Quaternary structure of ascorbate oxidase

Strothkamp, Kenneth G.; Dawson, Charles R.

doi:10.1021/bi00700a006

Cited by 48 publications

(27 citation statements)

References 31 publications

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“…This value lies in the range of the blue proteins containing only Type I Cu 2÷ [1], but is considerably lower than the values found for the laccases and ceruloplasmin [ 1 ]. The presence of only one Type 2 Cu 2 + in ascorbate oxidase is inconsistent with a symmetrical quaternary structure, consisting of two identical units, two 'laccase' halves, as proposed by Strothkamp and Dawson [15]. In fact all the blue oxidases seem to have only one Type 2 Cu 2÷ per molecule.…”

Section: Resultsmentioning

confidence: 83%

The stoichiometry of the three different types of copper in ascorbate oxidase from green zucchini squash

1974

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Section: Resultsmentioning

confidence: 83%

The stoichiometry of the three different types of copper in ascorbate oxidase from green zucchini squash

1974

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“…they are subunits or biological degradation products of the corresponding blue oxidases. Earlier work by Strothkamp and Dawson [38] on the quaternary structure of ascorbate oxidase led to the conclusion that the multicopper oxidase contains two subunits with a molecular weight of approximately 65000. Each of these two subunits can be subdivided into one component a (38000 M,) and one component /3 (28 000 Mr).…”

Section: Possible Physiological Role Of Mavicyaninmentioning

confidence: 99%

Mavicyanin, a Blue Copper Protein from Cucurbita pepo medullosa

Marchesini¹,

Minelli²,

Merkle³

et al. 1979

European Journal of Biochemistry

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The copper protein mavicyanin has been isolated and purified from the green squash Cucurbita pepo medullosa. Mavicyanin contains one type‐1 copper/18000 Mr, which can be characterized by: intense absorption maximum at 600 nm (ɛ= 5000 M−1 cm−1/Cu, A280/A600= 8.0 ± 0.5, A600/A403= 7.0 ± 0.25, maximum of fluorescence emission at 335 nm. In the oxidized state the copper of mavicyanin is 100% detectable by electron paramagnetic resonance (EPR). Computer simulation of the rhombic EPR signal gives gz= 2.287, gy= 2.077, gx= 2.025, Az= 3.5 mT, Ay= 2.9 mT and Ax= 5.7 mT. Like other simple type‐1 copper proteins, such as stellacyanin, azurin or plastocyanin, mavicy‐anin is readily reduced by hydroquinone or L‐ascorbic acid. Its midpoint potential E′m was determined to be + 285 mV. The reduced protein reacts rather slowly with dioxygen, but is rapidly reoxidized by ferricyanide.

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“…which is made of two identical subunits c $ as proposed by Strothkamp and Dawson [15]. On thc other hand so far all the multicopper oxidases described in the literature seem to have only 1 type-2 copperiactive site [16].…”

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confidence: 95%

Ascorbate Oxidase from Cucurbita pepo medullosa

Marchesini¹,

Kroneck²

1979

European Journal of Biochemistry

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1. Ascorbate oxidase has been isolated from the green squash Cucurbitapepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20 000 M, could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 f 0.11140000 MI) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 2. The pure enzyme is characterized by the following optical purity indices: A2so/Ablo = 25 +_ 0.5, A330/A610 = 0.65 & 0.05 and A6I0/A500 = 7.0 & 0.25. The molar absorption coefficient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts to 9700 M-' em-'.3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, g , = 2.058, g, = 2.036; A , = 5.0 mT, A , = A , = 0.5 mT, for the type-2 (non-blue) copper gli = 2.242, g l = 2.053; All = 19.0 mT, A1 = 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra.4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductate must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (%ax = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (l/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm ( A s = 1000 f 50 M-'

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Quaternary structure of ascorbate oxidase

Cited by 48 publications

References 31 publications

The stoichiometry of the three different types of copper in ascorbate oxidase from green zucchini squash

The stoichiometry of the three different types of copper in ascorbate oxidase from green zucchini squash

Mavicyanin, a Blue Copper Protein from Cucurbita pepo medullosa

Ascorbate Oxidase from Cucurbita pepo medullosa

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