Masahito Suiko

LOCALIZATION AND FUNCTIONAL ANALYSIS OF THE SUBSTRATE SPECIFICITY/CATALYTIC DOMAINS OF HUMAN M-FORM AND P-FORM PHENOL SULFOTRANSFERASES

Human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases (PSTs), which are greater than 93% identical in their primary sequences, were used as models for investigating the structural determinants responsible for their distinct substrate specificity and other enzymatic properties. A series of chimeric PSTs were constructed by reciprocal exchanges of DNA segments between cDNAs encoding M-form and P-form PSTs. Functional characterization of the recombinant wild-type M-form, P-form, and chimeric PSTs expressed in Escherichia coli and purified to homogeneity revealed that internal domain-spanning amino acid residues 84–148 contain the structural determinants for the substrate specificity of either M-form or P-form PST. Data on the kinetic constants (K m , V max, andV max/K m ) further showed the differential roles of the two highly variable regions (Region I spanning amino acid residues 84–89 and Region II spanning amino acid residues 143–148) in substrate binding, catalysis, and sensitivity to the inhibition by 2,6-dichloro-4-nitrophenol. In contrast to the differential sulfotransferase activities of M-form and P-form PSTs toward dopamine and p-nitrophenol, the Dopa/tyrosine sulfotransferase activities were found to be restricted to M-form, but not P-form, PST. Furthermore, the variable Region II of M-form PST appeared to play a predominant role in determining the Dopa/tyrosine sulfotransferase activities of chimeric PSTs. Kinetic studies indicated the role of manganese ions in dramatically enhancing the binding ofd-p-tyrosine to wild-type M-form PST. Taken together, these results pinpoint unequivocally the sequence encompassing amino acid residues 84–148 to be the substrate specificity/catalytic domain of both M-form and P-form PSTs and indicate the importance of the variable Regions I and II in determining their distinct enzymatic properties.

Tissue-specific and developmental stage-dependent expression of a novel rat dopa/tyrosine sulfotransferase

Tissue-specific and developmental stage-dependent expression of a novel Dopa/tyrosine sulfotransferase in Sprague-Dawley rats was examined. Both immunoblot and Northern blot analyses showed that the enzyme was expressed predominantly in liver and to a lesser extent in kidney. Its expression could not be detected in nine other organs tested. Livers from different age groups of male or female rats were examined for the developmental regulation of the expression of the Dopa/tyrosine sulfotransferase. Results from immunoblot and Northern blot analyses revealed that the enzyme was present at a very low level in livers of 1-day-old to 2-week-old rats, and gradually increased to a maximum level in rats older than 2 months. Data from the enzymatic assays also showed a similar trend of expression in both male and female rats. The Dopa/tyrosine sulfotransferase activities detected in liver samples of the 8-week-old male and female rats were, respectively, 8.6 and 6.6 times that of the activities detected in liver samples of the 1-day-old male and female rats. These data provide a foundation for the future investigation of the cis- and trans-acting factors involved in the regulation of the tissue-specific and developmental stage-dependent expression of this enzyme.

Purification and Characterization of two Molecular Forms of Bovine Complement Factor H

Complement factor H was highly purified from bovine serum using a combination of tyrosine-O-sulfate (TyrS)-Affi-Gel 10 affinity chromatography, DEAE-BioGel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified bovine factor H migrated as doublet protein bands with apparent molecular weights of ca. 160,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The two forms of the purified factor H were investigated with respect to the sensitivity to limited trypsin digestion. The high-molecular weight form was cleaved into two fragments with apparent molecular masses of, respectively, 45 kD and 125 kD. The low-molecular weight form was cleaved in a different manner to generate three major fragments with molecular masses of, respectively, 25 kD, 45 kD and 100 kD. Limited V8 protease mapping of the two forms of factor H yielded similar, although not identical, peptide band patterns. N-terminal amino acid sequencing revealed that the 13 amino acid residues of the two forms identified are identical, suggesting that they may in fact be isoforms of the same protein. Purified factor H appeared to bind agarose-bonded TyrS or heparin through its anion-binding domain. Furthermore, the binding to TyrS or heparin bonded to agarose was inhibited by the presence of free heparin or dextran sulfate.