM. The activity of purified hyaluronidase increased in the presence of low concentrations of the specific HA-binding glycoprotein hyaluronectin, or of bovine serum albumin or immunoglobulins, or of human albumin, transferrin, or hemoglobin, showing that proteins cooperate in enzyme activity. The ELSA technique showed that optimal pH was slightly lower in the presence of HN than that with BSA. The optimal BSA concentration was determined with the ELSA technique at 0.1 g/liter, and excess of either protein inhibited hyaluronidase. When measured with the Reissig technique, the activity 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 zymography. We conclude that the total protein content of hyaluronidase solutions must be considered to correctly interpret quantitation of the enzyme in sera or tissue extracts because protein concentrations above 200 microg/liter lead to underestimation of the enzyme.
Laboratoire d'Oncologie Moléculaire, Centre Henri-Becquerel, Rue d'Amiens, Rouen, 76000, France.
C
C
Maingonnat
R
R
Victor
P
P
Bertrand
M N
MN
Courel
R
R
Maunoury
B
B
Delpech
D000818:Animals; D002021:Buffers; D002417:Cattle; D002623:Chemistry Techniques, Analytical / Q000379:methods; D003470:Culture Media; D004789:Enzyme Activation; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009994:Osmolar Concentration; D011506:Proteins / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002021:Buffers; D003470:Culture Media; D011506:Proteins; D006821:Hyaluronoglucosaminidase