Allan G. Fischer

Purification and characterization of a purine-nucleoside phosphorylase from bovine thyroid

Abstract A purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from bovine thyroid tissue has been purified 670-fold utilizing the techniques of ammonium sulfate precipitation, ion-exchange and molecular-exclusion chromatography, and polyacrylamide-gel electrophoresis. The protein has an apparent molecular weight of 90,000, a single isoelectric point at 5.6, and a Michaelis constant of 0.028 m m for inosine. Double-reciprocal plots of the reaction rate for the phosphorylase-catalyzed reaction versus phosphate or arsenate concentration display a downward trend at high substrate concentrations. Two apparent Michaelis constants of 0.38 and 1.49 m m were determined for phosphate.

Xanthine oxidase from bovine thyroid glands

Abstract An organic inhibitor of peroxidative enzymes was isolated from the soluble fraction of bovine thyroid glands and identified as uric acid by spectroscopic and enzymatic methods and elemental analysis. The origin of uric acid within the thyroid gland was investigated. Xanthine oxidase which had been previously reported to be absent in the thyroid gland was detected and partially purified.

Bovine thyroidal xanthine oxidase

Abstract 1. 1. Xanthine oxidase from bovine thyroid glands was purified 1000-fold. Electrophoresis of the purified enzyme yielded a single enzymatically active band. 2. 2. A molecular weight of 260,000 ± 20,000 was estimated by gel filtration and by polyacrylamide gel electrophoresis. 3. 3. By electrofocusing the isoelectric point was shown to be 6.2 and the enzyme has a pH optimum at 7.8. 4. 4. Among the most common substrates, thyroid xanthine oxidase oxidizes only xanthine and hypoxanthine with oxygen as an electron acceptor. 5. 5. With dichloroindophenol as the electron acceptor the preferred substrates are xanthine and hypoxanthine compared to NADH. NADPH and acetaldehyde. 6. 6. Based on the isolation procedures and the K m and V max values, thyroidal xanthine oxidase appears to be present in sufficient concentration to supply the hydrogen peroxide requirement of the thyroid gland.

Characterization of the active site of homogeneous thyroid purine nucleoside phosphorylase

Purine nucleoside phosphorylase (purine-nucleoside : orthophosphate ribosyltransferase, EC 2.4.2.1) has been purified approx. 4000-fold and to electrophoretic homogeneity from bovine thyroid glands. The isolated enzyme has a specific activity of 17 mumol . min-1 . mg-1. The native enzyme appears to have a molecular weight of 92 000 as determined by sedimentation equilibrum ultracentrifugation and is comprised of three subunits having a molecular weight of 31 000 each as shown by sodium dodecyl sulfate gel electrophoresis. The enzyme is irreversibly denatured below pH 5 and the enzyme-substrate complex is shown to have an ionization constant (pKa) of 9.2 which influences catalytic activity. The pH dependence of the kinetic constants identifies three amino acid ionizable protons. The binding of inosine is effected by an imidazole ring of histidine (pKa 5.65) and a sulfhydryl group of cysteine (pKa 8.5) and the maximal velocity is restricted by an epsilon-amino group which is essential for phosphate binding. The requirement of these residues for activity was confirmed by group-specific chemical modification. The presence of phosphate protected only the lysyl residue while inosine protected all three residues from chemical titration. A model is proposed for the catalytic mechanism of purine nucleoside phosphorylase.

Xanthine oxidase: A source of hydrogen peroxide in bovine thyroid glands☆

Abstract Hydrogen peroxide produced by bovine thyroidal xanthine oxidase was found to yield protein bound iodine in vitro in the presence of a thyroidal peroxidase. The thyroid metabolites, mono- and diiodotyramine, which have very potent inhibitory effects on thyroid monoamine oxidase have very little effect on thyroid xanthine oxidase below 1 mM concentration. Allopurinol and formycin B reduced the level of iodination of protein in thyroid tissue slices. These data suggest that thyroid xanthine oxidase may be an important source of the hydrogen peroxide required for thyroxine biosynthesis.

Purine nucleoside and purine base concentrations in bovine thyroid and plasma

Concentrations of eight purine nucleosides and bases in bovine thyroid and plasma were determined by high pressure liquid chromatography. Plasma purines were metabolized to uric acid in the absence of inhibitors. The concentrations of these purines were 10-100 times greater in thyroid tissue than in plasma.

Formation of phenylalanylserine and cyclo-phenylalanylseryl by protoplasts of Gliocladium virens

Abstract Protoplasts of Gliocladium virens, when incubated with the amino acids, phenylalanine and serine, synthesized the dipeptide, phenylalanylserine and the diketopiperazine, cyclo-phenylalanylseryl.

Formation of protoplasts from Gliocladium virens.

Abstract 1. 1. Protoplasts were generated from 72-hr-old mycelium of Gliocladium virens, NRRL 1828 by using a lytic preparation produced by Spicaria violacea grown on a synthetic medium containing salts and 1% suspension of cell wall material from G. virens as the sole carbon source. 2. 2. Mannitol (0.6 M) was an excellent osmotic stabilizer and protoplasts could be stored 1–2 weeks at 4°C. 3. 3. The lytic enzymes from S. violacea were fractionated by ammonium sulfate precipitation and DEAE-cellulose chromatography. 4. 4. The major protein in the extracellular material appears to be an amylase and suggests that the cell wall of G. virens contains a significant amount of α(1 → 4) bonds. 5. 5. Chitinase also appears to be present and both enzymes are required for protoplast formation.

Kinetic studies on bovine thyroidal purine nucleoside phosphorylase

Abstract 1. 1. Bovine thyroid purine nucleoside phosphorylase shows negative cooperativity in the presence of phosphate with a Hill coefficient of 0.56 which is influenced by pH. 2. 2. Initial velocity and product inhibition studies show the enzyme reaction to be a Bi-Bi ordered mechanism. 3. 3. Of the substrates, phosphate binds first followed by the nucleoside and the release of nucleobase precedes the release of ribose-1-phosphate for the products.