Vol.
THE JOURNAL OF BIOLOGICAL CHEMKWRY 241, No. 12, Issue of June 25, pp. 28%2875, l’ri’nted
Enzymes
in
1966
U.S.A.
of Nucleic
I. FRACTIONATION AND 1112, 3’-NUCLEOTIDASE,
Acid
Metabolism
from
Mung
CONCENTRATION OF PHOSPHOMONOESTERASE, AND DEOXYRIBONUCLEASE* (Received
TOM
L. WALTERS$
AND
HUBERT
Bean
Sprouts
RIBONUCLEASES
for
publication,
December
Ml AND
8, 1965)
S. LORING
From the Department of Chemistry, Stanford University, Stanford, California 9&3’05
SUMMARY
In connection with studies of phosphomonoester end groups of ribonucleic acid, it became desirable to secure moderate amounts of a comparatively pure 3’nucleotidase. Extracts of mung bean sprouts (Phaseolus uureus Roxb.) were prepared, and their 3’nucleotidase activity was compared with that of extracts of sprouted rye grass. The former were relatively free from the dark pigments associated with our rye grass extracts and conThese factors, tained appreciably more 3’nucleotidase activity. * Aided by Research Grants E-128 and E-31B from the American Cancer Society and by Grant 05566 the Council on Allergy and Infectious Diseases, National Institutes of Health, United States Public Health Service. The data are taken from a dissertation submitted by T. L. Walters in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry, Stanford University. $ Present address, Department of Chemistry, La Sierra College, Riverside, California.
EXPERIMENTAL
PROCEDURE
AND
RESULTS
Materials The 2’- and 3’-AMP and the 3’-CMP ods developed in this laboratory and characteristic of the pure compounds (9, 10). The 3’-disodium UMP was 2870
were prepared gave optical as previously prepared by
by methrotations described standard
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Several enzymes involved in nucleic acid metabolism, phosphomonoesterase, ribonuclease Ml, 3’-nucleotidase, deoxyribonuclease, and ribonuclease Mz, were partially purified from mung bean sprouts. The phosphomonoesterasewas separated from the other enzymes and purified 130-fold. It was shown to be nonspecific, with a pH optimum of 6.5. RNaseMl wasfractionated from the other types of enzymes by two methods, one of which resulted in a 200-fold purification. Its action on ribonucleic acid was shown to produce a mixture of 2’,3’-cyclic nucleotides; the cyclic purine nucleotides were further hydrolyzed to 3’-phosphates, while the cyclic pyrimidine nucleotides were not. The enzyme was found to be stable to dialysis at pH 5, and to have optimum activity near pH 5.5. 3’-Nucleotidase, RNase Mz, and DNase, which were present in a commonfraction, were purified about SO-fold. Unlike the 3’nucleotidase and RNase Mz activities, the DNase activity declined considerably upon standing, and was therefore considered to be due to a unique enzyme. The specificities and other properties of the 3’-nucleotidase and RNase Mz are reported in two accompanying papers.
along with the availability of the sprouts as a fresh vegetable, led us to attempt the purification of the mung bean enzyme. The bean sprout extract was fractionated with ammonium sulfate and by alumina adsorption, and a number of the properties of the 3’-nucleotidase were determined (1). While these investigations were in progress, papers by Sung and Laskowski (2), Stockx and Van Parijs (3), and Stockx and Vandendriessche (4) appeared dealing with the occurrence of deoxyribonuclease, ribonuclease, 3’-nucleotidase, and phosphomonoesterase in mung bean sprouts. The DNase was purified and characterized as a nuclease by Sung and Laskowski (2), who also reported the presence of RNase and 3’-nucleotidase Stockx and Van Parijs examined activities in the preparations. aqueous extracts of mung bean sprouts for phosphomonoesterase and 3’-nucleotidase activities and studied a number of the properties of each enzyme (3). Stockx and Vandendriessche purified an acid-stable RNase 200-fold and identified its hydrolysis products as purine and pyrimidine 2’, 3’-cyclic mononucleotides (4, 5). 2’,3’-Cyclic AMP was decyclized by the enzyme, but whether the product was 2’- or 3’-AMP was not determined (6). This paper presents data on the fractionation and 130-fold purification of the phosphomonoesterase, on a 200-fold purification of the acid-stable RNase, and on a 50-fold purification of the 3’-nucleotidase, DNase, and acid-labile RNase components. The pH optima of the phosphomonoesterase and the acidstabile RNase, called RNase Mr, and the identification of cyclic purine and pyrimidine nucleotides as hydrolysis products of RNase M1 action on tobacco mosaic virus RNA confirm previous results of Stockx, Van Parijs, and Vandendriessche. The final hydrolysis products of RNase Mr were identified as 3’-AMP, 3’-GMP, 2’,3’-cyclic CMP, and 2’,3’-cyclic UMP. The specificity and some properties of the 3’-nucleotidase are presented in in a second paper (7). The nature of the linkages cleaved by the second RNase, called RNase MP, including the identification of 5’-nucleotides as hydrolysis products, is presented in a third paper @I.
T. L. Walters
Issue of June 25, 1966
and H. S. Loring
Methods Assay for Phosphomonoesterase and S’-Nucleotidase-Phosphomonoesterase activity was assayed with ,&glycerolphosphate in 0.1 M ammonium acetate, pH 5, and 3’-nucleotidase was assayed with 3’-AMP in 0.1 M Tris-acetate, pH 8. Buffered 0.008 M substrate (1 ml) and enzyme solution (1 ml) were incubated at 37” for 15 min. The reaction was stopped by the addition of 6 ml of phosphate color reagent (15) ,2 and phosphomolybdate color was developed by incubating the mixture at 60” for 20 min. The mixture was cooled to room temperature, and its absorbance at 820 mp was determined in a Beckman spectrophotometer. The release of Pi was measured by the increase of A~B in the test solution as compared to a control consisting of the same mixture with the enzyme solution added after addition of the phosphate color reagent. A solution containing 5 pg of Pi was used as the standard. Unit and Specijic Activities for S’-Nucleotidase and Acid Phosphatase-One unit of enzyme is defined as the amount that causes the release of 1 pmole of Pi per hour under the conditions of the standard assay. Specific activity is expressed as units per mg of protein as determined by the method of Wade11 and Hill (16). Assay for RNase or DNase-A solution (1 ml) containing 4 Azeo units of either RNA or DNA in 0.1 M acetate (NHJ at pH 5 was mixed with an equal volume of enzyme solution, and the mixture was incubated at 37” for 30 min. The reaction was stopped by the addition of 2 ml of acid-uranium reagent (0.003 M uranyl acetate in 0.2 M HCl). The mixture was centrifuged for 5 min, and the A260 of the supernatant fluid was determined in a Beckman DU spectrophotometer. The control consisted of 1 Prepared in this laboratory by Mrs. Maravene Edelstein. 2 The reagent of Chen, Toribara, and Warner (15) was modified slightly by making it 0.8 N in H2SOI, 0.2yo in ammonium molybdate, and 0.75% in ascorbic acid. If stored at 4’, it could be used satisfactorily for about 1 week.
the same mixture with the enzyme solution added after the addition of the acid-uranium reagent. Unit and Specific Activities for RNase and DNase-One unit of RNase or DNase activity is defined as the amount required to cause an increase in absorbance of the supernatant fluid of 1.00 per hour as compared to the control. Specific activity is expressed as units per mg of protein. Enzyme Puri$cation Preparation of Crude Enzyme Extract and Fractional Precipitation with Ammonium Sulfate-All steps in enzyme fractionation were carried out at 4”. About 8 ounces of frozen mung bean sprouts were minced for 5 min in a Waring Blendor with 300 ml of water. The suspension was clarified by centrifugation, and the extract was made 40% saturated with respect to ammonium sulfate (28 g of solid ammonium sulfate per 100 ml of extract). The suspension was stirred for 30 min, and the precipitate was removed by centrifugation and discarded. The supernatant fluid was filtered and then brought to 70% saturation with ammonium sulfate by the addition of 21 g of the solid salt per 100 ml of original extract. The suspension was stirred for 30 min and centrifuged, and the supernatant fluid was discarded. The precipitate was dissolved in 100 ml of water, and the resulting solution was dialyzed by continuous flow dialysis (17) against 8 liters of 0.01 M triethylamine-0.001 M Tris-acetate, pH 8. The dialyzed solution was centrifuged for 1 hour, and the precipitate was discarded. The supernatant fluid contained phosphomonoesterase, 3’-nucleotidase, RNase, and DNase activities. Fractionation on DEAE-cellulose-The dialyzed fraction was placed on a column (15 X 1.2 cm) of DEAE-cellulose that had been equilibrated with 0.01 M triethylamine-0.001 M Tris adjusted to pH 8 with acetic acid. The column was then treated with 2 liters of a linear gradient solution of 0.01 to 0.20 M triethylamine acetate in 0.001 M Tris, pH 8. The flow rate of the eluate was about 50 ml per hour, and fractions of approximately 18 ml each were collected. Phosphomonoesterase, 3’-nucleotidase, RNase, and DNase assays were made on every other fraction in the ranges where the respective enzyme activities were located. Three main bands of enzyme activity were separated, as shown in Fig. 1. Fraction A (tubes 18 to 26) contained phosphomonoesterase; Fraction B (tubes 46 to 72), RNase Mr activity; and Fraction C (tubes 76 to 116), nucleotidase, DNase, and RNase Mz activities. Further Purijication of Phosphomonoesterase-The phosphomonoesterase activity of Fraction A represented a 70-fold increase in concentration above that of the original extract. A further doubling of specific activity resulted when Fraction A was chromatographed on CM-cellulose. The pooled fraction was adjusted to pH 4.5 with acetic acid and placed on a column (15 x 1.2 cm) of CM-cellulose that had been equilibrated with 0.01 M ammonium acetate, pH 4.7. The column was treated with 2 liters of a linear gradient solution of 0.01 to 0.50 M ammonium acetate, pH 4.7, at a flow rate of about 100 ml per hour. Fractions of approximately 18 ml were collected. The phosphomonoesterase fractions that were eluted at concentrations of 0.24 to 0.35 M buffer were pooled and dialyzed. The purification procedure is summarized and specific activities and yields are shown in Table I. Alternative Fractionation of RNase Mr-RNase Mr was concentrated approximately 30-fold by DEAE-cellulose chromatography as described above and was free of phosphomonoesterase,
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procedures from its dibrucine uridylate, [(Y],, -59”, which in turn had been prepared by fractional recrystallization of the dibrucine uridylates separated from the mixed dibrucine uridylates and cytidylates by pyridine extraction (11). The 3’GMP was prepared by a chemical fractionation procedure involving separation and recrystallization of the more insoluble monocyclohexylammonium 3’-GMP from the more soluble 2’-GMP salt; the 3’-GMP decomposed at 191” (when placed in the bath at 180”), [(Y]:~ -25.5” for a 0.62% solution in 0.5 M disodium phosphate. The 2’-GMP was recovered, purified, and used as the monobrucine salt;’ [crJi5 -40.3 f 1.3” (c, 0.4, in 40% ethanol). Crystalline 5’-AMP-2Hz0 and disodium P-glycerolphosphate pentahydrate were purchased from P-L Biochemicals and Eastman Kodak, respectively. Tobacco mosaic virus RNA was prepared by phenol extraction (12) with 0.01 M sodium EDTA, pH 7, as buffer. The sodium RNA was precipitated from the aqueous fraction with ethanol and was further purified by redissolving it in a minimum volume of water and reprecipitating with ethanol three times. Thymus DNA was prepared by modification of the procedure of Kay, Simmons, and Dounce (13) involving treatment of the crude sodium DNA solution with sodium lauryl sulfonate to decrease accompanying protein impurities (14). Diethylaminoethyl cellulose and carboxymethyl cellulose were purchased from BioRad.
2872
of Nucleic Acid Metabolism from Mung Bean Sprouts. 1
Enzymes I
I
against 0.01 M triethylamine acetate, pH 9, and placed on a column (15 x 1.2 cm) of DEAE-cellulose that had been equilibrated with 0.05 M triethylamine acetate, pH 9. The column was then treated with 2 liters of a linear gradient solution of 0.05 to 0.30 M triethylamine acetate, pH 9, at a flow rate of approximately 50 ml per hour. Fractions of about 18 ml were collected and assayedfor 3’-nucleotidase,RNase, and DNase activities. Tubes 38 to 56 were pooled, dialyzed against 0.01 M Tris-acetate,pH 7.5, and storedin the cold. The elution pattern for the three types of enzyme activities, asshownin Fig. 3, wascloselycomparableto that found at pH 8 with the exception that the relative amounts of 3’-nucleotidase and DNase werereversed,suggestinga greater instability of the latter. This was confirmed when the solution was allowed to standin the refrigerator for 2 months. Assaysfor 3’nucleotidase and RNasegavevaluesalmostidentical with thosefound initially, but DNase activity had decreasedabout 90%. A summary of the purification procedure,specific activities, and yields of the three enzyme activities is given in Table III.
I Fraction
6
80
70 Fraction
C
60
-C
I
-C
20
40
80
60 number
Tuba
100
I
I
I
40
50
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600
Vol. 241, No. 12
1
1:
FIG. 1. Fractionation of phosphomonoesterase (Fraction A), RNaseM1 (Fraction B), anda fraction containing3’-nucleotidase,
RNase
Mz,
and
DNase
(Fraction
C)
on DEAE-cellulose.
- - -,
molarity of triethylamine acetate. TABLE Summary
I
of purification
d-ounce
of nonspecij2 package of rnuj Total activity
Fraction
units
Crude extract. (NH&S04
5300
CM-cellulose.
from
Total protein
Specific activity
Yield
w 2300
units/mg 2.3
% 100
160 300
69 10
190
precipitate.
DEAE-cellulose, Fraction A . .
acid phosphatase bean sprouts
3600 520
22
1.7
10
20 Tube
FIG. 2. Fractionation on CM-cellulose at pH
5.
3’-nucleotidase,and DNaseactivities. A product representinga 200-fold increasein activity but containing somephosphomonoesteraseactivity was prepared by chromatography on CM-celSummary of purification lulose. The protein fraction from an S-ouncepackageof sprouts of (precipitated between40 and 60% saturation with ammonium sulfate) wasredissolvedin 50 ml of water and dialyzed against8 Fraction liters of 0.01M ammoniumacetate, pH 5. This treatment eliminated RNase MS, DNase, and 3’-nucleotidaseactivities, and the resulting solution, containing RNase M1 and phosphomonoes- Crude extract”. precipitate”. terase, wasapplied to a column (30 x 1.2 cm) of CM-cellulose (NH&S04 Fraction that had been treated with 0.01 M ammoniumacetate, pH 5. DEAE-cellulose, B RNaseM1 and phosphomonoesterase wereeluted separatelyby a (alternative linear gradient solution of approximately 700 ml of 0.01 to 0.4 M CM-cellulose method of purification). ammoniumacetate, pH 5, as shownin Fig. 2. The recovery of RNaseM1 and the specificactivity at various stagesof purificaa In order to calculate tion
by the two
procedures
are recorded
in Table
II.
Further Purification of Enzymesin Fraction C-Fraction C from the first DEAE-cellulose fractionation was extensively dialyzed
crude
extract
and
in the
30 number
of RNase Ml and phosphomonoesterase - - -, molarity of ammonium acetate. TABLE
II
of RNase Ml from mung bean sprouts
g-ounce
package T
Total activity
units
.
Total protein
22,000 15,000
m 2,300 190
7,500
25 1.2
2,300
the RNase (NH&S04
SpifiC
activity
Yield
units/mg 9.6 79
%
100 68 34
300 1,900
Ml and M2 activities precipitate, it was
in the assumed
that the total RNaseactivity found in eachwasdue to RNaseMr and MZ in the same ratio
found
in Fractions
B and
C, respectively.
T. L. Walters and H. 8. Loring
Issue of June 25, 1966 Attempted. Purijication
of Y-Nwleotidase
with Alumina
2873
Gel-A
difference in the adsorption properties of the 3’nucleotidase of mungbeansproutsand thoseof rye grassand barley wasnoticed during attempts t.a purify the mung bean enzyme by adsorption and elution from alumina gel as used by Shuster and Kaplan. The 40 to 70% ammonium sulfate-insolublefraction from 9 ouncesof sproutswas dialyzed and adsorbedin a volume of 80 ml on 1 g of alumina. Washing the alumina with 0.5 M ammonium sulfate, asusedto remove impurities in the caseof the rye grass enzyme, extracted the mung bean enzyme. Conversely, only small amounts of mung bean 3’-nucleotidaseactivity were extracted by 1 M sodiumbicarbonate, which eluted the rye grass enzyme. Dialysis of the ammoniumsulfate extract and measurementof specificactivity showedabout a 30% enhancement of potency. As only about 50% of the original enzyme was recovered, this purification step was subsequently eliminated.
,I40
Enzyme Properties
5.0
c.5
5.5
6.5
60
7.0
75
Some Properties of Mung Bean Phosphomonocstcrase-The FIN. 4. 0, pH optimum of phosphomonoesterase; pH optimum of the phosphomonoesterase for @-glycerolphos- timum of R.NaaeMl. Buffers used: pH 4.0 to 5.5,0.1 acetate;
pH
6.0
PH
6.0 to 7.0, 0.1
M
0, pH opammonium pH 7.5 to 8.0, 0.1 M
Tris-malonate;
M
phate wasfound to be near pH 6.5, as shownin Fig. 4. Under the assay conditions &glycerol phosphate and 2’-, 3’-, and 5’AMP were hydrolyzed by the enzyme at pH 5 at similar rates; &glycerol phosphatehydrolysis was inhibited 90% in the presence of 0.01 M NaF. The phosphomonoesterase preparation
RNase U2
50
3’Nucleotidase
40
ii
was tested for RNase activity by increasing assay times to 2 and 24 hours. The results of both tests indicated the presence of
z if!30 :
about 0.003unit of RNaseper unit of acid phosphatase. SomeProperties of RNase Ml-The pH optimum of RNaseM,, as also shownin Fig. 4, was near pH 5.5. The enzyme, unlike 3’-nucleotidase,RNaseMz, and DNase of Fraction C, retained
20
activity IO-
)/* c
)/*
0
1 20
40 Tube
60
80
100
number
FIG. 3. Attempted fractionation of 3’-nucleotidase, RNase Mz, and DNase on DEAE-cellulose at pH 9. - - -, molarity of triethylamine acetate. TABLE
Summary
of purification
of
S’-nucleotidase,
RNase
after dialysis
Total protein
fu 2,300
Crude extract”.
(NHG~OI DEAE-cellulose, Fraction C. DEAE-cellulose,
a See footnote, specific
units
ammonium
activities from S-ounce package of mung bean sprouts RNase Ma
Specific activity
Total activity
Yield
units/mg
acetate, pH 5,
units
%
Specific activity
units/mg
DNase Yield
%
Total activity
Specific activity
units
--
units/mg
Yield
%
33,006
14
100
42,000
19
100
46,000
20
100
190
23,000
120
70
29,ooO
150
69
35,000
180
78
26
~,ooO
770
15
13,000
840b
61 40
14,000 12,000
540 800
33 29
17,006 10,000
650 690
37 23
precipi-
tate”
b The
Total actitity
M
III
Mz, and DNase
3’-Nucleotidase Fraction
against 0.01
or after treatment with cyanide or EDTA. The conditionsused in the last mentionedexperimentsand the resultsfound alsowith fluoride and tartrate are presentedin Table IV. RNase M1 Specijicity-The hydrolysis products of tobacco mosaicvirus RNA produced by the RNase M1 fraction (GMcellulosefractionation) were characterized&sa mixture of purine nucleosides,3’-nucleoticles,and 2’,3’-cyclic purine and pyrimidine nucleotidesby the R, values and Azao: Asso and Anso: AzM) ratios of fractions sepa&ed by thin layer cellulosechromatog-
pH 8, ... pH 9. Table II. activity
of the 3’-nucleotidase
fractionated
similarly
from
other
samples
of bean
sprouts
ranged
from
840 to about
3,100.
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Tris-acetate.
2874
Enzymes of Nucleic Acid Metabolism from Mung Bean Sprouts. TABLE
E$ect
of some common inhibitors RNase
MP, DNase,
and
IV on the activities Y-nucleotidase
at
of RNase pH 7.6
MI,
Solutions containing approximately 5 units of enzyme per ml (except for DNase, which was presentat about 1 unit per ml), 0.1 M Tris-acetate, and 0.01M inhibitor at pH 7.5 wereleft at 25” for 1Ghours and then assayedfor enzyme activity. Inhibitor
Control (Tris only). Cyanide (Na). Fluoride (Na) EDTA (Na) Tartrate (NHI).
100 70 90 90 80
100 0 90 10 50
100 0 60
0 20
100 0 110
0 110
Vol. 241, No. 12
amountsof adenosinein the 36-hourdigestshowedthat hydrolysisof the cyclic AMP had proceededto the mononucleotidestage as found by Stockx and Vandendriessche(6) and that sufficient phosphomonoesterase activity was also present to produce nucleosides. The presenceof guanosinewas demonstratedby treating a portion of the 36-hour digest with 0.1 N HCl as described previously and fractionating the componentson thin layer cellulosein Solvent 1. Three areas separated,with Ra values of 0.66, 0.38, and 0.06 as comparedto Rp values of 0.63 and 0.39 for adenosineand guanosine,respectively, and to an Rp valuesof 0.06for a mixture of the 2’- and 3’-mononucleotides. Chromatographyof the eluted Band 2 in Solvent 2 showedthe presenceof four componentswith RF values of 0.88, 0.79, 0.39, and 0.28 in comparison,respectively, with RF values of 0.80 for 3’-CMP, 0.75 for 3’-UMP, 0.41 for 3’-GMP, and 0.26 as mentioned before for 3’-AMP. These results indicated that the migration rates for the expected2’,3’-cyclic purine and pyrimidine nucleotideswerenot greatly different in Solvent 2 than were those for the 3’-nucleotides. Elution of the respective areas gave A250:A260and Azso:A26,,ratios at pH 3 of 0.53 and 1.8, respectively, for the RF 0.88 component, in agreement with cytidylic acid, and ratios of 0.79 and 0.33, respectively, for the Rp 0.79 component,in agreementwith uridylic acid. For confirmation of the presenceof cyclic cytidylic and cyclic uridylic acids, the componentswith RF values of 0.88 and 0.79 were eluted, allowedto standfor 30 min in 0.1 N HCI, and re-examined in Solvent 1. In each instancethe previously rapidly moving componentwaseliminatedand another with RF 0.08wasformed, indicating that the cyclic nucleotideshad been hydrolyzed to 2’- and 3’-CMP and 2’- and 3’-UMP, respectively. DISCUSSION
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raphy. Two solvent systemswere used: Solvent 1 (18) contained (NH&C03, 9.6 g; water, 250 ml; and 2-propanol, 750 ml; Solvent 2 (19) consistedof saturated (NH&SO+ 80ml; water, 18 ml; and 2-propanol, 2 ml. In Solvent 1 the 2’,3’-cyclic purine and pyrimidine nucleotidesmove at relatively rapid rates, in contrast to the 2’- and 3’-nucleotides,which are poorly fractionated and remain near the origin. On treatment at room temperature in 0.1 N HCl for 30 min, the cyclic nucleotidesare converted to 2’- and 3’-nucleotides(20). A C-hour hydrolysate containing 1.2 mg of tobacco mosaic virus RNA and 10units of RNaseM1 per ml (tubes 10through 19 from the CM-cellulosefractionation) in 0.1 M ammoniumacetate, pH 5, at 37” wasexaminedin Solvent 1 beforeand after treatment with 0.1 N HCl. Three clearly separatedareaswerefound before HCl treatment with Rp valuesof 0.46,0.38,and 0.26ascompared to values for cyclic adenylate (0.45), for cyclic uridylate and cyclic cytidylate (0.38 and 0.35, respectively), and for cyclic guanylate (0.29) found by paper chromatography (18). After acid treatment, only componentsgiving Rp valuesin the rangeof 0 to 0.06 were present. The occurrencein the 4-hour RNaseM1 digestof rapidly moving componentsthat were eliminated by treatment at room temperature with 0.1 N HCl indicated that 2’,3’-cyclic nucleotides were producedduring the digestion,in agreementwith the conclusionsof Stockx and Vandendriesschefor the acid-stable RNase component of mung bean sprouts. Examination of a 36-hour hydrolysate by thin layer chromatographyin Solvent 1, however, showedthe presenceof componentswith RF values of 0.66, 0.36, 0.09, and 0.02 (Bands 1 through 4, respectively). Thesefour areaswerescrapedfrom the glassplate and extracted with 0.01 N HCl; Bands1 (RF 0.66), 3 (RF 0.09), and 4 (RF 0.02) were identified from their &,,:A26,, and Azso:Azooratios and from Rp values of the extracts in Solvent 2. Band 1 (19% of the total Aneoeluted) gave respectiveratios of 0.85 and 0.27and an Rp value of 0.24 in Solvent 2, in agreementwith comparable values for adenosine. Band 3 (5.3% of the total A!& gave absorbanceratios of 0.85 and 0.29, respectively, and an RF value of 0.24 in Solvent 2, in agreementwith comparableratios and an RF value of 0.26for 3’-AMP. The Rp value of 2’-AMP under comparableconditions was 0.38. Band 4 (3.1% of the total AZ& gave absorbanceratios of 0.98 and 0.66,respectively, and an RF value of 0.45 in Solvent 2 as comparedto absorbance ratios of 0.93and 0.69 and an RF value of 0.41for 3’-GMP. The Rp value of 2’-GMP was0.55. The presenceof relatively large
I
The nonspecificmung bean phosphomonoesterase was previously studied in aqueousextracts containing both nonspecific phosphomonoesterase and 3’-nucleotidaseactivities (3). The present experimentsshow that thesetwo enzyme activities are readily separableby DEAE-chromatography and that the most active phosphomonoesterase contained only a trace of RNase activity. Previously the nonspecificenzyme showeda pH optimum of about 6.0 on monophenylphosphateand was characterized asan acidphosphatasebecauseits activity decreasedsharply at pH 7 but extendeddown to pH 4. The presentpreparations gave a pH optimum of 6.5 on P-glycerol phosphate,and appreciable enzyme activity waspresentat both pH 4 and pH 8. Previous classificationas an acid phosphataseappears,therefore, to have doubtful value. The enzyme may possibly be better described as a neutral phosphataseor phosphomonoesterase. Phosphomonoesterases with pH optima nearneutrality have been describedpreviously from severalsources,but, as far as we are aware, such enzymeshave been studied only in crude extracts and have beenseeminglyspecific(21, 22). The RNase Mi is very likely the sameenzyme or enzymes purified and describedby Stockx and Vandendriessche(4-6). Theseauthorsfound evidencefor three components,calledA, B, and C, that wereseparableby chromatographyon Amberlite and gave pH optima of 5, 5.2, and 5.6, respectively. Becauseof similar temperature optima, comparablethermostabilities,and specific activities, it was thought that two of the components were slightly degradedforms of the third, but no identification of the respective componentswas made. Chromatography of the presentpreparationon either DEAE- or CM-celluloseshowed
Issue of June 25, 1966
T. L. Walters and H. S. Loring
REFERENCES 1. WALTERS, T. L., MCLENNAN, J. E., AND LORING, tion Proc., 22, 349 (1963).
2. SUNG, S. C., AND LASKOWSKI, 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20.
H. S., Federa-
3 This conclusion is based on the chromatographic evidence that only one nuclease was present in the Ml fraction. As pointed out by a referee, the results do not definitely establish that the hydrolysis of the RNA and of the cyclic purine nucleotides was catalyzed by the same enzyme.
21. 22. 23. 24. 25.
M., SR., J. Biol. Chem., 237,506 (1962). STOCKX, J., AND VAN PARIJS, R., Arch. Intern. Physiol. Biochim., 69, 194 (1961). STOCKX, J., AND VANDENDRIESSCHE, L., Arch. Intern. Physiol. Biochim., 69, 493 (1961). STOCKX, J., AND VANDENDRIESSCHE, L., Arch. Intern. Physiol. Biochim., 69, 521 (1961). STOCKX, J., AND VANDENDRIESSCHE, L., Arch. Intern. Physiol. Biochim., 69, 545 (1961). LORING, H. S., MCLENNAN, J. E., AND WALTERS, T. L., J. Biol. Chem., 241, 2876 (1966). WALTERS, T. L., AND LORING, H. S., J. Biol. Chem., 241, 2881 (1966). REICHARD, P., TAKENAKA, Y., AND LORING, H. S., J. Biol. Chem., 198, 599 (1952). LORING, H. S., BORTNER, H. W., LEVY, L. W., AND HAMMELL, M. L., J. Biol. Chem., 196, 807 (1952). LORING, H. S., AND LUTHY, N. G., J. Am. Chem. Sot., 73, 421; (1951). LORING, H. S., AL-RAWI, S., AND FUJIMOTO, Y., J. Biol. Chem., 233, 1415 (1958). KAY, E. R. M., SIMMONS, N. S., AND DOUNCE, A. L., J. Am. Chem. Sot., 74, 1724 (1952). DERANLEAU, D. A., M. S. thesis, Stanford University, 1958. CHEN, P. S., JR., TORIBARA, T. Y., AND WARNER, H., Anal. Chem., 28, 1756 (1956). WADDELL, W. J., AND HILL, C., J. Lab. Clin. Med., 48, 311 (1956). KUNITZ, M., AND SIMMS, H. S., J. Gen. Physiol., 11, 641 (1928). STOCKX, J., AND VAN PARIJS, R., Naturwissenschaften, 47, 39 (1960). MARKHAM, R., AND SMITH, J. D., Biochem. J., 49, 401 (1951). BROWN, D. M., MAGRATH, D. I., AND TODD, A. R., J. Chem. Sot., 2708 (1952). SCHMIDT, G., AND LASKOWSKI, M., Enzymes, 6, 24 (1961). STADTMAN, T. C., Enzymes, 6, 56 (1961). SZER, W., AND SHUGAR, D., Biochem. Prepn., 10, 142 (1963). HOLDEN, M., AND PIRIE, N. W., Biochem. J., 60, 39 (1955). FRISCH-NIGGEMEYER, W., AND REDDI, K. K., Biochim. Biophys. Acta, 26, 40 (1957).
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only a single component, and it may be that the less acidic conditions now used avoided the probable partial degradation mentioned. As the pH optimum of RNase MI, pH 5.5, agreed most closely with the value of pH 5.6 for Component C of Stockx and Vandendriessche, our data suggest that Components A and B were the partly degraded ones. Our results on the nature of the hydrolysis products confirm the conclusions of Stockx and Vandendriessche that 2’,3’-cyclic purine and pyrimidine nucleotides were formed, and show further that the cyclic purine nucleotides were hydrolyzed to 3’-AMP and 3’-GMP. The previous investigators showed that the 2’,3’cyclic pyrimidine components were decyclized to a small extent after long incubation periods. No evidence for the formation of either 2’- or 3’-CMP or 2’- or 3’-UMP was obtained in the present experiments with RNase MI. Because the 2’,3’-cyclic CMP and UMP are hydrolyzed nonenzymatically to 2’- and 3’nucleotides on standing in neutral solution (23), the previous results may have been due to a nonenzymatic rather than to an enzymatic hydrolysis. Accordingly, the MI enzyme appears to be like pea and tobacco leaf RNases (24,25), which decyclize the 2’,3’-cyclic .purine ‘nucleotides to 3’nucleotides but have no effect on the 2’,3’-cyclic pyrimidine derivatives.3 Previously the DNase and RNase activities were believed to be due to the same enzyme, which was called a nuclease (2). The present results, which show that RNase activity can be maintained while DNase activity is lost, indicate that two different enzymes are involved.
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Enzymes of Nucleic Acid Metabolism from Mung Bean Sprouts: I. FRACTIONATION AND CONCENTRATION OF PHOSPHOMONOESTERASE, RIBONUCLEASES M1 AND M2, 3'-NUCLEOTIDASE, AND DEOXYRIBONUCLEASE Tom L. Walters and Hubert S. Loring J. Biol. Chem. 1966, 241:2870-2875.
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