Purification and properties of a phenol sulphotransferase from Euglena using l-tyrosine as substrate

Abstract

A purification procedure based on (NH4)2SO4 precipitation, and chromatography on Affi-Gel Blue, DEAE-cellulose, hydroxyapatite and Bio-Gel P-60 yields a stable 6400-fold-purified active monomeric phenol (tyrosine) sulphotransferase of 26 kDa from W10BSmL, an aplastidic mutant of Euglena gracilis var. bacillaris. The apparent Km for adenosine 3'-phosphate 5'-phosphosulphate (PAPS) is 15 microM (60 microM tyrosine as substrate); adenosine 5'-phosphosulphate is inactive. L-Tyrosine gave the lowest apparent Km (33 microM) (with PAPS at 30 microM), but tyrosine esters, tyrosinamide, L-p-hydroxyphenylglycine and a number of tyrosine dipeptides were also active, with higher Km values. Nitrophenols (m- and p-) and chlorophenols (o-, m- and p-) were active, with higher Km values than for tyrosine. D-Tyrosine was inactive as a substrate, as was D-p-hydroxyphenylglycine and a number of other tyrosine derivatives lacking the carboxy carbonyl or the amino group, or having extra ring substituents or the hydroxy group in the wrong position. Adenosine 3',5'-bisphosphate and tyrosine O4-sulphate, products of the enzyme reaction with PAPS and tyrosine as substrates, showed competitive (Ki = 20 microM) and uncompetitive (Ki = 500 microM) inhibition kinetics respectively. This appears to be the first phenol sulphotransferase to accept tyrosine as substrate. This membrane-bound enzyme may be involved in tyrosine transport as well as detoxification.

Purification and characterization of sulfotransferase specific to O-22 of 11-hydroxy saxitoxin from the toxic dinoflagellate Gymnodinium catenatum (dinophyceae)

“…The specific activity of the purified O‐ST (1691 pmol/mg per min) was 1–2 orders of magnitude lower than those of most ST 16,17 18 …”

“…We have reported elsewhere that N‐ST is bound to an affinity column with Cibacron blue as a ligand, 8,9 which is used in the purification of a phenol ST from Euglena gracilis. 17 However, O‐ST was not bound to this column.…”

“…Affinity chromatography with PAP‐agarose is used widely in purification of ST from mammals 16 . The reaction product, PAP, has been reported to be a competitive inhibitor of ST 17 …”

PURIFICATION AND CHARACTERIZATION OF A SULFOTRANSFERASE SPECIFIC TO N‐21 OF SAXITOXIN AND GONYAUTOXIN 2+3 FROM THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE)

“…Subsequent use of Mono Q column chromatography and Superdex 200HR to increase purity resulted in a marked decrease in the activity of N‐ST (Table 1). Purification of an aryl ST using Sephadex chromatography resulted in a loss of activity due to exposure of the enzyme to dextrans (Saidha and Schiff 1994). Therefore, the low specific activity of purified N‐ST may be due to inactivation through the Mono Q column and the Superdex 200HR column.…”

“…Therefore, the low specific activity of purified N‐ST may be due to inactivation through the Mono Q column and the Superdex 200HR column. The specific activity of most STs in mammals was reported to be in nmol· mg −1 ·min −1 (Saidha and Schiff 1994). The specific activity of the purified N‐ST (317 pmol·mg −1 ·min −1 ) was two to three orders of magnitude lower than that of most STs.…”