Molybdenum cofactor in nitrate reductase-deficient tobacco mutants. II. Release of cofactor by heat treatment

Mendel, Ralf R.; Alikulov, Z.A; Müller, Andreas J.

doi:10.1016/0304-4211(82)90208-5

Cited by 13 publications

(5 citation statements)

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“…The efficiency of this inactivation was dependent on tungstate concentration, so that at 50 mM tungstate MoCo activity of CP was significantly inactivated within minutes. Another important fact is that 5 mM molybdate included in the apoNR reconstitution assay only slightly activated MoCoCP activity (about 20%), whereas free Moco was strongly activated (about three times) by this concentration of molybdate, as previously reported [8, 26]. This different degree of activation might reflect that most MoCo in CP is already charged with molybdate, but free MoCo is present mainly in a demolybdo form as previously proposed [26, 27].…”

Section: Discussionsupporting

confidence: 73%

“…One unit of MoCo is defined as the amount of MoCo that yields one unit of reconstituted NR activity, defined as the amount of enzyme that catalyzes the reduction of 1 μmol of nitrate per min. Free MoCo was extracted from milk xanthine oxidase by heat treatment (80°C, 90 s) of enzyme preparations 50‐fold diluted in buffer M [8].…”

Section: Methodsmentioning

confidence: 99%

“…Addition of MoCo from different sources to extracts of this mutant results in the reconstitution of nitrate reductase (NR) activity from inactive apoNR. Heat (80°C, 90s) or acid treatment is needed to release MoCo from molybdoenzymes [7, 8]. Only low M r free MoCo and MoCo bound to carrier protein (CP) can directly complement in the nit‐1 assay without the releasing treatment [2, 9].…”

Section: Introductionmentioning

confidence: 99%

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The Chlamydomonas reinhardtii MoCo carrier protein is multimeric and stabilizes molybdopterin cofactor in a molybdate charged form

et al. 1998

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In Chlamydomonas reinhardtii, molybdopterin cofactor (MoCo) able to reconstitute active nitrate reductase (NR) with apoenzyme from the Neurospora crassa mutant nit-1 was found mostly bound to a carrier protein (CP). This protein is scarce in the algal free extracts and has been purified 520-fold. MoCoCP is a protein of 64 kDa with subunits of 16.5 kDa and an isoelectric point of 4.5. In contrast to free MoCo, MoCo bound to CP was remarkably protected against inactivation under both aerobic conditions and basic pH. MocoCP transferred active MoCo to apoNR in vitro without addition of molybdate, though reconstituted activity was 20% higher in the presence of molybdate. Incubation with tungstate specifically inhibited MoCoCP activity but had no effect on the activity of free MoCo released from milk xanthine oxidase. MoCoCP did not charge molybdate unless in the presence of N. crassa extracts. Our data support that MoCoCP stabilizes MoCo in an active form charged with molybdate to provide MoCo to apomolybdoenzymes.z 1998 Federation of European Biochemical Societies.

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Section: Discussionsupporting

confidence: 73%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Chlamydomonas reinhardtii MoCo carrier protein is multimeric and stabilizes molybdopterin cofactor in a molybdate charged form

et al. 1998

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“…the stabilization of the cofactor under anaerobic conditions and in the presence of reducing agents such as thiols or ascorbate), the quantitative data are equivocal. At variance with previous work, we find that heat treatment releases only 30% of molybdenum cofactor from xanthine oxidase (rather than 95%; Alikulov et al, 1980;Mendel et al, 1982) and that NADPH inhibits (rather than activates; Mc-Kenna et al, 1974;Claassen et al, 1982) the complementation of nit-i extracts by the cofactor. Our results place on a firmer footing earlier evidence (Johnson, 1980;Johnson et al, 1980c) that active xanthine oxidase, desulpho xanthine oxidase and sulphite oxidase all yield equivalent amounts of a single cofactor.…”

Section: Discussioncontrasting

confidence: 98%

“…Despite non-quantitative cofactor transfer in earlier work, the assay has been widely employed to make semi-quantitative comparisons of different procedures used in the extraction, and reagents used in the stabilization, of the cofactor (Lee et al, 1974;Pienkos et al, 1977;Alikulov et al, 1980;Mendel et al, 1982;Mendel, 1983). Not surprisingly, although there is broad agreement on qualitative phenomena (e.g.…”

Section: Discussionmentioning

confidence: 99%

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

Hawkes¹,

Bray²

1984

Biochemical Journal

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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)

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Auxotroph Complementation via Protoplast Fusion in Hyoscyamus Muticus and Nicotiana Tabacum

Potrykus

Shillito

Jia

et al. 1983

Genetic Engineering in Eukaryotes

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Molybdenum cofactor in nitrate reductase-deficient tobacco mutants. II. Release of cofactor by heat treatment

Cited by 13 publications

References 6 publications

The Chlamydomonas reinhardtii MoCo carrier protein is multimeric and stabilizes molybdopterin cofactor in a molybdate charged form

The Chlamydomonas reinhardtii MoCo carrier protein is multimeric and stabilizes molybdopterin cofactor in a molybdate charged form

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

Auxotroph Complementation via Protoplast Fusion in Hyoscyamus Muticus and Nicotiana Tabacum

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