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.
Kyossev
Zhetcho
Z
Department of Biochemistry & Molecular Biology and The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.
Weigel
Paul H
PH
D001426:Bacterial Proteins / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D008565:Membrane Proteins; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D018502:Streptococcus equi / Q000201:enzymology; D014166:Transferases / Q000737:chemistry / Q000235:genetics; D014530:Uridine Diphosphate / Q000737:chemistry
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C049538:streptavidin-agarose; D014530:Uridine Diphosphate; D006820:Hyaluronic Acid; D012685:Sepharose; D004492:Edetic Acid; D014166:Transferases; D014453:Glucuronosyltransferase; C523794:HAS1 protein, human; D000076002:Hyaluronan Synthases