Guanine and Xanthine Phosphoribosyltransfer Activities of Lactobacillus casei and Escherichia coli THEIR RELATIONSHIP TO HYPOXANTHINE AND ADENINE PHOSPHORIBOSYLTRANSFER ACTIVITIES

the of the of and phosphoribosyltransfer in extracts of a of xanthine the for


SUMMARY
The specificity and properties of the group of enzymes present in extracts of Lacfobacillus casei which catalyzed phosphoribosyltransfer from 5-phosphoribosyl I-pyrophosphate to purine bases were markedly different from those in extracts of Escherichia coli.
A comparison of the levels of hypoxanthine, guanine, and xanthine phosphoribosyltransfer activities in extracts of a wild strain and a 6-mercaptopurine-resistant strain of L. casei suggested the presence of a xanthine phosphoribosyltransferase which was distinct from the enzyme specific for hypoxanthine and guanine. This suggestion was substantiated by the finding that the activity toward xanthine was less stable at -21' and 60' than the activities toward hypoxanthine and guanine, and by its separation from these activities on Sephadex G-100. The hypoxanthine-guanine phosphoribosyltransferase present in the 6-mercaptopurine-resistant strain differed from that of the wild strain in its lower rate of ribonucleotide synthesis with hypoxanthine relative to that with guanine, and in its somewhat lower affinities for hypoxanthine and 6-mercaptopurine. When extracts of E. coli were treated at 60", the phosphoribosyltransfer activities toward guanine and xanthine decreased at the same rate, whereas the activity toward hypoxanthine decreased more rapidly. Chromatography on Sephadex G-100 did not resolve the activities toward hypoxanthine, guanine, and xanthine in extracts of E. coli. However, on Ecteola-cellulose, these activities were separated into two distinct peaks, the relative specificities of which indicated the presence of two different enzymes or enzyme forms. With one, hypoxanthine was the most efficient phosphoribosyl acceptor, whereas with the other, guanine was the most efficient acceptor.
No distinct xanthine phosphoribosyltransferase was detected in extracts of E. coli. In extracts of both L. casei and E. coli, the activity toward adenine was distinct from the activities toward hypoxanthine, guanine, and xanthine.
This was shown by separations on Sephadex G-100 columns and by the differences in rates of heat inactivation.
The major metabolic pathway for the conversion of purines to mononucleotides is phosphoribosyltransfer from 5-phosphoribosyl 1-pyrophosphate to the purine base. Two distinct enzymes capable of catalyzing this transfer are known to be present in mammalian tissues. One preferentially acts on g-amino purines (adenosine monophosphate : pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) (1) and the other on 6-oxopurines (inosine monophosphate : pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) (2). Studies with highly purified preparations from brewers' yeast (3, 4) and with less pure preparations from human blood cells (2, 5-7) indicate that a single enzyme catalyzes phosphoribosyltransfer to both hypoxanthine and guanine.
However, with enzyme preparations from other sources some differential effects on the activities toward hypoxanthine and guanine have been observed (8-11).
The enzymatic basis for phosphoribosyltransfer to xanthine is likewise somewhat unclear.
The hypoxanthine-guanine phosphoribosyltransferase from human blood cells has been shown to have low but detectable activity toward xanthine (2, 12). The binding constant and reaction rate of xanthine with this enzyme are poor as compared to those for hypoxanthine or guanine (2). Studies with microorganisms indicate the existence of a xanthine phosphoribosyltransferase which is distinct from that for hypoxanthine and guanine. The earliest suggestions of the existence of this enzyme came from studies on purine utilization by bacteria which were resistant to 6-mercaptopurine (13,14). Later, enzyme preparations from purine analogue-resistant strains of Xalmonella typhimurium (15) and Streptococcus faecalis (8) were reported to catalyze phosphoribosyltransfer preferentially to xanthine.
Previous studies with Lactobacillus casei have shown that a 6-mercaptopurine-resistant strain was able to utilize guanine, xanthine, and, to a lesser extent, adenine, but not hypoxanthine to support growth (13). The wild strain utilized all four purines equally well. These observations suggested t'hat a study of the phosphoribosyltransfer activities from both a wild and a 6mercaptopurine-resistant strain of L. casei might help to elucidate the multiplicity and specificity of the purine phosphoribosyltransferases of this organism.
To test the generality of the findings with L. casei, similar experimental procedures were applied to extracts of Escherichia coli. from the International Chemical and Nuclear Corporation. The dimagnesium salt of 5-phosphoribosyl I-pyrophosphate was purchased from P-L Biochemicals.
Proteins used to calibrate the Sephadex columns were purchased from Mann.
Ecteolacellulose (capacity 0.34 meq per g) was purchased from Bio-Rad Laboratories, Richmond, California. E. coli B (ATCC 11303), grown in Kornberg medium' and harvested in late log phase, was purchased from General Biochemicals, and stored at -21".
Enzyme Assays-Assay mixtures were prepared at a final volume of 0.4 ml and contained the following components: 14Cpurine, 0.1 rnhf (except with the kinetic studies when the concentrations ranged from 2 to 50 PM); dimagnesium 5-phosphoribosyl 1-pyrophosphate, 1.0 mM; magnesium sulfate, 2.5 mM; Tris hydrochloride buffer (pH 7.7), 0.225 M; and enzyme sufficient to catalyze nucleotide synthesis at a linear rate during the time of incubation.
Each reaction mixture contained approximately 150,000 cpm. Reactions were initiated by the addition of enzyme.
Assay mixtures were routinely incubated at 38" for 3 to 10 min. Reactions were stopped by the addition of 0.1 ml of a 0.1 M sodium EDTA solution (pH 8.2) to the reaction mixture.
In control assays, EDTA was added prior to the addition of enzyme. Aliquots (0.15 ml) of the reaction mixtures were applied to Whatman No. 3MM paper which had been previously spotted with a solution containing 0.1 Kmole of nonradioactive purine and the corresponding 5'-ribonucleotide. Chromatograms were developed as described in Table I. The extent to which the reactions had proceeded was determined by cutting out the ultraviolet light-absorbing spots containing the 14C-purine and the corresponding 14C-ribonucleotide from the paper chromatograms of the reaction mixtures. The radioactivity in each spot was counted by liquid scintillation as previously described (2). Paper chromatograms of the reaction mixtures containing crude extracts of L. casei did not possess significant radioactivity at the RF values of the ribonucleosides (Table I), indicating the absence of interfering phosphatase activity.
With the crude extracts of E. coli, however, relatively low but detectable ribonucleotide phosphatase activity was present.
With these chromatograms, therefore, the area of the chromatogram containing the ribonucleoside was included with that of the ribonucleotide to compensate for this phosphatase activity.2 Culture of L. case&The wild strain of L. casei (ATCC 7469) was maintained in Difco folic acid assay medium supplemented with 10 mpg per ml of folic acid. The 6-mercaptopurine-resistant strain was developed from the wild strain and maintained according to the procedures described by Elion,Singer,and Hitchings (13) except that the folic acid levels of the cultures were 0.2 mpg per ml.
Before preparation of the extracts, cells from maintenance cultures were subcultured twice.
The first subculture (10 ml) was seeded with 0.2 ml of a 5-to S-day-old maintenance culture. The second subculture (2 liters) was seeded with washed cells harvested from the first subculture.
2 When reactions in assay mixtures containing E. coli extracts were stopped by immersion in a boiling water bath for 3 min, the phosphatase activity was found to be appreciably higher than when the reactions were stopped by the addition of EDTA. were the same. These parameters are individually specified under "Results." Cells were harvested by centrifugation at 13,000 x g for 30 min. The cell pellet was resuspended in a volume of 0.14 M sodium chloride equal to the original culture volume and centrifuged as above. Preparation of Cell Extracts-L. casei cells harvested from the second subculture were resuspended in a minimal amount (2 to 10 ml) of 9 mM Tris hydrochloride-O.1 InM magnesium sulfate, pH 7.7 (Buffer A). This suspension was passed through a French Pressure Cell (American Instrument Company) at 17,000 psi. and then centrifuged at 80,000 x g for 20 min. These resulting supernatant fluids are hereafter referred to as extracts.
One gram of frozen E. coli cells was thawed and suspended in 5 or 10 ml of Buffer A. An extract of this cell suspension was prepared as described above.
Protein concentrations were determined by the method of Lowry et al. (16) with human serum albumin as the standard.

Studies with Extracts of L. casei
Levels of Activity- Table  II lists the phosphoribosyltransfer activities toward hypoxanthine, guanine, xanthine, and adenine in extracts of a wild strain and a 6-mercaptopurine-resistant strain of L. case? cultured under various conditions.
The levels of activity toward hypoxanthine and guanine in the extracts of the resistant strain were appreciably lower than those of the wild strain.
This difference between the wild and resistant strains was more pronounced when the cells were cultured in Medium 1, than when cultured in Medium 3 (see Table II for composition of media).
Under all conditions of cell culture used, the activity toward guanine in the extracts of the resistant strain was about twice that toward hypoxanthine; whereas these activities were nearly equal in the extracts of the wild strain.
The levels of activity toward xanthine and adenine, in contrast to those toward hypoxanthine and guanine, were similar in both wild and resistant strains and did not vary greatly with conditions of cell culture.
The presence of purines in the culture media did not induce higher phosphoribosyltransferase levels. Even with resistant cells cultured in Medium 4, where the primary source of purines was xanthine,* the level of activity toward this substrate was similar to that of cells cultured in other media (Table II).
On the other hand, the level of activity toward guanine was appreciably lower in cells of the resistant strain which were cultured in the presence of guanine (Medium 1) as compared with those cultured in its absence (Medium 3).
The wild or the resistant strain of L. casei could be stored at -21" as a cell suspension for at least several weeks without significant loss in the extractable phosphoribosyltransfer activities toward hypoxanthine, guanine, xanthine, or adenine. Extracts of these cells, however, when stored in this manner, suffered appreciable losses in their activity toward xanthine.
Only 2 to 5% of the original activity toward xanthine was detected after 16 days at -21".
The levels of the activity toward hypoxanthine, guanine, and adenine were essentially unchanged after this treatment.
Heat Inactivation-When an extract of the wild strain of L. casei was incubated at 60" the activities toward hypoxanthine and guanine were most stable and decreased at essentially identical rates (Fig. 1). The decrease in the activity toward adenine was faster. The activity toward xanthine was the most sensitive to this heat treatment. Product Characterization-A pyrimidine phosphoribosyltransferase from bovine erythrocytes has been shown to catalyze phosphoribosyltransfer to nitrogen atom 3 but not nitrogen atom 9 of xanthine (18,19). Hypoxanthine and guanine were not substrates for this enzyme.
It was therefore of interest to determine the position of phosphoribosyltransfer to xanthine catalyzed by the extracts of L. casei.
(3-Ribosylxanthine) 5'-phosphate is much more resistant to acid hydrolysis than is (9-ribosylxanthine) 5'-phosphate (19). The product of the reaction catalyzed by the L. casei extract was 90% hydrolyzed to xanthine after 2 min at 100" in N HCI. Hydrolysis was essentially complete after 20 min. Under these conditions, the 3-ribosyl derivative is much more stable. The enzymatic product obtained with extracts of L. casei therefore appears to be (9-ribosylxanthine) 5'-phosphate. Sephadex Column Chromatography-When a freshly prepared extract of the wild strain of L. cusei cultured in Medium 1 was chromatographed on Sephadex G-100 the peak of phosphoribosyltransfer activity toward xanthine was eluted first, followed by a peak of activity toward adenine, and then by the overlapping activity peaks toward hypoxanthine and guanine (Fig. 2). A similar sequence of elution was obtained with the 6-mercaptopurine-resistant strain cultured under the same to xanthine (Xan), adenine (Ade), guanine (Gus), and hypoxanthine (Hyp) at the respective rates of 62, 358,733, and 691 mpmoles per min. Extract (2 ml) was applied to a column (3 X 37.5 cm) which had been previously equilibrated with Buffer A at 23". The flow rate was 63 ml per hour. The void volume of the column was 68 ml. Phosphoribosyltransfer activities recovered were 53y0 with xanthine, 81% with adenine, 81% with guanine, and 94% with hypoxanthine. of the extract contained 21 mg of protein and was capable of catalyzing phosphoribosyltransfer to xanthine (Xan), adenine (Ade), guanine (Gus), and hypoxanthine (Hyp) at the respective rates of 100, 889, 69, and 33 mpmoles per min. Extract (2 ml) was applied to a column (3 X 34 cm) which had been previously equilibrated with Buffer A at 23". The flow rate was 43 ml per hour. The void volume of the column was 53 ml. Phosphoribosyltransfer activities recovered were 91% with xanthine, 86% with adenine, 115y, with guanine, and 106a/, with hypoxanthine. conditions (Fig. 3), except that two separate peaks of activity toward guanine were discernible. The peak eluted first contained 10% of the total activity toward guanine and was accompanied by the phosphoribosyltransfer activity toward

xanthine.
The second peak of activity toward guanine was accompanied by the activity toward hypoxanthine.
GMP synthesis catalyzed by material from the first activity peak toward guanine rapidly decreased as the reaction progressed (Fig. 4), although XMP synthesis followed a more usual time course. GMP synthesis catalyzed by the second activity peak was essentially linear until 20% of the substrate had been converted to ribonucleotide.
A time course for GMP synthesis similar to that shown in Fig. 4 was observed by Brockman et al. (8) with an extract of a 6-mercaptopurine-resistant strain of Streptococcus jaecalis which preferentially catalyzed phosphoribosyltransfer to xanthine. The peak of activity toward hypoxanthine in the chromatogram of the resistant strain (Fig. 3) catalyzed phosphoribosyltransfer to 6-mercaptopurine at one-third the rate of transfer to hypoxanthine, whereas the peak of activity toward xanthine lacked significant activity toward 6mercaptopurine.
The values for the hypoxanthine-guanine phosphoribosyltransferases from the wild and resistant strains were not significantly different. These particle weight values were estimated from the elution volumes of the phosphoribosyltransfer activities from Sephadex columns which were calibrated with proteins of known molecular weights (20). It. is important to point out that these values are not presented as molecular weight values, but rather as apparent particle weights which might be of some use for future comparisons with values obtained with purified preparations.  that from the resistant strain was 7 PM. The K, value for guanine for this enzyme from the wild strain was 9 PM, and for that from the resistant strain, 8 PM. 6-Mercaptopurine exhibited competitive inhibition with 14Chypoxanthine as the variable substrate.
The Ki values of 6-mercaptopurine obtained from the magnitude of this inhibition were 7 pM with the wild strain enzyme, and 12 pM with the resistant strain enzyme.
The K, value for xanthine for the xanthine phosphoribosyltransferase taken from the Sephadex eluate shown in Fig. 3  Studies with Extracts of E. coli Levels of Activity-Extracts of E. coli were capable of catalyzing phosphoribosyltransfer to hypoxanthine, guanine, xanthine, and adenine at 73, 35, 20, and 41 mpmoles per min per mg of protein, respectively.
These activities are in the same range as those observed with extracts of L. easei (Table II).
Xephadex Column Chromatography-When an extract of E. coli was chromatographed on Sephadex G-100, the activity toward hypoxanthine was eluted first, closely followed by the overlapping activities toward guanine and xanthine (Fig. 6). The activity toward adenine was eluted last. The apparent particle weights estimated from this chromatogram were 75,000 for the activity toward hypoxanthine, 62,000 for that toward guanine and xanthine, and 28,000 for that toward adenine.
Ecteola-cellulose Column Chromatography-When extracts of E.
coli were chromatographed on Ecteola-cellulose the phosphoribosyltransfer activities toward guanine and xanthine were resolved into two distinct peaks (Fig. 7). The activity peak eluted first had a 4-fold higher rate of phosphoribosyltransfer to hypoxanthine than to guanine.
Conversely, the second peak of activity had a 4-fold higher rate of phorphoribosyltransfer to guanine than to hypoxanthine.
Rechromatography of the latter peak on Ecteola-cellulose resulted in a single peak of overlapping activities toward guanine, xanthine, and hypoxanthine at the relative rates of 5,4, and 1, respectively.
Stability at --Ho--Fractions from the Sephadex and Ecteola columns (Figs. 6 and 7) containing high activity toward hypoxanthine lost much of this activity after storage at -21", whereas no or relatively small losses in the activity toward guanine were observed (Table III).
Both peaks of activity toward guanine from Dhe Ecteola column (Fig. 7) had similar stabilities.
Kinetic StudiesMichaelis constants were determined with fractions (stored for several days at -21") from both activity peaks from the Ecteola column (Fig. 7). The K, values for guanine (1 PM) and for xanthine (50 j&M) were the same for both activity peaks. The K, value for hypoxanthine determined with the first peak of activity was 4 PM. In contrast, the K, value for hypoxanthine determined with the second activity peak was 80 PM. DISCUSSION The levels of xanthine phosphoribosyltransfer activity in extracts of the wild and 6-mercaptopurine-resistant strains of L. casei were similar, whereas the levels of activity toward hypoxanthine and guanine in the resistant strain were appreciably lower than those in the wild strain (Table II). The activity toward xanthine also differed from that toward hypoxanthine and guanine in stability to storage at -21" (see "Results") and to heat treatment (Fig. 1). Most striking was the chromatographic separation of the activity toward xanthine from that toward hypoxanthine and guanine (Figs. 2 and 3). These observations show that L. casei has a xanthine phosphoribosyltransferase distinct from its hypoxanthine-guanine phosphoribosyltransferase.
In the elution profile of the column in Fig. 3, the xanthine phosphoribosyltransferase was accompanied by some activity toward guanine which exhibited a peculiar time course (Fig. 4). Inhibition of both activities by nonradioactive xanthine or guanine (see "Results") suggested that they are not catalyzed by the same enzyme. However, because of the low levels of activity involved, a final decision must await further purification of these activities.
The level of guanine phosphoribosyltransfer activity extractable from L. casei cultured in different media varied in parallel with the activity toward hypoxanthine (Table II). Further, both activities had similar heat stabilities (Fig. 1) and elution volumes from Sephadex columns (Figs. 2 and 3). These observations indicate that L. casei has a hypoxanthine-guanine phosphoribosyltransferase similar to those studied from other sources (2, 3, 5, 21). This enzyme from the 6-mercaptopurineresistant strain differed from that of the wild strain in the relative rates of reaction with hyposanthine and guanine and the affinity for hypoxanthine and 6-mercaptopurine. These differences suggest that a genetic alteration of the hypoxanthine-guanine phosphoribosyltransferase, similar to those described with X. typhimurium (21), has occurred. It is important to note that although the resistant strain enzyme had a somewhat reduced affinity for 6-mercaptopurine, it was still capable of catalyzing the synthesis of the ribonucleotide of this analogue. It seems probable that both the reduced levels of the enzyme activity (Table II) and its modified catalytic properties contributed to the resistance of this organism.
Another way of interpreting the differences between the hypoxanthine-guanine phosphoribosyltransfer activities in the wild and resistant strains of L. casei is to postulate the existence of multiple forms of this enzyme in the wild strain.
It is conceivable that the resistant strain possesses only that isoenzymic form which is least efficient toward 6-mercaptopurine.
Indeed, the broad peak and varying ratios of the activity toward hypoxanthine and guanine in the Sephadex elution profile of extracts of the wild strain (Fig. 2) contrasting with the relatively sharp peak and const,ant ratios in that of the resistant strain (Fig. 3) are consistent with this view. However, attempts to resolve any isoenzymic forms of the hypoxanthine-guanine phosphoribosyltransferase in extracts of the wild strain of L. casei by Sephadex and DEAE-cellulose chromatography have thus far been inconclusive.
The properties and relationships of the phosphoribosyltransfer activities toward hypoxanthine, guanine, and xanthine in E. coli were quite different from those in L. casei. At BO", the activities toward guanine and xanthine in E. coli extracts decreased at the same rate (Fig. 5), while the activity toward hypoxanthine decreased more rapidly. Chromatography on Sephadex G-100 did not resolve the hypoxanthine, guanine, and xanthine phosphoribosyltransfer activities (Fig. 6). However,