P. G. Pentchev

DISTRIBUTION OF A "MUTAROTASE" ACTIVITY IN RAT TISSUES AND POSSIBLE FUNCTION IN ACTIVE TRANSPORT OF SUGARS.

Abstract Homogenates of rat kidney, liver, intestine, stomach, lung, spleen, heart, brain, diaphragm and skeletal muscle were prepared both in EDTA and phosphate buffers. “Mutarotase” (EC 5.1.3.3) and lactate dehydrogenase (EC 1.1.1.27) activity were measured on the supernatant solutions following centrifugation. Mutarotase activity was highest in those tissues capable of active sugar transport. Enzyme from 1 g kidney cortex converted 380 mg of α to β-glucose per min. There was no correlation between either mutarotase, lactate dehydrogenase activity or Q -glucose values for the tissues, suggesting that enzyme is not involved merely as a component of normal glucose metabolism. Kidney, liver and small-intestinal homogenates were separated into nuclear, mitochondrial, microsomal and supernatant fractions in 0.2 M sucrose. Mutarotase activity was concentrated almost exclusively in supernatant fraction. Results are discussed in terms of a function for mutarotase protein as the hypothetical sugar “carrier” molecule of active transport theory.

[100] Mutarotase (aldose 1-epimerase) from kidney cortex

Publisher Summary Mutarotase (aldose 1-epimerase, EC 5.1.3.3) catalyzes the interconversion of the α- and β-anomers of certain sugars including D-glucose and D-galactose. This chapter examines the assay method, purification procedure, and properties of mutarotase (aldose 1-epimerase) from kidney cortex. The assay system for the enzyme is based upon the change in optical rotation of the substrate during the enzyme-catalyzed mutarotation reaction. The specific rotation of a-D-glucose used as substrate is 112.5° at 589 nm. The specific rotation of β-D-glucose is 19° and that of the equilibrium mixture produced is 52.5°. The manual polarimetric assay procedure, semiautomated assay procedure, and colorimetric procedure for mutarotase is described. The manual assay procedure involves calculation and plotting of the first-order rate curves for mutarotation of substrate directly from measurements of angular rotation displayed by the polarimeter. A semiautomated assay procedure uses a recording polarimeter, and gives values for the overall first-order rate constant of the enzyme-catalyzed mutarotation of a sugar at a fixed substrate concentration. Colorimetric procedure lacks the necessary precision for kinetic studies, but is suitable for routine assays of the enzyme where a polarimeter is not available. It is based upon the observation that glucose oxidase is specific for the β-anomer of D-glucose. The purification procedure mutarotase (aldose 1-epimerase) from kidney cortex involves ammonium sulfate fractionation, DEAE-cellulose chromatography (pH 6.5), chromatography on hydroxyapatite, gel filtration on bio-gel P-100, DEAE-cellulose chromatography (ph 7.7), and crystallization. The enzyme is a monomer, and the molecular weights from many sources are very similar and average 37,000. The enzyme has a broad pH optimum in the range pH 4–8. The purified enzyme is rapidly photoinactivated in the presence of methylene blue or rose Bengal.

Mutarotase in Higher Plants: Distribution and Properties

The enzyme mutarotase from mammalian tissues catalyzes interconversion of anomeric forms of glucose and structurally related sugars; it may be involved in transport of sugars. Isolation and species distribution of a similar enzyme in higher plants are described. The enzyme from green pepper (Capsicum frutescens) was purified 230-fold. It differs from the mammalian enzyme in both substrate specificity and lack of inhibition by 1-deoxyglucose and phloridzin.

Distribution of Mutarotase in Subfractions of Rat Intestinal Mucosa and Rat Kidney.

Summary 1. A method for assaying the enzyme mutarotase in rat kidney and rat intestine was developed. 2. Kidney contains considerably more enzyme than intestine and the activity appears to be concentrated in the tubules. 3. Intestinal mucosa was fractionated into brush border and supernatant fractions by the method of Crane. Brush border membrane contained most of the mucosal invertase but little of the mutarotase. Ninety per cent of the mutarotase remained in a supernatant fraction after removal of brush border. 4. The possible implications of these findings in connection with mechanism of sugar transport are discussed.

[101] Mutarotase from higher plants

Publisher Summary This chapter describes mutarotase from higher plants, and focuses on its assay method, purification procedure, and properties. Plant tissues are finely diced and homogenized in 3 volumes of EDTA buffer (5 mM, pH 7.4) for 2 min at 0° in a high-speed blender. The homogenates when necessary are adjusted to at least pH 6 with 1 M NaOH, and then centrifuged at 27,000g for 20 rain at 4°. An aliquot of the supernatant is then assayed for mutarotase activity by the standard method. A boiled aliquot is also assayed to measure any nonenzymic effects. Purification procedure includes steps, such as preparation of initial extract, ammonium sulfate fractionation, hydroxyapatite column chromatography, and dialysis. The substrate specificity of the plant enzyme is different from that of other mutarotases. Pentoses are poor substrates. Maltose is a poor substrate but an excellent inhibitor. D-fructose is not a substrate. A number of sugars inhibit the plant enzyme. Maltose, cellobiose, L-arabinose, D-galactose, and D-xylose are competitive inhibitors. Other properties are also discussed.