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Arginase (EC 3.5.3.1), the final enzyme in the urea cycle, catalyzes the cleavage of arginine to orthinine and urea. At least two forms of this enzyme, AI and AII, have been described and are probably encoded by discrete genetic loci. The expression of these separate genes has been studied in mammalian cells grown in culture. The permanent rat-hepatoma line H4-II-E-C3 contained exclusively the AI enzyme; the form in mammals comprising about 98% of the arginase activity in liver and erythrocytes but catalyzing only about one half of that reaction in kidney, gastrointestinal tract, and brain. By contrast, human-embryonic-kidney and -brain cells, after transformation with the human papovavirus BK, contained only the AII species of arginase, which form contributes the remaining half of that catalysis in those mammalian tissues in vivo. We report here the results of an extensive study on the properties of these two forms of arginase in the three cell lines, including Km values for arginine, behavior on polyacrylamide gels under non-denaturing conditions, and cross-reactivity with lapine antibodies against the arginases from either rat or human liver.

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We have found that the induction of phenylalanine hydroxylase by hydrocortisone and serum in confluent cultures of H4--E-C3 rat hepatoma cells is accompanied by an increase in polysomal mRNA specific for phenylalanine hydroxylase, as measured by translation in a cell-free protein-synthesizing system. Thus, the induction is mediated largely, if not entirely, by a pretranslational mechanism, possibly by stimulation of the transcription of the phenylalanine hydroxylase gene.It has been shown that two cultured rat hepatoma cell lines, H4-II-E-C3 (H4) and MH1C1, contain phenylalanine hydroxylase but at a very low level (1). Addition of hydrocortisone or dexamethasone at submicromolar concentrations and of steroid-free blood serum, from diverse mammalian species, to confluent cultures of the H4 cells caused an increase in the hydroxylase content of the cells to levels comparable with those found in normal rat liver (1, 2). This increase resulted from an increase in the amount of hydroxylase protein and not from activation of an inactive (pro)enzyme (3). The stimulation by glucocorticoids was shown to be mediated by an increase in the rate of the synthesis of the enzyme (4) without any change in the rate of its degradation (5).By the use ofa cell-free protein-synthesizing system ofwheat germ and specific antiserum against rat liver phenylalanine hydroxylase, we now show that the induction of the hydroxylase by hydrocortisone and serum in H4 cells is associated with an increase of polysomal mRNA specific for phenylalanine hydroxylase (mRNAph).METHODS AND MATERIALS Cell Cultures and Assay for Phenylalanine Hydroxylase. H4 cells were grown to confluency in 75-cm2 flasks (Lux Scientific, Thousand Oaks, CA) in modified Swim's medium S-77 supplemented with 5% (vol/vol) fetal bovine and 10% horse sera (1, 2). The harvesting and extraction of postconfluent cultures for assay of phenylalanine hydroxylase by the method of Ayling et al. (6) have been described (1-3). One enzyme unit is defined as the phenylalanine-dependent oxidation of 1 nmol of 6,7-dimethyltetrahydropterin to 6,7-dimethyldihydropterin or the formation of 1 nmol of tyrosine per min at 27°C. Protein concentrations were measured by dye-binding (7).To prepare the basal and fully induced cells used for the preparation of poly(A)+RNA (see Table 1 and Fig. 2), postconfluent cultures of H4 cells were first washed with modified S-77 medium and then given either the same medium without serum and hydrocortisone or serum-containing medium supple- The incorporation of 14C into total soluble cellular protein was 0.35 ,Ci per flask; ofthis, '3% could be precipitated by antiserum against rat liver phenylalanine hydroxylase.Isolation of Total Polysomal Poly(A)+RNA. Poly(A)+RNA was isolated from disrupted H4 cells by a combination of previously described methods (8-10) with minor modifications, such as the use ofHepes for all buffers. A total of 12 preparations was made; each was derived from cells pooled from sets of 10, 25, 30, or 32 similarly grown cultures. Nine prep...

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