Jack bean urease (EC 3.5.1.5). Metalloenzyme. Simple biological role for nickel

Dixon, Nicholas E.; Gazzola, T C; Blakeley, Robert L.; Zermer, B

doi:10.1021/ja00847a045

Cited by 793 publications

(411 citation statements)

References 5 publications

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“…The value for E280 given by Blakeley et al (1969a) is in good agreement with that reported by Reithel & Robbins (1967), but is considerably smaller than that reported by Gorin & Chang-Chen Chin (1966). Spectroscopic analysis ofurease concentrations is further complicated by the recent discovery that nickel is bound to the enzyme (see Dixon et al, 1975aDixon et al, ,b, 1976. For crude urease preparations and for urease derivatives, protein was determined by the biuret method and the Folin method respectively (see Layne, 1957).…”

Section: Protein Determinationssupporting

confidence: 70%

“…(Berger & Schechter, 1970). An intriguing aspect of the reactivity of these thiol groups is the possibility that it may be influenced by the presence of bound Ni2+ (Dixon et a!., 1975b). The reactivities of the class-I thiol groups at pH17.8 are not affected by incorporating 0.2M-urea into the reaction mixtures.…”

Section: Characterization Ofthe Class-iand Class-il Thiolgroups Ofurementioning

confidence: 99%

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A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

Norris

Brocklehurst

1976

Biochemical Journal

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1. A convenient method ofpreparation ofjack-bean urease (EC 3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5±1.47kat/kg, Km = 3.32±0.05mM; kcat. = 2.15 x 104±0.05 x 104s-1 at pH7.0 and 38°C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin. 11, 1535-1558 suggests that the urease molecule (assumed to have mol.wt. 483000 and e280=2.84x105 litre-molhl cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six 'essential' thiol groups of low reactivity (class-Il) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25'C (1= 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-Il thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 x 104M-l'S-1 and (b) pKa = 8.1, k = 8.05 x 102M-1 .-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60x 102M-I s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50 % class-Ia groups and 50 % class-lb groups. The ratio class Ia/class lb decreases as the pH is raised according to a pKa value > approx. 9.5, and at high pH the class-I thiol groups consist of at most 25 % class-Ia groups and at least 75 % class-lb groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the 'essential' thiol groups in urease appear to be reactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.Urease (EC 3.5.1.5) can be extracted fromjack-bean (Canavalia ensiformis) meal and reproducibly purified to high specific activity by the method of Blakeley et al. (1969a). A chloroform/acetone-dried powder of jack-bean meal is extracted with 30% (v/v) acetone containing 1 % 2-mercaptoethanol at 39°C for 5min. The filtrate is left for 48h at 4°C to provide crystalline material that is then dissolved in buffer, dialysed and further purified by recycling upward-flow gel filtration on Sephadex G-200.We have reported a simple general method (covalent chromatography by thiol-disulphide interchange) for the purification ofthiol enzymes and its application in the immobilization of commercial samples of urease

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Section: Protein Determinationssupporting

confidence: 70%

Section: Characterization Ofthe Class-iand Class-il Thiolgroups Ofurementioning

confidence: 99%

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

Norris

Brocklehurst

1976

Biochemical Journal

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“…In 1975, Dixon et al (1975) found that Ni is a component of urease, a ubiquitous metalloenzyme present in plants, and later on, based on works by Eskew et al (1983Eskew et al ( , 1984 and Brown et al (1987), Ni was classified as a micronutrient (Salisbury & Ross, 1992;Marschner, 2008). However, research with Ni has mainly addressed its toxic effects on plants and on how Ni-hyperaccumulator plants respond to high Ni concentrations.…”

Section: Introductionmentioning

confidence: 99%

Effects of nickel and nitrogen soil fertilization on lettuce growth and urease activity

Oliveira¹,

Fontes²,

Rezende³

et al. 2013

Rev. Bras. Ciênc. Solo

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SUMMARYNickel is a micronutrient involved in nitrogen metabolism and a constituent of the urease molecule. Plant growth and urease activity were evaluated in lettuce (Lactuca sativa L.) grown in soil-filled pots in a 2 x 8 factorial design with two nitrogen (N) sources and eight Ni rates, with five replications. Nitrogen was applied at 200 mg dm -3 (half the dose incorporated into the soil at seedling transplanting and half top-dressed later) using the sources NH 4 NO 3 (AN) and CO(NH 2 ) 2 (Ur). The Ni treatments (0, 2, 4, 8, 12, 16, 24 and 32 mg dm -3 ) were applied as NiCl 2 . The shoot dry-matter yield, leaf urease activity, Ni levels in the lettuce leaves and Ni levels extracted from soil with Mehlich-3 (M-3) and DTPA were determined. In the plants supplied with AN , the shoot dry-matter yield was higher than in those supplied with Ur. There was no difference in shoot dry matter in response to soil-applied Ni. The leaf urease activity increased with Ni application, regardless of the N source. The extractions with M-3 and DTPA were efficient to evaluate Ni availability for lettuce in the Red-Yellow Latosol. Ni (0, 2, 4, 8, 12, 16, 24

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“…The first identified nickel enzyme was Jack Bean urease which requires nickel as an essential element for its activity (Dixon et al, 1975). Urease converts urea to ammonia and carbamate by using water and while doing this it requires nickel in its active site.…”

Section: Introductionmentioning

confidence: 99%

Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast

Bozdag

Kaya

Koç

et al. 2014

Gene

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Nickel is an essential micronutrient due to its involvement in many enzymatic reactions as a cofactor. However, excess of this element is toxic to biological systems. Here, we constructed a cDNA library from Beta maritima and screened it in the yeast system to identify genes that confer resistance to toxic levels of nickel. A cDNA clone (NIC6), which encodes for a putative membrane protein with unknown function, was found to help yeast cells to tolerate toxic levels of nickel. A GFP fused form of Nic6 protein was localized to multivesicular structures in tobacco epidermal cells. Thus, our results suggest a possible role of Nic6 in nickel and intracellular ion homeostasis.

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Jack bean urease (EC 3.5.1.5). Metalloenzyme. Simple biological role for nickel

Cited by 793 publications

References 5 publications

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

Effects of nickel and nitrogen soil fertilization on lettuce growth and urease activity

Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast

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