PAPS-GENERATION AND PHENOLSULFOTRANSFERASE (PST) IN RELATION TO SULFATE CONJUGATION IN HUMAN PLATELETS AND LIVER

Abstract

Sulfate conjugation is a sequence of three enzymic reactions catalyzed by ATP sulfurylase, APS kinase (which, together constitute the PAPS-generating system) and phenolsulfotransferase (PST). The human platelets contain PST activity and are also capable of PAPS-generation. Thus they possess a full complement of the enzyme system for sulfate conjugation. The PAPS-generation by the human platelets was determined by the quantitative transfer of sulfuryl group from [35S]-PAPS formed in vitro to N-acetyldopamine (NADA), catalyzed by the rat liver PST. A prerequisite of this assay was the prior inhibition of the sulfate-activating system present in the rat liver extract by the addition of 10 mM EDTA and 14 mM pyrophosphate. An alternative HPLC-ECD procedure was developed which measured directly NADA-SO4 formed and the assay was not limited by the concentrations of S04 - or PAPS employed. PAPS-generation in 60 human platelet samples and 13 human liver extracts gave significantly higher correlations with the overall sulfation (r= 0.96 and 0.98, respectively) than the PST reaction (r= 0.82 and 0.31, respectively), indicating that PAPS-generation may control the rate of overall sulfation. The confidence of measurement of PAPS-generation by the HPLC-ECD procedure and fluorimetric assays was shown by similar values determined in the human liver samples. The rates of PAPS-generation in the human liver extracts approached that of APS kinase which is a very fast reaction, in spite of the thermodynamically unfavourable ATP sulfurylase reaction. Data obtained showed APS kinase activities were only 1.4 to 1.5 times higher than those for PAPS-generation suggesting that APS kinase can overcome the constraints imposed by the ATP sulfurylase reaction. The APS kinase activity correlated very well with the rate of PAPS-generation in the human liver extracts examined; r = 0.91 as determined by the HPLC-ECD and r = 0.97 by the continuous fluorimetric assay. The rates of the overall sulfate conjugation of NADA measured in the human liver extracts correlated significantly with those measured using dopamine, suggesting that NADA can be a surrogate substrate in the measurement of the overall sulfate conjugation. In addition, NADA was shown to be the only substrate of both the M- and P-PST at micromolar concentration. A biphasic Michaelis-Menten curve with two apparent Km values of 4.2 µM and 22.6 µM for NADA was obtained. These values were similar to those determined when either the M form was inactivated by heat at 44°C for 15 min (Km= 4.8 µM) or the P form was inhibited by 10-5M 2,6-dichloro-4-nitrophenol (Km= 18.8 µM). Thus it is a suitable substrate to use for measuring total PST activity. The total PST activities measured in human liver, platelets and whole blood differed by three orders of magnitude. The red blood cells were shown to possess low PST and negligible overall sulfate conjugating activities. Thus in the circulation platelets could be the major site for sulfate conjugation as demonstrated by the high PAPS-generating and PST activities. In addition, intact platelets were shown to accumulate NADA which was then sulfated and transported out of the platelets.