Abstract
The two sulfate-activating enzymes, ATP-sulfurylase (EC 2.7.7.4) and adenosine-5'-phosphosulfate kinase (adenylylsulfate kinase, EC 2.7.1.25), were each purified about 2000-fold from crude rat chondrosarcoma homogenate. Throughout a purification protocol which included Sephacryl S-300 gel filtration, DEAE-Sephadex ion exchange, hydroxylapatite, and ATP-agarose affinity chromatography, these two activities consistently co-purified. ATP-sulfurylase and adenosine-5'-phosphosulfate kinase each showed a pH optima of 7.0-7.4 and a bimodal temperature optima of 46 and 52-54 degrees C. Both activities preferred Mg2+ as their divalent cation source over Mn2+, Co2+, or Zn2+. The apparent Km values determined for adenosine 5'-phosphosulfate in both assays was 1-5 microM; the Km for pyrophosphate in the sulfurylase reaction was 40 microM and for ATP in the kinase reaction was 5 mM. Gel electrophoresis indicated major bands at Mr = 160,000 in nondenaturing systems and 35,000-37,000 and 60,000 under dissociative conditions, whereas gel filtration of the most highly purified fractions yielded a coincident peak in the molecular weight range 260,000.
Highlights
MATERIALS ANDMETHODSAPS PPi [35S]PAPS (1-3 Ci/mmol) and Na432P201(1-60 Ci/mmol) were obtained from Du Pont-New England Nuclear
We havepreviously studiedandreportedonthese two enzymes in the contexotf a defect in the productionof PAPS in the brachymorphic mutantmouse [21,22,23]
We have shown that reduced PAPS levels in this system are correlated with decreases in the activities of both ATP-sulfurylase (-50%)
Summary
APS PPi [35S]PAPS (1-3 Ci/mmol) and Na432P201(1-60 Ci/mmol) were obtained from Du Pont-New England Nuclear. The abbreviations used are: APS, adenosine 5’-phosphosulfate; saturation, left to stir for 4 h, and centrifuged for 30 min at PAPS, 3”phosphate adenosine 5’-phosphosulfate, SDS, sodium do- 10,000 X g in a Sorvall RC-5B with a Beckman SA600 rotor This decyl sulfate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesul- supernatant was precipitated to 80% ammonium sulfate with fonic acid MES, 2-N-(morpholino)ethanesulfonicacid BSA, bovine stirring overnight, and thetwo pellets were dialyzed overnight versus serum albumin. The hydroxylapatite pool was loaded onto an ATPagarose (5 ml) column in 5-ml batches, and washed with 30 ml of 0.025 M phosphate buffer, 10% glycerol; and enzyme activity was eluted with the phosphate buffer, 10% glycerol, and 0.05 M followed by 0.25 M KC1 (Fig. 3) This stepyielded an almost20-fold increase in specificactivity over the hydroxylapatitefraction with greaterthan 60% recovery. It should alsboe noted that early purification attempatlso employed a DEAE-Sephadex A-50 (24 X 3 cm) ion-exchange step after gel filtration, in which active fractions were eluted using a continuous gradient of 0-0.5 M KC1 in the standard phosphate BufferA, and these fractionswere dialyzed before application to hydroxylapatite(Fig. 4). this stepwas
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