Genetics of the mammalian phenylalanine hydroxylase system. IV. Evidence of phenylalanine hydroxylase in a cultured human hepatoma cell line

We report here the identification of a cultured human hepatoma cell line which possesses an active phenylalanine hydroxylase system. Phenylalanine hydroxylation was established by growth of cells in a tyrosine-free medium and by the ability of a cell-free extract to convert [¹⁴C]phenylalanine to [¹⁴C]tyrosine in an enzyme assay system. This enzyme activity was abolished by the presence in the assay system of p-chlorophenylalanine but no significant effect on the activity was observed with 3-iodotyrosine and 6-fluorotryptophan. Use of antisera against pure monkey or human liver phenylalanine hydroxylase has detected a cross-reacting material in this cell line which is antigenically identical to the human liver enzyme. Phenylalanine hydroxylase purified from this cell line by affinity chromatography revealed a multimeric molecular weight (estimated 275,000) and subunit molecular weights (estimated 50,000 and 49,000) which are similar to those of phenylalanine hydroxylase purified from a normal human liver. This cell line should be a useful tool for the study of the human phenylalanine hydroxylase system.     

Phytanoyl-CoA hydroxylase activity is induced by phytanic acid.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid present in various dietary products such as milk, cheese and fish. In patients with Refsum disease, accumulation of phytanic acid occurs due to a deficiency of phytanoyl-CoA hydroxylase, a peroxisomal enzyme containing a peroxisomal targeting signal 2. Recently, phytanoyl-CoA hydroxylase cDNA has been isolated and functional mutations have been identified. As it has been shown that phytanic acid activates the nuclear hormone receptors peroxisome proliferator-activated receptor (PPAR)alpha and all three retinoid X receptors (RXRs), the intracellular concentration of this fatty acid should be tightly regulated. When various cell lines were grown in the presence of phytanic acid, the activity of phytanoyl-CoA hydroxylase increased up to four times, depending on the particular cell type. In one cell line, HepG2, no induction of phytanoyl-CoA hydroxylase activity was observed. After addition of phytanic acid to COS-1 cells, an increase in phytanoyl-CoA hydroxylase activity was observed within 2 h, indicating a quick cell response. No stimulation of phytanoyl-CoA hydroxylase was observed when COS-1 cells were grown in the presence of clofibric acid, 9-cis-retinoic acid or both ligands together. This indicates that the activation of phytanoyl-CoA hydroxylase is not regulated via PPARalpha or RXR. However, stimulation of PPARalpha and all RXRs by clofibric acid and 9-cis-retinoic acid was observed in transient transfection assays. These results suggest that the induction of phytanoyl-CoA hydroxylase by phytanic acid does not proceed via one of the nuclear hormone receptors, RXR or PPARalpha.     

Intracellular enzymes of collagen biosynthesis in rat liver as a function of age and in hepatic injury induced by dimethylnitrosamine. Purification of rat prolyl hydroxylase and comparison of changes in prolyl hydroxylase activity with changes in immunoreactive prolyl hydroxylase.

Prolyl hydroxylase was purified from newborn rats by affinity chromatography using poly(L-proline), and antiserum to the enzyme was prepared in rabbits. The rat prolyl hydroxylase was similar to the chick and human enzymes with respect to specific activity, molecular weight and molecular weights of the polypeptide chains. The activity of prolyl hydroxylase and the content of immunoreactive enzyme were measured in rat liver as a function of age in experimental hepatic injury. Active prolyl hydroxylase comprised about 13.2% of the total immunoreactive protein in the liver of newborn rats and the value decreased to about 3.6% at the age of 420 days. This decrease was due to a decrease in the enzyme activity, whereas only minor changes were found in the content of the immunoreactive protein. In hepatic injury, a significant increase was found in the ratio of active enzyme to total immunoreactive protein, owing to an increase in the enzyme activity. The data indicate that prolyl hydroxylase activity in rat liver is controlled in part by a mechanism which does not involve changes in the content of the total immunoreactive protein.     

Molecular mechanisms of olfactory reception. IV. Some biochemical characteristics of the camphor receptor from rat olfactory epithelium.

Some parameters of the receptor element from the rat olfactory epithelium are evaluated; it is characterized by high affinity for camphor (KD = 1.5. x 10(-9) M). Triton X-100 has no marked effect on the binding of [3H]camphor. Neither RNAase nor phospholipase C affected [3H]camphor-binding activity. Pronase and trypsin abolished [3H]camphor binding activity by 65 and 40%, respectively. Sulfhydryl reagents decrease the binding of [3H]camphor by a factor of 5--8. The isoelectric point of the receptor solubilized with Triton X-100 is 4.8, as determined by isoelectric focusing. The molecular weight of the receptor as determined by gel electrophoresis is about 120 000. It is proposed that the camphor receptor is a membrane protein containing sulfhydryl groups and playing a key role in olfactory reception.     

An activatable form of prolyl hydroxylase in fibrobalast extracts.

An $\text{in vitro}$ increase in prolyl hydroxylase activity has been effected in sonicates of early log phase L 929 mouse skin fibroblasts from either monolayer or suspension cultures. The requirements for activation are identical to those needed for the hydroxylation reaction itself, i.e., ferrous ion, ascorbate and α-ketoglutarate. Catalase, which is not an absolute requirement for the hydroxylation, is also necessary for activation. The activation is time dependent and, under the conditions used, is complete in 3 hr at 30°. Since ferrous ion also appears necessary for the activation in intact cells and since the same level of activation is achieved in intact cells as in sonicates, it appears that the $\text{in vitro}$ activation proceeds in the same manner as that seen in cultured cells.     

Mixed function oxidation of hexobarbital and generation of NADPH by the hexose monophosphate shunt in isolated rat liver cells.

Mixed function oxidation of hexobarbital and the generation of NADPH by the hexose monophosphate shunt were studied in isolated rat liver parenchymal cells from phenobarbital-pretreated and untreated animals. In cells isolated from untreated rats, a maximal rate of hexobarbital oxidation of 17 μmol·g^?1 liver wet weight·(60 min)^?1 was observed, while in cells isolated from phenobarbital-pretreated rats a maximal rate of 29 μmol·g^?1 liver wet weight·(60 min)^?1 has been obtained. On the basis of the specific radioactivity at carbon atom 1 of glucose 6-phosphate, fructose 6-phosphate and 6-phosphogluconate, determined by enzymatic decarboxylation, a ratio between NADPH formation via the hexose monophosphate shunt and NADH utilization for hexobarbital oxidation of 6:1 in untreated and 9.5:1 in pretreated cells has been obtained. With phenazine methosulfate the stimulation of NADPH generation via the hexose monophosphate shunt exceeded that observed in the presence of hexobarbital by 329 and 160%, respectively, indicating that the capacity of this pathway is sufficient to provide more reducing equivalents than are required for maximal rates of mixed function oxidation.     

Molecular mechanisms of olfactory reception IV. Some biochemical characteristics of the camphor receptor from rat olfactory epithelium

Some parameters of the receptor element from the rat olfactory epithelium are evaluated; it is characterized by a high affinity for camphor (K_D = 1.5 · 10^?9 M). Triton X-100 has no marked effect on the binding of [³H]camphor. Neither RNAase nor phospholipase C affected [³H]camphor-binding activity. Pronase and trypsin abolished [³H]camphor binding activity by 65 and 40%, respectively. Sulfhydryl reagents decrease the binding of [³H]camphor by a factor of 5–8. The isoelectric point of the receptor solubilized with Triton X-100 is 4.8, as determined by isoelectric focusing. The molecular weight of the receptor as determined by gel electrophoresis is about 120 000. It is proposed that the camphor receptor is a membrane protein containing sulfhydryl groups and playing a key role in olfactory reception.     

Intracellular enzymes of collagen biosynthesis in rat liver as a function of age and in hepatic injury induced by dimethylnitrosamine. Changes in prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase activities.

The relationship between the changes in the four enzyme activities catalysing intracellular post-translational modifications in collagen biosynthesis were studied in rat liver as a function of age and in experimental hepatic injury induced by the administration of dimethylnitrosamine. During aging, relatively large changes were found in prolyl hydroxylase and lysyl hydroxylase activities, whereas only minor changes took place in collagen galactosyltransferase and collagen glucosyltransferase activities. In hepatic injury, the two hydroxylase activities increased earlier and to a larger extent than did the two glycosyltransferase activities, and the largest was found in lysyl hydroxylase activity. The data support previous suggestions that changes in the rate of collagen biosynthesis in the liver cannot be explained simply by a change in the number of collagen-producing cells, but regulation of the enzyme activities existed, so that the two hydroxylase activities altered considerably more than did the two collagen glycosyltransferase activities.