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Metabolism of 35S-sulphate and properties of APS-kinase and PAPS-reductase in Nitrobacter agilis

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Summary

The incorporation of [35S]-sulphate was followed into washed-cell suspensions of Nitrobacter agilis. Thus, bound sulphate, sulphite, sulphide, cysteine, glutathione, homocysteine, methionine and taurine were detected in the ethanol-soluble fraction as well as in the residual hydrolysed fraction. The reaction between thiol groups and N-ethylmaleimide has been successfully used to stabilize the SH-compounds in cell extracts, and the derivatives thus obtained were separated by paper chromatography.

A soluble enzyme system catalyzing the reduction of sulphate to sulphite has been prepared. As a result of DEAE-cellulose-11 column chromatography, the enzyme complex was cleaved into two protein bands, one containing ATP-sulphurylase and the other APS-kinase and PAPS-reductase. The last two enzymes were further purified by DEAE-sephadex and Sephadex G-150 column chromatography. At pH 7.6 the enzymes show maximal activity in the presence of ATP and an ATP-generating system (creatine phosphate and creatine phosphokinase), APS, NADP+, a NADP+-reducing system (glucose-6-phosphate and a glucose-6-phosphate dehydrogenase) and MgCl2. Addition of small amounts of 2,3-dimercaptopropan-1-ol (BAL) to the buffers stabilized the enzymes and enabled them to be dialyzed for 16 h, without loss of activity. Anaerobic conditions are required for maximal activity.

The optimum concentration of various cofactors for enzyme activity has been determined. The K m values are as follows: ATP, 1.3×10-3 M; APS, 1.6×10-4 M and NADP+, 1.8×10-3 M. The molecular weight of the APS-kinase and PAPS-reductase complex is about 280000. The PCMB inhibition of the two enzymes is reversed by adding GSH, L-cysteine and Cleland's reagent.

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Abbreviations

APS:

adenosine 5′-phosphosulphate

PAPS:

3′-phosphoadenosine 5′-phosphosulphate

PCMB:

p-chloromercuribenzoate

NEM:

N-ethylmaleimide

PPO:

2,5-diphenyloxazole

POPOP:

1,4-bis-(5-phenyloxazole-2)-benzene; Cleland's reagent, dithiothreitol

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  1. Department of Agricultural Biochemistry, Waite Agricultural Research Institute, University of Adelaide, 5064, South Australia, Australia

    A. K. Varma & D. J. D. Nicholas

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  1. A. K. Varma
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  2. D. J. D. Nicholas
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Varma, A.K., Nicholas, D.J.D. Metabolism of 35S-sulphate and properties of APS-kinase and PAPS-reductase in Nitrobacter agilis . Archiv. Mikrobiol. 78, 99–117 (1971). https://doi.org/10.1007/BF00424867

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