R.G. Hansen

An enzymic method for determination of inorganic pyrophosphate and its use as an assay for RNA polymerase

1. 1. An enzymic method for the determination of PP1 has been developed. PP1 can be quantitatively estimated from the amount of NADPH formed via the action of UDP-glucose pyrophosphorylase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. Neither P1 nor any of the organic phosphate compounds tested interfered in the PP1 determination. 2. 2. The method has been used for the assay of DNA-dependent RNA polymerase—by coupling the generation of PP1 by RNA polymerase with NADPH formation. The RNA polymerase catalyzed syntheses of RNA and polyriboadenylate have been assayed by this procedure.

[38] UDP-glucose pyrophosphorylase (crystalline) from liver

Publisher Summary This chapter discusses the determination of uridine diphosphate (UDP)-glucose pyrophosphorylase from liver. The formation of Glc-l-P is followed spectrophotometrically by coupling the pyrophosphorolysis of UDP-Glc with pyridine nucleotide reduction in the presence of (1) nicotinamide adenine dinucleotide phosphate (NADP), (2) phosphoglucomutase, and (3) Glc-6-P dehydrogenase. The reaction is made quantitative by measuring the appearance of NADPH speetrophotometrically at 340 mμ. UDP-Glc is the most active substrate for the crystalline pyrophosphorylase; however the enzyme is not highly specific for either the pyrimidine or hexose component of the nucleoside diphosphate hexose. On substitution of d-thymidine, cytidine, or guanosine for the uridine of UDP-Glc, the reaction rate is about 0.1–3%. With the substitution of inosine or adenosine for uridine, only a trace of activity remains. The twice recrystallized enzyme is essentially homogeneous on polyacrylamide gel electrophoresis and in the analytical ultracentrifuge. The steps involved in the fractionation of liver discussed in the chapter are first extract, ammonium sulfate, second ammonium sulfate fraction, and crystallization.

Ion-exchange paper chromatography of nucleoside diphosphate sugars and related nucleotides

Abstract A new method especially adapted for the characterization of nucleoside diphosphate sugars in mixtures containing nucleoside mono-, di-, and tri-phosphates is described. It is based on ion-exchange chromatography using papers impregnated with polyethyleneimine (PEI). Nucleoside diphosphate sugars are separated from related compounds in 40 to 50 min. Good separations of the four common ribo- and deoxyribo-nucleoside monophosphate are achieved in about 4 hr. Thus homologous compounds are readily separated by this procedure which differ in the purine or pyrimidine component or which differ in the number of moles of phosphate present in the molecule.

[127] Galactose 1-phosphate uridyl transferase

Publisher Summary This chapter discusses the determination of galactose 1-phosphate (Gal-l-P) uridyl transferase. The assay is based on the rate of Glc-l-P formed in the transferase reaction. Glc-l-P formation is followed by the rate of increase in optical density at 340 mμ which results from reduced triphosphopyridine nucleotide (TPNH) formation in a reaction coupled with phosphoglucomutase and Glc-6-P dehydrogenase. A unit is defined as the amount of enzyme that causes the formation of 1 micromole of products per minute under the conditions described. The calculation of product is based on a molar extinction coefficient of 6.22 × 10 3 for TPNH at 340 mμ. A modified and extended procedure for fractionation of calf liver, leading to a more purified enzyme, and an additional procedure for the partial purification of the enzyme from human red blood cells are described.

The biosynthesis of guanosine diphosphate mannose and phosphomannan by Hansanula holstii

The biosynthesis of phosphomannan by cultures of Hansenula holstii NRRL Y-2448 has been demonstrated to proceed from glucose with all carbons remaining intact. Enzymes were found in cell-free extracts capable of converting glucose to guanosine diphosphate mannose via mannose 1-phosphate. The isomerization of glucose to mannose occured as hexose phosphate and not as nucleotide hexose. The isolation and identification of guanosine diphosphate mannose from perchloric acid extracts gave further evidence for the proposal that guanosine diphosphate mannose is a precursor of phosphomannan.

Uridine Diphosphate Galactose Pyrophosphorylase from Calf Liver

Summary The biosynthesis of UDP-Gal from UTP and Gal-1-P is catalyzed by extracts of calf liver. At all stages of purification and after several recrystallizations, catalytic activity for UDP-Gal is inseparably connected with the enzyme catalyzing the biosynthesis of UDP-Glc. These findings are discussed from the standpoint of galactose metabolism in the galactosemic.

A spectrophotometric method for the determination of nucleoside triphosphates (pyrophosphorolysis of nucleoside diphosphate sugars)

Abstract The enzyme-catalyzed pyrophosphorolysis of nucleoside diphosphate sugars has been followed spectrophotometrically using 3-P-glycerate kinase (EC 2.7.2.3) and glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12) coupled to the oxidation of NADH. The method is rapid, and gives a direct recording of enzyme activity. Some limitations are discussed but these generally apply when crude systems are analyzed. The method has been used for measuring the adenosine, guanosine, inosine, and uridine triphosphates resulting from the pyrophosphorolysis of ADP-Man, GDP-Glc, GDP-Man, IDP-Glc, IDP-Man, UDP-Glc, UDP-Man, UDP-Gal, UDP-N-AGlem, and UDP-Xyl.

The pyrophosphorylysis of adenosine diphosphate glucose and adenosine diphosphate mannose

Abstract 1. 1. The biosynthesis of adenosine diphosphate glucose and adenosine diphosphate mannose from adenosine triphosphate and respectively glucose 1-phosphate and mannose 1-phosphate has been observed in extracts from mammalian tissues. The reaction is reversible in the presence of the nucleoside diphosphate sugar and pyrophosphate. 2. 2. Two different enzymes are probably responsible for the reactions with either adenosine diphosphate glucose or adenosine diphosphate mannose. 3. 3. Adenosine diphosphate mannose may be synthesized by the same enzyme which catalyzes the biosynthesis of guanosine diphosphate mannose and inosine diphosphate mannose. 4. 4. Evidence is presented that adenosine diphosphate glucose pyrophosphorylase (ATP: α- d -glucose-1-phosphate adenylyltransferase) may be a specific enzyme. 5. 5. The relative levels of nucleoside diphosphate hexose pyrophosphorylases have been measured in different extracts from mammals, plants or yeast. From these data the possible significance of adenosine diphosphate glucose pyrophosphorylase in mammals is discussed.

[128] Galactose 1-phosphate uridyl transferase (clinical aspects)

Publisher Summary This chapter describes the determination of clinical aspects of galactose 1-phosphate uridyl transferase. Galactosemia, a hereditary disorder in humans, is characterized by a low level or a complete absence of the enzyme galactose 1-phosphate uridyl transferase (transferase) in blood cells, liver, and other tissues. The symptoms of the fully manifested disease are jaundice, cataracts, failure to grow, and excess galactose, amino acids, and protein in the urine. If the disease is not detected early and treated in the infant, mental retardation and death may occur. Measurement of transferase in blood cells is the most direct way of establishing or excluding a diagnosis of galactosemia. The method is based on the uridine diphosphate (UDP)-Glc consumption test with suitable modifications to measure the transferase quantitatively. Red cells are incubated with Gal-l-P and UDP-Glc in suitable excess, the reaction is stopped, and the residual UDP-Glc is determined spectrophotometrically with UDP-Glc dehydrogenase by measuring the reduced nicotinamide adenine dinucleotide (NADH) which is formed. For an added check on the procedure, it is possible to measure the UDP-Gal which is formed in the reaction by addition of a purified UDP-Gal-4-epimerase, thus re-forming UDP-Glc which may again be quantitated with UDP-Glc dehydrogenase.

An enzymic method for the determination of the chondroitin sulfates

Abstract A method employing the enzyme chondroitinase has been described for measuring the chondroitin sulfates, and it has been applied to urine specimens from normal and dwarf cattle and normal man and patients with Hurlers syndrome. The method is sensitive and specific for the three chondroitin sulfates. Quantitative recoveries were achieved when chondroitin sulfate A or B were added to urine. With the recent interest in disorders of mucopolysaccharide metabolism, this method should have application for categorizing the various types of mucopolysaccharidosis.