BIOSYNTHESIS OF THE POLYGALACTURONIC ACID CHAIN OF PECTIN BY A PARTICULATE ENZYME PREPARATION FROM PHASEOLUS A UREUS SEEDLINGS* BY C. L. VILLEMEZ, TSAU-YEN LIN, AND W. Z. HASSID DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF CALIFORNIA, BERKELEY

Communicated October 11, 1965

Pectin is an important structural component of the cell walls of all higher plants and is formed primarily during the early stages of cellular growth.1 The basic building unit of this polymer is known to be D-galactopyranosyluronic acid in which the linear skeleton of these units is connected by a-1,4-D-glycosidic linkages.2 The carboxyl groups of this compound are methylated to various degrees, and polygalacturonate is associated with other carbohydrates, mainly with D-galactan and L-araban. Although a considerable amount of information is available pertaining to the hydrolytic action of a number of enzymes on pectin, virtually nothing is known concerning the mechanism of biosynthesis of this polymer. UDP-a-D-galacturonic acid was shown to exist in mung beans (Phaseolus aureus).3 A dehydrogenase capable of oxidizing UDP-D-glucose to UDP-D-glucuronic acid4 and a UDP-D-glucuronic acid 4-epimerase which interconverts UDP-D-glucuronic acid and UDP-D-galacturonic acid have been found in plants.5 Furthermore, enzymes that catalyze the formation of D-galacturonic acid 1-phosphate6 and UDP-D-galacturonic acid,7 leading to the formation of the same uronic acid nucleotide in plants by an alternate mechanism, have also been found in mung beans. The sequence of these enzymatic reactions has led to the hypothesis that UDP-Dgalacturonic acid is a precursor of pectin. In addition, preliminary experiments using nucleoside diphosphogalacturonic acid with different bases as precursors for pectin formation by particulate fractions of various plants indicated that UDP-Dgalacturonic acid was the most likely precursor.8 However, verification of this indication has been hindered thus far by the lack of a satisfactory cell-free system which would catalyze pectin formation. In this communication we wish to report the synthesis of the D-galacturonic acid chain of pectin with a cell-free enzyme preparation from mung beans. Methods.-Preparation of radioactive substrate: Uniformly labeled C14-D-galactose was obtained through the courtesy of D. McBrien and S. Abraham. It had a specific activity of 127 jsc/,umole and was prepared by a modification of the method described by Bean et al.9 C14-labeled a-Dgalactose 1-phosphate was prepared by incubation of a-D-galactose-CH with D-galactokinase from Saccharomycesfragilis.10 Conversion of C14-a-D-galactose 1-phosphate to labeled a-D-galacturonic acid 1-phosphate was accomplished with about 60% yield using a modification of the catalytic oxidation described by Marsh."1 The radioactive a-D-galacturonic acid 1-phosphate was quantitatively converted to UDP-D-galacturonic acid, labeled in the D-galacturonic acid moiety, by a mung bean pyrophosphorylase-catalyzed reaction with UTP.7 The final product was purified by paper electrophoresis in 0.05 M phosphate buffer, pH 7.5, and by paper chromatography with a developing solvent consisting of 95% ethanol-1 M ammonium acetate (7:3), pH 3.8. Radioactive substrate purity: Hydrolysis of the UDP-D-galacturonic acid-C14 with snake venom phosphodiesterase produced galacturonic acid 1-phosphate as the sole radioactive product. A combination of phosphodiesterase and seminal phosphatase degraded this uronic acid nucleotide to galacturonic acid as the only radioactive compound. These results, in conjunction with the single radioactive bands obtained by subjecting the UDP-D-galacturonic acid-C14 to paper electrophoresis at pH 3.7 and 7.5, indicated that the radioactive substrate used in these experiments was pure with respect to radioactivity. 1626

VOL. 54, 1965

BIOCHEMISTRY: VILLEMEZ ET AL.

1627

Enzyme preparation: Mung beans were germinated for 3 days at room temperature in a closed compartment, the relative humidity of which was maintained near 100%. In a typical preparation 20 gm seedlings, from which the cotyledons had been removed, were chilled and ground for a few minutes with sand in a cold mortar. All subsequent operations were carried out between 0 and 4°. The semisolid mass produced by homogenization was strained through two layers of cheesecloth and the liquid (10-15 ml) centrifuged at 500 X g for 10 min. The 500 X g sediment was discarded and the supernatant solution centrifuged at 10,000 X g for 15 min. The supernatant liquid resulting from this centrifugation was discarded and the pellet resuspended in 10 ml medium with a loose-fitting Potter-type homogenizer. One ml of the resuspended particles, which contained about 0.5 mg of protein, was used as an enzyme source. The resuspension medium of 0.01 M cacodylate (pH 7.2), 0.4 M sucrose, and 1% polyvinylpyrrolidine. Assay: Approximately 0.1 mumole UDP-D-galacturonic acid-C14 in 10 IAl water was added to 1 ml of the enzyme suspension and the mixture incubated at 250 for 10 min. The reaction was terminated by adding 1 ml of 20% trichloroacetic acid (TCA). The mixture was cooled to 00 and the TCA precipitate separated by centrifugation. After washing with 3 ml cold water, 1 mg citrus pectic acid in 0.2 ml sodium acetate buffer, pH 4.5, was added to the TCA precipitate. The washed TCA precipitate was then extracted three times with 2-ml portions of boiling water. The extracts were separated from the sediment by centrifugation, combined, and 6 ml 95% ethanol added. The mixtures were kept at room temperature for at least 1 hr, and the coagulum was separated by centrifugation. The coagulum was washed successively with 3-ml portions of 50% ethanol, methanol, and acetone; it was then resuspended in 0.5 ml water, placed on an aluminum planchet, and dried. Radioactivity was determined with a thin-window counter. Characterization reactions: The material to be characterized was extracted from the TCA precipitate with 0.5% ammonium oxalate and purified by ethanol fractionation; it was dissolved in a NaOH solution, pH 10, and allowed to stand at room temperature for 30 min to hydrolyze any esterified carboxyl groups. The solution was then adjusted to pH 6 with HCl. Cetyl pyridinium chloride was used in the salt precipitation procedures,12 which served as an alternate method of purifying the radioactive polygalacturonate. The C'4-polygalacturonate was partially hydrolyzed by incubating at room temperature for 18 hr at pH 8.0 with a purified transeliminase isolated from Clostridium multifermentans culture medium.13 Complete hydrolysis was accomplished by incubation at 300 for 24-36 hr at pH 4.5 with filtered, dialyzed culture fluid from Penicillium chrysogenum, which contained a polygalacturonase. The hydrolysates were subjected to paper electrophoresis at pH 3.7 in 0.2 M ammonium formate, and the zones of radioactivity detected by cutting the electrophoretograms into strips of 0.5-cm width. The strips were counted at 00 in a liquid scintillation counter, using 15 ml of a counting medium consisting of 0.4% 2.5diphenyloxazole and 0.01% 1-4-bis-2-(5-phenyloxazolyl)-benzene in toluene (w/v). Electrophoretograms of the authentic materials used as standards were treated with silver nitrate to detect the position of the spots. Identification of polygalacturonase hydrolysis product: Confirmation of the identity of the radioactive material produced by complete hydrolysis of polygalacturonic acid-C14, presumably Dgalacturonic acid, could be obtained by reduction to the corresponding sugar alcohol. The uronic acid could be converted to an aldonic acid by treatment with sodium borohydride. The carboxyl group of this aldonic acid could then be esterified with methanol, and the methyl ester further reduced with sodium borohydride. This series of reactions would produce a hexitol with a configuration corresponding to that of the parent uronic acid. Since all of the known hexitols, except glucitol and iditol, separate well electrophoretically, identical electrophoretic mobilities of the radioactive hexitol and authentic galactitol would indicate that the original compound is galacturonic acid. In accordance with this rationale the methanol-extractable material resulting from the treatment of the radioactive pectin with P. chrysogenum polygalacturonase was dissolved in water and reacted with approximately a tenfold molar excess of sodium borohydride for 30 min at room temperature. The reaction mixture was adjusted to pH 3.5 with Dowex 50 (H+) suspended in methanol, and evaporated to dryness. The residue was dissolved in methanol and evaporated to dryness again; this procedure was repeated several times. The dry material was dissolved in 1 N HC1 in methanol and heated at 1000 in a sealed tube for 1 hr. Subsequently, several volumes of methanol were added, and the reaction mixture was evaporated to dryness under vacuum. The residue was dissolved in water, the solution adjusted with NaOH to pH 9, and treated again

PROC. N. A. S.

BIOCHEMISTRY: VILLEMEZ ET AL.

1628

'

60

--

'

18

24

500

400

300

200

100

00

t

ORIGIN 0

6

12

30

CENTIMETERS

FIG. 1.-Paper electrophoresis of radioactive polygalacturonic acid. The radioactive polygalacturonic acid obtained from the incubation of UDP-D-galacturonic acid-C14 with mung bean particulate fraction was subjected to paper electrophoresis in 0.2 M ammonium formate of pH 3.7. The electrophoretogram was cut into 0.5-cm strips which were analyzed for radioactivity with a scintillation counter.

with sodium borohydride. After acidification and evaporation with methanol, the residue was dissolved in water and subjected to paper electrophoresis in 0.05 M sodium borate at pH 9.7. Galactitol, glucitol, and mannitot standards were made visible by the periodate-benzidine procedure of Gordon et al.14

Results.-Polygalacturonic acid-C14: Precipitation of the radioactive material isolated by the assay procedure, presumably polygalacturonic acid, as the cetyl pyridinium salt resulted in no loss of specific activity. Four additional precipitations with ICO ethanol and cetyl pyridinium chloride (alternating) also failed to reduce the specific activity. The radioactive polygalacturonic acid was immobile upon paper electrophoresis at pH 3.7 l l - (Fig. 1). Extensive incubation of the radioactive polygalacturonic acid with P. chrysogenum polygalacturonase produced a radioactive prod___ 24 3 0 uct which moved as a single spot when the reac12 1. 6t* CENTIMETERS tion mixture was subjected to paper electroFIG. 2.-Hydrolysis of radioactive phoresis at pH 3.7 (Fig. 2). The mobility of polygalacturonate with P. chrysogenum this radioactive spot coincided with the movepolygalacturonase. The reaction mixture from the treatment of radioactive ment of authentic D-galacturoniic acid, the pectin with pectinase was subjected to expected product of complete hydrolysis of paper electrophoresis and analyzed for polygalacturonic acid. radioactivity as described in Fig. 1. 1500

1O00

V

0

VOL. 54, 1965

BIOCHEMISTRY: VILLEMEZ ET AL.

1629

,

300 (

EX

FIG. 3.-Identification of P. chrysogenum polygalacturonase hydrolysis product. Theofmethanol-soluble material radioactive polygalactufrom the incubation resulting ronic acid with P. chrysogenum polygalacturonase was reduced with sodium borohydride in a three-step reaction (see text). The reaction mixture was subjected to paper elecI _ trophoresis in 0.05 M borate buffer of pH 9.7, and anaas in Fig. 1. A faster-moving peak Ilyzed for radioactivity coinciding with the movement of authentic D-galacturonic acid is not shown.

200

S 100 -Z t

0

1' 0

20

10

30

CENTIMETERS

The methanol-extractable material, presumably galacturonic acid, produced by the incubation of the radioactive polygalacturonic acid with P. chrysogenum polygalacturonase was subjected to borohydride reduction followed by methyl esterification and an additional borohydride reduction. Electrophoresis of the reaction products in 0.05 M borate buffer, pH 9.7, resulted in a radioactive spot coincident with the mobility of authentic galactitol (Fig. 3) and a smaller, faster-moving spot (not shown) coincident with authentic galacturonic acid, presumably unreduced carboxylic acid. Treatment with a bacterial exopolygalacturonate transeliminase'2 produced a single mobile radioactive spot when the reaction mixture was subjected to paper electrophoresis at pH 3.7 (Fig. 4). This spot 500 was coincident with authentic unsaturated diA galacturonic acid, the expected degradation Z~~~~~~~~~~~~~~~~~~~o 2 product of polygalacturonic acid when treated with this enzyme. 9

300

Properties of enzyme preparation: (1) Frac-> tionation: When the mung bean seeds were germinated for 3 days in the dark, essentially all the enzyme activity was found in preparations from the root and hypocotyl portions of the seedlings. Centrifugal fractionation of seedling homogenates indicated that, within the limits of the assay procedure, the total enzyme activity resided in the fraction sedimenting between 500 anld 10,000 X g. Wheii this fraction was resuspended in pH 7.2 buffer (0.05 M phosphate-0.4

sucrose-1%

albumin)

and

sedi-

mented again at 10,000 X g, no loss of enzyme activity occurred, although additional activity was found in the fraction of the supernatant solution sedimenting between 10,000 and 35,000 X g. (2) Stability: Crude homogenates, 10,ooo X g pellets, and resuspended particles lost the ability to catalyze the incorporation of galacturonic acid into pectin within a few

o

o

0

0

C

X _ i n

-

30 -

Z

°

> 200 100

00

C

0

e.

__< 30 2

CENTIMETERS

FIG. 4.-Partial hydrolysis of radioactive polygalacturonic acid with exopolygalacturonic acid transeliminase.

After incubation for (A) 1.5 hr and (B) 18 hr,tothe reaction mixture was and electrophoresis subjected as in Fig. 1. analyzed radioactivity paper

PROC. N. A. S.

BIOCHEMISTRY: VILLEMEZ ET AL.

1630

hours when they were stored at 00 or - 15°. However, if the particles were resuspended in a medium containing 1 per cent bovine serum albumin (0.05 M phosphate-0.4 M sucrose-1% albumin), the preparation could be stored at -15° for at least 2 weeks with only a 20 per cent loss in activity. This loss occurred within 4 days. However, if the albumin concentration was reduced to 0.1 per cent, the activity loss was 35-40 per cent within 4 days at -15° and continued until only 20 per cent of the original activity remained after 2 weeks. Storage at 00, even with 1 per cent albumin, resulted in a total loss of activity within 4 days. (3) Reaction: Incorporation of radioactivity into the polymer increased with time for 10 min to 1 hr depending upon the preparation. Regardless of the time at which incorporation ceased, no decrease in pectin radioactivity was observed during 8 hr of incubation. Extraction of the TCA precipitate with hot water solubilized only a portion of the radioactive pectin. It was found that a much larger quantity of radioactive pectin could be solubilized if the TCA precipitate was extracted with 0.5 per cent ammonium oxalate at 95°. Thus, a particulate enzyme preparation from 80 gm mung bean seedlings containing about 20 mg protein catalyzed the incorporation of 0.5 manmole of UDP-D-galacturonic acid-C'4 into hot water-soluble polygalacturonic acid, while extraction of the hot water residue with 0.5 per cent ammonium oxylate produced the main fraction, 13 m~imoles, of polygalacturonic acid-C'4. The incubation time was 1 hr at 25° and pH 7.2. The radioactivity found in the pectin-C'4 represented 67 per cent of the original UDP-D-galacturonic acid-C14 present in the incubation mixture. The incorporation of D-galacturonic acid from UDP-D-galacturonic acid-C'4 into pectin exhibited a pH optimum within the range 6.3-7.0. At pH 7.4 and 5.4 the rate was half as great; at pH 8.0 the preparation was totally inactive. Thymidine triphosphate at 1 mM inhibited the reaction approximately 42 per cent, whereas the same concentration of UTP, UDP, or UMP inhibited the reaction 72, 84, and 89 per cent, respectively (Table 1). No significant inhibition was noted with 1 mM CTP, GTP, ATP, or inorganic phosphate. Discussion.-Figures 1-4 show that D-galacturonic acid from UDP-D-galacturonic acid-C'4 is incorporated into pectin by cell-free preparations of mung bean seedlings. TABLE 1 EFFECT OF NUCLEOTIDE ADDITIONS ON THE INCORPORATION OF RADIOACTIVITY FROM UDP-D-GALACTURONIC ACID-C14 INTO PECTIN Material added

Control

ATP CTP

GTP Inorganic phosphate TTP UTP UDP UMP

Radioactive pectate (cpm)

485 573 489 470 451 277 136 76 52

A reaction mixture consisting of the 500-10,000 X g sediment from 1.7 gm mung bean seedlings was incubated at 250 for 15 min with 3500 cpm UDP-Dgalacturonic acid-C'4 and 0.5 ;&mole of the test materials in a total volume of 0.5 ml. The boiling water extract of the washed TCA precipitate was treated with cetyl pyridinium chloride and the precipitated pectate washed with water and

counted.

VOL. 54, 1965

BIOCHEMISTRY: VILLEMEZ ET AL.

1631

Although there is no definite proof, on the basis of our results and the currently available information regarding biosynthesis of polysaccharides, it can be assumed that the pectin chain is synthesized by a direct transglycosylation reaction from UDP-D-galacturonic acid. The particulate enzyme preparation that was used in these studies undoubtedly contained preformed polygalacturonate molecules. Therefore, there existed the possibility that the incorporation of radioactive uronic acid into polygalacturonate was not indicative of biosynthesis but was the result of a single transglycosylation reaction between nonradioactive polygalacturonate and UDP-D-galacturonic acid-C14. This possibility was eliminated by the results of partial hydrolysis of the radioactive polygalacturonate by a purified clostridial polygalacturonate transeliminase (Fig. 4). The partial hydrolysis of the radioactive polygalacturonate by this transeliminase produced radioactive unsaturated digalacturonic acid, the quantity of which was proportional to the incubation time. It is known that this enzyme begins the degradation from the reducing end of a polygalacturonic acid molecule.'5 A transfer of individual D-galacturonic acid-C'4 units from UDP-Dgalacturonic acid-C'4 to unlabeled pectin molecules would label only the nonreducing ends of the molecules, assuming that the polygalacturonate chain is synthesized via transglycosylation reactions. It is also known that the uronic acid moiety which occurs at the nonreducing end of a polygalacturonic acid molecule appears, upon complete hydrolysis by this clostridial transeliminase, as galacturonic acid or diagalacturonic acid.'5 No radioactive galacturonic acid or diagalacturonic acid was produced. Therefore, the product of hydrolysis and progress of this transeliminase degradation support the conclusion that the radioactive pectin resulting from the incorporation of UDP-D-galacturonic acid-C'4 is labeled extensively throughout the chain. Summary.-A particulate preparation from Phaseolus aureus seedlings which catalyzes the formation of radioactive pectin from UDP-D-galacturonic acid-C'4 is described. The biosynthetic polygalacturonate was found to be completely hydrolyzed with Penicillium chrysogenum polygalacturoilase to D-galacturonic acid-C'4. Partial degradation of the radioactive biosynthetic polygalacturonic acid by the action of an exopolygalacturonic acid transeliminase produced radioactive unsaturated digalacturonic acid. Since the pattern of action of this transeliminase is known, the production of this radioactive degradation product implies that the radioactive pectin is formed by successive additions of radioactive D-galacturonic acid residues. Approximately 65 per cent of the radioactive D-galacturonic acid from UDP-Dgalacturonic acid-C'4 could be incorporated into pectin by this mung bean enzyme system. The authors wish to express their gratitude to Dr. J. D. Macmillan and Professor H. J. Phaff who generously provided the Clostridium multifermentans transeliminase and the Penicillium chrysogenum pectinase, as well as the oligogalacturonides used as standards. The excellent technical assistance of Miss A. L. Swanson is also gratefully acknowledged. * This research was supported in part by research grant A-1418 from the National Institutes of Health, U.S. Public Health Service, and by research grant G-23763 from the National Science Foundation. 1 Northcote, D. H., Symp. Soc. Exptl. Biol., 17, 157 (1963).

1632

BIOCHEMISTRY: J. H. NORTHROP

PROC. N. A. S.

2Beavan, G. H., and J. K. N. Jones, J. Chem. Soc., 1218 (1947). 3 Neufeld, E. F., and D. S. Feingold, Biochim. Biophys. Acta, 53, 589 (1961). 4Strominger, J. L., and L. W. Mapson, Biochem. J., 66, 567 (1957). 6 Feingold, D. S., E. F. Neufeld, and W. Z. Hassid, J. Biol. Chem., 235, 910 (1960). 6 Neufeld, E. F., D. S. Feingold, S. M. Ilves, G. Kessler, and W. Z. Hassid, J. Biol. Chem., 236, 3102 (1961). 7Feingold, D. S., E. F. Neufeld, and W. Z. Hassid, Arch. Biochem. Biophys., 78, 401 (1958). 8 Lin, T. Y., A. D. Elbein, and J. C. Su, unpublished results. 9 Bean, R. C., E. W. Putman, R. E. Trucco, and W. Z. Hassid, J. Biol. Chem., 204, 169 (1953). 10Trucco, R. E., R. Caputto, L. F. Leloir, and N. Mittelman, Arch. Biochem., 18, 137 (1948). 11 Marsh, C. A., J. Chem. Soc., 1578 (1952). 12 Scott, J. E., Methods Biochem. Anal., 8, 145 (1960). 13 Macmillan, J. D., and R.. H. Vaughn, Biochemistry, 3, 564 (1964). 14 Gordon, H. T., Anal. Chem., 28, 849 (1956). 15 Macmillan, J. D., H. J. Phaff, and R. H. Vaughn, Biochemistry, 3, 572 (1964).

PRODUCTION OF A NEW BACTERIAL VIRUS BY PROLONGED GROWTH OF LYSOGENIC E. COLI CULTURES IN THE PRESENCE OF TRIETHYLENE MELAMINE* BY JOHN H. NORTHROP LABORATORY OF THE ROCKEFELLER UNIVERSITY, THE DONNER LABORATORY OF BIOPHYSICS AND MEDICAL PHYSICS, AND DEPARTMENT OF BACTERIOLOGY, UNIVERSITY OF CALIFORNIA, BERKELEY

Communicated October 11, 1965

Mutagens usually increase the proportion of all mutants in the population to approximately the same extent (Muller, 1954). They probably act by increasing the number of errors made by the cell during replication of the nucleic acids, rather than by a specific reaction of the mutagen with the nucleic acid. The same effect results from exposure to high temperatures. Exposure of B. megatherium (Northrop, 1962) or K12 X (Northrop, in press) cultures, for instance, to a series of mutagens results in an increase of 2-20 times in the proportion of several mutants and also of the virus-producing cells. In these experiments the cultures were exposed to the mutagens for a short time only. If P22 or K12 X cultures are grown continuously, instead of for a few hours, in the presence of triethylene melamine (TEM) either in the automatic cell (Northrop, 1960) or by repeated transfer, an entirely different result is obtained. [This TEM mutagen possesses the unusual ability to cause bacterial cells to mutate without growth or cell division (Szybalski, 1960; Northrop, 1963); it probably reacts directly with the DNA (Lorkiewicz and Szybalski, 1961).] Under these conditions, after 4-15 days, there is a great increase in the production of X phage. P22 cultures, which are noninducible and which normally produce about 1 X phage particle per 1010 cells, now produce about 108 particles per 109 cells, an increase of more than 109 times. Two to five days after this occurs, a new virus (PTEM), which attacks the parent culture itself, appears and increases rapidly to 108 or 109 per 109 cells. The results of an experiment of this kind with coli P22 are shown in Figure 1. For the first 12 transfers, the culture produced less than 1 X or PTEM plaque per 10