ABSTRACT
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.
Subject(s)
Phosphotransferases (Alcohol Group Acceptor) , Phosphotransferases/metabolism , Fucose , In Vitro Techniques , Oxygen , Phosphates , Phosphorylation , Stereoisomerism , Substrate SpecificityABSTRACT
The enzyme GTP:alpha-D-mannose-1-phosphate guanylyltransferase from porcine thyroid tissue has been purified 69 900-fold on columns of blue-Sepharose, DEAE-Sepharose, phenyl-Sepharose and agarose-GTP affinity materials. Although it exhibits a tendency to aggregate, the enzyme travelled, upon sucrose velocity sedimentation, as a single oligomer with a molecular mass of 412 kDa. Michaelis constants were determined to be 1.0 microM, 1.0 mM, 3.5 microM and 0.4 microM for GDP-alpha-D-mannose, pyrophosphate, GTP and mannose-1-phosphate, respectively. The enzyme appears to be specific for the mannose moiety but will accept an inosine replacement for guanine and a deoxyribose replacement for ribose in GTP.
Subject(s)
Nucleotidyltransferases/isolation & purification , Thyroid Gland/enzymology , Animals , Centrifugation, Density Gradient , Chemical Phenomena , Chemistry , Chromatography, Affinity , Chromatography, DEAE-Cellulose , Electrophoresis , Hydrogen-Ion Concentration , Kinetics , Substrate Specificity , SwineABSTRACT
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.
Subject(s)
Hydro-Lyases/metabolism , Guanosine Diphosphate Mannose , Tritium , WaterABSTRACT
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.
Subject(s)
Thyroid Gland/enzymology , alpha-L-Fucosidase/metabolism , Animals , Kinetics , Molecular Weight , Monosaccharides/pharmacology , Substrate Specificity , Swine , alpha-L-Fucosidase/isolation & purificationABSTRACT
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.
Subject(s)
Fucose/biosynthesis , Thyroid Gland/metabolism , Animals , Carbohydrate Dehydrogenases/isolation & purification , Chromatography, Affinity , Guanosine Diphosphate Fucose/metabolism , Guanosine Diphosphate Mannose/metabolism , SwineSubject(s)
Fucose/analysis , Animals , Carbon Radioisotopes , Glycoproteins/analysis , Hydrolysis , Microchemistry , Swine , Thyroglobulin/analysisABSTRACT
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.
Subject(s)
Phosphotransferases/isolation & purification , Thyroid Gland/enzymology , Animals , Fucose , Guanosine Diphosphate Sugars/pharmacology , Hydrogen-Ion Concentration , Kinetics , Methods , Molecular Weight , Nucleotides/pharmacology , Phosphotransferases (Alcohol Group Acceptor) , Substrate Specificity , SwineABSTRACT
Thyroid fucokinase is responsive to a number of metabolites which might serve in a regulatory capacity. In addition to inhibition by ADP and stimulation by GMP, fucokinase responds selectively to a series of nucleotide sugars. Of those studied, only guanine nucleotide sugars moderate the activity of the enzyme. GDP-alpha-D-mannose, GDP-alpha-D-glucose, GDP-alpha-D-rhamnose, and GDP-alpha-L-fucose on the other hand is strongly inhibitory. In the case of GDP-alpha-D-mannose stimulation, a physiological role seems possible, but the rationale is not entirely clear. The effects of GDP-beta-L-fucose, on the other hand may represent physiological control effected through feedback inhibition by and end product.
Subject(s)
Fucose/metabolism , Phosphotransferases/metabolism , Thyroid Gland/enzymology , Animals , Dogs , Kinetics , Nucleoside Diphosphate Sugars/pharmacology , SwineSubject(s)
Adipose Tissue/enzymology , Diabetes Mellitus, Experimental/enzymology , Fasting , Pituitary Gland/physiology , Pyruvate Kinase/metabolism , Adipose Tissue/drug effects , Animals , Cycloheximide/pharmacology , Diabetes Mellitus, Experimental/drug therapy , Hypophysectomy , Male , Phosphates/pharmacology , RatsABSTRACT
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.
