Hydrophobic interactions in the hinge domain of DNA polymerase beta are important but not sufficient for maintaining fidelity of DNA synthesis.

We previously described a general mutator form of mammalian DNA polymerase beta containing a cysteine substitution for tyrosine 265. Residue 265 localizes to a hydrophobic hinge region predicted to mediate a polymerase conformational change that may aid in nucleotide selectivity. In this study we tested the hypothesis that van der Waals and hydrophobic contacts between Y265 and neighboring residues are important for DNA synthesis fidelity and catalysis, by altering interactions in the hinge domain via substitution at position 265. Consistent with the importance of hydrophobic interactions, we found that phenylalanine, leucine, and tryptophan substitutions did not alter significantly the steady-state catalytic efficiency of DNA synthesis, relative to wild type, while the polar serine substitution decreased catalytic efficiency 6-fold. However, we found that all substitutions other than phenylalanine increased the error frequency, relative to wild type, in the order serine > tryptophan = leucine. Therefore, maintenance of the hydrophobicity of residue 265 was not sufficient for maintaining fidelity of DNA synthesis. We conclude that while hydrophobic interactions in the hinge domain are important for fidelity, additional factors such as electrostatic and van der Waals interactions contributed by the tyrosine 265 aromatic ring are required to retain wild-type fidelity.

Stabilization of RNA tertiary structure by monovalent cations.

The effects of monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg(2+) compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg(2+) concentration is zero and the other where it is maximally stabilizing ("saturating"). The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg(2+) from the RNA, its ability to stabilize tertiary structure in the absence of Mg(2+), and its ability to stabilize tertiary structure at saturating Mg(2+) concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg(2+), there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K(+) is the most effective of the tested cations, and Mg(2+) appears ineffective at this site. At high ionic strength, and in the absence of Mg(2+), stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations ( approximately eight) are released upon RNA tertiary structure denaturation, and NH(4)(+) appears to be the most effective cation in stabilizing tertiary structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes tertiary but not secondary structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg(2+) competition.

Formation and fate of tyrosine. Intracellular partitioning of newly synthesized tyrosine in mammalian liver.

Tyrosine in an hepatocyte is transported from the plasma, synthesized from phenylalanine, or released during protein turnover. Effects of phenylalanine and tyrosine on the formation and fate (partitioning) of tyrosine from the different sources were examined in primary rat hepatocyte cultures. Rates of tyrosine degradation, transport, incorporation into and release from protein, and synthesis from phenylalanine were measured as well as the intracellular dilution of labeled tyrosine and phenylalanine incorporated into protein. We found tyrosine had little effect on phenylalanine hydroxylation over a wide range of conditions, that transported tyrosine and tyrosine from phenylalanine are in different metabolic pools, and that there appears to be channeling of newly synthesized tyrosine during degradation. In addition, under some conditions, intracellular partitioning of tyrosine is determined by tyrosine concentration. Specifically, if extracellular tyrosine is low and phenylalanine is at a normal plasma level, tyrosine use in protein synthesis takes precedence over tyrosine degradation or export. It is proposed that the mechanism controlling this is kinetic, based on relative rates of tyrosyl-tRNA formation and tyrosine degradation and export. A quantitative model of tyrosine and phenylalanine in-flow and out-flow in hepatocytes is given, incorporating tyrosine synthesis, degradation, plasma membrane transport, and tyrosine and phenylalanine use and release during protein turnover.

Coordinate regulation of tetrahydrobiopterin turnover and phenylalanine hydroxylase activity in rat liver cells.

This work had two purposes: (i) to determine in vivo whether liver phenylalanine hydroxylase (PAH) is regulated by its substrates phenylalanine and tetrahydrobiopterin (BH4) as studies with purified enzyme suggest and (ii) to investigate in vivo the relationship between PAH activity and BH4 turnover. We found there are two BH4 pools in hepatocytes, one that is metabolically available (free BH4) and one that is not (bound BH4). Bound BH4 appears bound to PAH; the PAH-BH4 complex has much less catalytic activity and is less readily phenylalanine activated than uncomplexed enzyme. Interconversion of activated and unactivated PAH and bound and free BH4 is driven by phenylalanine; and free BH4 concentration is determined by the state of activation and activity of PAH. In hepatocytes, BH4 and PAH (subunit) concentrations are equal, all intracellular BH4 appears to be available to PAH, and free BH4 turns over rapidly (t1/2 approximately 1 hr). There is no evidence for feedback inhibition of BH4 synthesis; the BH4 synthetic rate appears high when free BH4 concentration is high and low when free BH4 is low. The data provide support in vivo that phenylalanine and BH4 are positive and negative regulators of the activity and activation state of PAH in the proposed manner; they also imply that regulation of BH4 turnover and PAH activity are linked processes, which are both controlled by phenylalanine concentration.

Regulation of rat liver phenylalanine hydroxylase. II. Substrate binding and the role of activation in the control of enzymatic activity.

Activation by phenylalanine and reduction by the co-factor (6R)-tetrahydrobiopterin (BH4) are required for formation of active liver phenylalanine hydroxylase. This work describes effects of the activation and redox state on substrate and effector recognition of this enzyme, it establishes relationships among the pterin and phenylalanine binding sites on the different forms of the enzyme, and it provides a quantitative description of the enzyme's presumptive regulatory and catalytic sites. BH4, 7,8-dihydrobiopterin (BH2), 6-methyltetrahydropterin, and 5-deaza-6-methyltetrahydropterin were found to bind to unactivated phenylalanine hydroxylase with a stoichiometry of 1/enzyme subunit and with hyperbolic kinetics; all appear to compete for the same binding site on the enzyme, and all appear to bind in the proximity of, but not to, the enzyme's non-heme iron. In the transition from unactivated to activated enzyme, phenylalanine and pterin binding is modified, a new site for phenylalanine is formed, and the pterin site is replaced by a site of greatly decreased affinity for BH4 and BH2, one which does not appear to recognize the dihydroxypropyl side chain of BH4 and BH2. The pterin- and phenylalanine-binding sites on activated phenylalanine hydroxylase appear to be part of the enzyme's active site. Despite large effects on substrate binding, neither chelator binding ability nor solvent accessibility of the iron are affected by activation; activation appears to affect the nearby environment of the enzyme's iron but not the iron itself. Studies of oxidized and reduced phenylalanine hydroxylase indicate that the redox state is not a major determinant of pterin and phenylalanine association with enzyme.

Regulation of rat liver phenylalanine hydroxylase. I. Kinetic properties of the enzyme's iron and enzyme reduction site.

Tetrahydropterins react with phenylalanine hydroxylase at a redox site, a regulatory site, and the catalytic site, but neither the properties of nor relationships among these sites are well understood. We have studied the redox site using the fluorescent iron chelators 2,3-dihydroxynaphthalene and bathophenanthroline; these compounds act as site-specific reporter groups for reactions on oxidized and reduced enzyme, respectively. The chelators bind reversibly and specifically to the enzyme's iron with 1:1 stoichiometry, high affinity (Kd values approximately 1 nM), and complete quenching of their own fluorescence. The kinetic behavior of these and other iron chelators indicates that the enzyme's iron is solvent accessible and in a hydrophobic pocket of the protein. Both ferrous and ferric chelators inhibit phenylalanine hydroxylase activity. Bathophenanthroline inhibits by binding to Fe2+ on reduced, active enzyme. 2,3-Dihydroxynaphthalene inhibits by binding to Fe3+ on enzyme that is oxidized during catalysis. This oxidation occurs approximately 1/150 enzyme turnovers, and its rate is increased when p-chloro- or p-fluorophenylalanine is used as the reaction substrate. Studies of the reaction of tetrahydrobiopterin (BH4) at the enzyme's redox site showed that BH4 reduces the enzyme more slowly than 6-methyltetrahydropterin under catalytic and non-catalytic conditions. Reduction occurs at a distinct site whose binding determinants and reaction characteristics are different from those of the BH4 regulatory or catalytic sites, and phenylalanine-activated enzyme is reduced more rapidly than unactivated enzyme. In reducing phenylalanine activated enzyme, BH4 donates one electron/subunit (1/iron atom); the reduction kinetics suggest a trihydrobiopterin-free radical as a reaction intermediate.

Regulation of rat liver phenylalanine hydroxylase. III. Control of catalysis by (6R)-tetrahydrobiopterin and phenylalanine.

Effects of phenylalanine and di- and tetrahydropterins on presteady-state and steady-state catalytic behavior of rat liver phenylalanine hydroxylase are analyzed. From this and previous work (Shiman, R, Xia, T., Hill, M., and Gray, D.(1994) J. Biol. Chem. 269, 24647-24656), which analyzed binding of the same compounds to the enzyme in the absence of catalysis, a model of phenylalanine hydroxylase regulation is proposed. The mechanism appears novel in that 1) one substrate, phenylalanine, is a positive effector (activator), 2) a second substrate, (6R)-tetrahydrobiopterin (BH4), is a negative effector that blocks phenylalanine activation by forming an inactive BH4.enzyme complex, and 3) the BH4.enzyme complex sequesters BH4 and controls its metabolic availability. Reaction progress curves showing regulatory effects of BH4, 7,8-dihydrobiopterin (BH2), and phenylalanine are fit by the model with high precision. Data are presented that the high affinity pterin-binding site on unactivated phenylalanine hydroxylase is the pterin site that regulates catalysis. Occupancy of this site by BH4 or BH2 causes non-cooperative, linear inhibition of phenylalanine activation of the enzyme. All inhibitory effects of BH4 appear due to its binding at the pterin regulatory site on unactivated enzyme. BH2 inhibits by binding at the active site as well as the pterin regulatory site. 6-Methyltetrahydropterin also appears to bind at the pterin regulatory site, but its effect is only seen at high phenylalanine concentrations. Using kinetic constants measured in this and earlier work, quantitative effects of phenylalanine and BH4 regulation on the rate of the phenylalanine hydroxylase reaction in vitro and in vivo are calculated. The effects of formation of the BH4.enzyme complex on free BH4 concentration, on enzyme activity, and on regulation of the rate of phenylalanine hydroxylation in liver are discussed.

Characterization of a beta-lactamase from Clostridium clostridioforme.

A beta-lactamase-producing strain of Clostridium clostridioforme isolated from human peritoneal fluid was examined by MIC testing and enzyme characterization. MICs of penicillins (64-512 mg/L) were higher than those of cephalosporins (8-128 mg/L); the strain was susceptible to cefoxitin (8 mg/L) and imipenem (1 mg/L). No enhancement of cephalosporin activity occurred when clavulanate was also added, but a limited degree of enhancement of penicillin activity (resulting in beta-lactam MICs higher than available NCCLS breakpoints) occurred when clavulanate, sulbactam or tazobactam was added simultaneously. By contrast, addition of BRL 42715 with amoxycillin, ticarcillin or piperacillin led to a drop in beta-lactam MICs from 512 to < or = 1 mg/L, with a drop from 64 to 1 mg/L when BRL 42715 was added with cefotaxime. All inhibitors were added at fixed concentrations of 2 mg/L. As determined spectrophotometrically, the enzyme hydrolysed penicillin G, cloxacillin and piperacillin (Vmax values (%) 372, 1816, 1001, respectively relative to cephaloridine) more efficiently than cephalosporins (69-191, with cephaloridine as 100%). Km values (microM) varied between 30-308 microM (penicillins) and 2-20 microM (cephalosporins). Relative enzyme efficiency (relative Vmax/Km with cephaloridine as 100) varied from 21-100 (cephalosporins) and 8-77 (penicillins). IC50 values (microM) with nitrocefin, piperacillin and penicillin G substrates (concentrations 20, 100 and 20 microM, respectively) were > 1000, 7, 3.5 (clavulanate); > 1000, 300, 59 (sulbactam), > 1000, 29, 7.7 (tazobactam); 0.0004, 0.001, 0.0018 (BRL 42715). The enzyme was not inhibited by EDTA, cefoxitin, cloxacillin or aztreonam, but was inhibited by pCMB.(ABSTRACT TRUNCATED AT 250 WORDS)

Susceptibilities of 540 anaerobic gram-negative bacilli to amoxicillin, amoxicillin-BRL 42715, amoxicillin-clavulanate, temafloxacin, and clindamycin.

Agar dilution MIC testing of amoxicillin, amoxicillin-BRL 42715, amoxicillin-clavulanate, temafloxacin, and clindamycin against 496 beta-lactamase-producing anaerobic gram-negative rods revealed MICs for 90% of the strains tested of 256.0 (amoxicillin), 2.0 (amoxicillin-BRL 42715 and amoxicillin-clavulanate), and 4.0 (temafloxacin and clindamycin) microgram/ml. Amoxicillin, temafloxacin, and clindamycin inhibited all 44 beta-lactamase-negative strains (MICs for 90% of the strains tested, less than or equal to 2.0 micrograms/ml). BRL 42715 will not be developed, but temafloxacin merits clinical evaluation.

Mechanism of phenylalanine regulation of phenylalanine hydroxylase.

The mechanism of phenylalanine regulation of rat liver phenylalanine hydroxylase was studied. We show that phenylalanine "activates" phenylalanine hydroxylase, converting it from an inactive to active form, by binding at a true allosteric regulatory site. One phenylalanine molecule binds per enzyme subunit; it remains at this site during catalytic turnover and, while there, cannot be hydroxylated. Loss of phenylalanine from the site causes a loss of enzymatic activity. The rate of loss of activation is dramatically slowed by phenylalanine, which kinetically "traps" activated enzyme during relaxation from the activated to unactivated state. An empirical equation is presented which allows calculation of relaxation rates over a wide range of temperatures and phenylalanine concentrations. Kinetic trapping by phenylalanine is a novel effect. It was analyzed in detail, and its magnitude implied that phenylalanine activation involves cooperativity among all four subunits of the enzyme tetramer. A regulatory model is presented, accounting for the properties of the phenylalanine activation reaction in the forward and reverse directions and at equilibrium. Fluorescence quenching studies confirmed that activation increases the solvent accessibility of the enzyme's tryptophan residues. Physical and kinetic properties of purified phenylalanine hydroxylase from rat, rabbit, baboon, and goose liver were compared. All enzymes were remarkably alike in catalytic and regulatory properties, suggesting that control of this enzyme is similar in mammals and birds.

Reaction of rat liver phenylalanine hydroxylase with fatty acid hydroperoxides. Characterization and mechanism.

Rat liver phenylalanine hydroxylase must be in a reduced form to be catalytically active (Marota, J.J. A., and Shiman, R. (1984) Biochemistry 23, 1303-1311). In this communication we show that a fatty acid hydroperoxide, 13-hydroperoxylinoleic acid (LOOH), can efficiently oxidize the reduced enzyme. In the process, the hydroperoxide is decomposed, oxygen consumed, and hydrogen peroxide formed. Enzyme reduction by the tetrahydropterin cofactor and reoxidation by LOOH can occur as two single steps or, when the enzyme concentration is low compared to that of the substrates, as part of a catalytic cycle. In this latter case, phenylalanine hydroxylase is a hydroperoxide-dependent tetrahydropterin oxidase. The reaction requires 1.0 mol of O2, 1.0 mol of tetrahydropterin, and 0.5 mol of LOOH to yield 1.0 mol of quinonoid dihydropterin, 0.4 mol of H2O2, and fatty acid products. Thus far, the catalytic and single-step reactions appear the same in all properties, consistent with the steady-state reaction following a ping-pong mechanism. Phenylalanine hydroxylase is an excellent catalyst for this reaction: the turnover number with LOOH is slightly greater than with phenylalanine; the Km(app) for LOOH is 11 +/- 4 microM; and the kcat/Km ratio for LOOH is about 25 times greater than for phenylalanine. LOOH and phenylalanine appear to react at different sites on phenylalanine appear to react at different sites on phenylalanine hydroxylase, and the reaction of LOOH is inhibited only slightly by phenylalanine and not at all by 5-deaza-6-methyltetrahydropterin, a competitive inhibitor of phenylalanine hydroxylation. The reaction of LOOH with phenylalanine hydroxylase strongly resembles the nonenzymatic reaction of LOOH with hematin, implying similar mechanisms for the two reactions and implicating the enzyme's non-heme iron as both the site of reaction of LOOH and of electron transfer during oxidation and reduction. The formation of hydrogen peroxide during a reaction of phenylalanine hydroxylase is unusual. Indirect evidence indicates a reduced oxygen species, formed on the enzyme during the reduction step, is (partially) released as H2O2 when the hydroperoxide reacts.

Regulation of albumin synthesis by hormones and amino acids in primary cultures of rat hepatocytes.

Culture conditions necessary for optimizing albumin secretion were studied in rat hepatocytes maintained in a chemically defined, serum-free medium. Amino acid analysis of the culture medium, which was based on a 1:1 mixture of Ham's F12:Dulbecco's modified Eagle's medium (unsupplemented medium), revealed that certain essential amino acids were depleted from this medium over a 24-h incubation. Rates of albumin secretion were significantly higher and better maintained when the medium was supplemented with additional amino acids (supplemented medium). Moreover, selective removal of an essential amino acid resulted in an immediate decrease in total protein and albumin synthesis and after 48 h a further selective decrease in albumin synthesis. Linear rates of albumin secretion were observed over a wide variety of experimental conditions, but secretion was not strictly proportional to cell number. Maximal rates of secretion were obtained at plating densities of 2-3 X 10(6) cells/60 mm culture dish. Albumin secretion also increased with time in culture reaching a maximum on days 3 and 4. When added singly, either insulin or dexamethasone increased rates of albumin secretion in a dose-dependent manner, but both hormones and an adequate supply of amino acids were necessary for maximal rates of secretion as well as long-term maintenance of the hepatocytes (greater than 3-4 days). In the presence of dexamethasone the dose-response curve for insulin was shifted toward physiological insulin concentrations. Changes in rates of albumin secretion in response to added hormones in supplemented media were found to parallel changes in albumin synthesis and relative amounts of albumin mRNA. Changes in gene transcription were probably involved.

Direct effect of insulin on albumin gene expression in primary cultures of rat hepatocytes.

The purpose of this study was to identify a cell culture system in which the role of insulin in regulating albumin gene expression could be investigated. The system selected was rat hepatocytes maintained in primary culture in a chemically defined, serum-free medium. Under control conditions albumin secretion was nearly the same as the rate recorded in vivo and in perfused liver and was reasonably well maintained during 8 days of culture. Deletion of insulin from the culture medium for 3-6 days resulted in 40-60% reductions in albumin secretion. Furthermore, albumin secretion relative to the rate of total protein synthesis was reduced by approximately 50% as a result of insulin deficiency. Readdition of the hormone to insulin-deficient cultures restored secretion to the control rate. A maximal effect of insulin was observed within 3 days after readdition of the hormone, and a half-maximal response was obtained with a hormone concentration of approximately 3.0 nM. The relative abundance of albumin mRNA, as measured by solution hybridization using a complementary DNA probe, responded in a parallel fashion to the changes in albumin secretion. Thus rat hepatocytes maintained under appropriate culture conditions reflect the effects of diabetes and insulin treatment on albumin gene expression observed in vivo and provide an excellent model system in which to study the mechanism(s) of insulin action.

Stoichiometric reduction of phenylalanine hydroxylase by its cofactor: a requirement for enzymatic activity.

We have found that rat liver phenylalanine hydroxylase oxidizes a stoichiometric amount of its cofactor, 6-methyl-5,6,7,8-tetrahydropterin (6MPH4), in a reaction that is independent of phenylalanine. The reaction requires oxygen, and one 6MPH4 is oxidized per subunit of enzyme. A quinonoid dihydropterin is directly produced in the reaction, and there is no evidence for the intermediate formation of a 4a-hydroxydihydropterin. Neither hydrogen peroxide nor superoxide anions were detected as products of the oxidation, and phenylalanine hydroxylase appears to be the sole electron acceptor from 6MPH4. Therefore, in a functional sense, phenylalanine hydroxylase is reduced by its cofactor. The reduced state of the enzyme is stable to activation by phenylalanine and during catalytic turnover, and the electrons on the reduced enzyme cannot be directly used to drive phenylalanine hydroxylation. Of greatest importance, enzyme reduction appears to be required for the formation of a catalytically active enzyme species. Phenylalanine hydroxylase is chemically and physically altered by reduction. Reduced enzyme exhibits (1) a greatly increased fluorescence, which is quantitatively related to the extent of reduction, (2) an altered UV-visible absorbance spectrum, (3) a greatly increased sensitivity to inactivation by hydrogen peroxide, and (4) a greatly decreased sensitivity to inhibition by Dopa which quantitatively correlates with the increase in enzyme fluorescence. Second-order rate constants, kr, for the reduction of the enzyme by 6MPH4 have been determined and found to vary with pH, temperature, buffer, and enzyme activation: at pH 6.8, 25 degrees C, and in phosphate buffer, for phenylalanine-activated enzyme kr = 15 X 10(6) min-1 M-1. Tris is a competitive inhibitor with respect to 6MPH4 of enzyme reduction and also of catalysis.

Regulation of phenylalanine hydroxylase activity by phenylalanine in vivo, in vitro, and in perfused rat liver.

We show that phenylalanine is able to control the extent of activation and, as a result, the catalytic activity of rat liver phenylalanine hydroxylase in vivo, in perfused liver, and in vitro. Both phosphorylated and unphosphorylated enzyme activities are controlled by phenylalanine activation and, overall, this mechanism appears to be a major means of regulating the enzyme's activity in rat liver. At normal phenylalanine levels in vivo, phenylalanine hydroxylase is at most 1-4% activated, and phosphorylated enzyme (glucagon-induced) appears at most 5-7% activated under similar conditions. In both cases, a phenylalanine load increased the percentage of activated enzyme found in vivo to about 40% of maximal. In perfused rat livers, a plasma phenylalanine concentration of only 4 times normal induced a 4-fold increase in the amount of activated enzyme present and a corresponding functional increase in the rate of phenylalanine hydroxylation by the tissue. Under the latter conditions, more than 25% of the amino acid could be hydroxylated in a single pass through the organ. Purified phosphorylated phenylalanine hydroxylase must be activated to be catalytically active. The activation with phenylalanine, at equilibrium, is a cooperative process, and the phosphorylated enzyme is activated more rapidly at pH 6.8 and 8.0 and at lower phenylalanine concentration than the unphosphorylated species. Overall, phosphorylation appears to allow phenylalanine hydroxylase to be more easily activated at relatively low phenylalanine concentrations.

The mechanism of phenylalanine hydroxylase.

The site of oxygen binding during phenylalanine hydroxylase (PAH)-catalyzed turnover of phenylalanine to tyrosine has been tentatively identified as the 4a position of the tetrahydropterin cofactor, based on the spectral characteristics of an intermediate generated from both 6-methyltetrahydropterin and tetrahydrobiopterin during turnover. The rates of appearance of the intermediate and tyrosine are equal. Both rates exhibit the same dependence on enzyme concentration. PAH also requires 1.0 iron per 50,000-dalton subunit for maximal activity. A direct correlation between iron content and specific activity has been demonstrated. Apoenzyme can be reactivated by addition of Fe(II) aerobically or Fe(III) anaerobically and can be repurified to give apparently native protein. Evidence from electron paramagnetic resonance implicates the presence of high spin (5/2) Fe(III). As a working hypothesis we postulate that a key complex at the active site may be one containing iron in close proximity to a 4a-peroxytetrahydropterin.

Phenylalanine hydroxylase. Correlation of the iron content with activity and the preparation and reconstitution of the apoenzyme.

Phenylalanine hydroxylase requires 1.0 mol of iron/Mr = 50,000 subunit for maximal activity. A direct correlation between iron content and specific activity has been demonstrated through a comparison of enzyme activity and iron bound per subunit for various enzyme preparations and a measurement of the remaining activity upon partial and total removal of the iron by chelation. Apoenzyme has been prepared that can be reactivated by addition of Fe(II) aerobically or of Fe(III) anaerobically. A comparison of the native and reconstituted phenylalanine hydroxylase shows the latter behaves identically upon affinity and Chelex column chromatography as well as precipitation with ammonium sulfate supporting its close similarity to the native enzyme.

Immunocytochemical identification of phenylalanine hydroxylase and albumin in cultured hepatoma cells and isolated rat hepatocytes.

Rhodamine-conjugated antibodies specific for phenylalanine hydroxylase and serum albumin were employed as cytochemical probes to identify these two proteins in H4 hepatoma cells and in isolated rat hepatocytes. Each fluorescent antibody stained the cells specifically and in a distinctive manner. In both cell types, albumin staining was discretely localized in cytoplasmic and in H4 cultures varied somewhat from cell to cell. Evidence from cultures of REB15 cells, a strain derived by cloning H4 cells in tyrosine-free medium, suggested that the staining variability of H4 cells could reflect a variability in phenylalanine hydroxylase content. Hydrocortisone-treated H4 cells and REB15 cultures contain increased amounts of phenylalanine hydroxylase; and all cells in the culture appear to be induced by the hormone. Evidence was presented to show that the albumin visualized within the isolated hepatocytes had been synthesized by these cells, and, furthermore, that quantitatively nearly all intracellular albumin in the isolated rat hepatocytes appeared to be entrained in the secretion pathway (analogous data already exist for H4 cells [Baker, R.E., and R. Shiman. 1979. J. Biol. Chem. 254:9633-9639]). By scoring specific fluorescence, 86 and 98% of the H4 cells and 89 and 98% of the isolated hepatocytes were found to contain phenylalanine hydroxylase and albumin, respectively. Therefore, almost all cells in each population appeared to synthesize both proteins. An implication of these findings is that in rat virtually all liver parenchymal cells must synthesize both phenylalanine hydroxylase and albumin.