UDPG PYROPHOSPHORYLASE FROM MUSCLE
at 60 ° and centrifuged after cooling. The clear pale-yellow supernatant solution, after dialysis against cold distilled water for 16 hours, is centrifuged at 100,000 X g for 2 hours in the Spinco Model L preparative ultracentrifuge. The supernatant solution is decanted, and the transparent sediment remaining is suspended evenly in 5 ml. of water. The enzyme is stored at + 3 ° and is stable for several weeks. The enzyme can be further purified 3 with alumina CT. Reaction. The D P N H - X to DPNH reaction is conveniently followed by using the decrease in absorption at 290 m/z or the increase at 340 m~. AE29o = 11.4 and --AE34o ---- 6.0 for a DPNH-X concentration of 1 X 10-3 M. If any D P N H oxidase is present, reduced DPNH formed will be oxidized to DPN, which can be readily determined. A typical reaction mixture for carrying out this reaction is made up in a 3.0-ml. volume to contain 5 X 10-5 M DPNH-X, 1.3 X 10-2M MgS04, 5 X 10-4M ATP, and Tris-acetate buffer, 2.5 X 10-a to 2.5 X 10-3 M, pH 6.5. Initial absorption readings are made, and enzyme diluted in water is added to start the reaction. Suitable corrections are applied for enzyme absorption. Observations on the Structure of DPNH-X D P N H - X differs from D P N H through changes in the reduced nicotinamide portion of the molecule.~,3 From spectral evidence~,7 and results of catalytic hydrogenation2 it appears that DPNH-X is a derivative of the 1,4,5,6-tetrahydronicotinamide analog of DPN. DPNH-X formed in the presence of D20 incorporates D in the 5-position2 ,9 Thus DPNH-X may be the 6-hydroxy derivative formed by the addition of water to DPNH. The conversion of DPNH-X to DPNH would consist of an energy-requiring dehydration reaction. "M. Von Marti, M. Viscontini, and P. Karrer, Helv. Chim. Acta 39, 1451 (1956). K. Wallenfels and If. Schiily, Biochem. Z. 329, 75 (1957). 8j. O. Meinhart and M. C. Hines, Federalion Proc. 16, 425 (1957). oj. O. Meinhart, M. C. Hines, A. F. Fluharty, and E. G. Krebs, in preparation.
[ 46] U D P G
Pyrophosphorylase from Muscle
UTP A- G-1-P ~ UDPG -4- PP
By C. VILLAR-PALASI and J. LARNER Assay Method
Principle. The most commonly used method of determination of the enzymatic activity is based on the spectrophotometric measurement of
ENZYMES OF COENZYME AND VITAMIN METABOLISM
the glucose 1-phosphate formed from U D P G on addition of inorganic pyrophosphate (see Vol. II ). Isolation by paper chromatography of the nucleotides formed in the reaction has been used in studies of specificity. 1
Reagents a. Prepare daily a mixture of: 0.025 M Tris, pH 7.45, 0.002 M MgC12, 0.0004M UDPG, 2 X 10-6M glucose 1,6-diphosphate, 0.0005M TPN, 0.05 mg. of lyophilized glucose 6-phosphate dehydrogenase 2 per milliliter, and 0.02 ml. of freshly dialyzed phosphoglucomutase '~ per milliliter. b. 0.02 M potassium pyrophosphate.
Procedure. To 0.95 ml. of the reaction mixture (a), in a quartz cell with a 1-cm. light path, the sample of enzyme is added (about 0.01 to 0.05 ml.), and the reaction is started by addition of 0.05 ml. of pyrophosphate solution (b). Optical density readings at 340 m~ are recorded every 30 to 60 seconds for 5 to 10 minutes. Definition o] Unit and Specific Activity. One unit of enzymatic activity is defined as that amount which will form 1 micromole of glucose 1-phosphate per minute at 30 °, an increase of optical density of 6.200 being equivalent under the conditions of assay to the split of 1 micromole of UDPG. Specific activity is expressed as units per milligram of protein, as measured by the biuret method. 4 Application o] Assay Method to Crude Tissue Preparations. This method appears to be valid for the assay of crude muscle extracts. As muscle contains about ten times as much phosphoglueomutase as U D P G pyrophosporylase, 5 the addition of phosphoglueomutase to the assay system appears to be necessary only when highly purified samples of the enzyme are tested. Glucose 6-phosphate dehydrogenase, as usually prepared, 2 is contaminated with trace amounts of U D P G pyrophosphorylase; this contaminating activity, however, disappears slowly after the lyophilized enzyme has dissolved. Pur~catJon Procedure
Step 1. Preparation of Crude Extract. Rabbit muscle is used as starting material. The animals are killed by a blow on the head and deeapiI C. Villar-Palasi and J. Lamer, Arch. Biochem. Biophys. 86, 61 (1960). SA. Kornberg and B. L. Horecker, Vol. I . ' V. A. Najjar, Vol. I . ~H. W. Robinson and C. G. Hogden, J. Biol. Chem. 135, 727 (1940). 'C. Villar-Palasi and J. Lamer, Arch. Biochcm. Biophys. 86, 270 (1960).
UDPG PYROPHOSPHORYLASE FROM MUSCLE
tated; the muscles of the back and hind legs are removed, ground twice in a preehilled meat grinder, and weighed. All subsequent manipulations take place in the cold room at 3 °. The muscle is extracted with 2 vol. (w/v) of cold 0.03 N KOH-0.005 M EDTA for 30 minutes, with occasional stirring. The suspension is filtered through four layers of gauze. The gauT,e bag is squeezed, such that the recovery of fluid is over 90~'o of the added extraction medium. About 50% of the UDPG pyrophosphorylase activity is extracted in this way. The enzyme is unstable at this stage, and it is necessary to proceed to the next step without delay. Step 2. Ammonium Sul]ate Fractionation. The pH of the extract is adjusted to 6.8 measured at 15 ° with N acetic acid (about 10 ml./1, of extract). Solid ammonium sulfate, 277 g./1. of extract, is slowly added, the suspension is stirred, and after 3 hours at 0 ° the precipitate formed is collected by centrifugation (10 minutes at 8000 X g). The fatty layer floating at the top is separated by filtration through glass wool. Ammonium sulfate, 134 g./1., is then added to the clear filtrate, and the precipitate formed after 3 hours at 0 ° is collected by centrifugation, as above. The precipitate is dissolved with 0.03 N KOH-0.005M EDTA and dialyzed overnight against 100 vol. of 0.005 N KOH-0.001 M EDTA. The dialyzed enzyme is stable for at least a week at 3 °. Step 3. Acetone Fractionation. The dialyzed solution is made 0.005 M in pyrophosphate with neutral 0.1 M K4P20,, and the pH is adjusted to 8.0. The solution is cooled to 0 °, and acetone, prechilled at --20 °, is added dropwise to a 37% final concentration, the mixture being stirred in an ice-salt bath at --10% After the addition is completed, the suspension is kept at --20 ° for 30 minutes and centrifuged in prechilled tubes. The precipitate is rapidly suspended in 0.01 M K4P,Or0.005 M EDTA, pH 9.6, and dialyzed for several hours, with mechanical stirring, against 200 vol. of 0.005 M K4P~07-0.001 M EDTA,. pH 9.6. The dialyzed fluid is adjusted to pH 6.0 with N acetic acid and centrifuged at 8000 X g for 10 minutes; after the protein content of the supernatant fluid has been measured, the following step is carried out without delay. Step 4. Alumina C~ Treatment. To the solution from step 3, a suspension of alumina C,r (20 mg./ml.) and distilled water are added to bring the protein concentration to 7.5 mg./ml, and the alumina C~ to 5.0 mg./ ml. After 10 to 15 minutes the suspension is centrifuged, and the precipitate is washed with distilled water and centrifuged. The enzyme is eluted with several portions of 250 to 300 ml. of 0.01 M K4P20,, pH 9.6, and the eluates with higher specific activity are collected. Solid ammonium sulfate is added (351 g./1. of pooled eluates), the suspension is kept at 0 ° overnight, and the precipitate is collected by centrifugation at 8000 X g for 10 minutes. The precipitate is dissolved in 0.01 M K,P207-
ENZYMES OF COENZYME AND VITAMIN METABOLISM
0.005 M EDTA, pH 9.6, and dialyzed against the same solution for 3 hours; an inactive precipitate that forms is removed by centrifugation. The supernatant fluid is adjusted to pH 8.0 and dialyzed overnight against 140 vol. of 0.005 M K4Pz07, pH 8.0. Step 5. DEAE-CeUulose Chromatography. The columns are prepared by allowing a slurry of 10 g. of DEAE-cellulose (Type 20, capacity 0.86 meq./g.), equilibrated with 0.005 M K4P207, pH 8.0, to pack in a column 2.5 X 14 cm. first by gravity, and then, to obtain the desired flow rate (2 ml./min.), with pressure of N2. The columns are washed with several liters of 0.005 M K4P207, pH 8.0, the dialyzed enzyme of step 4 (about 100 ml.) is applied, and the effluent liquid is collected in 25-ml. fractions. The columns are then first washed with 300 ml. of 0.005 M K,P207, pH 8.0, and the enzyme is eluted by applying a gradient increase in the concentration of pyrophosphate (to 0.1 M). The fractions with higher specific activity are pooled; practically all the enzymatic activity is recovered in one peak. The collected fractions contain very diluted protein, and the recovery of the enzyme in a concentrated form appears difficult. The enzyme loses activity after some weeks at --20 ° in this dilute solution. A summary of the purification procedure is given in the table. SUMMARY OF PURIFICATION OF
Step 1. 2. 3. 4. 5.
Extract (NH4)~SO~fraction Acetonefraction Alumina C~ treatment DEAE-cellulosechromatography
Specific Volume, Protein, U n i t s , activity, ml. mg. gmoles/min,units/rag. 2250 650 250 752 50
60,400 21,200 4,000 3,000 16
3800 3800 2420 2580 1320
0.06 0.18 0.55 0.86 82.50
Specificity. Several hexose and pentose phosphates have been tested as substrates for the purified enzyme.1 Only a-D-glucose-l-phosphate appears to be a reasonable substrate, fl-D-glucose 1-phosphate, a- and fl-Larabinose 1-phosphate, a- and fl-n-galactose 1-phosphate, a- and fl-Dxylose 1-phosphate, and a-D-ribose 1-phosphate did not react with UTP when incubated in the presence of the purified enzyme. Activators and Inhibitors. The enzyme has a total requirement of Mg ++ for activity. On the other hand, excess Mg ++ is inhibitory. Equilibrium. Preliminary determinations of the Keq of the reaction appear to be close to 1.
A BACTERIAL PTERIN DEAMINASE
Kinetic Properties. The K,, for UDPG is 4.5 X 10-'~ M. The K~ for both Mg ÷÷ and P20, -4 are of the order of 1 >< 10-4 M. Ef]ect o/pH. The pH of maximal stability is 9.8. The pH of optimal activity in short incubation periods is 7.45.
 A Bacterial Pterin D e a m i n a s e ~
HO\ N... /N\ C~" C ~'~.C + NH 3 t i t Nb" C\ ~ C \ C/ N/ COOH t
By 0SAMU HAYAISHI Away Method
Principle. The reaction can be followed either by measuring the formation of ammonia or by the spectral change of pterin carboxyli¢ acid 2 accompanying the conversion of the 2-amino group to the 2hydroxyl group at neutral pH. Reagents Pterin carboxylic acid, 0.002 M. 0.1 M potassium phosphate buffer, pH 6.3. Pterin deaminase preparation.
Procedure. In the standard assay, the mixture contains 0.05 ml. of pterin carboxylic acid, 0.8 ml. of phosphate buffer, and 0.02 to 0.15 ml. of enzyme in a final volume of 1.0 ml. Incubation is carried out at 23 ° in a 1-ml. Beckman quartz cuvette with a light path of 1.00 cm. Reaction is begun by the addition of enzyme, and readings of optical density are taken every 3 minutes thereafter. (The control cuvette contains buffer and enzyme, but no pterin carboxylic acid.) With crude preparations, light measurement at 360 mg is employed. However, with more purified fractions of the deaminase, it is possible to follow the course of the reaction at 290 m~, the wavelength at which there is observed the largest AE value between substrate and product. Definition o/ Unit and Specific Activity. A unit of activity is defined as that quantity of enzyme which, under these conditions, will catalyze lB. Levenberg and O. YIayaishi,J. Biol. Chem. 234, 955 (1959). =Abbreviation: Pterin carboxylic acid ----2-amino-g-hydroxypteridine-6-carboxylic acid.