The arginase enzyme is the sixth and final enzyme in the urea cycle. The arginase is found in the cytosol and mitochondria. It catalyzes the amino acid arginine and water to produce ornithine and urea. The urea cycle is essential for countless species of terrestrial organisms to excrete excess nitrogen in the form of ammonia. Reptiles and birds use a different cycle known as the uric acid cycle to excrete excess nitrogen, while fish excrete nitrogen in the form of ammonia into water in small amounts. For land animals, the ammonia is used to make urea and for those that fly, uric acid is produced.

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It is known that the fungus Neurospora Crassa (N. crassa), a common species of bread mold, produces arginase as well. This form of the enzyme is a protein currently being studied in Stockton’s Biochemistry Lab Methods class. The enzyme must be extracted from N. Crassa. In order to do this, the cell walls of the fungus must be broken down mechanically to create a crude extract. In order to extract the arginase alone, an antibody tailored specifically to bind to arginase based on its structure. There is no commercially available antibody that recognizes N. Crassa arginase, however there is a human antibody that will bind to the enzyme. This suggests a high degree of similarity in the primary, secondary, tertiary, and even quaternary structures between arginase used by homo sapiens and N. Crassa. If this is true, we hypothesized that arginase enzyme amino acid sequences and metabolic functions among more complex eukaryotic species would display an even higher degree of similarity among many other diverse eukaryotic organisms, even among the organisms who use different nitrogenous waste cycles. These species include humans, other mammals, fishes, birds, reptiles, amphibians.

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Mammals are the primary producers of urea. They produce urea as the primary nitrogenous waste product. They are known as ureotelic organisms because they secrete urea, the main nitrogen-containing substance in the urine. Urea is also found in the urine of amphibians (tadpoles excrete ammonia then convert to urea post metamorphosis) and some fish (“Nitrogenous Wastes”, n.d.). Fish (ammonotelic) can excrete toxic ammonia directly into the aquatic environment around them. The large amount of water dilutes the ammonia enough so it won’t change the pH of the environment. However, Gulf toadfish is one of the few adult teleostean fish that use the urea cycle to excrete urea as their predominant nitrogenous waste cycle (Barimo and Walsh, 2006) . Teleost is the major group of ray-finned vertebrae fish. Tilapia fish Oreochromis secretes urea almost exclusively. It is hypothesized that they do this to survive in alkaline conditions (Randall et al., 1989). Birds, reptiles, and terrestrial invertebrates such as insects and arthropods excrete uric acid or guanine. These are known as uricotelic organisms. In comparison to urea, uric acid production requires more energy, but is less toxic and reduces water loss for the organism (“Nitrogenous Wastes”, n.d.). Uric acid is excreted in the feces as a white powder. The urea cycle uses five intermediate steps to convert ammonia to urea.

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There are two types of Arginase: Arginase I and Arginase II. Arginase I is found in the liver while Arginase II is found in the mitochondrial kidney. Arginase I is primarily expressed in the liver and plays a role in urea synthesis. This type of Arginase eliminates nitrogen that is generated when the amino acids are metabolized. It also serves to eliminate nucleotides that are generated in the urea cycle. On the other hand, arginase II is a mitochondrial enzyme that is expressed in most tissues. Extra hepatic Arginase II primarily functions to produce ornithine as a precursor for the biosynthesis of polyamines, glutamate, and proline, as well as the synthesis of urea for osmoregulation and regulation of arginine for nitrous oxide (NO) formation. Arginase is mostly confined to the kidney in uricotelic organisms. Its main function in birds is supplying ornithine for detoxification, however it does play a role in producing a very low level of urea as well. Arginase catalyzes the fifth and final step of the urea cycle, converting the amino acid arginine into ornithine and urea, a chemical reaction that occurs in the liver. Arginase I and Arginase II are related by 58% sequence identity (Ash, 2004).

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The overall goal of this study was to compare and contrast the similarity of arginase primary structure from 15 different species as well as to assess the metabolic function of two isoforms of arginase in ureotelic, ammonotelic, and uricotelic organisms.