Sperm adhesion molecule 1 (SPAM1), also known as PH20 hyaluronidase (1), is encoded by one of the six hyaluronidaselike genes (1, 2, 3). SPAM1 is a GPIanchored enzyme located on the sperm surface and inner acrosomal membrane (4). SPAM1 degrades hyaluronic acid (HA), a major structural glycosaminoglycan found in extracellular matrices and basement membranes. The enzyme activity enables sperm to penetrate through the HArich cumulus cell layer surrounding the oocyte and therefore facilitates the fertilization process (5). However, detailed enzymatic analysis of this enzyme is hindered by the limited techniques/methods available to monitor the HA degradation products (6). A novel method for analyzing SPAM1 activity was utilized here. Because of the structural similarity between HA (repeating units of GlcAbeta13GlcNAc) and chondroitin sulfate (repeating units of GlcAbeta13GalNAc), the enzyme is also able to hydrolyze chondroitin sulfate. In this assay, radiolabeled chondroitin sulfate was digested with recombinant SPAM1. Degradation products were then separated using a polyacrylamide electrophoresis and visualized with an Xray film (7). The bovine SPAM1 is 63% identical to human homologue in sequence. Source: Recombinant corresponding to Met1-Thr497 from bovine SPAM1, C-terminal, 6-His tag, expressed in CHO cells. Molecular Weight: ~53kD Biological Activity: Measured by its ability to hydrolyze S35 labeled bovine cartilage chondroitin sulfate. <20 ng of rhSPAM1 is required for the 50% hydrolysis of 10ug chondroitin sulfate, as measured under the described conditions. Endotoxin Level: <1EU/ug (LAL method) Storage and Stability: Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
