THE AMYLASES OF PSEUDOMONAS SACCHAROPHILA

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1 THE AMYLASES OF PSEUDOMONAS SACCHAROPHILA PHILIP S. THAYER' Department of Bacteriology, Univer8ity of California, Berkeley, California Received for publication June 15, 1953 In his original study of Pseudomonas saccharophila, Doudoroff (1940) found that an inducible extracellular amylase was formed when cells were grown with soluble starch or maltose. He also observed indications of intracellular amylase activity. The present study was undertaken in order to characterize further the amylase(s) of P. saccharophila. Two enzymes with different physical and enzymatic properties have been found. MATERIALS AND METHODS The strain of P. saccharophila used has been maintained in this laboratory for a number of years. The medium used for growing the organism contains 0.10 per cent NH4Cl, 0.05 per cent MgSO4 7H20, per cent ferric ammonium citrate, per cent CaCl2, and 0.2 per cent carbon source, all in M phosphate buffer, ph 6.8. Cultures were grown at 30 C with shaking. The potato starch used for enzyme adsorption was Baker's (powdered, for iodometry), and the soluble starch was Pfanstiehl (reagent grade). Dr. W. Z. Hassid kindly furnished generous samples of purified potato amylose and potato amylopectin. Amylase activity has been determined routinely by iodimetric assay, using 0.04 per cent soluble starch as substrate. In some cases, amylose and amylopectin have been used as substrates. Digestion mixtures are prepared by adding a suitable volume of enzyme solution to 5 ml of 0.1 M phosphate, ph 5.4, with water to make 10 ml total volume. To start the reaction, 0.2 ml of 2 per cent soluble starch (boiled and filtered) is added. Samples are removed after various periods of incubation at 30 C. When whole cells are being assayed, the reaction mix ture is shaken mechanically. For each analysis, a 2 ml aliquot is added to 1 ml of I2-KI solution and diluted to 10 ml. (The iodine solution used contains 0.2 per cent I2 and 0.3 per cent KI.) Optical density of the assay solution is measured in the Klett colorimeter using filter 54 (green) or, for reasons presented below, filters 54 and 66 (red). The decrease in optical density is nearly linear as a function of time until a value about one-half the original is reached and is proportional to enzyme concentration. The amount of enzyme used is one which will produce an optical density difference of not more than 100 units between the zero-time and 10 minute samples. One enzyme unit is defined as the amount of enzyme which causes the loss of 100 optical density units in 30 minutes as measured with the green filter on samples taken according to the above procedure. In practice, samples have been taken at several times during hydrolysis, and the activity calculated from the one which shows a decrease of 20 to 100 units in optical density. Analysis of the products of starch hydrolysis has been performed in three ways: (1) by determination of reducing groups released; (2) by determination of the decreases in reducing power after successive fermentations of digests with glucose adapted Torula monosa and maltose adapted Saccharomyces cerevisiae (Doudoroff et al., 1949); and (3) by paper chromatography. Reducing sugar values have been determined by a modification of the method of Schales and Schales (1949), using 0.5 ml of undiluted ferricyanide reagent and 5 ml of sample containing 25 to 150 micrograms of reducing sugar. After 15 minutes in boiling water, the decrease in optical density from the reagent blank is measured in the Klett colorimeter with blue filter 42. Reproducibility of glucose and maltose standards is I Merck Postdoctoral Research Fellow in the Natural Sciences, Present address: Department of Chemistry, University of California, is expressed as maltose. Thus, in digests where excellent. The reducing sugar content of digests Los Angeles. appreciable amounts of glucose are present there 656

2 O.:a"1 ': :,::,.k, 1953] AMYLASES OF PSEUDOMONAS SACCHAROPHILA 657 may be more than 100 per cent conversion of same buffer and a magnetic stirring rod. The starch to "maltose". side arm of the flask is made of 8 or 10 mm pyrex Paper chromatography has been used for the tubing. The lower end of the column is drawn separation and identification of carbohydrates. out to facilitate collection of eluate fractions in Chromatograms were run on Whatman no. 1 test tubes placed inside the suction flask. To start paper by the descending technique, using a elution, the stopcock of the separatory funnel is solvent of n-butanol, pyridine, and water opened, and vacuum applied. Each fraction of (Jeanes et al., 1951). Each chromatogram was the eluate is tested with iodine immediately, and developed twice to obtain better separation of elution terminated when intact starch is found mono- and disaccharides. Carbohydrate spots on to be coming off the bottom of the column. If the dried chromatograms were detected by spraying with saturated p-anisidine hydrochloride in starch digest is used, each fraction is assayed n-butanol and heating at 100 to 150 C (Hough et al., 1950). Protein determinations have been made photometrically using the Beckman Model DU Quartz spectrophotometer. The E20/E260 ratio has been used to measure contamination with nucleic acids, and the formula given by Kalckar (1947) has -A been employed for determination of piotein concentration. Specific enzymatic activity is expressed as units of enzyme per mg of protein. i SX fw S For the preparation of cell-free extracts, cells have been disrupted by grinding with levigated W : alumina (Norton) by the procedure of Hayaishi F: t..x. r and Stanier (1951) or by sonic disintegration in the Raytheon 9 kc magneto-striction oscillator. h \\ For the lattei, washed cells are suspended to 5 to 10 per cent wet weight, and 20 to 25 ml aliquots ff treated in the oscillator for 15 minutes. Both j Z alumina and sonic extracts are made with phosphate buffer, ph 7. The supernatant after centrif- r >...,.>s..s ugation of the homogenate for 20 minutes at approximately 20,000 G is designated a "crude extract". Experiments described below led to the development of the following procedure for purification of the extracellulai enzyme by adsorption and elution. As adsorbant, a 4:1 mixture of Figure 1. Apparatus for continuous gradient potato starch and celite is packed dry in an 8 to elution of adsorbed enzyme from starch column. 12 mm tube to a height of 40 to 60 mm and washed with buffer (ph 6 oir 7) and water. The column, fitted with a rubber stopper, is put on a suction flask, and cell-free medium run through. Most adsorptions have been performed at 4 C to minimize digestion of the potato starch. When adsorption is completed, the column is washed with cold buffer and set up on the "gradient elution" apparatus shown in figure 1. The separatoiy funnel contains 50 ml of a 0.5 or 1.0 per cent solution of soluble starch (oir equivalent digest), in 0.01 M phosphate, ph 6. The 125 ml Erlenmeyer flask contains 20 to 25 ml of the semiquantitatively to locate the peak. To one (diop of enzyme (in a spot-plate) is added one drop of 0.1 per cent soluble starch in 0.1 M phosphate, ph 5.4. After 30 seconds to two minutes' incubation, one drop of iodine reagent is added. Relative activities of the fractions are indicated by differences in the loss of iodine staining capacity. RESULTS Intact washed cells from starch or maltose medium show amylase activity. The activity of

3 658 PHILIP S. THAYER [VOL. 66 staichl giown cells is usually several times higher than that of maltose grown cells (Doudoroff, 1940). It has been found, however, that this activity is not due to intracellular enzyme but to extr acellular enzyme associated with the cells. The higher activity of starch "cells" is produced by enzyme adsorbed onto a precipitate which is formed during partial degradation of starch and is difficult to separate from the cells. A demonstration of true intracellular amylase activity depends, therefore, on preparation of cell-free extracts with removal of contaminating extracellular enzyme. When this is done, amylase activity is found, differing in several respects from the activity exhibited by the extracellular enzyme. The differences between these two enzymes will be presented in the subsequent sections. Purification of the extracellular amylase. The extracellular amylase produced by P. saccharophila is adsorbed by (insoluble) potato starch as are some other amylases (e.g., Schwimmer and Balls, 1949). It is not released to any extent by treatment with buffers of different ph or with calcium salt solutions. It has been found, however, that adsorbed enzyme can be eluted by suspending the potato starch in a solution of soluble starch (0.5 to 1.0 per cent). After centrifugation, the enzyme is found in the supernatant and soon digests the soluble substrate. One digestion product, an insoluble dextrin (to be described below), may be removed by centrifugation, and the soluble products, by dialysis. Adsoibed enzyme is also eluted by the soluble partial breakdown products formed from soluble starch by the action of the enzyme. Maltose is inactive in elution so that the active components of the digest appear to be oligosaccharides. The use of such a digest is preferable, particularly in the chromatographic procedure described above. When intact soluble starch is used, the insoluble material is formed in the column and may nearly block the flow of the eluting solution. The enzyme can be adsorbed from the medium in which the bacteria have been grown without preliminary treatment except for the removal of cells by centrifugation. A convenient method for adsorption is to run the medium thiough a starch column. Sectional elution of such a column after extrusion has shown that the enzyme is adsorbed in the top one-fifth to one-quarter of the column. Complete removal of the enzyme from such column sections requiies seveial elutions. In order to avoid this, the procedure for continuous "gradient elution" has been devised. As described above, the elution method utilizes a closed system designed to supply a constant flow of soluble starch (or ligest) to the top of the column at a steadily incieasing concentration, and to allow fractional collection of eluted enzyme at the bottom. This "gradient elution" gives fractions of somewhat lower activity per ml but of higher specific activity than if starch of constant concentration is used. When fractions of 3 ml are taken, peak activity is localized in two or three fractions which contain 75 per cent or more of the total activity adsorbed. Amylase activity of peak fractions obtained by the "gradient elution" procedure is one to two thousand units pei ml, a 100-fold concentration over the activitv of the original medium. The increase in specific activity cannot be determined easily because of the small amounts of protein involved (1 to 2 mg of amylase piotein per liter of medium) and the analytical complications introduced by the relatively high amounts of iron salts in the growth medium. Fractions of highest specific activity and Em0/260 ratio were obtained when fractions from previous columns were combined and adsorbed and eluted a second time. For example, three fractions (3 ml each) from the peak of such a column had specific activities of 7,750, 10,600, and 6,900 units per mg of protein, and E20/E260 ratios of 1.59, 1.42, and 1.31, respectively. Several columns have yielded a peak fraction with a specific activity of approximately 10,000 which may represent the maximum purification obtainable by this method. Preparation of the intracellular arnylase. Protamine precipitation and ammonium sulfate fractionation have been used to give some degree of purification of the intracellular amylase, which is not adsorbed by potato starch under any conditions tested. Data for a t-pical example in which a twofold increase in specific activity was obtained after protamine treatment are presented in table 1. The homogenate resulting from sonic disintegration of cells is mixed with potato starch to adsorb extracellular enzyme and centrifuged to remove the starch as well as whole cells, cell walls, and other particulate material. The clear supernatant is treated with protamine to remove nucleic acids according to the procedure of Korkes et al. (1950). Further inert material precipitates

4 19531 AMYLASES OF PSEUDOMONAS SACCHAROPHILA 659 from the protamine soluble supernatant during dialysis against 0.01 M phosphate, ph 7, and is removed by centrifugation. Fractional ammonium sulfate precipitation of the final protamine soluble material gives considerable fractionation of amylase activity. The most active fraction, soluble at 0.4 saturation and precipitated at 0.5 saturation with ammonium sulfate, is contaminated only very slightly with nucleic acid. By analysis of the "D ratios" obtained during hydrolysis of starch (see section on iodimetry below), it has been found that the amylase precipitated at 0.4 saturation is largely of the extracellular type in a form not adsorbed by potato starch. FRACTION TABLE 1 Purification of intracellular amylase the acid side of ph 5.4, activity falling only to 80 to 90 per cent of the maximum at ph 4.2, at which point the activity of the intracellular enzyme is 20 to 30 per cent of the maximum. Iodimetric comparison. In an early iodimetric experiment, when the two amylases were being tested in parallel, it was noticed that the colors of corresponding iodine assay tubes were quite different although the color decrements (measured with the green filter) were nearly identical. When the absorption of the same samples was determined with the red filter (no. 66), it was found that, for identical decreases measured with the green filter, the decrease measured with the red filter is less for the extracellular enzyme than ENZYME PROTEIN ACTIVITY Total Per cent Eno Total Units mg units recovery E2oo protein mg Crude homogenate 790 _ Adsorbed and centrifuged Protamine soluble Ammonium sulfate fractions: 0.4 saturated saturated saturated * Corrected for volume of samples used for analyses. Starting material: 4 g (wet weight) starch grown cells in 40 ml M phosphate, ph 7. Twenty ml aliquots treated in Raytheon sonic oscillator. The partially purified intracellular enzyme is not adsorbed by potato starch, suggesting that the lack of adsorption from crude extracts is not attributable to the presence of interfering substances or to similar effects. Further evidence for this conclusion was obtained by passing different mixtures of extracellular enzyme and crude extract (containing the intracellular enzyme) through starch columns. The activity of the effluents was equivalent to that of the intracellular enzyme used, while the activity adsorbed (measured by loss) was equivalent to the amount of extracellular enzyme added. ph maxima. When tested iodimetrically in acetate and phosphate buffers in the ph range 4.2 to 7.4, both enzymes show ma around ph 5.4. The maxal ph range of the extracellular enzyme is much broader, especially on for the intracellular enzyme. When the ratio of density decreases (D66/D54) is calculated for the early stages of hydrolysis, values for the extracellular enzyme are found in the range 0.8 to 1.3 and for the intracellular enzyme in the range 1.7 to 2.2. This difference is consistent and characteristic with the most commonly found values being very nearly 1.0 and 2.0. Such "D ratios" have been useful in characterizing amylase activity in various preparations. For instance, adsorption of intracellular enzyme preparations with potato starch has often resulted in a considerable increase of the "D ratio", indicating the removal of contaminating extracellular enzyme. Similarly, it has been possible to show by "D ratios" that the amylase activity of intact, washed cells is caused by extracellular enzyme. The explanation for the difference in "D ratios"

5 660 PHILIP S. THAYER [VOL. 66 seems to lie in differences in relative affinities of the two enzymes for amylose and amylopectin (or their partially degraded derivatives in soluble starch). The absorption peak of the blue amyloseiodine complex lies near 650 m,u, whereas for the red amylopectin-iodine complex it is found near 550 m,u. Changes in absorption measured with the red and green filters, therefore, may be considered to represent (roughly) relative activities on amylose and amylopectin, respectively. A direct test, using amylose and amylopectin as substrates, confirms this supposition, showing that, relative to activity on amylopectin, the intracellular enzyme is 2½ times more active on amylose than is the extracellular enzyme (table Relative activities of intracellular and ENZYME SUBSTRATE (an a-amylase) and that the intracellular enzyme is saccharogenic (f8-amylase), mixed with other enzymes which contribute to the activity observed. When soluble starch is digested to the "achroic point" by the two enzymes, the intracellular enzyme produces approximately 80 per cent reducing sugar (expressed as maltose), and the extracellular enzyme 35 to 40 per cent reducing sugar. Further incubation results in values of 100 to 140 per cent for the intracellular enzyme while values for the extracellular enzyme do not exceed 75 per cent and are usually about 60 per cent. When amylose, amylopectin, or glycogen is used as substrate, the results are similar. TABLE 2 extracellular amylases on amylose and amylopectin LOSS OF IODINE COLOR ACIVTY RTOS (OD DECREMENT IN 20MmI)ACITYRIO Filter 54 Filter 66 De6 D(amylose)ss (green) (red) D64 D(amylopectin)i Extracellular Soluble starch Amylopectin Amylose Amylose and amylo pectin Intracellular Soluble starch Amylopectin Amylose Amylose and amylo pectin Iodimetric assay. Enzyme concentration: 0.33 units/ml. Substrate concentrations: soluble starch, 0.04 per cent; amylose and amylopectin, 0.02 per cent each. Amylose dissolved in 0.5 N NaOH, neutralized with 1.0 N acetic acid just before use. Reaction started by addition of enzyme. 2). The use of a mixture of the two substrates, analogous to soluble starch but in an undegraded form, further accentuates the difference between the two enzymes. In this case, competitive or other interactions occur with the result that: (1) the activity of either enzyme on the mixture is not equal to the sum of the activities on the individual substrates, and (2) the "D ratio" of the intracellular enzyme (i.e., the ratio of activity on amylose to activity on amylopectin) is 5½ times larger than the "D ratio" of the extracellular enzyme. Products of digestion. The results of chromatographic and reductimetric experiments indicate that the extracellular amylase is dextrinogenic Paper chromatography shows the presence of maltose and glucose in digests produced by the action of the intracellular enzyme. Fermentation tests account for 94 per cent of the original substrate as these two sugars (table 3). The presence of a large amount of glucose and the absence of limit dextrin formation indicate the combined action of two or more enzymes. Of several possible combinations, perhaps the most likely is: (1) an amylae of the 13-type; (2) a maltase; and (3) an enzyme which either splits the 1,6 cross-linkages of amylopectin or allows the by-passing of such linkages. The hydrolysis of starch to glucose by a "maltase" of the type found in Clostridium acetobutylicum by French

6 19531 AMYLASES OF PSEUDOMONAS SACCHAROPHILA 661 and Knapp (1950) could not be responsible for the total activity observed in the present case since the loss of iodine straining properties occurs concomitantly with the production of reducing groups instead of lagging behind it. Chromatographic analysis has shown that these partially purified amylase preparations are capable of producing glucose from maltose. Similar maltase activity is also present in extracts of sucrose grown cells which produce very little intracellular amylase. Whether this represents a different enzyme, a less specific a-glucosidase, or the same enzyme has not been determined. The seeming absence of a,8-limit dextrin may be matograms of such digests have shown spots with Rf values lower than that of maltose, also suggesting the presence of oligosaccharides and possibly isomaltose. An additional product of the action of the extracellular enzyme is an insoluble material formed when initial substrate concentrations are 0.1 per cent or higher. To prepare some of this material for analytical purposes, 5 g of soluble starch were digested with 1,000 units of purified extracellular enzyme in 150 ml of M phosphate, ph 5.4. After 17 hours' incubation, the digest was chilled and centrifuged, and the gelatinous precipitate washed with water, frozen, TABLE 3 Glucose and maltose produced from 8oluble starch by amylase action REMOVE FROM- REDUING -D1CRFSE IN ENZYME SUG IMOVED FROM DUCING REDUCING VALLUE SUGAR PER CENT lnzy DIGECST VALVZ/I{L DUE TO REMOVAL MICROGRAMS/LL OF SUBSTRATE Intracellular 1. None 2,080 I 2. Glucose 1, (from 1) 3. Maltose (from 2) Extracellular 1. None 1, Glucose 1, (from 1) 3. Maltose (from 2) Digest: 0.15 per cent soluble starch, 1.5 units of enzyme per ml, incubated 25 hours at ph 5.4 and 30 C. Fermentation: 1.0 ml sample, 1.0 ml 0.1 M KH2PO4, and 0.5 ml washed yeast suspension. Shaken 45 minutes under nitrogen at 30 C. One ml aliquot of supernatant from Torula monosa fermentation fermented with Saccharomyces cerevisiae. Reducing values converted to original volume. Reducing values for 100 micrograms of sugar: maltose, 110; glucose, 160; measured as loss of color with Klett, filter 42. attributed to the presence of (1) traces of the extracellular enzyme (not removed by adsorption with potato starch) which would permit bypassing of the branch points of amylopectin, or (2) an additional "R enzyme" (Hobson et al., 1951) which splits 1,6 linkages. The extracellular enzyme produces only small amounts of maltose and even less glucose. Most of the products of the action of this enzyme on soluble starch are not fermented by T. monosa or S. cerevisiae, maltose and glucose representing only 14 per cent of the original substrate (table 3). The high reducing value of the remaining material indicates the presence of a large proportion of tri- and tetrasaccharides. Paper chroand dried in vacuo. The yield of coarse, white powder was 288 mg (5.8 per cent). Since some losses occur during preparation, the true yield may be as much as 10 per cent of the original substrate. The isolated material stains purple with iodine and is largely soluble in hot water. Tests performed by Dr. W. Z. Hassid indicate that it is a linear oligosaccharide of 8 glucose residues (an a-dextrin), which aggregates by hydrogen bonding to produce an insoluble complex. Combined action of the two amylases. In a culture of P. saccharophila, the utilization of starch must depend on the consecutive action of the two amylases. Experiments with the separate

7 662 PHILIP S. THAYER [VOL. 66 enzymes have shown that the products of hydrolysis by the extracellular enzyme are attacked by the intracellular enzyme. The soluble products, after digestion of soluble starch to 57 per cent reducing sugar, (as maltose) were attacked further by the intracellular enzyme to give 120 per cent conversion, indicative of the formation of maltose and glucose. In addition, a solution of the material precipitated during action of the extracellular enzyme was attacked by the intracellular enzyme with a large increase in reducing value and the production of glucose and maltose (identified chromatographically). EXPERI- MENT TABLE 4 Induction of amylase formation GROWTH SUhBSRATE AMYLASE ACTIVITY Intracellular (UNITS/UL) Extracellular 1 Maltose Amylopectin Glycogen Soluble starch Sucrose Raffinose Sodium lactate Maltose Melibiose Cellobiose Trehalose Cultures grown to stationary phase. Iodimetric assay. Enzyme preparations: intracellular, sonic extracts of washed cells, adsorbed with potato starch to remove extracellular enzyme; activities converted to original culture volume; extracellular, aliquots of supernatant medium after removal of cells. Further evidence for the necessity of combined action of the two amylases has been obtained in a few preliminary Warburg experiments on the oxidation of starch by washed starch grown cells. With starch as substrate, the final rate of oxygen uptake is the same as the rate of oxygen uptake with maltose. Oxygen uptake on starch does not become linear until after approximately one-half hour of incubation. This initial lag is eliminated, however, by the addition of extracellular enzyme to the reaction mixture. Presumably, if it were possible to remove adsorbed extracellular enzyme from the cells, the lag would be prolonged, perhaps indefinitely. This conclusion is supported by the observation of Doudoroff (1940) that washed, glycogen grown cells did not oxidize glycogen except in the presence of added supernatant medium. The extracellular enzyme appears to be essential for the initial stages in the breakdown of starch or glycogen in cultures of P. 8accharophila, producing fragments which enter the cells and are hydrolyzed further by the intracellular enzyme to yield the oxidizable products, maltose and glucose. Induction of enzyme formation. As noted previously, the formation of both amylases is dependent largely on the nature of the growth substrate. Only small amounts of either enzyme are formed when cells are grown with lactate or with sugars not containing the maltose moiety (table 4). Cellobiose is an exception in that it contains a p3-linkage and induces intracellular enzyme activity but not extracellular activity. Maltose is the most active inductor of extracellular enzyme formation. The five substrates which induce intracellular enzyme formation possess more or less equal inductive capacity. Doudoroff (1940) suggested that the extracellular enzyme formed during growth on glycogen and essential for the oxidation of glycogen by intact cells was a specific glycogenase. However, it has been found that the amylases formed during growth on glycogen do not differ from those formed when other carbohydrates are used with respect to activity on soluble starch or the "D ratios" observed. ACKNOWLEDGMENT The author wishes to acknowledge with pleasure the assistance and helpful criticism given him by all members of the microbiology group of the Department of Bacteriology, particularly Drs. Michael Doudoroff and Roger Y. Stanier. SUMMARY P8eudomonas 8accharophila produces two amylases, one extracellular, the other intracellular. Their formation is induced by growth with soluble starch, glycogen, amylopectin, or maltose as carbon source. Cellobiose induces intracellular activity only. Cells grown at the expense of other compounds produce very small amounts of both enzymes. The extracellular amylase is of the dextrinogenic or a-amylase type, hydrolyzing amyloid

8 1953] AMYLASES OF PSEUDOMONAS SACCHAROPHILA 663 substrates principally to oligosaccharides, which include an insoluble straight chain dextrin of eight glucose units. The amounts of maltose and glucose produced are small. Preparations of the intracellular enzyme hydrolyze amyloid substrates almost entirely to maltose and glucose. This activity is probably caused by the combined action of two or more enzymes, including an amylase of the saccharogenic (3-amylase) type. Only the extracellular enzyme is adsorbed by potato starch. It may be eluted therefrom with a solution of soluble starch. A method for purifying the enzyme, based on this observation, has been described. The two enzymes differ in their effects on the iodine staining properties of soluble starch. Both enzymes show maximal activity at ph values near 5.4. REFERENCES DOUDOROFF, M The oxidative assimilation of sugars and related substances by Pseudomonas saccharophila. Enzymologia, 9, DOUDOROFF, M., HASSID, W. Z., PUTMAN, E. W., POTTER, A. L., AND LEDERBERG, J Direct utilization of maltose by Escherichia coli. J. Biol. Chem., 179, FRENCH, D., AND KNAPP, D. W The maltase of Clostridium acetobutylicum. Its specificity range and mode of action. J. Biol. Chem., 187, HATAISHI, O., AND STANIER, R. Y The bacterial oxidation of tryptophan. III. Enzymatic activities of cell-free extracts from bacteria employing the aromatic pathway. J. Bact., 62, HoBSON, P. N., WHELAN, W. J., AND PEAT, S The enzymic synthesis and degradation of starch. Part XIV. R-enzyme. J. Chem. Soc., HOUGH, L., JONES, J. K. N., AND WADMAN, W. H Quantitative analysis of mixtures of sugars by the method of partition chromatography. Part V. Improved methods for the separation and detection of the sugars and their methylated derivatives on the paper chromatogram. J. Chem. Soc., JEANES, A., WIsE, C. S., AND DIMLER, R. J Improved techniques in paper chromatography of carbohydrates. Anal. Chem., 23, KALCEAR, H. M Differential spectrophotometry of purine compounds by means of specific enzymes. III. Studies of the enzymes of purine metabolism. J. Biol. Chem., 167, KORKES, S., DEL CAMPILLO, A., AND OCHOA, S Biosynthesis of dicarboxylic acids by carbon dioxide fixation. IV. Isolation and properties of an adaptive "malic" enzyme from Lactobacillus arabinosus. J. Biol. Chem., 187, SCHALES, O., AND SCHALES, S. S A simple method for the determination of glucose in blood. Arch. Biochem., 8, SCHWIMMER, S., AND BALLS, A. K Isolation and properties of crystalline a-amylase from germinated barley. J. Biol. Chem., 179,