Proteins urease

A model enzyme (protein), urease, did not lose much of its activity until the eompaetion pressure exeeeded 474 mPa (63 mm Hg) above which 50% of the relative aetivity was lost [95],... 

The protein nature of enzymes was established through the seminal work of James Sumner. In 1926, Sumner succeeded in isolating the enzyme urease in crystalline form from jack bean meal. This was the first time in history that an enzyme had been obtained in crystalline, though not completely pure, form. Subsequently, Sumner established that the crystalline enzyme was a protein. Urease is an enzyme that degrades one of the human end products of nitrogen metabolism, urea, to ammonia and carbon dioxide ... 

Table 1 summarizes the general characteristics of representative urease, hydrogenase and CODHs. As it will be further discussed below, the X-ray structures of only two Ni-containing proteins, urease and hydrogenase, are known [16, 17]. The former has the well known triose phosphate isomerase (TIM) barrel topology (Fig. 1) whereas the latter displays a so far unique folding (Fig. 2). The next challenge will be the elucidation of the crystal structures of the CODH/ACS enzyme of Clostridium thermoaceticum and of the simpler CODH from Rhodospirillum rubrum. 

FITC-BSA (Model Immunization Protein), Urease potential study... 

Urease is one of the enzymes which have been obtained in the crystalline state. This has been done by stirring jack bean meal with 30°o aqueous acetone, filtering and allowing the filtrate to remain at o for several hours. The urease which crystallises out is separated by centrifuging and is then recrystallised. Like crystalline pepsin and trypsin, it is a protein. 

Arachin, the counterpart of glycinin in peanuts, consists of subunits of 60,000—70,000 mol wt which on reduction with 2-mercaptoethanol yield polypeptides of 41,000—48,000 and 21,000 mol wt (17) analogous to the behavior of glycinin. In addition to the storage proteins, oilseeds contain a variety of minor proteins, including trypsin inhibitors, hemagglutinins, and enzymes. Examples of the last are urease and Hpoxygenase in soybeans. 

Enzymes are proteins of high molecular weight and possess exceptionally high catalytic properties. These are important to plant and animal life processes. An enzyme, E, is a protein or protein-like substance with catalytic properties. A substrate, S, is the substance that is chemically transformed at an accelerated rate because of the action of the enzyme on it. Most enzymes are normally named in terms of the reactions they catalyze. In practice, a suffice -ase is added to the substrate on which die enzyme acts. Eor example, die enzyme dial catalyzes die decomposition of urea is urease, the enzyme dial acts on uric acid is uricase, and die enzyme present in die micro-organism dial converts glucose to gluconolactone is glucose oxidase. The diree major types of enzyme reaction are ... 

Bacterial catabolism of oral food residue is probably responsible for a higher [NHj] in the oral cavity than in the rest of the respiratory tract.Ammonia, the by-product of oral bacterial protein catabolism and subsequent ureolysis, desorbs from the fluid lining the oral cavity to the airstream.. Saliva, gingival crevicular fluids, and dental plaque supply urea to oral bacteria and may themselves be sites of bacterial NH3 production, based on the presence of urease in each of these materials.Consequently, oral cavity fNTi3)4 is controlled by factors that influence bacterial protein catabolism and ureolysis. Such factors may include the pH of the surface lining fluid, bacterial nutrient sources (food residue on teeth or on buccal surfaces), saliva production, saliva pH, and the effects of oral surface temperature on bacterial metabolism and wall blood flow. The role of teeth, as structures that facilitate bacterial colonization and food entrapment, in augmenting [NH3J4 is unknown. 

In contrast to the abundance of Fe-proteins, there are only six known nickel-containing enzymes hydrogenase, CO dehydrogenase (CODA), acetyl-CoA synthase (ACS), superoxide dismutase, urease, and S-methyl-CoM methylreductase. Among these enzymes, it exists in very diverse environments, including a dinickel site (urease), a Ni-Fe heterobinuclear site (hydrogenase), a Ni-Fe4S4 heterometallic... 

Recently, the old alkaline phenol method has been revived, and is being widely used in clinical laboratories, without protein preclpltatlon(27). In this procedure, the serum is added to an alkaline phenol reagent, and the ammonia generated from urea is determined either after the action of urease or after strong alkaline treatment of the serum. The objection to this procedure is first, that all urease is rich in ammonia, and second, the color produced with alkaline phenol is not specific for ammonia. It will react with other compounds, especially for those that liberate ammonia. By this procedure one obtains a useful number from the point of view of determining whether the patient has nitrogen retention, but a value which is somewhere between a urea and an N.P.N. determination. 

Urease assay. When Proteus mirabilis grows in a urea-containing medium it hydrolyses the urea to ammonia and consequently raises the pH of the medium. This production of urease is inhibited by aminoglycoside antibiotics (inhibitors of protein synthesis Chapter 8). In practice, it is difficult to obtain reliable results by this method. 

Enzymes modified with N -carbonyldiimidazole (CDI) include horseradish peroxidase 761 /1-lactamase after nitration and reduction,[771 lysozyme, and urease.[781 Ref. [77] describes how the tyrosine side chain of a protein was nitrated, reduced with dithionite to an amino group, and then treated with CDI or A/-(2,2,2-trifluoro-ethoxycarbonyl)imidazole to give the benzoxazolinonyl alanine moiety ... 

The results of most model studies for Ni-mediated urea degradation reported to date are consistent with a cyanate intermediate. While this differs from the most likely mechanism of urease activity as deduced from protein crystallography, there is still no definitive evidence ruling out a transient Ni-bound cyanate intermediate for the enzyme. 

Laufberger had tried to obtain the protein from horse liver, but it did not crystallize, and as he described to me when I met him in Prague some years ago, in those days everyone wanted to have protein crystals as a criteria of purity. Although James Sumner had crystallized jack bean urease in 1926, his preparations were somewhat impure, and it was only in the mid-1930s, when John Northrop and Moses Kubnitz showed that there is a direct correlation between the enzymatic activities of crystalline pepsin, trypsin and chymotrypsin that the protein nature of enzymes was generally accepted. 

For other, larger proteins, the release was very slow - in 100 hours no more than 5% of loaded catalyse, urease, glucose oxidase or hemoglobin were released. 

As the one of the main end products of protein metabolism in living organisms, urea is a primary source of organic nitrogen in soil (from animal urine, fertilizers, etc.). Monitoring the level of urea is important for medicine, as well as for environmental protection. Urease is an enzyme that breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia and water. This enzyme is widely used for determination of urea in... 

Benoit, S.L., Mehta, N., Weinberg, M.V., Maier, C., and Maier, R.J. (2007) Interaction between the Helicobacter pylori accessory proteins HypA and UreE is needed for urease maturation. Microbiology 153,1474-1482. 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

Given that hydroxylamine reacts rapidly with heme proteins and other oxidants to produce NO [53], the hydrolysis of hydroxyurea to hydroxylamine also provides an alternative mechanism of NO formation from hydroxyurea, potentially compatible with the observed clinical increases in NO metabolites during hydroxyurea therapy. Incubation of hydroxyurea with human blood in the presence of urease results in the formation of HbNO [122]. This reaction also produces metHb and the NO metabolites nitrite and nitrate and time course studies show that the HbNO forms quickly and reaches a peak after 15 min [122]. Consistent with earlier reports, the incubation ofhy-droxyurea (10 mM) and blood in the absence of urease or with heat-denatured urease fails to produce HbNO over 2 h and suggests that HbNO formation occurs through the reactions of hemoglobin and hydroxylamine, formed by the urease-mediated hydrolysis of hydroxyurea [122]. Significantly, these results confirm that the kinetics of HbNO formation from the direct reactions of hydroxyurea with any blood component occur too slowly to account for the observed in vivo increase in HbNO and focus future work on the hydrolytic metabolism of hydroxyurea. 

James Sumner received the Nobel Prize for Chemistry in 1946 for the crystallization of proteins. Richard Willstatter, the 1915 Chemistry prizewinner, had proposed that proteins were not enzymes, and that the protein in urease was simply a scaffold for the veritable catalyst. Since urease is inactive without Ni, he was not so far wrong ... 

Hydrogenases are not the only nickel-containing enzyme, and researchers must therefore compare the maturation of different nickel proteins to obtain an integrated picture of nickel metabolism. Indeed, similarities between some of the hydrogenase-related nickel-processing proteins with urease and carbon monoxide dehydrogenase maturation factors have been noted, and this has facilitated interpretations of the results for... 

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