Urease molecular weight

Conjugation of the nucleotides to urease was confirmed by ultraviolet (UV) spectra between 300 and 240 nm. Based on the change in the urease spectra before and after conjugation and the known molar extinction coefficients of the cyclic nucleotides, an estimate as to the degree of conjugation can be made. Typically, a conjugation between 2 and 7 mol of nucleotide per mole of enzyme is obtained based on a urease molecular weight of 480,000. 

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 ... 

Several investigators have presented evidence for low molecular weight forms exhibiting urease activity. Hand (33), in 1939, obtained diffusion data indicating particles of 17,000 daltons or less that retained enzymic activity. More recently, sucrose density gradient ultracentrifugation (34)... 

Using quantitative gel electrophoresis (37) and a simple activity stain (37), Blattler examined urease preparations showing evidences of aging changes (38, 38a). When urease, initially electrophoretically homogeneous, was allowed to age in buffer or 50% diol, enzymically active species appeared that had electrophoretic mobilities between the usual bands. These intermediate bands corresponded to a variety of species that differed by approximately 60,000 molecular weight between 240,000 and 480,000, and between 480,000 and 960,000. 

Urease activity persists unaltered when the enzyme is dissolved in SM urea although the ultracentrifuge data indicate a molecular weight of about 90,000 (7). This form of urease has not been sufficiently characterized but does indicate that neither enzymic activity nor specific activity is dependent upon very high molecular weight aggregates. 

Urease is remarkably resistant to unfolding in urea. No change in optical rotatory dispersion or specific activity of the enzyme occurs in concentrations of urea up to 8 M (7). Between 8 and 9M, unfolding occurs and the molecular weight becomes 60,000, but some activity persists. 

Solution in 0.1 M acetate buffer, pH 3.5, resulted in an enzymically active species (8n) of 240,000 daltons (46). Tanis and Naylor (47) have reported that at low concentration of protein the 18 S form predominated above pH 5.3 and the 12 S form below pH 4.8. Between these pH values a rapid equilibrium of the 12 S and 18 S species was observed The dissociation behavior of urease at low pH depends on the buffer used. In 0.1 M potassium phosphate buffer, adjusted to pH 2.0 with HC1, a heterogeneous mixture of dissociated forms was obtained (d) with an Mw of about 150,000. In acetate buffer at pH 3.5 dissociation into a 120,000 molecular weight species (4n) was observed (48). In 34% acetic acid at pH 2.2 there is effected a dissociation to subunits (n) of 30,000 daltons (7). This same value was obtained for urease ultracentrifuged in 8 M urea -f- 0.5 M thiol and in performic acid oxidized urease (48). 

A partially purified enzyme from P. mirabilis (72) was found to have a molecular weight of 151,000. The urease of P. rettgeri is an inducible enzyme that appears only when urea, but not its analogs, are present in the media (73). Proteus vulgaris urease was found to be inhibited in vitro by thiourea and two derivatives (74), and by hydroxamic acids (93). 

Ureases from several sources have been examined for enzymically active low molecular weight forms (47). It was noted that the 12 S forms from jack bean did not hybridize with that from B. pasteurii. It now seems probable that gastric urease is bacterial in origin (85). The oc-... 

Urease activity in soils has been found to reflect the bacterial count and content of organic matter. The urease isolated from an Australian forest soil (87) was crystallized and found to have a specific activity of 75 Sumner units (S.U.) per mg. The molecular weight species were estimated (sedimentation velocity) to be 42, 131, and 217 X 103. That urease activity persists in soils is shown by the finding that enzymic activities, including urease, could be demonstrated in soil samples over 8000 years old (88). 

Second, the correlation of change in enzymic activity with the titration of essential sulfhydryl groups has led to a postulation of eight active sites per 480,000 (56). Unfortunately, the possibility of structural changes during such titrations makes interpretation of such data equivocal. However, the observation that urease retained its activity in 8 M urea, where the molecular weight has been reduced at least to 90,000 (7), supports the conclusion above. 

Much uncertainty reigned over the nature of proteins, the best known of which were hemoglobin, the digestive enzymes, and later, insulin. Properties of individual amino acids and the peptide bond were studied early in this century, but it was not until urease was crystallized by Sumner1 in 1926, followed by the isolation of other pure enzymes, that it was finally accepted in the 1930s that enzymes were proteins and that their catalytic properties were not the function of some adsorbed low molecular weight entity. Somewhat later, towards the end of the 1930s, coenzymes were isolated and their roles established. 

Enzymes are the reaction catalysts of the biological systems. They are protein in nature (With exception of small group of catalytic RNA molecules). Molecular weight ranges from 12000 to over million. They are specific in action e.g. Urease. Their catalytic activity depends upon the integrity of their native protein conformation. If enzyme is denatured or dissociated into subunits catalytically activity is usually lost. The enzymes carry out transformation of molecules and also mediate transformation of energy e.g. PHOTOSYNTHESIS. 

Nickel is required for the synthesis of active urease in plant and other cells. The enzyme catalyzes the hydrolysis of urea to carbon dioxide and ammonia, via the intermediate formation of carbamate ion (equation 46). The molecular weight has been redetermined recently as 590 000 30 000, with six subunits. Each subunit has two nickel centres and binds one mole of substrate. The activity of the enzyme is directly proportional to the nickel content, suggesting an essential role for nickel in the enzyme. Several approaches, including EXAFS measurements, suggest that histidine residues provide some ligands to nickel, and that the geometry is distorted octahedral. There appears to be a role for a unique cysteine residue in each subunit out of the 15 groups present. Covalent modification of this residue blocks the activity of the enzyme. 

Urease (E.C.3.5.1.5) catalyzes the hydrolytic decomposition of urea into ammonium, bicarbonate, and hydroxide ions. It has a molecular weight of 483 kDa, and preparations with specific activities of 10,000 U/mg at 37 °C and pH 7.0 are commercially available. 

Figure 12 shows a possible set-up for hemodialysis monitoring. Patients blood is pumped through a dialysis cell, and low molecular weight substances including urea are removed by a semipermeable membrane (cut off 10 kD) and dialysis buffer. The urea enriched dialysate passes through an injection valve and enters a waste container. Due to switching the valve, a defined sample volume is pumped to the ET. Here, enzymatic conversion takes place via immobilized urease and provides information about the current urea concentration. Thus, the hemodialysis effect is automatically monitored via urea analysis and makes an individual treatment possible. 

Urease has a molecular weight of590 000 30 000 and consists of six identical subunits. Each subunit contains two Ni ions of different valency which are involved in substrate binding and conversion. The isoelectric point of the protein is at pH 5 and the temperature optimum of the catalysis at 60°C. The kinetic constants for urea hydrolysis have been determined to be k+2 = 5870 s"1 and Km = 2.9 mmol/1. Other amides, such as formamide and semicarbazide, react much more slowly than urea. The pH optimum of urease depends on the nature of the buffer used and, with the exception of acetate buffer, equals the pJTs value of the buffer. The active center of urease contains an SH-group that is essential for the stability of the enzyme. Complexing agents, such as EDTA and reductants, are required for stabilization. 

FIGURE 8. Determination of the native molecular weight of the purified PSPBP from Acanthocardia tuberculatum foot by native gel electrophoresis of various concentrations of polyacrylamide (6, 8, 10 and 12 %). Molecular weight marker proteins were commercial tetrameric urease (545 kDa), dimeric urease (272 kDa), dimeric BSA (132 kDa), monomeric BSA (66 kDa) and ovalbumin (45 kDa) (Sigma). (A) represents the relative mobilities of proteins plotted as Log (Rf x 100) vs. gel concentration. A plot of the obtained slopes vs. molecular weight was linear and used to determine native PSPBP molecular weight (B). 

The link between enzymes and substrates is so strong that enzymes often are named after the substrate involved, simply by adding ase to the name of the substrate. Eor example, lactase is the enzyme that catalyzes the breakdown of lactose, while urease catalyzes the chemical breakdown of urea. Enzymes bind their reactants or substrates at special folds and clefts, named active sites, in the structure of the substrate. Because numerous interactions are required in then-work of catalysis, enzymes must have many active sites, and therefore can have molecular weights as high as one million. 

Sumner s analytical studies convinced him that urease was a protein. This conclusion was resisted by the chemical community but John H. Northrop s (1891-1987) crystallization of pepsin in 1930 at the Rockefeller Institute in New York City and its unambiguous decomposition into amino acids fully vindicated Sumner. Sumner and Northrop were able to make use of the ultracentrifiige developed by Svedberg and the electrophoresis technique developed by his student Tiselius to fully establish purities and molecular weights of their enzymes. Sumner and his coworkers then crystallized trypsin and chymotrypsin. Sumner and Northrop shared the 1946 Nobel Prize in chemistry with Wendell M. Stanley (1904—71), who in 1935 crystallized the tobacco mosaic virus in his laboratory at the Rockefeller Institute. 

Urease is a simple protein with a molecular weight of 473,000 and an isoelectric point of 5.0-5.1. It contains 31 sulfhydryl groups per mole, not all of which are required for activity. Urease is sensitive, however, to both heavy metals and oxidizing agents. 

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