Purification of a Multipotent Antideath Activity from Bovine Liver and Its Identification as Arginase: Nitric Oxide-Independent Inhibition of Neuronal Apoptosis

Abstract

Catalase is an antioxidant enzyme that has been shown to inhibit apoptotic or necrotic neuronal death induced by hydrogen peroxide. We report the purification of a contaminating antiapoptotic activity from a commercial bovine liver catalase preparation by following its ability to inhibit apoptosis when applied extracellularly in multiple death paradigms. The antiapoptotic activity was identified by protein microsequencing as arginase, a urea cycle and nitric oxide synthase-regulating enzyme, and confirmed by demonstrating the presence of antiapoptotic activity in a >97% pure preparation of recombinant arginase. The pluripotency of recombinant arginase was demonstrated by its ability to inhibit apoptosis in multiple paradigms including rat cortical neurons induced to die by glutathione depletion and oxidative stress, by 100 nM staurosporine treatment, or by Sindbis virus infection. The protective effects of arginase in these apoptotic paradigms, in contrast to previous studies on excitotoxic neuronal necrosis, are independent of nitric oxide synthase inhibition. Rather, arginase-induced depletion of arginine leads to inhibition of protein synthesis, resulting in cell survival. Because inhibitors of nitric oxide synthesis and of protein synthesis have been shown to decrease necrotic and apoptotic death, respectively, in animal models of stroke and spinal cord injury, arginine-depleting enzymes, capable of simultaneously inhibiting protein synthesis and nitric oxide generation, may be propitious therapeutic agents for acute neurological diseases. Furthermore, our results suggest caution in attributing the cytoprotective effects of some catalase preparations to catalase.

Decreased Intracellular Superoxide Levels Activate Sindbis Virus-induced Apoptosis

“…levels and not increased or decreased peroxide levels. Furthermore and consistent with our previous observations (36,47), these data demonstrate that H 2 O 2 is not a regulator of SV-induced apoptosis. 2 .…”

Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis

“…In brief, murine arginase I cDNA in plasmid pCMV-SPORT6 (Incyte Genomics, St. Louis, Missouri, USA) was linearized by EcoRI or Not I digestion, and antisense and sense RNA probes, respectively, were generated by T7 and SP6 RNA polymerase (Riboprobe Gemini Core System II transcription kit; Promega, Madison, Wisconsin, USA). The radiolabeled (αS 35 -UTP) probes were hybridized and washed under high-stringency conditions.…”

“…We were interested in proving that products downstream from arginase were actually overproduced in the allergic lung. Recent studies have shown that arginase is the rate-limiting enzyme for biosynthesis of putrescine, and that arginase expression can inhibit cellular apoptosis (35). We therefore examined putrescine levels in the lungs of asthmatic and control mice.…”

Increased Synthesis of Spermidine as a Result of Upregulation of Arginase I Promotes Axonal Regeneration in Culture andIn Vivo

“…Protein concentration was measured with a Bio-Rad kit. Normalized lysates were boiled for 5 min, after which they were subjected to SDS-PAGE in a 10% polyacrylamide gel, transferred to nitrocellulose membranes, and immunostained for Arg I (polyclonal) (Esch et al, 1998) at 1:5000 overnight at 4°C. After two washes with PBS-0.05% Tween 20, the membranes were incubated with HRP-conjugated anti-rabbit IgG (1:5000) at room temperature for 1 h. After an additional three washes with PBS-0.05% Tween 20, HRP was visualized with ECL Western blotting reagent (GE Healthcare).…”