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Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase.
Biochem J. 1984 Apr 15; 219(2):481-93.BJ

Abstract

An assay method is described for measurement of absolute concentrations of the molybdenum cofactor, based on complementation of the defective nitrate reductase ('apo nitrate reductase') in extracts of the nit-1 mutant of Neurospora crassa. A number of alternative methods are described for preparing, anaerobically, molybdenum-cofactor-containing solutions from sulphite oxidase, xanthine oxidase and desulpho xanthine oxidase. For assay, these were mixed with an excess of extract of the nit-1 mutant, incubated for 24 h at 3.5 degrees C then assayed for NADPH:nitrate reductase activity. In all cases, the specific activity of the molybdenum cofactor, expressed as mumol of NO2-formed/min per ng-atom of Mo added from the denatured molybdoenzyme , was 25 +/- 4, a value that agrees with the known catalytic activity of the nitrate reductase of wild-type Neurospora crassa. This indicates that, under our conditions, there was quantitative transfer of the molybdenum cofactor from denatured molybdoenzyme to yield fully active nitrate reductase. Comparable cofactor assay methods of previous workers, apparently indicating transfer efficiencies of at best a few per cent, have never excluded satisfactorily the possibility that cofactor activity arose, not from stoichiometric constituents of the molybdoenzymes , but from contaminants. The following factors were investigated separately in developing the assay:the efficiency of extraction of the cofactor from the original enzyme, the efficiency of the complementation reaction between cofactor and apo nitrate reductase, and the assay of the resultant nitrate reductase, which must be carried out under non-inhibitory conditions. Though the cofactor is unstable in air (t1/2 about 15 min at 3.5 degrees C), it is stable when kept anaerobic in the presence of sodium dithionite, in aqueous solution or in dimethyl sulphoxide (activity lost at the rate of about 3%/24 h at 20-25 degrees C). Studies of stabilities, and investigations of the effect of added molybdate on the assay, permit conclusions to be drawn about the ligation of molybdenum to the cofactor and about steps in incorporation of the cofactor into the apoenzyme. Though the development of nitrate reductase activity is slow at 3.5 degrees C (t1/2 1.5-3 h) the complementation reaction may be carried out in high yield, aerobically. This is ascribed to rapid formation of an air-stable but catalytically inactive complex of the cofactor, as a precursor of the active nitrate reductase.(

ABSTRACT

TRUNCATED AT 400 WORDS)

Authors

No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

6234882

Citation

Hawkes, T R., and R C. Bray. "Quantitative Transfer of the Molybdenum Cofactor From Xanthine Oxidase and From Sulphite Oxidase to the Deficient Enzyme of the Nit-1 Mutant of Neurospora Crassa to Yield Active Nitrate Reductase." The Biochemical Journal, vol. 219, no. 2, 1984, pp. 481-93.
Hawkes TR, Bray RC. Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase. Biochem J. 1984;219(2):481-93.
Hawkes, T. R., & Bray, R. C. (1984). Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase. The Biochemical Journal, 219(2), 481-93.
Hawkes TR, Bray RC. Quantitative Transfer of the Molybdenum Cofactor From Xanthine Oxidase and From Sulphite Oxidase to the Deficient Enzyme of the Nit-1 Mutant of Neurospora Crassa to Yield Active Nitrate Reductase. Biochem J. 1984 Apr 15;219(2):481-93. PubMed PMID: 6234882.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase. AU - Hawkes,T R, AU - Bray,R C, PY - 1984/4/15/pubmed PY - 1984/4/15/medline PY - 1984/4/15/entrez SP - 481 EP - 93 JF - The Biochemical journal JO - Biochem J VL - 219 IS - 2 N2 - An assay method is described for measurement of absolute concentrations of the molybdenum cofactor, based on complementation of the defective nitrate reductase ('apo nitrate reductase') in extracts of the nit-1 mutant of Neurospora crassa. A number of alternative methods are described for preparing, anaerobically, molybdenum-cofactor-containing solutions from sulphite oxidase, xanthine oxidase and desulpho xanthine oxidase. For assay, these were mixed with an excess of extract of the nit-1 mutant, incubated for 24 h at 3.5 degrees C then assayed for NADPH:nitrate reductase activity. In all cases, the specific activity of the molybdenum cofactor, expressed as mumol of NO2-formed/min per ng-atom of Mo added from the denatured molybdoenzyme , was 25 +/- 4, a value that agrees with the known catalytic activity of the nitrate reductase of wild-type Neurospora crassa. This indicates that, under our conditions, there was quantitative transfer of the molybdenum cofactor from denatured molybdoenzyme to yield fully active nitrate reductase. Comparable cofactor assay methods of previous workers, apparently indicating transfer efficiencies of at best a few per cent, have never excluded satisfactorily the possibility that cofactor activity arose, not from stoichiometric constituents of the molybdoenzymes , but from contaminants. The following factors were investigated separately in developing the assay:the efficiency of extraction of the cofactor from the original enzyme, the efficiency of the complementation reaction between cofactor and apo nitrate reductase, and the assay of the resultant nitrate reductase, which must be carried out under non-inhibitory conditions. Though the cofactor is unstable in air (t1/2 about 15 min at 3.5 degrees C), it is stable when kept anaerobic in the presence of sodium dithionite, in aqueous solution or in dimethyl sulphoxide (activity lost at the rate of about 3%/24 h at 20-25 degrees C). Studies of stabilities, and investigations of the effect of added molybdate on the assay, permit conclusions to be drawn about the ligation of molybdenum to the cofactor and about steps in incorporation of the cofactor into the apoenzyme. Though the development of nitrate reductase activity is slow at 3.5 degrees C (t1/2 1.5-3 h) the complementation reaction may be carried out in high yield, aerobically. This is ascribed to rapid formation of an air-stable but catalytically inactive complex of the cofactor, as a precursor of the active nitrate reductase.(ABSTRACT TRUNCATED AT 400 WORDS) SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/6234882/Quantitative_transfer_of_the_molybdenum_cofactor_from_xanthine_oxidase_and_from_sulphite_oxidase_to_the_deficient_enzyme_of_the_nit_1_mutant_of_Neurospora_crassa_to_yield_active_nitrate_reductase_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/bj2190481 DB - PRIME DP - Unbound Medicine ER -