A two-component affinity chromatography purification of Helix pomatia arylsulfatase by tyrosine vanadate.
The inhibition of Helix pomatia arylsulfatase by the synergistic combination of N-acetyl-l-tyrosine ethyl ester and vanadate has been extended to affinity chromatography for purification. In the presence of vanadate, l-tyrosine ethyl ester (TEE), immobilized on CH-Sepharose 4B retained arylsulfatase from the digestive juice or lyophilized powder of H. pomatia. No enzyme was retained without vanadate or with arsenate or phosphate. Arylsulfatase was eluted from the column matrix by removing the vanadate to less than 50 microM with buffer containing EDTA to chelate the vanadate. Escherichia coli alkaline phosphatase and potato acid phosphatase, two enzymes which are inhibited by vanadate but not by the vanadate-TEE complex, were not retained by the immobilized TEE under any conditions used. The sulfatase activity was completely separated from contaminating glucuronidase activity present in the crude enzyme extracts. The Ki for the immobilized vanadate-TEE system was found to be 5.0 x 10(-7) M with a capacity of 25 mg/ml swollen gel. A purification of greater than 40-fold from the lyophilized powder of H. pomatia (Sigma Type H-5) was achieved using this technique. The Ki/Keq of other phenols with vanadate were determined in a 96-well plate format as an example of a rapid screening technique that could be extended to other phosphoryl and sulfuryl-transfer enzyme classes.
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe-Claire Dorval, Quebec, H9R 4P8, Canada.
K I
KI
Skorey
N A
NA
Johnson
G
G
Huyer
M J
MJ
Gresser
D000818:Animals; D001192:Arylsulfatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D006372:Helix, Snails / Q000201:enzymology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014638:Vanadates / Q000378:metabolism* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D014638:Vanadates; D014443:Tyrosine; C051772:ethyl tyrosine ester; D001192:Arylsulfatases