3 -Phosphoadenosine 5 -phosphosulfate preparation

Phosphonate analogues of nucleotides continue to attract attention. S -Hydrogen-phosphonates and 5 -methylphosphonates of various anti-HIV nucleosides have been made, and the H-phosphonates from AZT and 3 -deoxy-3 -fluorothymidine were highly active. 5 -Fluoro-methyl- and -difluoromethyl-phosphonatcs have been prepared from d4T,26 AZT,264,265 gnj various other nucleosides and deoxynucleosides,265 and phosphonates 199 (n = 1,2) were prepared from the 5 -bis(TMS)phosphite by Arbusov or Michael reaction. The non-hydrolysable analogue 200 of phosphoadenosine phosphosulfate ( active sulfate ) has been described. The 3 -phosphale was put on in the last step using trimetaphosphate, and the desired product was sq>arated from die 2 -phosphate by hplc.267... 

The reaction mixture in a total volume of 250 yL contained 50 /xL of 10 mM sodium phosphate buffer (pH 7.2), 50 /xL of enzyme preparation, and 100 /xL of 50 fiM 3 -phosphoadenosine 5 -phosphosulfate. The reaction was started by adding 50 /xL of 12.5 mM p-nitrophenol (when assaying the thermo-labile form) or 50 /xL of 50 yM p-NP (when determining the thermostable form). After incubation for 30 minutes at 37°C, the reaction was stopped by the addition of 25 /xL of 4 M perchloric acid. After centrifugation, a 20 /xL aliquot was injected into the HPLC system. Formation of product was linear with time for both forms of enzyme for up to 45 minutes, and with protein amount up to 0.5 mg. 

The enzyme was assayed in a final volume of 200 /xL containing 73 fiM 3 -phosphoadenosine 5 -phosphosulfate, 50 fiM A-acetyldopamine, and 50 mM phosphate buffer (pH 6.0). The reaction was started by addition of 10 to 60 /xL of the enzyme preparation. The reaction was stopped after 15 minutes of incubation at 37°C by boiling for 1 minute, followed by the addition of 50 fiL of 2 M Tris-HCl buffer (pH 8.6). Unreacted A-acetyldopamine was extracted twice by adsorption onto 10 mg of activated alumina. The supemate obtained after centrifugation was filtered, and 5 to 100 /xL aliquots were analyzed by HPLC. 

Phosphoadenosine 5 -phosphosulfate (PAPS) is the well-known sulfate donor that occupies a central position in sulfate biochemistry (Balasubrama-nian and Bachhawat, 1970 DeMeoi, 1975 Farooqui, 1980b). It is hydrolyzed into PAP and sulfate by PAPS sulfohydrolase (Balasubramanian and Bachhawat, 1962). Nothing has been reported on the purification of this enzyme, but the crude enzyme preparation shows optimal activity at pH 6.0. It is activated by Co " " and Mn " " and inhibited by ADP, fluoride, and sulfhydryl compounds (Balasubramanian and Bachhawat, 1962). The desulfation of PAPS is a specific reaction. Arylsulfohydrolase A from chicken brain and rabbit kidney cortex does not hydrolyze PAPS to PAP (Farooqui and Bachhawat, 1972 Farooqui and Helwig, unpublished). Because PAPS sulfohydrolase has never been purified from any source, nothing is known about its physicochemical properties. 

A cytosolic sulfotransferase has been identified in rat liver and kidney which utilizes 3 -phosphoadenosine-5 -phosphosulfate (PAPS) and shows a greater rate of sulfation for glycolithocholate than lithocholate. In an assay with the enzyme preparation, PAPS, and conjugated bile acids, 3 unidentified products were formed from taurocholate suggesting multiple sulfation of more polar conjugated bile acids [64]. The enzyme from liver, proximal intestine or adrenals of hamster produced only glycochenodeoxycholate 7-sulfate. Comparable results with the enzyme from kidney will be discussed in Section VI.3. Hepatic enzyme from the female hamster shows 4-fold greater activity than that of the male [65]. 

Vitamin A deficiency characteristically results in hypoplasia of mucus-secreting tissues and keratinizing metaplasia of epithelial surfaces (171). The inhibition of mucus secretion appears to be related to a defect in mucopolysaccharide synthesis which can be attributed to a diminished incorporation of sulfate into 3 -phosphoadenosine-5 -phosphosulfate (active sulfate) (172). The impaired synthesis of active sulfate can be demonstrated in cell-free colon preparations from vitamin A deficient rats and can be restored to normals by a metabolite of vitamin A. 

Sulfation of carbohydrates is a topic of growing interest. The first recycling system used for the sulfation of carbohydrates was reported by Lin et al. The regeneration of 3 -phosphoadenosine 5 -phosphosulfate (PAPS), the universal biological sulfuryl donor, was accomplished by a multi-enzyme cascade (Scheme 15) [39]. In conjunction with a Nod factor sulfotransferase, A,A -diacetylchitobiose was sul-fated at the reducing terminal GlcNAc 6-hydroxyl. This sulfation system was also used to generate 6-sulfo-LacNAc, a key intermediate in the preparation of 6-sulfo-sLe. ... 

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