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[20] Enzymes of the Leloir pathway
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ENZYMES OF CARBOHYDRATE METABOLISM
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Properties p H Ef]ect. The curve representing the p H dependence of the reaction shows t h a t the m a x i m u m velocity is reached between p H 8.1 and p H 8.5. At p H 7.5 and at p H 9.2 the reaction velocities are 70% of t h a t at p H 8.5. Specificity. T h e purified fraction was free of inorganic pyrophosphatase. I t contained traces of U D P G and U D P A G pyrophosphorylases. Activator. A divalent metal ion such as M g ++ is required in the reaction. A M g ++ ion concentration of 5 X 10 - 3 M has been used throughout. Dissociation Constant. The Michaelis constant for G D P M has been found to be 1.1 X 10 -5 M at p H 7.5.
[20]
Enzymes
of the Leloir Pathway
I-4
B y ELIZABETH S. MAXWELL, KIYOSHI KURAHASHI, and HERMAN M. t~ALCKAR
I. Q u a n t i t a t i v e D e t e r m i n a t i o n of E n z y m a t i c A c t i v i t y in B r o k e n Cell Preparations The following methods are specific enzymatic assays designed especially for use with crude bacterial extracts. In cases where less rigorous controls are required the assays described later in the sections on purification of enzymes are more suitable. Galactokinase 5-7 Principle. The enzyme catalyzes the reaction A T P + G a l - ~ A D P +a-Gal-l-P. The G a l - I - P produced in a preincubation mixture is quantitatively determined. Fluoride is added in order to protect A T P and G a l - I - P from being dephosphorylated by phosphatase. Addition of magnesium ions serves to ensure t h a t the reaction, which depends on
1L. F. Leloir, Arch. Biochem. Biophys. 33, 186 (1951), see also Vol. I [35]. R. Caputto, L. F. Leloir, R. E. Trucco, C. E. Cardini, and A. J. Paladini, .l. Biol. Chem. 179, 497 (1949). I-I. M. Kalckar, B. Braganca, and A. Munch-Petersen, Nature 172, 1036 (1953). 4A. Muneh-Petersen, H. M. Kalckar, and E. E. B. Smith, Kgl. Da~ske Videnslcab. Selslcab, Biol. Medd. 22, 3 (1955). 5 t:i. W. Kosterlitz, B~ochem. J. 37, 322 (1943). ~R. E. Trucco, R. Caputto, L. F. Leloir, and N. Mittelman, Arch. Biochem. 18, 137 (1948). 'C. E. Cardini and L. F. Leloir, Arch. Biochem. Biophys. 45, 55 (1953).
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ENZYMES OF THE LELOIR PATHWAY
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these ions, is not slowed down by a depletion of magnesium due to the presence of fluoride. The Gal-l-P formed is determined in the proteinfree filtrate by an analytical incubation mixture; the basis for the method rests on the ability of Gal-l-P to interact with UDPG in the presence of the specific enzyme, Gal=l-P uridyl transferase 8,~ (used here as indicator enzyme). The sequence of enzymatic reactions used for the analysis of GaI-1-P is as shown. The absolute molar amount of T P N H Gal-I-P + UDPG ~- G-1-P + UDPGal (catalyzed by Gal-l-P uridyl transferase) G-1-P ~ G-6-P (catalyzed by phosphoglucomutase) G-6=P + T P N --~ 6-Phosphogluconate + T P N H (catalyzed by G-6-P dehydrogenase) formed is a direct measure of Gal-l-P and is thus a measure of galacto= kinase activity. This is provided that the substrates used in the analytical reactions are in considerable excess of the Gal=l-P stemming from the preincubation mixture. The preincubation mixture has the following composition: galactose (recrystallized), 25 ~l. of a stock solution containing 200 micromoles/ml., i.e., 5 micromoles of galactose per milliliter of incubation mixture; ATP (0.1 M), 20/~l. corresponding to 2 micromoles; Tris buffer (1 M, pH 7.5), 100 ~l.; sodium fluoride (0.5M), 50 ~l.; MgC12 (0.1M), 50 ~l.; H20, 655 ~l.; bacterial extract (galactokinase to be determined), 100 ~l, The incubation period is 0, 10, 20, and 30 minutes. The temperature is 37% The reaction is stopped by placing the incubation tube in a boiling-water bath for 90 seconds. After spinning, part of the protein=free clear supernatant fluid is analyzed enzymatically for Gal-l-P. Usually a 100-/A. filtrate is analyzed. The analysis mixture is composed as follows: UDPG, 20 /~l. or 0.2 micromole; G-6-P dehydrogenase,8 10 ~l. of a solution containing 10 mg. of protein per milliliter; TPN, 20 ~l. of a solution containing 20 mg./ml.; cysteine (200 micromoles/ml., pH 8.5), 100 ~l.; phosphoglueomutase9 (1 mg./ml.), 20 ~l.; MgC12 (1 M), 10 ~l.; glucose-l,6-diphosphate (0.5 micromole/ml.), 5 ~l.; glycine (1M, pH 8.7), 100 ~l.; H20, 610 ~l.; fractionated Gal-l-P uridyl transferase from liver 1° or from E. coli K12 mutant 30921~ (see procedures later in this chapter). The amount of filtrate for the assay is 100 FL1.In order to calibrate the system, Gal-l=P SA. Kornberg, J. Biol. Chem. 182, Horecker, Vol. I [42]. 9V. A. Najjar, J. Biol. Chem. 175, 1oK. Kurahashi and E. P. Anderson, 11K. Kurahashi and A. Sugimura, J.
805 (1950); see also A. Kornberg and B. L. 281 (1948); see also Vol. I [36]. Biochim. e~ Biophys. Acta 29, 498 (1958). Biol. Chem. 235, 940 (1960).
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ENZYMES OF CARBOHYDRATE METABOLISM
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in amounts of 0.05 to 0.075 mieromole is added. For each 0.1 micromole of Gal-l-P per milliliter a density increase of 0.620 at 340 m~ take~ place. Usually the G-6-P dehydrogenase is added first in order to determine small amounts of G-1-P or G-6-P present in the filtrate. Subsequently the Gal-l-P uridyl transferase is added, and the total density increase at 340 ms minus the value obtained with a zero time control of preincubation mixture is a measure of the Gal-l-P which accumulates in the preincubation period and hence of the activity of the galactokinase. The method is highly specific but not highly sensitive. In its present design it can detect activities of 0.1 to 0.2 micromole/hr./mg, of protein. The sensitivity can be increased by analyzing a larger fraction of the preincubation mixture. The latter can also be incubated for longer periods of time (1 and 2 hours). Fluoride has not been found to inhibit galactokinase significantly. Simpler and more-sensitive methods for galactokinase can be devised. However, for projects in which it is essential to demonstrate that the hereditary blocks affect the steps of the Leloir pathway, the specific enzymatic methods are deemed most essential. A new sensitive specific method for Gal-l-P determination1~ has been worked out. In this method human hemolyzate is used as transferase source. The incubation mixture is heat-inactivated and analyzed for consumption of UDPG by means of UDPG dehydrogenase. This method is being modified with the purpose of assaying galactokinase in bacterial extracts. G a l - l - P Uridyl Trans]erase 3, lo, 11 Principle. The enzyme catalyzes the reaction
Gal-l-P ~- UDPG ~ G-1-P -~ UDPGal. The rate of G-1-P formation is in this case analyzed by adding the two indicator enzymes and coenzymes in excess, phosphoglueomutase9 (plus glucose-l,6-diphosphate) and G-6-P dehydrogenase8 plus TPN. The initial rate of formation of T P N H is used as a direct measure for the Gal-l-P uridyl transferase activity of the disrupted bacterial cell. Controls having either Gal-l-P or UDPG omitted from the incubation are always used. Reoxidation rate in the extracts is much slower than that of TPN reduction. Yet, for determination of low transferase activities it is necessary specifically to determine the rate of reoxidation of TPNH. 1~H. N. Kirkman and E. S. Maxwell, J. Lab. Clin. Med. 56, 161 (1960).
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The enzyme activity is determined directly in the Beckman spectrophotometer (i.e., "generation incubation" and "analysis incubation" are combined) by the rate by which T P N is reduced. All constituents, substrates, indicator coenzymes, and indicator enzymes are added in considerable excess over the transferase to be determined. An incubation mixture for the assay of Gal-l-P uridyl transferase has the following composition: cysteine (33 mg./ml., pH 8.5), 30 ~l.; magnesium chloride (0.1 M) 10 ~l.; glycine (1 M, pH 8.7), 60 ~l.; phosphoglucomutase together with glucose-l,6-diphosphate; T P N (20 mg./ml.), 10 ~l.; G-6-P dehydrogenase; U D P G (10 micromoles/ml.), 20 ill.; water, 360 ~l. To this mixture is added the K12 extract in amounts of 50 to 100 ~g. of protein and, as the last component, Gal-l-P (10 micromoles/ml.), 30 ~1. The total volume is 620 td. In order to ensure that the indicator enzymes are in excess it may be necessary to add more G-6-P dehydrogenase or phosphoglucomutase or glucose-l,6-diphosphate and ascertain that the rate of release of G-1-P from U D P G and Gal-l-P is not increased. Control mixtures with UDPG without Gal-l-P, or with Gal-l-P without UDPG, should only give very slow release of G-1-P. The release of G-1-P is measured by the rate of increase in optical density change at 340 m~, with 400 m~ as isobestic point. Another complication which might obscure comparison on a quantitative basis is the fact that 6-phosphogluconate dehydrogenase 13 present in the lysate (or in some preparations of indicator enzyme glucose-6phosphate dehydrogenase) will alter the scale. That is, the activity of transferase will be overestimated because each mole of U D P G exchanged will give a deflection at 340 mt~ which is twice as high. It is therefore necessary always to calibrate the enzyme mixture with a known amount of G-1-P or G-6-P. A direct assay with 6-phosphogluconate is to be recommended too. In most cases the activity of phosphogluconate dehydrogenase is low. Hence the calibration with G-1-P is most important in extracts in which transferase activity is moderate or low. These difficulties are less likely to be encountered if one is dealing with extracts having highly active transferase. If several factors contribute to complicate the quantitative analysis it might be helpful to perform the assay by a two-step procedure. The reaction mixture would in such a case consist of E. coli extract, phosphoglucomutase and glueose-l,6-diphosphate, cysteine, MgC12, Gal-l-P, and UDPG. After a brief incubation (for instance, 5 and 10 minutes) the reaction is stopped and the filtrate is analyzed for G-6-P by means of T P N and G-6-P dehydrogenase (which should be free of transferase). ~3B. L. Horecker and P. Z. Smyrniotis, J. Biol. Chem. 193, 371 (1951); see also Vol. I [42].
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ENZYMES OF CARBOHYDRATE METABOLISM
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The sensitivity of the direct transferase assay is of the order of magnitude of 0.01 micromole/hr./mg, protien. UD PGal_4.epimerase 1,2 Principle. The enzyme catalyzes the reaction UDPGal ,-~-~UDPG. UDPG formed is determined in a protein-free filtrate by means of a DPN-dependent specific dehydrogenase (UDPG dehydrogenase 14) which catalyzes the reaction U D P G + 2DPN--> Uridine diphosphoglueuronie acid + 2DPNH. The latter is performed in an analytical incubation. The absolute molar amount of D P N H formed divided by 2 provides a direct measure for UDPGal-4-epimerase activity in the disrupted cells. Since the disrupted K12 cells do not contain any enzymes that hydrolyze or oxidize U D P G (or UDPGal), it is possible by this method to determine quantitatively even very low activities of UDPGal-4-epimerase. The two-step procedure is performed as follows: The reaction mixture contains glycine (1 M, pH 8.7), 50 ~1.; E. coli extract, 20 t~l; UDPGa115,~6 (2.2 micromoles/ml.), 30 ~1. in a volume of 0.3 ml. The mixture is incubated for 5 and 10 minutes at room temperature (25°). At termination the mixture is best inactivated by placing the tubes (with a marble on top) in a boiling-water bath for 11/~ minutes. It is then cooled on ice and spun down, and 200 ~l. of the clear supernatant is used for the analytical mixture. The latter contains glycine (1 M, pH 8.7), 70 td.; D P N (25 micromolcs/ml.), 20 #l.; supernatant of the preincubated sample, 200 #l.; water, 15 ~l.; and finally U D P G dehydrogenase, 15 or 20 ~l. The control contains the same mixture except that the supernatant stems from a reaction mixture to which UDPGal was added after boiling. The sensitivity of the epimerase test is of the order of magnitude of 0.005 micromole/hr./mg, of protein. The one-step direct procedure can be used occasionally for crude extracts but preferentially for fractionated UDPGal-4-epimerase preparations free of hydrogen donors and acceptors. The procedure for crude bacterial extracts is executed as follows: The reaction mixture contains glycine (1M, pH 8.7), 50 ~l.; D P N (25 micromoles/ml.), 20 /~l.; UDPGal (2.2 micromoles/ml.), 30 ~l. ;. water, 475 td.; U D P G dehydrogenase, 25 #l. ; and E. coli extract, 5 or 10 ~I. On the addition of extract the rate of increase of optical density at 340 mfi is recorded (the isobestie region at 400 to 410 m# is used to check for extraneous optical ~4j. L. Strominger, E. S. Maxwell, J. Axelrod, and It. M. Kalckar, J. Biol. Chem. 224, 79 (1957); see also Vol. I I I [143A]. 1~E. P. Anderson and E. S. Maxwell. J. Am. Chem. Soc. 81, 6514 (1959). t~ff. G. Moffatt, YI. G. Khorana, and H. M. Kalckar, unpublished work (1958).
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ENZYMES OF THE LELOIR PATHWAY
179
density changes at reasonable intervals). I t is i m p o r t a n t t h a t U D P G dehydrogenase is in excess. This can be ascertained by lowering the amount of E. coli extract to see if the rate decreases proportionally. The control tube contains the same ingredients with the exception of U D P G a l . Reoxidation of D P N H interferes, and a direct assay with D P N H as substrate should be performed. Each millimicromole of U D P G a l converted to U D P G corresponds to an optical density change of 0.020 at 340 m~. The sensitivity of the direct epimerase test is as high as t h a t of the indirect. I I . Purification of G a l a c t o s e - l - p h o s p h a t e Uridyl Transferase U D P G ~ G a l - l - P ¢~-UDPGal ~ G-1-P This enzyme was first found in galactose-adapted yeast2 The enzyme from calf liver and from an Escherichia coli galactokinase-less m u t a n t has been partially purified. 1°,11
Assay Method Principle. G a l - l - P uridyl transferase activity is measured from T P N H formation in a coupled reaction with phosphoglucomutase and G - 6 - P dehydrogenase. Reagents 0.01 M U D P G , sodium salt. 0.0175 M G a l - l - P , sodium salt. 1.0 M glycine buffer, p H 8.75 when it is diluted to 0.1 M. 0.16 M cysteine hydrochloride, p H 8.5. 1.0 M MgC12. 0.02 M T P N . 0.0002 M G - l , 6-P27 Phosphoglucomutase, 70 units/ml2 s in 1% bovine albumin solution. Prepared according to the method of N a j j a r 2 ~7Some of the phosphoglucomutase preparations contain enough G-1,6-P, especially when freshly prepared. However, since Gal-l-P was found to be inhibitory for the phosphoglucomutase reaction [V. Ginsburg and E. F. Neufeld, Abstr. Papers, 132nd Meeting Am. Chem. Soc., New York 27C (1957); and J. B. Sidbury, ibid., 27C (1957)], G-1,6-P and phosphoglucomutase are used routinely in excess. It is also necessary to avoid prolonged contact of phosphoglucomutase with Gal-l-P. 18One unit of G-6-P dehydrogenase is defined as that amount of enzyme which causes the reduction of 1 micromole of TPN per minute under the assay condition used for Gal-l-P uridyl transferase except that 0.4 micromole of G-6-P is used as substrate. One unit of phosphoglucomutase is similarly defined as that amount of enzyme which causes the reduction of 1 micromole of TPN per minute when 0.4 micromole of G-1-P (free of G-1,6-P) is used as substrate.
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ENZYMES OF CARBOHYDRATE METABOLISM
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G-6-P dehydrogenase, 14 units/ml? s in 1% bovine albumin solution. Prepared according to the method of Kornberg and Horecker. 8 The gel adsorption and acid precipitation steps in their method are repeated twice, in order to remove 6-phosphogluconate dehydrogenase and Gal-l-P uridyl transferase. Gal-I-P uridyl transferase. Diluted with 1% bovine albumin to give an optical density change at 340 m~ between 0.03 and 0.05 per minute under the assay conditions described below.
Procedure. To a 1-ml. quartz cell having a light path of 1 cm. are added 425 ~l. of water, 60 ~l. of glycine buffer, 30 ~l. of cysteine hydrochloride, 5 ~l. of MgCl~, 20 t~l. of TPN, 25 ~l. of UDPG, l0 ~l. of G-1,6-P, 10 ~l. of phosphoglucomutase, 5 t~l. of G-6-P dehydrogenase, and 10 tL1. of diluted Gal-l-P uridyl transferase. Readings are taken at 340 mt~ at 1-minute intervals after addition of 20 ~1. of Gal-l-P in a Beckman DU spectrophotometer with a pinhole attachment. At the beginning of the reaction there is always a short lag period, and then the reaction proceeds linearly with time. The linear part of the curve is utilized for the calculation of the rate of the reaction. It is important to set up control ceils without Gal-l-P uridyl transferase in order to check the presence of Gal-l-P uridyl transferase in G-6-P dehydrogenase and the presence of G-1-P in Gal-l-P. It is also advisable to set up a control cell without Gal-l-P, when crude enzyme preparations are utilized. Definition of Unit and Specific Activity. A unit of enzyme is defined as that amount which causes the incorporation of 1 micromole of Gal-l-P into the uridine nucleotide per minute, and thus causes the formation of 1 micromole of T P N H per minute in the assay. Purification Procedure from Calf Liver Calf liver acetone powder is prepared as described by Horecker. 19 All the following steps are carried out at 2 ° . Step I. Preparation of Crude Extract. One hundred grams of acetone powder is extracted with 200 1111.of potassium phosphate buffer (0.002 M, pH 7.0) for 30 minutes. The residue is removed by centrifugation at 20,000 )K g for 10 minutes. Step 2. Protamine Sul]ate Precipitation. To the crude extract, 1/100 vol. of 0.5 M potassium phosphate (pH 6.0 when diluted to 0.05 M) and 1/100 vol. of protamine sulfate (20 mg./ml.) are added. After 5 minutes of stirring, the precipitate is collected by centrifugation at 20,000 X g for 5 minutes and discarded. 19For the preparation of acetone powder, see Vol. I I [123].
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ENZYMES OF THE LELOIR PATHWAY
Step 3. Fractionation with Ammonium Sulfate. To the supernatant solution enough cold ammonium sulfate solution (saturated at 2 °, pH 6.0) is added to give 42.5% saturation. After 10 minutes of stirring, the precipitate is removed by centrifugation at 20,000 X g for 5 minutes. To the supernatant solution ammonium sulfate solution is again added to give 52.5% saturation. After 10 minutes of stirring, the precipitate is collected by centrifugation and dissolved in 65 ml. of cold 0.9% NaC1 solution. This fraction is dialyzed overnight at 2 ° against 15 1. of 0.005 M potassium phosphate (pH 6.0). Step 4. Fractionation with Calcium Phosphate Gel. After removal of precipitate formed during dialysis by centrifugation, the dialyzed fraction is diluted to give a protein concentration of 10 mg./ml, by addition of 0.005M potassium phosphate (pH 6.0). Then calcium phosphate gel 2° (17.9 mg. dry weight per milliliter) equivalent to 1 mg. in dry weight per milligram of protein is added. After 15 minutes of stirring, the gel is removed by centrifugation and discarded. To the supernatant solution the same amount of gel is again added. After 15 minutes of stirring, the gel is collected by centrifugation and the supernatant solution is discarded. The gel is eluted by stirring for 15 minutes with 0.01 M potassium phosphate (pH 7.4, one-third of the volume of the discarded supernatant solution). The residue is removed by centrifugation and discarded. Step 5. Lyophilization. After addition of 1/20 vol. of reduced glutathione (15 mg./ml.), the eluate is lyophilized. A summary of the procedure is given in Table I. TABLE I SUMMARY OF PURIFICATION PROCESS FROM CALF LIVER
Step and fraction 1. 2. 3. 4.
Crude extract Protamine sulfate, supernatant (NH4)2S04fraction, 0.425-0.525 Ca3(PO4):gel eluate
Total T o t a l T o t a l Specific volume, protein, activity, activity, Yield, ml. nag. units units/mg. % 1670 1760 82 175
17,400 11,900 2,420 116
225 182 99 74
0. 013 0. 015 0. 041 0.64
100 81 44 33
Purification Procedure from Eschericl~ia colt
E. colt K12 W3092 (galactokinase-less) mutant21, 22 was obtained from Drs. E. M. and J. Lederberg at Stanford University. The cells are S°For the preparation, see Vol. I [12]. .~1M. L. Morse, E. M. Lederberg and J. Lederbcrg, Genetics 41, 143, 758 (1956). ~':K. Kurahashi, Science 125, 114 (1957).
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ENZYMES OF CARBOHYDRATE METABOLISM
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grown at 37 ° with aeration by rotatory shaking in a medium of the following composition: 10 g. of casamino acid (Difco), 5 g. of yeast extract (Difco, 3 g. of K~PO,, l g. of KH2P04, 5 g. of D-galactose, and 1 g. of D-glucose per liter. A loopful of bacteria from a 24-hour culture of E. coli at 37 ° on a nutrient agar slant (Difco) is used to inoculate 50 ml. of medium. After 8 to 10 hours, the entire culture is transferred into 1 1. of fresh medium, and incubation is continued for 16 hours. Step 1. Preparation of Crude Extract. The cells are harvested by centrifugation at 6000 X g for 10 minutes and washed with 200 ml. of cold water (yield: 4 to 5 g. of packed cells per liter). For each gram of packed cells 4 ml. of potassium phosphate (0.04M, pH 7.4) is added, and the cells are disintegrated in a sonic disintegrator (Raytheon 10 kc) for 10 minutes. The broken cell residues are removed by centrifugation at 20,000 X g for 10 minutes. Step 2. Incubation at 37 ° and Heat Denaturation. The crude extract is adjusted to contain 20 mg./ml, of protein (determined by the biuret method 23) and 100 micromoles of potassium phosphate per milliliter by the addition of enough water and 0.5 M potassium phosphate (pH 7.4). Sixty milliliters of this extract is worked up at one time for the following purification. The diluted solution is incubated at 37 ° for 2 hours to enhance the breakdown of nucleic acid ~4 (this fraction can be stored at --10 ° for several days without much loss of activity). After cooling it is placed in a hot-water bath at 65 ° to 70 °, and the temperature is brought to 53 ° rapidly (1.5 to 2 minutes). The solution is maintained at 53 ° for 11 minutes and then chilled in an ice bath. All the following steps are carried out at 2 ° . Step 3. Fractionation with Ammonium SuI]ate. To the heat-denatured solution ammonium sulfate solution (pH 7.0, saturated at 2 ° and containing 0.006 M Versene) is added to give 45% saturation of ammonium sulfate. After stirring for 10 minutes, the precipitate is removed by centrifugation at 20,000 X g for 5 minutes. To the supernatant solution ammonium sulfate solution is added to give 67% saturation. After 10 minutes of stirring, the precipitate is collected by centrifugation. The supernatant solution is removed completely by inverting the centrifuge tube for 5 minutes, because excess ammonium sulfate interferes with the effectiveness of the following gel step. The precipitate is dissolved in 10 ml. of water, and the protein concentration is determined by the biuret method. 23 Step 4. Calcium Phosphate Gel Adsorption o] Inactive Protein. The '3It. W. Robinson and C. G. ttogden, J. Biol. Chem. 13~, 707 (1940). 2*Personal communication from Dr. P. Berg of Stanford University (1958).
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solution is adjusted to contain 7.5 mg. of protein and 50 micromoles of potassium phosphate per milliliter by the addition of enough water and potassium phosphate (0.5 M, pH 5.8 when diluted to 0.05M). Then calcium phosphate gel2° (21.9 mg. dry weight per milliliter) equivalent to 1 mg. in dry weight per milligram of protein is added. After 10 minutes of stirring, the gel is removed by centrifugation and discarded. Step 5. Removal o] Residual Nucleic Acid by Nuchar. After the protein concentration has been determined spectrophotometrically,2~ the supernatant solution is treated with Nuchar (Grade XXX), 1 mg./mg. of protein, and stirred for 4 minutes with a stirring rod. Then another equivalent amount of Nuchar is added and stirred for another 4 minutes. The charcoal is removed by centrifugation and discarded. Step 6. Treatment with Alumina C-f Gel. After determination of the protein concentration, alumina C~ gel2° (12.2 mg. dry weight per milliliter) is added to the supernatant solution, 0.3 mg. dry weight per milligram of protein. After 10 minutes of stirring, the gel is removed by centrifugation and discarded. Step 7. Fractionation with Ammonium Sulfate. The supernatant solution is refractionated with saturated ammonium solution. The precipitate between 50% and 60% saturation is collected and dissolved in 4 ml. of water. The fraction is dialyzed for 5 hours at 2 ° against 0.005 M potassium phosphate (pH 7.4). When this fraction is stored overnight in an ice bath, about a quarter of the activity is lost. Step 8. DEAE-Column ChromatographyY 6'~7 The column (1.1 cm. in diameter and 6 cm. in height) is prepared by pouring 600 mg. of DEAE-cellulose (Eastman Kodak Company) suspended in 20 ml. of 1 M NaC1 solution and allowing it to pack by gravity. The column is washed successively with 100 ml. of 1 M NaC1, 40 ml. of water, 100 ml. of 0.5 M potassium phosphate (pH 7.4), 40 ml. of water, and 60 ml. of 0.005 M potassium phosphate (pH 7.4). After addition of the dialyzed enzyme solution, the column is washed through with 20 ml. of 0.005 M potassium phosphate (pH 7.4) and 60 ml. of 0.005 M potassium phosphate (pH 7.4) which contains 0.05 M NaCl. Then active enzyme fractions are eluted from the column by 0.005 M potassium phosphate (pH 7.4) which contains 0.12 M NaC1. Two-milliliter fractions are collected. Ordinarily the bulk of the enzymatic activity is recovered in fractions 3to7. Step 9. Lyophilization. The most active fractions are combined and 2~See Vol. I I I [73]. ~6E. A. Peterson and R. A. Sober, J. Am. Chem. Soc. 78, 751 (1956). 'TH. A. Sober, F. J. Gutter, M. M. Wyckoff, and E. A. Peterson, J. Am. Chem. Soc. 78, 756 (1956).
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ENZYMES OF CARBOHYDRATE METABOLISM
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dialyzed for 1 hour against 0.005 M potassium phosphate ( p H 7.4) a t 2 °. The dialyzed fraction is then lyophilized after addition of ~ o vol. of reduced glutathione (15 mg./ml.) and an equal volume of 0.25 M glycylglycine ( p H 7.4). The a m o u n t of enzyme protein in this lyophilized powder is determined b y the method of L o w r y et al. after precipitation of the enzyme protein by trichloroacetic acid. 2~ A s u m m a r y of the purification procedure is given in T a b l e I I . TABLE II PROCESS FROM E. coli
SUMMARY OF PURIFICATION
Step and fraction 1. 2. 3. 4. 5. 6. 7. 8.
Crude extract Incubation at 37 ° (NH4)2SO4fraction, 0.45-0.67 Ca3(PO4)2gel supernatant Nuchar supernatant Alumina C~ gel supernatant (NH4)~SO4fraction 0.5-0.6 DEAE chromatography Fraction 3 Fraction 4 Fraction 5 Fraction 6
Total Total Total Specific volume, protein, activity, activity, Yield, ml. mg. units units/rag. % 54.3 60.0 10.6 37 33 33 4.4 2.0 2.0 2.0 2.0
1275 1200 222 144 121 94 40 2.2 i. 4 1.0 0.7
347 366 301 288 248 231 169 44 24 11 8
0.27 0.30 1.4 2.0 2.1 2.5 4.2 20 16 11 11
100 105 87 83 71 66 49 13 7 3 2
Properties 10,n
Stability. The lyophilized enzyme preparations can be stored at - - 1 0 ° in a desiccator for several months without loss of activity. The enzyme solution prepared by dissolving the powder in 1% bovine albumin solution can be stored at - - 1 0 ° for a week with gradual loss of activity. Presence of Other Enzymes. The calf liver enzyme preparation is contaminated by U D P G pyrophosphorylase and phosphoglucomutase, and to a much smaller extent by U D P G a l - 4 - e p i m e r a s e and galactokinase. I t is free of U D P G dehydrogenase. The E. coli enzyme preparation is free of all the above enzymes. p t t Optimum. Both enzyme preparations have their p H optima between 8.5 and 8.9. Activators and Inhibitors. The enzyme activities of both p r e p a r a tions are partially protected by reduced glutathione. Calf liver enzyme is inhibited approximately 50% by p-chloromercuribenzoate at a concentration of 2 X 10-~ M. E. coli enzyme shows an absolute requirement
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ENZYMES OF THE LELOIR PATHWAY
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of cysteine for its activity. Mg ÷÷ ions and NaF are found slightly inhibitory for E. coli enzyme. K,~ Values. The K~ values of E. coli enzyme for UDPG, Gal-l-P, UDPGal, and G-1-P are estimated to be 1.5 X 10 ~, 4.1 X 10-4, 2.5 X 10-', and 2.4 X 10-*, respectively. III. Purification of UDPgalactose-4-epimerase UDPgal ~- UDPG Assay Method Principle. UDPgalactose-4-epimerase 1,2,-~,~9 is assayed spectrophotometrically by the rate of increase in optical density at 340 m~ when UDPgal is present as the substrate, and the reaction is coupled with an excess of UDPG dehydrogenase and DPN. UDPgal ~ UDPG UDPG + 2DPN -~ UDPGA + 2DPNH + 2H + Reagents 0.005 M UDPgal (prepared either enzymatically from UDPG and Gal-l-P 1~ or chemically by the method of Moffat et al2G). 0.05 M DPN. 1.0 M glycine buffer adjusted to pH 8.8 with NaOH. UDPG dehydrogenase, approximately 104 units/ml., carried through step 5 of the method of Strominger et al. 14 Procedure. The following reagents are added to a silica cuvette with 1.3-ml. capacity and a light path of 1 cm.: water to a final volume of 0.5 ml., 50 ~l. of glycine buffer, 10 /tl. each of UDPgal and DPN, and 20 ~l. of UDPG dehydrogenase. Optical density readings are taken at 340 m~ in a Beckman DU spectrophotometer equipped with a pinhole attachment so that 0.5-ml. volumes may be used in 1.3-ml. cuvettes. Because of small amounts of UDPG which may be present in the enzymatically prepared UDPgal, a small increase in optical density may be observed at this stage. When no further increase occurs, usually within 5 minutes, an aliquot of UDPgalactose-4-epimerase is added and the rate of increase in optical density is followed for 4 minutes. During the early stages of purification it is desirable to include a second cuvette with all the reagents except UDPgal. The rate of the reaction is proportional to enzyme concentration up to an increase in optical density of :SE. S. Maxwell, J. Biol. Chem. 229, 139 (1957). -'~E. S. Maxwell and H. de Ribichon-Szulmajster, J. Biol. Chem. 235, 308 (1960).
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ENZYMES OF CARBOHYDRATE METABOLISM
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about 0.050 per minute. A unit of activity is defined as an increase in optical density at 340 m s of 0.001 per minute under the conditions described. Purification from Calf Liver Acetone Powder ~8 Acetone powder from liver of freshly slaughtered calves is prepared according to Horecker. 1~ The enzyme is stable in the dry powder stored at --10 ° for at least a month. The purification procedure is carried out at 0 ° to 5 °, except as otherwise noted. Step 1. Water Extract o] Acetone Powder. Fifty grams of acetone powder is extracted with 1000 ml. of water by light grinding in a mortar, followed by stirring for 30 minutes. The suspension is centrifuged, and the residue discarded. Step 2. Fractionation with Acetone (0 to ~5%). The water extract (910 ml.) is adjusted to pH 5.4 with 1 M acetic acid. The precipitate is centrifuged immediately and discarded. The solution is cooled to 0 °, and 295 ml. of acetone (--15 °) is added dropwise over a period of 30 minutes, the solution being stirred continuously during the addition, and the temperature kept between 0 ° and --2 °. The precipitate is centrifuged and dissolved in 300 ml. of 0.1 M glyeine buffer, pH 8.0. Step 3. Precipitation with Alkaline Ammonium Sulfate (between ~5 and 65% Saturated). To 300 ml. of the dissolved precipitate from step 2 is added 245 ml. of alkaline ammonium sulfate (pit 8.0, when diluted fivefold; prepared by adding 3.7 ml. of concentrated NH40H to 1 1. of saturated ammonium sulfate). The precipitate is centrifuged and discarded. Then 315 ml. of alkaline ammonium sulfate is added to the supernatant fraction. The precipitate is centrifuged and dissolved in 80 ml. of distilled water; the solution can be stored at --20 ° overnight. Step ~. Precipitation at pH ~.6. To 80 ml. of the solution from step 3 is added 80 ml. of 0.5 M acetate buffer, pH 4.6, in 50% saturated ammonium sulfate (prepared by adding 50 ml. of 1 M sodium acetate buffer, pH 4.6, to 50 ml. of saturated ammonium sulfate). After standing for 10 minutes, the precipitate is centrifuged and dissolved in 80 ml. of 0.05 M glycine, pH 8.7. Step 5. Adsorption and Elution from Calcium Phosphate Gel. The solution from step 4 is diluted to 160 ml. with distilled water, and 65 ml. of calcium phosphate gel (17.9 mg./ml.) is added to the diluted solution. The suspension is stirred for 10 minutes and centrifuged. The supernatant solution is discarded, and the gel is suspended in 100 ml. of 0.1 M phosphate buffer, pit 7.5. After being stirred for 10 minutes, the gel is centrifuged and discarded. Step 6. Precipitation with Ammonium Sulfate (between ~5 and 65%
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ENZYMES OF THE LELOIR PATHWAY
187
Saturated). Twenty-six grams ammonium sulfate is added to the clear solution from step 5, and the precipitate is centrifuged and discarded. Then 14 g. of ammonium sulfate is added to the supernatant solution, and the precipitate is centrifuged and dissolved in 20 ml. of 0.25 M glycylglycine buffer, pH 7.5. The solution is lyophilized and stored at --20 ° . The results of the purification procedure are shown in Table III. TABLE III PURIFICATION OF UDPGALACTOSE-4-EPIMERASE FROM CALF LIVER ACETONE POWDER
Step 1 2 3 4 5 6
Total activity, units, X 10-3 840 1000 900 560 650 520
Units/mg, protein ~ 77 410 1,200 2,800 6,880 16,200
Protein is determined by the method of Lowry et al., Vol. III [73]. The purified enzyme can be kept frozen in 0 . 2 5 M glycylglycine buffer at p H 7.5 for about a week without significant loss of activity. The lyophilized preparation stored at --20 ° is stable for at least 3 months. The pH optimum lies between 8.7 and 9.5. At pH 7.0 the rate is about 3% of that at p H 9.0. The purified enzyme has an absolute requirement for catalytic amounts of D P N and is inhibited by D P N H . The Michaelis constants are 2 X 10-7 M for D P N and 5 X 10-' M and 9 X 10 -5 M, respectively, for U D P g a l and U D P G . At equilibrium the ratio of U D P G to UDPgal is about 3: 1. Purification from Galactose-Orown Yeast 29
Growth o] Yeast. Saccharomyces ]ragilis (American T y p e Culture Collection No. 10022) is adapted to galactose by growth in a medium containing 15 g. of yeast extract (Difco), 1.8 g. of (NH4):SO.,, 1.5 g. of KH_~PO4, and 75 g. of galactose in a final volume of 1500 ml. of medium. About 100 ml. of a 24-hour culture of adapted cells is inoculated into each of twelve 6-I. flasks containing 1500 ml. of the same medium. The cultures are incubated at room temperature with shaking for 24 hours. Cells are harvested in a Sharples centrifuge. Step I. Preparation o] Cell-Free Extract. The wet cell mass (about
188
ENZYMES OF CARBOHYDRATE METABOLISM
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310 g.) is suspended in a final volume of 1 I. of 0.1 M glycine, pH 6.5. Ceils are broken by a 30-second exposure in a Nossal disintegrator. Nine milliliters of the cell suspension and 8 g. of acid-washed glass beads (Minnesota Mining and Mfg. Co., St. Paul, Minnesota, No. 150 [75 t~]) are used in each Nossal tube. This procedure is carried out in a --10 ° room, and the cell suspension is kept just above freezing. All subsequent procedures are carried out at 3 ° to 5 ° except where otherwise noted. The broken-cell suspension is centrifuged at high speed in a Lourdes Model SL centrifuge. The yield of cell-free supernatant fluid is about 590 ml. Step 2. Removal of Nucleic Acid. Nucleic acid is removed by autolytie digestion as suggested by P. Berg and W. E. Razzell. To the cellfree extract is added 60 ml. of 1 M potassium phosphate buffer, pH 7.0. The preparation is incubated at 37 ° for 2 hours. The preparation may be frozen and stored overnight at this stage.
Step 3. Precipitation with Ammonium Sulfate (0 to 50% Saturated). The solution is made 50% saturated by the addition of 175 g. of ammonium sulfate to 600 ml. of solution. The precipitate is centrifuged, dissolved in distilled water, and made up to a final volume of 100 ml.
Step 4. Precipitation with Ammonium Sulfate (35 to 50% Saturated). The concentration of ammonium sulfate in the dissolved precipitate is determined with a Barnstead purity meter (Barnstead Still and Sterilizer Company), and the solution is usually found to be about 9% saturated. To the solution is added 13.9 g. of ammonium sulfate. The precipitate is centrifuged and discarded. An additional 8.7 g. of ammonium sulfate is added to the supernatant fluid. The precipitate is centrifuged and dissolved in 15 ml. of distilled water. Step 5. Chromatography on DEAE Cellulose. The solution is dialyzed for 21/~ hours against 4 1. of distilled water containing 1 ml. of mercaptoethanol. The dialyzed solution, which contains about 225 mg. of protein, is put on a column 1 inch in diameter containing 5 g. of water-washed DEAE cellulose. Continuous gradient elution of the protein is carried out. The mixing flask contains 500 ml. of a solution of 0.05 M glycylglycine, pH 6.0, in 0.05 M NaC1. The reservoir contains 500 ml. of 0.05 M glycine, pH 9.0, in 0.6M saline. The rate of flow is adjusted to about 1.5 ml./min., and 10-ml. fractions are collected. The fractions from the columns are examined' for fluorescence at 450 m~ with an activation wavelength of 350 mt~. Fractions 34 through 50 generally contain the bulk of the fluorescent protein. It is not necessary to assay the separate fractions for enzymatic activity in preparative runs because of the close correlation between fluorescence and activity. 29,8° mE. S. Maxwell, H. de Robichon-Szulmajster, and tt. M. Kalckar, Arch. Biochem. Biophys. 78, 407 (1958).
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ENZYMES OF THE LELOIR PATHWAY
189
The fluorescent fractions are pooled and concentrated by lyophilization to a volume of 45 ml. Step 6. Precipitation with A m m o n i u m Sulfate. A saturated solution of ammonium sulfate (140 ml.) is added to the concentrated pooled sample. The precipitate is centrifuged and dissolved in 6 ml. of 0.25 M glycylglycine buffer, pH 7.5. The results of the purification procedure are shown in Table IV. Although the degree of purification is about the same as that achieved from calf liver, the specific activity of the enzyme from yeast is about five times as great. TABLE IV PURIFICATION OF UDPGALACTOSE-4-EPIMERASE FROM GALACTOSE-ADAPTED Saccharomycesfragilis
Step and fraction 1. 3. 4. 5. 6.
Crude extract Ammonium sulfate I Ammonium sulfate II DEAE ceUuloseeluate Ammonium sulfate III
Total units, × 10-s
Specific activity, units/mg, protein, × 10-3
22 16 17 23 22
0.5 1.8 7.1 50 75
The purified enzyme can be stored at --20 °, either as a solution in glycylglycine buffer or as the lyophilized powder, for at least 4 months without significant loss of activity. The preparation is sufficiently free of contaminating enzymes to be used, in conjunction with UDP-glucose dehydrogenase and D P N , for the determination of UDP-galactose in crude preparations such as red blood cell homogenates. In this connection it has been used successfully in the assay for congenital galactosemia 31,32 and in a method 12 for the determination of galactose-l-P in red blood cell hemolyzates from galactosemic patients. In these procedures it is preferable to the previously described liver enzyme because of its relative purity and stability. The enzyme has a broad pH optimum between 8 and 9.5. At equilibrium the ratio of U D P G to UDPgal is, as previously reported by Leloir, about 3: 1, The Michaelis constants for U D P G and UDPgal have not been determined accurately, but they appear to be somewhat higher than the values obtained with the liver enzyme. The purified yeast enzyme, unlike the liver enzyme, does not require exogenous D P N , nor is it inhibited by D P N H . The lack of response to added D P N can be explained by the presence of tightly bound D P N in the purified yeast preparationY 9 ~ E. P. Anderson, H. M. Kalckar, K. Kurahashi, and K. J. Isselbacher, J. Lab. Clin. Med. 50, 469 (1957). ~"E. S. Maxwell, It. M. Kalckar, and E. Bynum, J. Lab. Clin. Med. 50, 478 (1957).
ENZYMES OF CARBOHYDRATE METABOLISM
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Properties p H Ef]ect. The curve representing the p H dependence of the reaction shows t h a t the m a x i m u m velocity is reached between p H 8.1 and p H 8.5. At p H 7.5 and at p H 9.2 the reaction velocities are 70% of t h a t at p H 8.5. Specificity. T h e purified fraction was free of inorganic pyrophosphatase. I t contained traces of U D P G and U D P A G pyrophosphorylases. Activator. A divalent metal ion such as M g ++ is required in the reaction. A M g ++ ion concentration of 5 X 10 - 3 M has been used throughout. Dissociation Constant. The Michaelis constant for G D P M has been found to be 1.1 X 10 -5 M at p H 7.5.
[20]
Enzymes
of the Leloir Pathway
I-4
B y ELIZABETH S. MAXWELL, KIYOSHI KURAHASHI, and HERMAN M. t~ALCKAR
I. Q u a n t i t a t i v e D e t e r m i n a t i o n of E n z y m a t i c A c t i v i t y in B r o k e n Cell Preparations The following methods are specific enzymatic assays designed especially for use with crude bacterial extracts. In cases where less rigorous controls are required the assays described later in the sections on purification of enzymes are more suitable. Galactokinase 5-7 Principle. The enzyme catalyzes the reaction A T P + G a l - ~ A D P +a-Gal-l-P. The G a l - I - P produced in a preincubation mixture is quantitatively determined. Fluoride is added in order to protect A T P and G a l - I - P from being dephosphorylated by phosphatase. Addition of magnesium ions serves to ensure t h a t the reaction, which depends on
1L. F. Leloir, Arch. Biochem. Biophys. 33, 186 (1951), see also Vol. I [35]. R. Caputto, L. F. Leloir, R. E. Trucco, C. E. Cardini, and A. J. Paladini, .l. Biol. Chem. 179, 497 (1949). I-I. M. Kalckar, B. Braganca, and A. Munch-Petersen, Nature 172, 1036 (1953). 4A. Muneh-Petersen, H. M. Kalckar, and E. E. B. Smith, Kgl. Da~ske Videnslcab. Selslcab, Biol. Medd. 22, 3 (1955). 5 t:i. W. Kosterlitz, B~ochem. J. 37, 322 (1943). ~R. E. Trucco, R. Caputto, L. F. Leloir, and N. Mittelman, Arch. Biochem. 18, 137 (1948). 'C. E. Cardini and L. F. Leloir, Arch. Biochem. Biophys. 45, 55 (1953).
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ENZYMES OF THE LELOIR PATHWAY
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these ions, is not slowed down by a depletion of magnesium due to the presence of fluoride. The Gal-l-P formed is determined in the proteinfree filtrate by an analytical incubation mixture; the basis for the method rests on the ability of Gal-l-P to interact with UDPG in the presence of the specific enzyme, Gal=l-P uridyl transferase 8,~ (used here as indicator enzyme). The sequence of enzymatic reactions used for the analysis of GaI-1-P is as shown. The absolute molar amount of T P N H Gal-I-P + UDPG ~- G-1-P + UDPGal (catalyzed by Gal-l-P uridyl transferase) G-1-P ~ G-6-P (catalyzed by phosphoglucomutase) G-6=P + T P N --~ 6-Phosphogluconate + T P N H (catalyzed by G-6-P dehydrogenase) formed is a direct measure of Gal-l-P and is thus a measure of galacto= kinase activity. This is provided that the substrates used in the analytical reactions are in considerable excess of the Gal=l-P stemming from the preincubation mixture. The preincubation mixture has the following composition: galactose (recrystallized), 25 ~l. of a stock solution containing 200 micromoles/ml., i.e., 5 micromoles of galactose per milliliter of incubation mixture; ATP (0.1 M), 20/~l. corresponding to 2 micromoles; Tris buffer (1 M, pH 7.5), 100 ~l.; sodium fluoride (0.5M), 50 ~l.; MgC12 (0.1M), 50 ~l.; H20, 655 ~l.; bacterial extract (galactokinase to be determined), 100 ~l, The incubation period is 0, 10, 20, and 30 minutes. The temperature is 37% The reaction is stopped by placing the incubation tube in a boiling-water bath for 90 seconds. After spinning, part of the protein=free clear supernatant fluid is analyzed enzymatically for Gal-l-P. Usually a 100-/A. filtrate is analyzed. The analysis mixture is composed as follows: UDPG, 20 /~l. or 0.2 micromole; G-6-P dehydrogenase,8 10 ~l. of a solution containing 10 mg. of protein per milliliter; TPN, 20 ~l. of a solution containing 20 mg./ml.; cysteine (200 micromoles/ml., pH 8.5), 100 ~l.; phosphoglueomutase9 (1 mg./ml.), 20 ~l.; MgC12 (1 M), 10 ~l.; glucose-l,6-diphosphate (0.5 micromole/ml.), 5 ~l.; glycine (1M, pH 8.7), 100 ~l.; H20, 610 ~l.; fractionated Gal-l-P uridyl transferase from liver 1° or from E. coli K12 mutant 30921~ (see procedures later in this chapter). The amount of filtrate for the assay is 100 FL1.In order to calibrate the system, Gal-l=P SA. Kornberg, J. Biol. Chem. 182, Horecker, Vol. I [42]. 9V. A. Najjar, J. Biol. Chem. 175, 1oK. Kurahashi and E. P. Anderson, 11K. Kurahashi and A. Sugimura, J.
805 (1950); see also A. Kornberg and B. L. 281 (1948); see also Vol. I [36]. Biochim. e~ Biophys. Acta 29, 498 (1958). Biol. Chem. 235, 940 (1960).
176
ENZYMES OF CARBOHYDRATE METABOLISM
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in amounts of 0.05 to 0.075 mieromole is added. For each 0.1 micromole of Gal-l-P per milliliter a density increase of 0.620 at 340 m~ take~ place. Usually the G-6-P dehydrogenase is added first in order to determine small amounts of G-1-P or G-6-P present in the filtrate. Subsequently the Gal-l-P uridyl transferase is added, and the total density increase at 340 ms minus the value obtained with a zero time control of preincubation mixture is a measure of the Gal-l-P which accumulates in the preincubation period and hence of the activity of the galactokinase. The method is highly specific but not highly sensitive. In its present design it can detect activities of 0.1 to 0.2 micromole/hr./mg, of protein. The sensitivity can be increased by analyzing a larger fraction of the preincubation mixture. The latter can also be incubated for longer periods of time (1 and 2 hours). Fluoride has not been found to inhibit galactokinase significantly. Simpler and more-sensitive methods for galactokinase can be devised. However, for projects in which it is essential to demonstrate that the hereditary blocks affect the steps of the Leloir pathway, the specific enzymatic methods are deemed most essential. A new sensitive specific method for Gal-l-P determination1~ has been worked out. In this method human hemolyzate is used as transferase source. The incubation mixture is heat-inactivated and analyzed for consumption of UDPG by means of UDPG dehydrogenase. This method is being modified with the purpose of assaying galactokinase in bacterial extracts. G a l - l - P Uridyl Trans]erase 3, lo, 11 Principle. The enzyme catalyzes the reaction
Gal-l-P ~- UDPG ~ G-1-P -~ UDPGal. The rate of G-1-P formation is in this case analyzed by adding the two indicator enzymes and coenzymes in excess, phosphoglueomutase9 (plus glucose-l,6-diphosphate) and G-6-P dehydrogenase8 plus TPN. The initial rate of formation of T P N H is used as a direct measure for the Gal-l-P uridyl transferase activity of the disrupted bacterial cell. Controls having either Gal-l-P or UDPG omitted from the incubation are always used. Reoxidation rate in the extracts is much slower than that of TPN reduction. Yet, for determination of low transferase activities it is necessary specifically to determine the rate of reoxidation of TPNH. 1~H. N. Kirkman and E. S. Maxwell, J. Lab. Clin. Med. 56, 161 (1960).
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The enzyme activity is determined directly in the Beckman spectrophotometer (i.e., "generation incubation" and "analysis incubation" are combined) by the rate by which T P N is reduced. All constituents, substrates, indicator coenzymes, and indicator enzymes are added in considerable excess over the transferase to be determined. An incubation mixture for the assay of Gal-l-P uridyl transferase has the following composition: cysteine (33 mg./ml., pH 8.5), 30 ~l.; magnesium chloride (0.1 M) 10 ~l.; glycine (1 M, pH 8.7), 60 ~l.; phosphoglucomutase together with glucose-l,6-diphosphate; T P N (20 mg./ml.), 10 ~l.; G-6-P dehydrogenase; U D P G (10 micromoles/ml.), 20 ill.; water, 360 ~l. To this mixture is added the K12 extract in amounts of 50 to 100 ~g. of protein and, as the last component, Gal-l-P (10 micromoles/ml.), 30 ~1. The total volume is 620 td. In order to ensure that the indicator enzymes are in excess it may be necessary to add more G-6-P dehydrogenase or phosphoglucomutase or glucose-l,6-diphosphate and ascertain that the rate of release of G-1-P from U D P G and Gal-l-P is not increased. Control mixtures with UDPG without Gal-l-P, or with Gal-l-P without UDPG, should only give very slow release of G-1-P. The release of G-1-P is measured by the rate of increase in optical density change at 340 m~, with 400 m~ as isobestic point. Another complication which might obscure comparison on a quantitative basis is the fact that 6-phosphogluconate dehydrogenase 13 present in the lysate (or in some preparations of indicator enzyme glucose-6phosphate dehydrogenase) will alter the scale. That is, the activity of transferase will be overestimated because each mole of U D P G exchanged will give a deflection at 340 mt~ which is twice as high. It is therefore necessary always to calibrate the enzyme mixture with a known amount of G-1-P or G-6-P. A direct assay with 6-phosphogluconate is to be recommended too. In most cases the activity of phosphogluconate dehydrogenase is low. Hence the calibration with G-1-P is most important in extracts in which transferase activity is moderate or low. These difficulties are less likely to be encountered if one is dealing with extracts having highly active transferase. If several factors contribute to complicate the quantitative analysis it might be helpful to perform the assay by a two-step procedure. The reaction mixture would in such a case consist of E. coli extract, phosphoglucomutase and glueose-l,6-diphosphate, cysteine, MgC12, Gal-l-P, and UDPG. After a brief incubation (for instance, 5 and 10 minutes) the reaction is stopped and the filtrate is analyzed for G-6-P by means of T P N and G-6-P dehydrogenase (which should be free of transferase). ~3B. L. Horecker and P. Z. Smyrniotis, J. Biol. Chem. 193, 371 (1951); see also Vol. I [42].
178
ENZYMES OF CARBOHYDRATE METABOLISM
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The sensitivity of the direct transferase assay is of the order of magnitude of 0.01 micromole/hr./mg, protien. UD PGal_4.epimerase 1,2 Principle. The enzyme catalyzes the reaction UDPGal ,-~-~UDPG. UDPG formed is determined in a protein-free filtrate by means of a DPN-dependent specific dehydrogenase (UDPG dehydrogenase 14) which catalyzes the reaction U D P G + 2DPN--> Uridine diphosphoglueuronie acid + 2DPNH. The latter is performed in an analytical incubation. The absolute molar amount of D P N H formed divided by 2 provides a direct measure for UDPGal-4-epimerase activity in the disrupted cells. Since the disrupted K12 cells do not contain any enzymes that hydrolyze or oxidize U D P G (or UDPGal), it is possible by this method to determine quantitatively even very low activities of UDPGal-4-epimerase. The two-step procedure is performed as follows: The reaction mixture contains glycine (1 M, pH 8.7), 50 ~1.; E. coli extract, 20 t~l; UDPGa115,~6 (2.2 micromoles/ml.), 30 ~1. in a volume of 0.3 ml. The mixture is incubated for 5 and 10 minutes at room temperature (25°). At termination the mixture is best inactivated by placing the tubes (with a marble on top) in a boiling-water bath for 11/~ minutes. It is then cooled on ice and spun down, and 200 ~l. of the clear supernatant is used for the analytical mixture. The latter contains glycine (1 M, pH 8.7), 70 td.; D P N (25 micromolcs/ml.), 20 #l.; supernatant of the preincubated sample, 200 #l.; water, 15 ~l.; and finally U D P G dehydrogenase, 15 or 20 ~l. The control contains the same mixture except that the supernatant stems from a reaction mixture to which UDPGal was added after boiling. The sensitivity of the epimerase test is of the order of magnitude of 0.005 micromole/hr./mg, of protein. The one-step direct procedure can be used occasionally for crude extracts but preferentially for fractionated UDPGal-4-epimerase preparations free of hydrogen donors and acceptors. The procedure for crude bacterial extracts is executed as follows: The reaction mixture contains glycine (1M, pH 8.7), 50 ~l.; D P N (25 micromoles/ml.), 20 /~l.; UDPGal (2.2 micromoles/ml.), 30 ~l. ;. water, 475 td.; U D P G dehydrogenase, 25 #l. ; and E. coli extract, 5 or 10 ~I. On the addition of extract the rate of increase of optical density at 340 mfi is recorded (the isobestie region at 400 to 410 m# is used to check for extraneous optical ~4j. L. Strominger, E. S. Maxwell, J. Axelrod, and It. M. Kalckar, J. Biol. Chem. 224, 79 (1957); see also Vol. I I I [143A]. 1~E. P. Anderson and E. S. Maxwell. J. Am. Chem. Soc. 81, 6514 (1959). t~ff. G. Moffatt, YI. G. Khorana, and H. M. Kalckar, unpublished work (1958).
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ENZYMES OF THE LELOIR PATHWAY
179
density changes at reasonable intervals). I t is i m p o r t a n t t h a t U D P G dehydrogenase is in excess. This can be ascertained by lowering the amount of E. coli extract to see if the rate decreases proportionally. The control tube contains the same ingredients with the exception of U D P G a l . Reoxidation of D P N H interferes, and a direct assay with D P N H as substrate should be performed. Each millimicromole of U D P G a l converted to U D P G corresponds to an optical density change of 0.020 at 340 m~. The sensitivity of the direct epimerase test is as high as t h a t of the indirect. I I . Purification of G a l a c t o s e - l - p h o s p h a t e Uridyl Transferase U D P G ~ G a l - l - P ¢~-UDPGal ~ G-1-P This enzyme was first found in galactose-adapted yeast2 The enzyme from calf liver and from an Escherichia coli galactokinase-less m u t a n t has been partially purified. 1°,11
Assay Method Principle. G a l - l - P uridyl transferase activity is measured from T P N H formation in a coupled reaction with phosphoglucomutase and G - 6 - P dehydrogenase. Reagents 0.01 M U D P G , sodium salt. 0.0175 M G a l - l - P , sodium salt. 1.0 M glycine buffer, p H 8.75 when it is diluted to 0.1 M. 0.16 M cysteine hydrochloride, p H 8.5. 1.0 M MgC12. 0.02 M T P N . 0.0002 M G - l , 6-P27 Phosphoglucomutase, 70 units/ml2 s in 1% bovine albumin solution. Prepared according to the method of N a j j a r 2 ~7Some of the phosphoglucomutase preparations contain enough G-1,6-P, especially when freshly prepared. However, since Gal-l-P was found to be inhibitory for the phosphoglucomutase reaction [V. Ginsburg and E. F. Neufeld, Abstr. Papers, 132nd Meeting Am. Chem. Soc., New York 27C (1957); and J. B. Sidbury, ibid., 27C (1957)], G-1,6-P and phosphoglucomutase are used routinely in excess. It is also necessary to avoid prolonged contact of phosphoglucomutase with Gal-l-P. 18One unit of G-6-P dehydrogenase is defined as that amount of enzyme which causes the reduction of 1 micromole of TPN per minute under the assay condition used for Gal-l-P uridyl transferase except that 0.4 micromole of G-6-P is used as substrate. One unit of phosphoglucomutase is similarly defined as that amount of enzyme which causes the reduction of 1 micromole of TPN per minute when 0.4 micromole of G-1-P (free of G-1,6-P) is used as substrate.
180
ENZYMES OF CARBOHYDRATE METABOLISM
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G-6-P dehydrogenase, 14 units/ml? s in 1% bovine albumin solution. Prepared according to the method of Kornberg and Horecker. 8 The gel adsorption and acid precipitation steps in their method are repeated twice, in order to remove 6-phosphogluconate dehydrogenase and Gal-l-P uridyl transferase. Gal-I-P uridyl transferase. Diluted with 1% bovine albumin to give an optical density change at 340 m~ between 0.03 and 0.05 per minute under the assay conditions described below.
Procedure. To a 1-ml. quartz cell having a light path of 1 cm. are added 425 ~l. of water, 60 ~l. of glycine buffer, 30 ~l. of cysteine hydrochloride, 5 ~l. of MgCl~, 20 t~l. of TPN, 25 ~l. of UDPG, l0 ~l. of G-1,6-P, 10 ~l. of phosphoglucomutase, 5 t~l. of G-6-P dehydrogenase, and 10 tL1. of diluted Gal-l-P uridyl transferase. Readings are taken at 340 mt~ at 1-minute intervals after addition of 20 ~1. of Gal-l-P in a Beckman DU spectrophotometer with a pinhole attachment. At the beginning of the reaction there is always a short lag period, and then the reaction proceeds linearly with time. The linear part of the curve is utilized for the calculation of the rate of the reaction. It is important to set up control ceils without Gal-l-P uridyl transferase in order to check the presence of Gal-l-P uridyl transferase in G-6-P dehydrogenase and the presence of G-1-P in Gal-l-P. It is also advisable to set up a control cell without Gal-l-P, when crude enzyme preparations are utilized. Definition of Unit and Specific Activity. A unit of enzyme is defined as that amount which causes the incorporation of 1 micromole of Gal-l-P into the uridine nucleotide per minute, and thus causes the formation of 1 micromole of T P N H per minute in the assay. Purification Procedure from Calf Liver Calf liver acetone powder is prepared as described by Horecker. 19 All the following steps are carried out at 2 ° . Step I. Preparation of Crude Extract. One hundred grams of acetone powder is extracted with 200 1111.of potassium phosphate buffer (0.002 M, pH 7.0) for 30 minutes. The residue is removed by centrifugation at 20,000 )K g for 10 minutes. Step 2. Protamine Sul]ate Precipitation. To the crude extract, 1/100 vol. of 0.5 M potassium phosphate (pH 6.0 when diluted to 0.05 M) and 1/100 vol. of protamine sulfate (20 mg./ml.) are added. After 5 minutes of stirring, the precipitate is collected by centrifugation at 20,000 X g for 5 minutes and discarded. 19For the preparation of acetone powder, see Vol. I I [123].
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ENZYMES OF THE LELOIR PATHWAY
Step 3. Fractionation with Ammonium Sulfate. To the supernatant solution enough cold ammonium sulfate solution (saturated at 2 °, pH 6.0) is added to give 42.5% saturation. After 10 minutes of stirring, the precipitate is removed by centrifugation at 20,000 X g for 5 minutes. To the supernatant solution ammonium sulfate solution is again added to give 52.5% saturation. After 10 minutes of stirring, the precipitate is collected by centrifugation and dissolved in 65 ml. of cold 0.9% NaC1 solution. This fraction is dialyzed overnight at 2 ° against 15 1. of 0.005 M potassium phosphate (pH 6.0). Step 4. Fractionation with Calcium Phosphate Gel. After removal of precipitate formed during dialysis by centrifugation, the dialyzed fraction is diluted to give a protein concentration of 10 mg./ml, by addition of 0.005M potassium phosphate (pH 6.0). Then calcium phosphate gel 2° (17.9 mg. dry weight per milliliter) equivalent to 1 mg. in dry weight per milligram of protein is added. After 15 minutes of stirring, the gel is removed by centrifugation and discarded. To the supernatant solution the same amount of gel is again added. After 15 minutes of stirring, the gel is collected by centrifugation and the supernatant solution is discarded. The gel is eluted by stirring for 15 minutes with 0.01 M potassium phosphate (pH 7.4, one-third of the volume of the discarded supernatant solution). The residue is removed by centrifugation and discarded. Step 5. Lyophilization. After addition of 1/20 vol. of reduced glutathione (15 mg./ml.), the eluate is lyophilized. A summary of the procedure is given in Table I. TABLE I SUMMARY OF PURIFICATION PROCESS FROM CALF LIVER
Step and fraction 1. 2. 3. 4.
Crude extract Protamine sulfate, supernatant (NH4)2S04fraction, 0.425-0.525 Ca3(PO4):gel eluate
Total T o t a l T o t a l Specific volume, protein, activity, activity, Yield, ml. nag. units units/mg. % 1670 1760 82 175
17,400 11,900 2,420 116
225 182 99 74
0. 013 0. 015 0. 041 0.64
100 81 44 33
Purification Procedure from Eschericl~ia colt
E. colt K12 W3092 (galactokinase-less) mutant21, 22 was obtained from Drs. E. M. and J. Lederberg at Stanford University. The cells are S°For the preparation, see Vol. I [12]. .~1M. L. Morse, E. M. Lederberg and J. Lederbcrg, Genetics 41, 143, 758 (1956). ~':K. Kurahashi, Science 125, 114 (1957).
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ENZYMES OF CARBOHYDRATE METABOLISM
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grown at 37 ° with aeration by rotatory shaking in a medium of the following composition: 10 g. of casamino acid (Difco), 5 g. of yeast extract (Difco, 3 g. of K~PO,, l g. of KH2P04, 5 g. of D-galactose, and 1 g. of D-glucose per liter. A loopful of bacteria from a 24-hour culture of E. coli at 37 ° on a nutrient agar slant (Difco) is used to inoculate 50 ml. of medium. After 8 to 10 hours, the entire culture is transferred into 1 1. of fresh medium, and incubation is continued for 16 hours. Step 1. Preparation of Crude Extract. The cells are harvested by centrifugation at 6000 X g for 10 minutes and washed with 200 ml. of cold water (yield: 4 to 5 g. of packed cells per liter). For each gram of packed cells 4 ml. of potassium phosphate (0.04M, pH 7.4) is added, and the cells are disintegrated in a sonic disintegrator (Raytheon 10 kc) for 10 minutes. The broken cell residues are removed by centrifugation at 20,000 X g for 10 minutes. Step 2. Incubation at 37 ° and Heat Denaturation. The crude extract is adjusted to contain 20 mg./ml, of protein (determined by the biuret method 23) and 100 micromoles of potassium phosphate per milliliter by the addition of enough water and 0.5 M potassium phosphate (pH 7.4). Sixty milliliters of this extract is worked up at one time for the following purification. The diluted solution is incubated at 37 ° for 2 hours to enhance the breakdown of nucleic acid ~4 (this fraction can be stored at --10 ° for several days without much loss of activity). After cooling it is placed in a hot-water bath at 65 ° to 70 °, and the temperature is brought to 53 ° rapidly (1.5 to 2 minutes). The solution is maintained at 53 ° for 11 minutes and then chilled in an ice bath. All the following steps are carried out at 2 ° . Step 3. Fractionation with Ammonium SuI]ate. To the heat-denatured solution ammonium sulfate solution (pH 7.0, saturated at 2 ° and containing 0.006 M Versene) is added to give 45% saturation of ammonium sulfate. After stirring for 10 minutes, the precipitate is removed by centrifugation at 20,000 X g for 5 minutes. To the supernatant solution ammonium sulfate solution is added to give 67% saturation. After 10 minutes of stirring, the precipitate is collected by centrifugation. The supernatant solution is removed completely by inverting the centrifuge tube for 5 minutes, because excess ammonium sulfate interferes with the effectiveness of the following gel step. The precipitate is dissolved in 10 ml. of water, and the protein concentration is determined by the biuret method. 23 Step 4. Calcium Phosphate Gel Adsorption o] Inactive Protein. The '3It. W. Robinson and C. G. ttogden, J. Biol. Chem. 13~, 707 (1940). 2*Personal communication from Dr. P. Berg of Stanford University (1958).
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183
solution is adjusted to contain 7.5 mg. of protein and 50 micromoles of potassium phosphate per milliliter by the addition of enough water and potassium phosphate (0.5 M, pH 5.8 when diluted to 0.05M). Then calcium phosphate gel2° (21.9 mg. dry weight per milliliter) equivalent to 1 mg. in dry weight per milligram of protein is added. After 10 minutes of stirring, the gel is removed by centrifugation and discarded. Step 5. Removal o] Residual Nucleic Acid by Nuchar. After the protein concentration has been determined spectrophotometrically,2~ the supernatant solution is treated with Nuchar (Grade XXX), 1 mg./mg. of protein, and stirred for 4 minutes with a stirring rod. Then another equivalent amount of Nuchar is added and stirred for another 4 minutes. The charcoal is removed by centrifugation and discarded. Step 6. Treatment with Alumina C-f Gel. After determination of the protein concentration, alumina C~ gel2° (12.2 mg. dry weight per milliliter) is added to the supernatant solution, 0.3 mg. dry weight per milligram of protein. After 10 minutes of stirring, the gel is removed by centrifugation and discarded. Step 7. Fractionation with Ammonium Sulfate. The supernatant solution is refractionated with saturated ammonium solution. The precipitate between 50% and 60% saturation is collected and dissolved in 4 ml. of water. The fraction is dialyzed for 5 hours at 2 ° against 0.005 M potassium phosphate (pH 7.4). When this fraction is stored overnight in an ice bath, about a quarter of the activity is lost. Step 8. DEAE-Column ChromatographyY 6'~7 The column (1.1 cm. in diameter and 6 cm. in height) is prepared by pouring 600 mg. of DEAE-cellulose (Eastman Kodak Company) suspended in 20 ml. of 1 M NaC1 solution and allowing it to pack by gravity. The column is washed successively with 100 ml. of 1 M NaC1, 40 ml. of water, 100 ml. of 0.5 M potassium phosphate (pH 7.4), 40 ml. of water, and 60 ml. of 0.005 M potassium phosphate (pH 7.4). After addition of the dialyzed enzyme solution, the column is washed through with 20 ml. of 0.005 M potassium phosphate (pH 7.4) and 60 ml. of 0.005 M potassium phosphate (pH 7.4) which contains 0.05 M NaCl. Then active enzyme fractions are eluted from the column by 0.005 M potassium phosphate (pH 7.4) which contains 0.12 M NaC1. Two-milliliter fractions are collected. Ordinarily the bulk of the enzymatic activity is recovered in fractions 3to7. Step 9. Lyophilization. The most active fractions are combined and 2~See Vol. I I I [73]. ~6E. A. Peterson and R. A. Sober, J. Am. Chem. Soc. 78, 751 (1956). 'TH. A. Sober, F. J. Gutter, M. M. Wyckoff, and E. A. Peterson, J. Am. Chem. Soc. 78, 756 (1956).
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ENZYMES OF CARBOHYDRATE METABOLISM
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dialyzed for 1 hour against 0.005 M potassium phosphate ( p H 7.4) a t 2 °. The dialyzed fraction is then lyophilized after addition of ~ o vol. of reduced glutathione (15 mg./ml.) and an equal volume of 0.25 M glycylglycine ( p H 7.4). The a m o u n t of enzyme protein in this lyophilized powder is determined b y the method of L o w r y et al. after precipitation of the enzyme protein by trichloroacetic acid. 2~ A s u m m a r y of the purification procedure is given in T a b l e I I . TABLE II PROCESS FROM E. coli
SUMMARY OF PURIFICATION
Step and fraction 1. 2. 3. 4. 5. 6. 7. 8.
Crude extract Incubation at 37 ° (NH4)2SO4fraction, 0.45-0.67 Ca3(PO4)2gel supernatant Nuchar supernatant Alumina C~ gel supernatant (NH4)~SO4fraction 0.5-0.6 DEAE chromatography Fraction 3 Fraction 4 Fraction 5 Fraction 6
Total Total Total Specific volume, protein, activity, activity, Yield, ml. mg. units units/rag. % 54.3 60.0 10.6 37 33 33 4.4 2.0 2.0 2.0 2.0
1275 1200 222 144 121 94 40 2.2 i. 4 1.0 0.7
347 366 301 288 248 231 169 44 24 11 8
0.27 0.30 1.4 2.0 2.1 2.5 4.2 20 16 11 11
100 105 87 83 71 66 49 13 7 3 2
Properties 10,n
Stability. The lyophilized enzyme preparations can be stored at - - 1 0 ° in a desiccator for several months without loss of activity. The enzyme solution prepared by dissolving the powder in 1% bovine albumin solution can be stored at - - 1 0 ° for a week with gradual loss of activity. Presence of Other Enzymes. The calf liver enzyme preparation is contaminated by U D P G pyrophosphorylase and phosphoglucomutase, and to a much smaller extent by U D P G a l - 4 - e p i m e r a s e and galactokinase. I t is free of U D P G dehydrogenase. The E. coli enzyme preparation is free of all the above enzymes. p t t Optimum. Both enzyme preparations have their p H optima between 8.5 and 8.9. Activators and Inhibitors. The enzyme activities of both p r e p a r a tions are partially protected by reduced glutathione. Calf liver enzyme is inhibited approximately 50% by p-chloromercuribenzoate at a concentration of 2 X 10-~ M. E. coli enzyme shows an absolute requirement
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ENZYMES OF THE LELOIR PATHWAY
185
of cysteine for its activity. Mg ÷÷ ions and NaF are found slightly inhibitory for E. coli enzyme. K,~ Values. The K~ values of E. coli enzyme for UDPG, Gal-l-P, UDPGal, and G-1-P are estimated to be 1.5 X 10 ~, 4.1 X 10-4, 2.5 X 10-', and 2.4 X 10-*, respectively. III. Purification of UDPgalactose-4-epimerase UDPgal ~- UDPG Assay Method Principle. UDPgalactose-4-epimerase 1,2,-~,~9 is assayed spectrophotometrically by the rate of increase in optical density at 340 m~ when UDPgal is present as the substrate, and the reaction is coupled with an excess of UDPG dehydrogenase and DPN. UDPgal ~ UDPG UDPG + 2DPN -~ UDPGA + 2DPNH + 2H + Reagents 0.005 M UDPgal (prepared either enzymatically from UDPG and Gal-l-P 1~ or chemically by the method of Moffat et al2G). 0.05 M DPN. 1.0 M glycine buffer adjusted to pH 8.8 with NaOH. UDPG dehydrogenase, approximately 104 units/ml., carried through step 5 of the method of Strominger et al. 14 Procedure. The following reagents are added to a silica cuvette with 1.3-ml. capacity and a light path of 1 cm.: water to a final volume of 0.5 ml., 50 ~l. of glycine buffer, 10 /tl. each of UDPgal and DPN, and 20 ~l. of UDPG dehydrogenase. Optical density readings are taken at 340 m~ in a Beckman DU spectrophotometer equipped with a pinhole attachment so that 0.5-ml. volumes may be used in 1.3-ml. cuvettes. Because of small amounts of UDPG which may be present in the enzymatically prepared UDPgal, a small increase in optical density may be observed at this stage. When no further increase occurs, usually within 5 minutes, an aliquot of UDPgalactose-4-epimerase is added and the rate of increase in optical density is followed for 4 minutes. During the early stages of purification it is desirable to include a second cuvette with all the reagents except UDPgal. The rate of the reaction is proportional to enzyme concentration up to an increase in optical density of :SE. S. Maxwell, J. Biol. Chem. 229, 139 (1957). -'~E. S. Maxwell and H. de Ribichon-Szulmajster, J. Biol. Chem. 235, 308 (1960).
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ENZYMES OF CARBOHYDRATE METABOLISM
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about 0.050 per minute. A unit of activity is defined as an increase in optical density at 340 m s of 0.001 per minute under the conditions described. Purification from Calf Liver Acetone Powder ~8 Acetone powder from liver of freshly slaughtered calves is prepared according to Horecker. 1~ The enzyme is stable in the dry powder stored at --10 ° for at least a month. The purification procedure is carried out at 0 ° to 5 °, except as otherwise noted. Step 1. Water Extract o] Acetone Powder. Fifty grams of acetone powder is extracted with 1000 ml. of water by light grinding in a mortar, followed by stirring for 30 minutes. The suspension is centrifuged, and the residue discarded. Step 2. Fractionation with Acetone (0 to ~5%). The water extract (910 ml.) is adjusted to pH 5.4 with 1 M acetic acid. The precipitate is centrifuged immediately and discarded. The solution is cooled to 0 °, and 295 ml. of acetone (--15 °) is added dropwise over a period of 30 minutes, the solution being stirred continuously during the addition, and the temperature kept between 0 ° and --2 °. The precipitate is centrifuged and dissolved in 300 ml. of 0.1 M glyeine buffer, pH 8.0. Step 3. Precipitation with Alkaline Ammonium Sulfate (between ~5 and 65% Saturated). To 300 ml. of the dissolved precipitate from step 2 is added 245 ml. of alkaline ammonium sulfate (pit 8.0, when diluted fivefold; prepared by adding 3.7 ml. of concentrated NH40H to 1 1. of saturated ammonium sulfate). The precipitate is centrifuged and discarded. Then 315 ml. of alkaline ammonium sulfate is added to the supernatant fraction. The precipitate is centrifuged and dissolved in 80 ml. of distilled water; the solution can be stored at --20 ° overnight. Step ~. Precipitation at pH ~.6. To 80 ml. of the solution from step 3 is added 80 ml. of 0.5 M acetate buffer, pH 4.6, in 50% saturated ammonium sulfate (prepared by adding 50 ml. of 1 M sodium acetate buffer, pH 4.6, to 50 ml. of saturated ammonium sulfate). After standing for 10 minutes, the precipitate is centrifuged and dissolved in 80 ml. of 0.05 M glycine, pH 8.7. Step 5. Adsorption and Elution from Calcium Phosphate Gel. The solution from step 4 is diluted to 160 ml. with distilled water, and 65 ml. of calcium phosphate gel (17.9 mg./ml.) is added to the diluted solution. The suspension is stirred for 10 minutes and centrifuged. The supernatant solution is discarded, and the gel is suspended in 100 ml. of 0.1 M phosphate buffer, pit 7.5. After being stirred for 10 minutes, the gel is centrifuged and discarded. Step 6. Precipitation with Ammonium Sulfate (between ~5 and 65%
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ENZYMES OF THE LELOIR PATHWAY
187
Saturated). Twenty-six grams ammonium sulfate is added to the clear solution from step 5, and the precipitate is centrifuged and discarded. Then 14 g. of ammonium sulfate is added to the supernatant solution, and the precipitate is centrifuged and dissolved in 20 ml. of 0.25 M glycylglycine buffer, pH 7.5. The solution is lyophilized and stored at --20 ° . The results of the purification procedure are shown in Table III. TABLE III PURIFICATION OF UDPGALACTOSE-4-EPIMERASE FROM CALF LIVER ACETONE POWDER
Step 1 2 3 4 5 6
Total activity, units, X 10-3 840 1000 900 560 650 520
Units/mg, protein ~ 77 410 1,200 2,800 6,880 16,200
Protein is determined by the method of Lowry et al., Vol. III [73]. The purified enzyme can be kept frozen in 0 . 2 5 M glycylglycine buffer at p H 7.5 for about a week without significant loss of activity. The lyophilized preparation stored at --20 ° is stable for at least 3 months. The pH optimum lies between 8.7 and 9.5. At pH 7.0 the rate is about 3% of that at p H 9.0. The purified enzyme has an absolute requirement for catalytic amounts of D P N and is inhibited by D P N H . The Michaelis constants are 2 X 10-7 M for D P N and 5 X 10-' M and 9 X 10 -5 M, respectively, for U D P g a l and U D P G . At equilibrium the ratio of U D P G to UDPgal is about 3: 1. Purification from Galactose-Orown Yeast 29
Growth o] Yeast. Saccharomyces ]ragilis (American T y p e Culture Collection No. 10022) is adapted to galactose by growth in a medium containing 15 g. of yeast extract (Difco), 1.8 g. of (NH4):SO.,, 1.5 g. of KH_~PO4, and 75 g. of galactose in a final volume of 1500 ml. of medium. About 100 ml. of a 24-hour culture of adapted cells is inoculated into each of twelve 6-I. flasks containing 1500 ml. of the same medium. The cultures are incubated at room temperature with shaking for 24 hours. Cells are harvested in a Sharples centrifuge. Step I. Preparation o] Cell-Free Extract. The wet cell mass (about
188
ENZYMES OF CARBOHYDRATE METABOLISM
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310 g.) is suspended in a final volume of 1 I. of 0.1 M glycine, pH 6.5. Ceils are broken by a 30-second exposure in a Nossal disintegrator. Nine milliliters of the cell suspension and 8 g. of acid-washed glass beads (Minnesota Mining and Mfg. Co., St. Paul, Minnesota, No. 150 [75 t~]) are used in each Nossal tube. This procedure is carried out in a --10 ° room, and the cell suspension is kept just above freezing. All subsequent procedures are carried out at 3 ° to 5 ° except where otherwise noted. The broken-cell suspension is centrifuged at high speed in a Lourdes Model SL centrifuge. The yield of cell-free supernatant fluid is about 590 ml. Step 2. Removal of Nucleic Acid. Nucleic acid is removed by autolytie digestion as suggested by P. Berg and W. E. Razzell. To the cellfree extract is added 60 ml. of 1 M potassium phosphate buffer, pH 7.0. The preparation is incubated at 37 ° for 2 hours. The preparation may be frozen and stored overnight at this stage.
Step 3. Precipitation with Ammonium Sulfate (0 to 50% Saturated). The solution is made 50% saturated by the addition of 175 g. of ammonium sulfate to 600 ml. of solution. The precipitate is centrifuged, dissolved in distilled water, and made up to a final volume of 100 ml.
Step 4. Precipitation with Ammonium Sulfate (35 to 50% Saturated). The concentration of ammonium sulfate in the dissolved precipitate is determined with a Barnstead purity meter (Barnstead Still and Sterilizer Company), and the solution is usually found to be about 9% saturated. To the solution is added 13.9 g. of ammonium sulfate. The precipitate is centrifuged and discarded. An additional 8.7 g. of ammonium sulfate is added to the supernatant fluid. The precipitate is centrifuged and dissolved in 15 ml. of distilled water. Step 5. Chromatography on DEAE Cellulose. The solution is dialyzed for 21/~ hours against 4 1. of distilled water containing 1 ml. of mercaptoethanol. The dialyzed solution, which contains about 225 mg. of protein, is put on a column 1 inch in diameter containing 5 g. of water-washed DEAE cellulose. Continuous gradient elution of the protein is carried out. The mixing flask contains 500 ml. of a solution of 0.05 M glycylglycine, pH 6.0, in 0.05 M NaC1. The reservoir contains 500 ml. of 0.05 M glycine, pH 9.0, in 0.6M saline. The rate of flow is adjusted to about 1.5 ml./min., and 10-ml. fractions are collected. The fractions from the columns are examined' for fluorescence at 450 m~ with an activation wavelength of 350 mt~. Fractions 34 through 50 generally contain the bulk of the fluorescent protein. It is not necessary to assay the separate fractions for enzymatic activity in preparative runs because of the close correlation between fluorescence and activity. 29,8° mE. S. Maxwell, H. de Robichon-Szulmajster, and tt. M. Kalckar, Arch. Biochem. Biophys. 78, 407 (1958).
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ENZYMES OF THE LELOIR PATHWAY
189
The fluorescent fractions are pooled and concentrated by lyophilization to a volume of 45 ml. Step 6. Precipitation with A m m o n i u m Sulfate. A saturated solution of ammonium sulfate (140 ml.) is added to the concentrated pooled sample. The precipitate is centrifuged and dissolved in 6 ml. of 0.25 M glycylglycine buffer, pH 7.5. The results of the purification procedure are shown in Table IV. Although the degree of purification is about the same as that achieved from calf liver, the specific activity of the enzyme from yeast is about five times as great. TABLE IV PURIFICATION OF UDPGALACTOSE-4-EPIMERASE FROM GALACTOSE-ADAPTED Saccharomycesfragilis
Step and fraction 1. 3. 4. 5. 6.
Crude extract Ammonium sulfate I Ammonium sulfate II DEAE ceUuloseeluate Ammonium sulfate III
Total units, × 10-s
Specific activity, units/mg, protein, × 10-3
22 16 17 23 22
0.5 1.8 7.1 50 75
The purified enzyme can be stored at --20 °, either as a solution in glycylglycine buffer or as the lyophilized powder, for at least 4 months without significant loss of activity. The preparation is sufficiently free of contaminating enzymes to be used, in conjunction with UDP-glucose dehydrogenase and D P N , for the determination of UDP-galactose in crude preparations such as red blood cell homogenates. In this connection it has been used successfully in the assay for congenital galactosemia 31,32 and in a method 12 for the determination of galactose-l-P in red blood cell hemolyzates from galactosemic patients. In these procedures it is preferable to the previously described liver enzyme because of its relative purity and stability. The enzyme has a broad pH optimum between 8 and 9.5. At equilibrium the ratio of U D P G to UDPgal is, as previously reported by Leloir, about 3: 1, The Michaelis constants for U D P G and UDPgal have not been determined accurately, but they appear to be somewhat higher than the values obtained with the liver enzyme. The purified yeast enzyme, unlike the liver enzyme, does not require exogenous D P N , nor is it inhibited by D P N H . The lack of response to added D P N can be explained by the presence of tightly bound D P N in the purified yeast preparationY 9 ~ E. P. Anderson, H. M. Kalckar, K. Kurahashi, and K. J. Isselbacher, J. Lab. Clin. Med. 50, 469 (1957). ~"E. S. Maxwell, It. M. Kalckar, and E. Bynum, J. Lab. Clin. Med. 50, 478 (1957).