In , Krebs and Henseleit conducted a series of experiments using liver slices and manometric assays to show that in the presence of arginase, ornithine produced urea Jenkinson et al.
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Crude preparations of arginase were reported as early as Salaskin and Solowjew , and Waldschmidt-Leitz et al. Hellerman and Perkins first showed activation by bivalent metal ions including cobalt, nickel, manganese, and iron Hellerman and Perkins They demonstrated that the activity of arginase was dependent upon manganese, and found the optimal pH to be 9. Much work was done investigating inhibitors in the late s and early s Rosenfeld et al.
Recent research has investigated the roles of arginases in vascular disease, pulmonary disease, infectious disease, and cancer Zimmerman and Rothenberg , Maarsingh et al. Varying levels of arginase have been found in the reproductive system of cattle Razmi et al. Arginase is highly specific for its substrate. Class I and Class II enzymes contain carboxy-terminal tyrosine residues required for maximal catalytic activity.
Arginase has the highest level of specific activity of the urea cycle enzymes in the liver Jenkinson et al. Arginase I and arginase II are each encoded by a different gene. Arginase I is located in the cytoplasm and expressed in the liver as part of the urea cycle. Arginase II is a mitochondrial enzyme, and is expressed primarily in the kidney Romero et al.
Based on sequence analysis, arginase is probably a primordial enzyme that was present in the universal common ancestor Ouzounis and Krypides The arg1 gene is conserved in several species including human, dog, mouse, rat, zebrafish, and S. The gene is induced in the late fetal period and is upregulated by high nutritional protein uptake Morris et al. This is mediated by glucocorticoids and glucagon Morris et al.
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A cis element is present around to relative to the start site and is overlapped by two protein binding regions. These regions have been identified as footprint areas A and B. An enhancer region is also present 11 kb downstream from the start site Gotoh et al. Arginase is a homotrimer.
Each subunit contains an active site, and the two essential manganese ions are bridged by oxygens and separated by approximately 3. The mechanism is proposed to be a nucleophilic attack by the metal bridging hydroxide at the guanidinium carbon of the arginine substrate Ash At timed intervals start reaction by adding: Arginine 0. Stop reaction at timed intervals by adding: B.
Protein concentrations were determined by the Bradford dye binding method [ 34 ] using bovine serum albumin as a standard. The gels were stained with 0. The quality of peptide and protein assignments was assessed using PeptideProphet and ProteinProphet [ 36 ]. A plot of relative activity against pH was created to determine the optimum pH for the reaction. Samples were taken at different time intervals and the residual enzyme activity was determined as described above.
Typical of haloarchaeal proteins, the bga gene product contains a high percentage Since blue colonies were indeed observed data not shown , we proceeded to assay for breakdown of ONPG in crude extracts of H. The enzyme activity in Miller units MU for the former haloarchaeon is shown on the left and the latter is shown on the right. Values are the average of triplicate experiments with standard deviation shown with error bars. In order to study the H. NRC-1, and origins of replication for both E. Plasmid pMC2 was transformed into Halobacterium sp. The Halobacterium sp.
The results clearly demonstrated that the recombinant Halobacterium sp. A nearly identical temperature profile was observed for the enzyme produced in both H. Next, induction of H. Induction of H. NRC-1 pMC2. Activity levels are shown in Miller units MU on the left for cell lysate and the right for supernatant, and are the average of triplicate experiments, with standard deviation shown with error bars. Gel-filtration chromatography of cell lysate led to 4.
The higher apparent molecular mass was expected based on previous results with haloarchaeal proteins [ 41 ].
Thirteen unique peptides corresponding to H. Purification of H. Proteins are shown electrophoresed on an 8. The results were similar with either NaCl or KCl, tested up to 4. The enzyme is most active at the high salt concentrations similar to that reported intracellularly in haloarchaea. The enzyme was also found to be active near neutral pH in the 6. Properties of purified H. The effect of addition of organic solvents on the activity and stability of the H. These results show that the H. Effect of organic solvents on purified H.
Values are the average of triplicate experiments, with standard deviation shown as error bars. We have established that the glycoside hydrolase GH family bga gene in the cold-adapted Antarctic haloarchaeon H. In order to characterize the salient properties of this novel enzyme, we developed a cold-inducible, cold shock protein csp D2 gene promoter-based expression plasmid in the genetic model system, Halobacterium sp. NRC-1, and overexpressed the H.
The enzyme also serves as an excellent model for potential enzymatic activity in extraterrestrial conditions, such as those found on Mars [ 9 ]. In the past, a barrier to such studies has been the requirement of high salt concentrations to obtain enzyme activity during overexpression in a foreign host, since low ionic strength conditions generally lead to misfolding or inactivation [ 8 , 47 ]. To avoid problems overproducing active H. NRC-1, for overexpression. NRC-1, we introduced a cold-active promoter for the cold shock protein gene, csp D2, into a haloarchaeal expression vector [ 40 ].
The csp D2 gene was selected based on previous transcriptomic studies of Halobacterium sp.
NRC-1 [ 33 ]. Another challenge in studies of haloarchaeal proteins has been the development of a purification method, as a result of interference of many analytical and chromatographic techniques by high salinity levels [ 47 ]. For purification of the H. NRC-1, we used a combination of ion exclusion chromatography and hydrophobic interaction chromatography, since both methods are distinguished by their ability to be applied at high salinity. Similarly, ion exclusion chromatography has been successful over a wide range of ionic strength buffers, even those at high salinity [ 50 ].
For the H. As previously observed for highly acidic haloarchaeal proteins, anomalous migration was expected during electrophoreses in SDS-PAGE gels, because of the binding of detergents with electrostatic and hydrophobic interactions slows the rate of migration [ 41 , 51 ]. Consequently, the bga polypeptide displayed an anomalous molecular mass of ca. The breakdown of the chromogenic substrates, X-gal on agar plates by Halobacterium sp. The purified H. It exhibited maximal activity in the presence of 4.
Halophilic enzymes usually feature an increase in the number of charged amino acids, especially acidic residues at the protein surface and the negative surface charge is critical to their solubility and prevents aggregation at high salt concentrations [ 8 , 37 , 47 ]. A reason for the observed difference could be that the purified enzyme was used without prior addition of stabilizer. BA [ 53 ], indicating that extremophilic enzymes frequently function suboptimally under physiological conditions.
In general, non-halophilic enzymes lose most of their activity in the presence of organic solvents [ 22 ]. Karan et al. A protease from halophilic archaeon Natrialba magadii was found to be active and stable in aqueous-organic solvent mixtures containing 1. In other studies, halophilic enzymes have been reported to be active and stable in biphasic solutions of water and hydrocarbon organic solvents, such as benzene.
These include an amylase of a haloarchaeon, Haloarcula sp. EMB2 [ 49 ]. These studies indicate that organic solvent stability is a general property of halophilic enzymes, owing to their ability to work at low water activity. However, this is the first report of retention of high levels of enzyme activity in short and long chain alcohols, which reflect the polyextremophilic character of the enzyme. Polyextremophilic characteristics make the H. For example, the solvent stability of H.
Maugard et al.
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Recently, Bridiau et al. Vic et al. These genes reflect a substantial resource for directing the conversion of biomass into valuable commodities, such as biofuels. Moreover, our ability to genetically manipulate and shuttle these and other genes represents a substantial resource for the future. Halophilic Archaea offer an incomparable resource of polyextremozymes which are active and stable in high concentrations of salt, a broad range of temperatures, and organic solvents. At high salinity, water is sequestered in hydrated ionic structures, limiting the availability of free water molecules for protein hydration [ 37 ].
Since halophilic enzymes are adapted to function at high salt concentrations, they are also found to be active at low water availability. Such conditions are also encountered by enzymes in cold temperatures due to the freezing of water molecules and consequent formation of structured ice-like lattices [ 8 ]. Therefore, structure-function studies of cold-active haloarchaeal enzyme, including H. Since H. Images from the Mars Reconnaissance Orbiter showed evidence for seasonal emergence of liquid flows in summer, findings consistent with briny liquid water emerging from underground reservoirs on the planet [ 63 ].
The Jovian satellite Europa is covered by frozen water-ice and the presence of liquid water beneath the surface has been hypothesized [ 64 , 65 ]. The genome sequence of H. NRC-1, provided a polyextremophilic enzyme for investigation. The purified enzyme was found to be active in high salt concentrations 4—4. The enzyme provides a platform for improving our understanding of biocatalysis under extreme water limiting conditions and for potential applications in synthetic chemistry. The system provides the basis for better understanding of life in a perennially cold, hypersaline environment, with relevance to life elsewhere.
All authors read and approved the final manuscript. National Center for Biotechnology Information , U. BMC Biotechnol. Published online Jan Author information Article notes Copyright and License information Disclaimer.
Strategies for Protein Purification and Characterization: A Laboratory Course Manual
Corresponding author. Ram Karan: ude. Received Oct 3; Accepted Jan This article has been cited by other articles in PMC. Abstract Background Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Results Analysis of the genome sequence of H. Conclusion The H. Background The genome sequence of a cold-adapted extremely halophilic archaeon, Halorubrum lacusprofundi , was recently completed and analyzed by comparative genomics [ 1 - 3 ].
Open in a separate window. Figure 1.
Bioinformatic analysis Bioinformatic analysis of the H. Figure 2. Table 1 Oligonucleotides used in this study as primers for PCR. NRC-1 Halobacterium sp. Results H. Figure 3. Cloning and overexpression of H. NRC-1 In order to study the H.
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Figure 4. Purification and identification of H. Table 2 Purification of H. Figure 5. Characterization of purified H. Figure 6. Figure 7. Discussion We have established that the glycoside hydrolase GH family bga gene in the cold-adapted Antarctic haloarchaeon H. Conclusions The genome sequence of H. Competing interests The authors declare that they have no competing interests. The information transfer system of halophilic archaea.
The core and unique proteins of haloarchaea. BMC Genomics. Novel insights into the diversity of catabolic metabolism from ten haloarchaeal genomes. PLoS One. Halobacterium lacusprofundi , sp. Syst Appl Microbiol. Terrestrial models for extraterrestrial life: methanogens and halophiles at Martian temperatures.
Intl J Astrobiol. London: Wiley; Encyclopedia of life sciences. Genome-wide responses of the model archaeon Halobacterium sp. J Bacteriol. Function and biotechnology of extremophilic enzymes in low water activity.
Aquat Biosyst. Extreme halophiles are models for astrobiology.