An enzyme capture assay for analysis of active hyaluronan synthases.

We describe a sensitive assay for detection of active hyaluronan synthases (HASs) capable of synthesizing hyaluronan (HA) without use of radioactive uridine 5'-diphosphate sugar precursors. The HAS capture assay is based on the binding of a biotinylated HA binding protein (bHABP) to HA chains that are associated with HAS and the subsequent capture of bHABP-HA-HAS complexes with streptavidin-agarose. Specific HAS proteins (e.g., HAS1, not HAS2 or HAS3) captured in this pull-down approach are readily immunodetected by Western blot analysis using appropriate antibodies. The assay was used to detect active HAS proteins in cell membranes, purified recombinant Streptococcus equisimilis HAS (SeHAS), and in vitro translated human HAS1 or SeHAS. The HAS capture assay was also used to assess the fraction of HAS molecules that were active, which cannot be done using standard assays for synthase activity. Assay sensitivity for detection of purified SeHAS is <1 pmol.

Phospholipid dependence and liposome reconstitution of purified hyaluronan synthase.

Previous radiation inactivation and enzyme characterization studies demonstrated that the Streptococcus equisimilis hyaluronan synthase (seHAS) is phospholipid-dependent and that cardiolipin (CL) is the best phospholipid for enzyme activation. Here we investigated the ability of seHAS, purified in the absence of added lipid, to be activated by synthetic phosphatidic acid (PA), phosphatidylserine, or CL lipids containing fatty acyl chains of different length or different numbers of double bonds. The most effective lipid was tetraoleoyl CL (TO-CL), whereas tetramyristoyl CL (TM-CL) was ineffective. None of the phosphatidylserine species tested gave significant activation. PAs containing C10 to C18 saturated acyl chains were not effective activators, and neither were oleoyl lyso PA, dilinoleoyl PA, or PA containing one oleoyl chain and either a palmitoyl or stearoyl chain. In contrast, dioleoyl PA stimulated seHAS approximately 10-fold, to approximately 20% of the activity observed with TO-CL. The tested acidic lipids such as PA and CL activated the enzyme most efficiently if they contained only oleic acid. Mixing experiments showed that the enzyme interacts preferentially with TO-CL in the presence of TM-CL. Similarly, seHAS incorporated into phosphotidylcholine-based liposomes showed increasing activity with increasing TO-CL, but not TM-CL, content. Inactivation of membrane-bound seHAS by solubilization with Nonidet P-40 was prevented by TO-CL, but not TM-CL. The pH dependence of seHAS in the presence of synthetic or naturally occurring CLs showed the same pattern of lipid preference between pH 6 and 10.5. Unexpectedly, HAS showed lipid-independent activity at pH 11.5. The results suggest that Class I HAS enzymes are lipid-dependent and that assembly of active seHAS-lipid complexes has high specificity for the phospholipid head group and the nature of the fatty acyl chains.