Thioredoxin, a known catalyst for reducing protein disulfides, was shown to catalyze efficiently the refolding of pancreatic RNase either from the reduced, denatured form or from the scrambled form containing oxidized but incorrectly paired disulfides. Thioredoxin was 1000-fold more efficient on a molar basis than the model dithiol, dithiothreitol, in reactivating reduced, denatured RNase, suggesting that thioredoxin acts as an efficient catalyst for disulfide interchange. Starting with reduced, denatured RNase, enzyme activity was recovered quantitatively with a til2 of 30 hr with 100 ,AM thioredoxin compared to only a 10-20% recovery of activity in the control using air oxidation. Oxygen further stimulated the effectiveness of thioredoxin severalfold. Thioredoxin was most effective in reactivating inactive scrambled RNase, which contained mispaired disulfides, showing a til2 of 2 hr. Reduced thioredoxin was optimal for catalyzing disulfide interchange in scrambled RNase, whereas oxidized thioredoxin was required for reactivation of the reduced, denatured species. Optimal reactivation of scrambled RNase required a mixture of reduced and oxidized thioredoxin. Addition of reduced thioredoxin after initiating refolding of reduced denatured RNase with oxidized glutathione effected a rapid reactivation of RNase, suggesting a two-step model for protein refolding in which the monothiol catalyzes the rapid initial formation of protein disulfides and thioredoxin catalyzes the second step of disulfide interchange. Arguments are presented suggesting that thioredoxin may serve an in vivo role analogous to the protein disulfide-isomerase (EC 5.3.4.1).Thioredoxin(s) have been implicated in a variety of physiological roles, including serving as the hydrogen donor for the reduction of ribonucleotides and sulfoxides (1,2) and for the activation of enzymes (3) and serving as a co-factor for T7 phage DNA polymerase (4) and assembly of filamentous phage (5

During in vitro incubation, nuclei from polyoma-infected cells elongate the daughter strands of the replicative intermediate of pblyoma DNA. This process is now shown to involve the transient formation of short ft-agments (4-5 S), a process that is stimulated by the addition of ribonucleoside triphosphates. The prresence of stretches of RNA at the 5'-end of short DNA chains was determined from Cs2SO4 equilibrium centrifugation and from the finding that isotope from a-3"P-labeled deoxynucleoside triphosphates was recovered in 2'(3')-ribonucleotides after alkaline hydrolysis. Recent work with microbial systems by Kornberg and coworkers (4, 5) and Sugino et al. (6) has implicated RNA as a primer of DNA replication. We will present evidence for a similar participation of RNA during polyoma DNA replication in isolated nuclei. This process appears to involve the intermediate formation of short fragments, which sediment at about 4-5 S, consisting of mixed RNA-DNA polynucleotides. Short chains also accumulate in vivo during deoxynucleotide starvation after addition of hydroxyurea. MATERIALS AND METHODSLabeled nucleoside triphosphates were purchased from New England Nuclear Corp. or were synthesized by the procedure of Symons (7). Other sources of materials, the infection of cells, the preparation of polyoma DNA, and the centrifugation procedures were as described (1-3). Nuclei were prepared from cells suspended in "isotonic Hepes" buffer (2) by Perry's procedure with 0.5% NP42 (8), rather than by homogenizaAbbreviation: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid. * Present address: Institut fur Genetik, Universitat zu Kln, D-5 Koln-Lindenthal, Germany. t Present address: Department of Biochemistry, Faculty of Arts and Sciences, University of Pittsburgh, Pittsburgh, Pa. 15213. 412 tion in "hypotonic Hepes" buffer (2). After centrifugation, the nuclei were resuspended in 1-4 volumes of isotonic buffer and incubated under standard conditions (2). In some experiments ribonucleoside triphosphates were added, as indicated in the legends. RESULTS Intermediate formation of short fragmentsInfected nuclei were incubated under standard conditions with ['H]dTTP, and the radioactive polyoma DNA formed was analyzed by centrifugation in alkaline sucrose gradients (Fig. 1). At short incubation times, radioactivity was distributed bimodally, with a sharp peak around 4-5 5 and a second, broader, peak at higher S-values. The relative amount of radioactivity present in this second peak increased with time. The different labeled species were shown to be polyoma specific by hybridization studies.In a pulse-chase experiment, the radioactivity from the short chains formed during the first 1.5 min was converted into longer chains on prolonged incubation (Fig. 2). These results suggest that the DNA sedimenting at 4-5 S represents an intermediate in the synthesis of longer chains.Involvement of RNA in the synthesis of short fragments ATP enhances the incorporation of ['H]dTTP into polyoma DNA (2). Additiori of a mixt...

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Aspartate transcarbamylase (ATCase) from Escherichia coli was found to contain zinc in the amount of 6 ± 1 atoms per molecule of protein (3.1 X 105 daltons). Zn2+ was not removed from the protein by prolonged dialysis against solutions of strong chelators and did not exchange detectably with free 66Zn2+ during an exposure period of 40 days. The requirement of metal ions for the formation of intact ATCase could be demonstrated in vivo. E. coli, forced to synthesize enzyme in Zn2+-deficient medium, contained only 30% of their transcarbamylase activity as ATCase, with the remaining 70 % present as catalytic subunit, whereas bacteria grown with the normal excess concentration of Zn2+ had less than 5 % of their transcarbamylase activity as the catalytic subunit. When purified ATCase was treated with the mercurial p-mercuribenzoate (PMB), dissociation into catalytic and regulatory subunits occurred and Zn2+ became easily removable from the protein by dialysis against chelators, or during the standard procedure employed for the isolation of subunits (Gerhart, J. C., and Holoubek, H. (1967), J. Biol. Chem. 242, 2881). Regulatory subunit, obtained by this procedure, contained no detectable Zn2+ but contained tightly bound mercuric ion in variable amounts ranging from 0.1 to 1.0 atom per polypeptide chain (1.7 X 104 daltons). Mercuric ion appeared to be released from PMB during subunit isolation. The mercury content of regulatory subunit preparations correlated positively with their capacity to aggregate with catalytic subunit to form reconstituted ATCase. The isolated catalytic subunit also lacked Zn2+ and, unlike the regulatory subunit, contained no detectable Hg2+. This finding indicates that the catalytic activity of the enzyme does not depend on metal ions such as zinc or mercury. A modified procedure for T X he regulatory enzyme, aspartate transcarbamylase (ATCase)1 from Escherichia coli, contains two types of protein subunits, one the catalytic subunit which possesses active

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