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Abstract
We have purified an enzymatically active form of arginase from a wild-type strain of Neurospora crassa to homogeneity. The enzyme has a subunit molecular weight of 38,300 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein migrated as a hexamer during gel-filtration chromatography with an apparent molecular weight of 266,000. The enzyme exhibited hyperbolic kinetics at pH 9.5 with an apparent Km for arginine of 131 mM. Antiserum was prepared against the purified enzyme and used to demonstrate the existence of three cross-reactive proteins in crude extracts of wild-type N. crassa. One of these proteins corresponded to the purified protein, whereas the other two were of molecular weights 41,700 and 26,800, respectively. Using the same antiserum, we found that rat liver, but not rat kidney, contains immunoreactive material. We also detected two proteins in extracts of Saccharomyces cerevisiae that were weakly cross-reactive with the antiserum. These data provide evidence for the existence of multiple forms of arginase in fungi as well as in mammals.
Highlights
Since the cytoplasmic concentration of ornithine to ornithine and urea. In ureotelic organisms, such as mam- is 0.2 mM under normal conditions, ornithine may funcmals, the liver enzyme participates in the urea cycle [1].The tion to inhibit the activity of arginase i n uiuo [21]
Taken kidney of ureoteles does not contain an active urea cycle
The same case of heat instability has beenobservedfor the S. cerevisiae arginase [34]
Summary
6) DE52-cellulosep H Gradient Chromatography.The enzymatically active fractions from Step 5 were concentrated at 4 "C (Amicon Ultrafiltration Cell, YM-10 membrane) and thendiluted to the same ionic strength as a buffer containing 25 mM K,HP04, 20 mM arginine, 1 mM EDTA, 1 mM 2-mercaptoethanol, 0.1 mM PMSF, and 20% glycerol, pH 8.0. Samples were treated with 1/10 volume of 10% (v/v) 2-mercaptoethanol, 62.5% (v/v) Laemmli upper gel buffer [29] and 15%(w/v) sodium dodecyl sulfate a t 100 "C for 5 min to denature the proteins. All extracts (approximately 10-ml total volume) were centrifuged at 15,000 rpm for 15 min in an SS 34 rotor at 2 “C to remove cell debris and aggregated proteins. After air drying a t room temperature, the nitrocellulose filter was autoradiographed at -70 ‘C with preflashed film [33] and intensifying screens
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