Enzymic sulphation of dopa and tyrosine isomers by HepG2 human hepatoma cells: stereoselectivity and stimulation by Mn2+.
HepG2 human hepatoma cells, labelled with [35S]sulphate in media containing L-3,4-dihydroxyphenylalanine (L-dopa), (D-dopa), DL-m-tyrosine or D-p-tyrosine, were found to produce the [35S]sulphated forms of these compounds. Addition to the labelling media of m-hydroxybenzylhydrazine, an aromatic amino acid decarboxylase inhibitor, greatly enhanced the production of L-dopa O-[35S]sulphate and DL-m-tyrosine O-[35S]sulphate, with a concomitant decrease in the formation of dopamine O-[35S]sulphate and m-tyramine O-[35S]sulphate. With 3'-phosphoadenosine 5'-phospho[35S]sulphate as the sulphate donor., HepG2-cell cytosol was shown to contain enzymic activity catalysing the sulphation of L-dopa, D-dopa, L-m-tyrosine, D-m-tyrosine, L-p-tyrosine and D-p-tyrosine. The pH optimum of the enzyme, designated dopa/tyrosine sulphotransferase, was determined to be 8.75 with D-m-tyrosine as the substrate. The enzyme exhibited stereoselectivity for the D-form of dopa or tyrosine isomers. Addition of 10mM MnCl2 to the reaction mixture resulted in a remarkable stimulation of dopa/tyrosine sulphotransferase activity, being as high as 267.8 times with D-p-tyrosine as the substrate. Quantitative assays revealed L-dopa, D-dopa and D-m-tyrosine to be better substrates than L-p-tyrosine. When the HepG2-cell cytosol was subjected to DEAE Bio-Gel and hydroxyapatite column chromatography, dopa/tyrosine sulphotransferase was co-eluted with the thermolabile 'M-form' phenol sulphotransferase. Furthermore dopa/tyrosine sulphotransferase displayed properties similar to that of the M-form phenol sulphotransferase with respect to thermostability and sensitivity to 2,6-dichloro-4-nitrophenol. Whether the M-form phenol sulphotransferase is truly (solely) responsible for the dopa/tyrosine sulphotransferase activity present in HepG2 cells remains to be clarified.
Suiko
M
M
Department of Biochemistry, University of Texas Health Center at Tyler, TX 75710, USA.
Sakakibara
Y
Y
Nakajima
H
H
Sakaida
H
H
Liu
M C
MC
D001792:Blood Platelets / Q000201:enzymology; D002855:Chromatography, Thin Layer; D004295:Dihydroxyphenylalanine / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008345:Manganese / Q000494:pharmacology*; D009596:Nitrophenols / Q000494:pharmacology; D013237:Stereoisomerism; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*
D007527:Isoenzymes; D009596:Nitrophenols; D013431:Sulfates; C005662:tyrosine O-sulfate; D014443:Tyrosine; D008345:Manganese; C015802:2,6-dichloro-4-nitrophenol; D004295:Dihydroxyphenylalanine; C097311:Dopa-tyrosine sulfotransferase; D015238:Sulfotransferases