George S. Serif

Porcine thyroid fucosidase

An alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) has been isolated from porcine thyroid tissue and purified 10,800-fold using a combination of ion exchange, affinity and molecular sieve chromatography. The enzyme appears homogeneous by SDS electrophoresis but isoelectric focusing procedures detect considerable heterogeneity. The enzyme is a glycoprotein and this fact interferes with accurate molecular weight estimates by SDS electrophoresis or molecular sieve techniques. The enzyme appears, however, to be a tetramer and density gradient measurements set its molecular weight at 192,000 +/- 3,000. The enzyme exhibits an optimum at a pH of 5.1 and shows a high order of specificity for L-fucose units linked through alpha bonds. Both sulfhydryl and carboxyl groups appear necessary for enzyme activity. The enzyme does not attack intact thyroglobulin directly but will remove fucosyl residues from the glycone moiety if the protein portion is largely removed. The enzyme thus functions in a salvage role as thyroglobulin is degraded.

Isolation and properties of porcine thyroid fucokinase

A 23 000-fold purification of porcine fucokinase (ATP:6-deoxy-L-galactose 1-phosphotransferase, EC 2.7.1.52) has been achieved using a combination of ion-exchange, hydrophobic ligand, affinity, hydroxyapatite and molecular sieve chromatography. The enzyme was determined to have a subunit molecular weight of 78 180 +/- 4260 by sodium dodecyl sulfate chromatography and a tetrameric molecular weight of 309 200 +/- 4100 in the active state as determined by molecular sieve chromatography. The enzyme exhibits a single pH optimum at a pH value of 6.5 and gives evidence of a high order of specificity for L-fucose and ATP. The enzyme requires a divalent metal ion and this need is best satisfied by Mg2+. The activity of the enzyme is modified by a number of nucleotides. ADP is an enzyme inhibitor competitive with ATP. GDP-beta-L-fucose is also an inhibitor and appears to compete with L-fucose. GDP-alpha-D-mannose stimulates the enzyme. A possible role for the actions of these nucleotide sugars is discussed.

Synthesis of l-fucose in thyroid tissue

A de novo pathway for L-fucose synthesis has been detected in porcine thyroid tissue. This system uses guanosine diphospho-alpha-D mannose as a precursor and forms guanosine diphospho-beta-L-fucose as product. The system seems similar to those reported by others to exist in microorganisms and plants in that the first step of the pathway involves a 4-keto sugar nucleotide intermediate. The first enzyme of the pathway, guanosine diphospho-alpha-D-mannose oxidoreductase has been purified 57-fold from crude extracts by virtue of its affinity for Blue Sepharose.

Canine thyroid fucokinase

A radiometric assay was developed for fucokinase (ATP:6-deoxy-L-galactose 1-phosphotransferase, EC 2.7.1.52) based on the conversion of L-[14C]fucose to L-[14C]fucose 1-phosphate which is trapped and counted on ion exchange paper. This assay was used to detect the presence of a fucokinase in canine thyroid tissue which was subsequently purified 2754-fold over the crude tissue extracts. The product of the fucokinase was identified as the beta-anomer. The pH versus activity curve for the enzyme appears biphasic with optima at pH 6.5 and pH 8.25. The enzyme was shown to be highly specific for L-fucose with a Km of 2.6 - 10(-5) M at pH 8.25. It was shown to be absolutely specific for ATP as a phosphate donor with a Km of 6.3 - 10(-4) M at pH 8.25. The enzyme requires a divalent cation. Mg2+ is slightly more effective than Mn2+ in meeting this need. The molecular weight of the enzyme has been determined to be 494 000 +/- 12 400.

Pyridine nucleotides: II. Radiometric determination of minute quantities of triphosphopyridine nucleotides

Abstract This paper reports the development of radiometric assays for DPN + and DPNH with assay samples of the order of 10 −12 mole. These methods utilize enzymic cycling of DPN + and DPNH by l -glutamate dehydrogenase and l -lactate dehydrogenase to convert (1- 14 C)-α-ketoglutarate to l -glutamate, which is decarboxylated to 14 CO 2 and counted as a measure of pyridine nucleotide concentration.

Fucokinase, its anomeric specificity and mechanism of phosphate group transfer☆

Fucokinase phosphorylates L-fucose at the anomeric position and, as such, might use either the alpha or beta anomer as its substrate. Examination of the utilization of radiolabelled alpha and alpha,beta mixtures established beta-L-fucose as the required substrate. Phosphorylation at the anomeric center might involve either the loss or retention of the anomeric oxygen. The mechanism has been shown to involve anomeric oxygen retention through mass spectrometric analysis of the product phosphate derived from 18O-labelled L-fucose.

Catabolism and excretion of the antithyroid substance, diacetyl-2,6-diiodohydroquinone

Abstract The metabolism of the antithyroid compound, diacetyl-2,6-diiodohydroquinone, and its related iodoquinones and iodohydroquinones was studied in rats by means of 131 I-labeled derivatives. Chromatography, radioautography, and enzyme studies of the excreted products indicated that the major portion of injected diacety 12, 6-diiodohydroquinone is deacetylated to form free diiodohydroquinone which is conjugated with glucuronic or sulfuric acid and excreted via the urine. Deiodination of diacetyl-2,6-diiodohydroquinone does occur as a catabolic step since free iodide ion was observed. However, the deiodination does not yield monoiodohydroquinone or its excretion forms. This fact suggests the simultaneous removal of two atoms of iodine from a given molecule. Toxicity studies of the various derivatives were also conducted.

An assay for GDP-D-mannose-4,6-dehydratase

The mechanism of GDP-D-mannose-4,6-dehydratase action with respect to loss of the C5 hydrogen has been established using GDP-D-[5-3H]-mannose as a substrate. This observation has been incorporated into a rapid assay for the enzyme based on the equilibration of 3H with the aqueous medium.

Radiometric assay for minute quantities of l-fucose

Abstract A radiometric assay has been developed for l -fucose which is capable of measuring quantities of this deoxysugar as low as 25 pmol. The assay couples l -fucose dehydrogenase to l -glutamate dehydrogenase and l -glutamate decarboxylase to yield radioactive CO2 which is collected and counted as a measure of l -fucose content. The assay eliminates high backgrounds observed with fluorescence assays.

Pyridine nucleotides: III. Response of dog thyroid pyridine nucleotides to hormonal control

Abstract TPN + levels in dog thyroid slices appear to be elevated above control values when these tissues are exposed to thyroid-stimulating hormone (TSH) and cholinergic agents. It has been uncertain as to whether these hormone effects are due to an increased synthesis de novo of TPN + or an inhibition of TPN + degradation. This question has been evaluated through a study of the rate of incorporation of [ 32 P]orthophosphate and [ 14 C]nicotinic acid into DPN + and TPN + in the presence and absence of TSH and cholinergic agents. The specific activity data show an enhanced synthesis de novo of TPN + with no apparent concomitant enhancement of DPN + formation. Although these data suggest a specific stimulation of the DPN + kinase step, the possibility of pool dilution effects does not permit such an absolute assignment.