THE PURIFICATlON AND
CHARACTERIZATION OF A. NUCLEASE.
FROM THE SEEDS 6F MUSKMELON

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
LAWRENCE D. ,MUSCHEK
197.0

  
     

  

THEE. ’

LIB RA R Y
Michigan 5' can:
University

This is to certify that the

thesis entitled
THE PURIFICATION AND CHARACTERIZATION
OF A NUCLEASE FROM THE
SEEDS OF MUSKMELON
presented by

Lawrence D. Muschek

has been accepted towards fulfillment
of the requirements for

E; degree in

Biochemistry

   

Major professor

Date 8//¢/70
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ABSTRACT

THE PURIFICATION ANIDCHARACTERIZATION OF
A NUCLEASE FROM THE SEEDS 0F MUSKMEIDN

by Lawrence D. Muschek

A nuclease, i.e. an enzyme capable of degrading both
RNA and DNA, has been purified about 2900-fold from the
seeds of muskmelon. The purification procedure includes
ammonium sulfate fractionation, DEAE-cellulose and CM-cellu-
lose column chromatography, and gel filtration on.P-100.

Purified nuclease catalyzes the hydrolysis of dena-
tured DNA, native DNA, RNA, and the 3'-ph08phoryl linkages of
3'-AMP. 3'-GMP, and 3'-UMP, but not of 3'-CMP. The enzyme
preparation also catalyzes the dephOSphorylation of the cor-
reSponding 2t-deoxyribonucleoside-B'-monoph08phates (except
3'-dCMP) but at a significantly slower rate.

The various activities are inseparable and remain in
constant ratios to each other throughout the purification
procedure subsequent to the DEAE-cellulose step. Furthermore,
the activities remain together through polyacrylamide disc
gel electrophoresis and in isoelectric focusing eXperiments.

The enzyme preparation is free of nonSpecific phOSphO-
diesterase activities as indicated by a lack of activity on
a variety of 3'- and S'gpgnitrOphenyl nucleotide derivatives.
Slight contamination of the preparation by nonSpecific phos-

phomonoesterase, however, has been observed.

Tm “me-

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Lawrence D. Muschek

The DNase activity is preferential towards denatured
DNA, has two separate pH optima, 5.7 and 8.0, exhibits endo-
nucleolytic hydrolysis at both pH Optima, and at both pH's
forms 5'-ph08phoryl terminated products.

Activity on 3'-AMP is maximal at pH 8.0.

The RNase activity is maximal at pH 5.7, proceeds in
an endonucleolytic manner at about twice the rate of that of
the DNase, and forms 5'-ph08phory1 terminated products.

Activity on native DNA appears to be endonucleolytic
in nature and proceeds at pH 5.7 at about twice the rate
observed at pH 8.0. The rate of hydrolysis of native DNA
at pH 8.0 is less than 10 percent of that observed at the
same pH with denatured DNA.

Cytosine mononucleotide is absent from hydrolyzates
of denatured DNA at pH 8.0, and present only in minor amounts
in hydrolyzates of both RNA and denatured DNA at pH 5.7.

All activities described migrate together through
Sephadex G-100 with an elution pattern of a Species having
a molecular weight of about 50,000.

At pH 8.0 tre enzyme preparation appears to be capable
of hydrolyzing polyadenylic acid and polyuridylic acid, but
not polycytidylic acid. The structures of the products of
hydrolysis of the copolymer, polyuridylic-cytidylic acid,
indicate that the 3' oxygen to phoSphorus bond of cytidylate
is not hydrolyzed.

An.extensive hydrolysis of denatured DNA by the nuclease

appears to form approximately equal quantities of mono-, di-,

31.. and tetr
The enZ
gtbqlwemlic

aligomcle ot ic‘:

 

Lawrence D. Muschek
tri-, and tetranucleotides.
The enzyme preparation is able to hydrolyze poly-2'-O-
methyladenylic acid to 5'-phosphoryl terminated mono- and

oligonucleotides.

THE PURIFICATION ANI>CHARACTERIZATION OF
A NUCIEASE FROM THE SEEDS OF MUSKMELON

By

_\
Lawrence D; Muschek

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

IDCTOR OF PHILOSOPHY
Department of Biochemistry

1970

 

 

1'. James L. F

the course of
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Anderson, Dr.
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ACKNOWLEDGMENTS

The author would like to express deep gratitude to
Dr. James L. Fairley for his help and guidance throughout
the course of this work and for constantly transmitting the
excitement of learning pure science. Thanks are extended
to Dr. Fritz M. Rottman, Dr. John C. Speck, Dr. Richard L.
Anderson, Dr. John E. Wilson, an‘l Dr. Theodore M. Brody who
kindly and patiently served on his guidance committee.
Appreciation is also extended to Mrs. Norma Green, Mrs.
Shirley Randall, Mrs. Linda Hartwig, Miss Jane Shoal, and
Mrs. Joann Seltz for their assistance in the preparation of
this manuscript. Most of all thanks are due his wife,
Nancy, who patiently endured and supported a not-so-pleasant
husband for the last few months of this work.

Appreciation is also expressed to the National
Institutes of Health for providing the funds to support
this investigation.

L.D.M.

***********

ii

Dedicated to the light of my life . . .

NANCY, WENDY, AMY, DEBBY . . .
and CHRISTIAN

iii

 

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TABLE OF CONTENTS
Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
EXPERIMENTAI.PROCEDURE

Materials . . . . . . . . . . . . . . . . . . . . . 6
Enzyme Source . . . . . . . . . . . . . . . . . 6
Nucleic Acids and Derivatives . . . . . . . . . 6
Enzymes . . . . . . . . . . . . . . . . . . . . 7
Resins . . . . . . . . . . . . . . . . . . . . . 7
Reagents . . . . . . . . . . . . . . . . . . . . 7

Methods . . . . . . . . . . . . . . . . . . . . . . 8
Preparation of Buffers . . . . . . . . . . . . . 8
Preparation of RNA from Yeast . . . . . . . . . 9
Preparation of Dialysis Tubing . . . . . . . . . 9
Preparation of Ion Exchange Resins . . . . . . . 9
Thin-Layer Chromatography . . . . . . . . . . . 12
Paper Chromatography . . . . . . . . . . . . . . 12
Polyacrylamide Disc Electrophoresis . . . . . . 13
DNase Assays . . . . . . . . . . . . . . . . . . 1n
BNase Assay . . . . . . . . . . . . . . . . . . 15
3'-AMPase Assay . . . . . . . . . . . . . . . . 16
Protein Determination . . . . . . . . . . . . . 18
Determination of Average Chain Length of Mixed
Oligonucleotides . . . . . . . . . . . . . . . . 18

iv

 

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Further At
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pH Opt.
pH Opt
pH Opt
Effect

Bat 10 S
Ul‘lase

Stabll
Studies 0

EXPERIMENTAL RESULTS
Purification of Enzyme . . . . . . . . . .
Preparation of Muskmelon Seeds . . . .
Homogenization of Seeds . . . . . . . .
Ammonium Sulfate Fractionation . . . .
LEAE-Cellulose Chromatography . . . . .
CM-Cellulose Chromatography . . . . . .

Chromatography on Bio-Rad P-iOO (DEAE-
C6 11111039) 0 o o c o o o o o o o o o o 0

Further Attempts to Separate the Four Activities

Isoelectric Focusing . . . . . . . . .
Polyacrylamide Disc Electrophoresis . .
PrOperties of the P-100 Enzyme Preparation
Contaminating Enzyme Activities . . . .
pH optima of DNase Activity . . . . . .
pH Optimum of RNase Activity . . . . .
pH Optimum of 3'-AMPase Activity . . .
Effect of Metal Ions . . . . . . . . .

Ratios of DNase A, RNase, and 3'-AMPase
DNase B Through Purification . . . . .

Molecular Weight Determination . . . .
Stability of P-100 Enzyme Preparation .
Studies on Enzyme Specificity . . . . . .
Relative 3'-Nuc1eotidase Activities . .
Activity Toward Cyclic Mononucleotides

Relative Hydrolysis Rates of Native DNA,
Denatured DNA, and RNA . . . . . . . .

Mode of Action on.RNA . . . . . . . . .

Page

21
21
21
22
23
24

27
33
33
37
38
38
1+1
47
47
47

54
57

59
62

62
65

67
68

Mode of
Node of

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BIBLIOGHAP HY
APPENDIX

 

Page
Mode of Action on Denatured DNA . . . . . . . . 7“
Mode of Action on Native DNA . . . . . . . . . 7“

Determination.of Phosphate Position in.Mono-
nucleotides Obtained from a Hydrolyzate onRNA. 77

Determination of Phosphate Position in Mono-
nucleotides Obtained from a Hydrolyzate of
muturOdDM‘tpHSa'leeeeeeeeeeee 8“
Determination oijhosphate Position in Oligo-
nucleotides Obtained from a Hydrolyzate of
Pclyadenylic Acid by P-iOO Enzyme . . . . . . . 88
Susceptibility of Pclyadenylic Acid, Poly-

uridylic Acid, and Polycytidylic Acid to

aural-’81! DIP-100E112!” e e e e e e e e e e 93
Characterization of the Products of Hydrolysis

of Polyuridylic-cytidylic Acid (Poly U,C)

Produced by P-iOO Enzyme . . . . . . . . . . . 95
Characterization of the Products of Hydrolysis

of Denatured DNA Produced by P-iOO Enzyme at
pHBeoeeeeeeeeeeeeeeeeeeee 105

The Hydrolysis of Poly-2'-O-methy1adenylio
Acid by P-iOO Enzyme . . . . . . . . . . . . . 11“

DISCUSSION . . . . . . . . . . . . . . . . . . . . . 123
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 1““
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 1H8
APPENDIX . . . . . . . . . . . . . . . . . . . . . . 151

vi

'n‘

 

Table

XI

XII.

XIII

 

Table
I.

II.
III.

VI.

VII.

VIII.

XI.

XII.

XIII.

LIST OF TABEES

Summary of purification of DNase B from one
kilogram (dry weight) of muskmelon seeds . . .

Effect of cations on activity . . . . . . . .

Ratios of DNase A, RNase, and 3'-AMPase to
DNase B through purification . . . . . . . . .

Protein standards used in molecular weight
determlmtion O O O O O O O O O 0 O O O O O 0

Relative 3'-nucleotidase activities toward
ribonucleoside-3'-mon0ph08phates . . . . . . .

Relative 3i-nuc1eotidase activities toward
2i-deoxyribonucleoside-B'-monoph03phates . . .

Characterization of the products of hydroly-
sis of polyuridylic-cytidylic acid (poly U,C)
produced by P-100 enzyme . . . . . . . . . . .

Summary of the characterization of peaks
C and D from a DEAR-Sephadex A-25 column
(Figure 18)e e e e e e e e e e e e e e e e e e

Recovery of the products of hydrolysis of
dDNA from a DEAE—Sephadex A-25 column . . . .

Relative amounts of mononucleotides in
fraction I (Figure 19) . . . . . . . . . . . .

Distribution of products in a hydrolyzate of
dDNA O O C O O O O O O O O O O O O 0 O O O O 0

Recovery from a Whatman Advanced DE-23 column
of the products of hydrolysis of polyAm as
determined by A260 e e e e e e e e e e e e e e

Characterization of the products of hydroly-

sis of polyAm eluted from a Whatman.Advanced
DE-23 Calumn O O O O O C O O O O O O O O O O 0

vii

Page

3a
55

56

58

64

66

100

10“

110

112

113

120

122

Figure

1.

10.
11.

12.

13.

LIST OF FIGURES

Elution pattern from a DEAR-cellulose column
of protein, DNase, RNase, and 3'-AMPase
BOtIVIty e e e e e e e e e e e e e e e' e e e e

Elution pattern from a CM-cellulose column

of protein, DNase, RNase, and 3'-AMPase
a°t1v1ty O O O O O O O O O O O O O O O O O O O
Elution pattern from a Bio-Gel.P-100 DEAE-
cellulose combination column of protein,
DNase, RNase, and 3'-AMPase activity ... . . .

Elution.pattern of DNase, RNase, and 3'-AMPase
from an electrofocusing column . . . . . . . .

Staining pattern of protein and distribution

of enzyme activities in.a polyacrylamide gel

after electrophoresis . . . . . . . . . . . .
pH—activity curve for DNase . . . . . . . . .
pH-activity curve for DNase using a pH stat .
pH-activity curve for RNase . . . . . . . . .
pH-activity curve for RNase . . . . . . . . .

pH-aCt1v1ty curve for 3'-AFP386 e e e e e e 0

Calibration curve for molecular weight deter-
mination on Sephadex G-100 . . . . . . . . . .

Relative hydrolysis rates of native DNA,
denatured DNA, and RNA . . . . . . . . . . . .

a. Chromatography on Sephadex 0-25 of a mix-
t‘lre or RNA am 5".AMP e e e e e e e e e e

b. Chromatography on Sephadex 6-25 of a
reaction containing RNA and venom phos—
phodiesterase, a known exonuclease, at
various stages in the hydrolysis . . . . .

viii

Page
25
28

31
35

39
42

1+5
48
50
52

60
69

71

71

Figure
13.

14.

15.

16.

17.

18.

19.

20.

Page

0. Chromatography on Sephadex 0—25 of a
reaction containing RNA and pancreatic
RNase, a known endonuclease, at various
stages in the hydrolysis . . . . . . . . . 71

d. Chromatography on Sephadex 6-25 of a
reaction containing RNA and P-100 puri-
fied muskmelon nuclease at various stages
in the hydrolysis . . . . . . . . . . . . 71

a. Chromatography on Sephadex 0-25 of a
mixture of dDNA and dAMP . . . . . . . . . 75

b. Chromatography on Sephadex 6—25 of a
reaction, at pH 5.7, containing dDNA and
P-100 enzyme at various stages in the
hydrOIYSIB e e e e e e a e e e e e e e e e 75

c. Chromatography on Sephadex G—25 of a
reaction, at pH 8.0, containing dDNA and
P-100 enzyme at various stages in the
hYd-I‘Olysis e e e e e e e e e e e e e e e e 75

a. Chromatography on Sephadex 0-25 of a mix-
ture of nDNA and dAMP . . . . . . . . . . 78

b. Chromatography on Sephadex 6-25 of a
reaction, at pH 5.7, containing nDNA and
P-100 enzyme at various stages in the
hydrOJ-ysls O O O O O O O O O O O O O O O O 78

c. Chromatography on Sephadex 6-25 of a
reaction, at pH 8.0, containing nDNA and
P-100 enzyme at various stages in the
hydrolysis . . . . . . . . . . . . . . . . 78

Chromatography on DEAE-Sephadex A-25 of a
hydrolyzate of RNA produced by P-100 enzyme . 81

Chromatography on DEAF-Sephadex A-25 of a
hydrolyzate of dDNA produced by P-100 enzyme
at DB 507 e e e e e e e e e e e e e e e e e e 86

Elution pattern from a DEAR-Sephadex A-25
column of the products of hydrolysis of poly
U,C produced by P-iOO enzyme . . . . . . . . . 97

Elution pattern.from a DEAR-Sephadex A-25
column of the products of hydrolysis of dDNA
produced by P-100 enzyme at pH 8.0 . . . . . . 107

Elution pattern from a Whatman.Advanced DE-23

column of the products of hydrolysis of poly-
2'-O-methy1adenylic acid produced by P-100

enzyme 0 C I O O O O O O O O O O O O O O O O O 118

ix

INTRODUCTION

We wish-to suggest a structure for the
salt of deoxyribose nucleic acid (D.N.A.).
This structure has novel features which
are of considerable biological interest.

To say that the communication (1) introduced by these
words set off a virtual bomb in the fields of biochemistry
and genetics would certainly be an understatement. Watson
and Crick's (1, 2) helical model for the structure of DNA
made an impact that was felt not only by the chemist but
also by the geneticist. Whenever a new and radical idea is
introduced, feverish work ensues to attempt to support or
disprove such an idea. Thus, the field of nucleic acid
chemistry called for new technology to meet the needs of
the biochemist who was attempting to delineate nucleic acid
structure.

Certainly, the key to elucidating the functions of
the nucleic acids lies partly in the determination of the
primary, secondary, and higher orders of structure. When
one considers the difficult question of the primary sequence
of nucleotides along a DNA or RNA chain, it is not surpris-
ing to find that the first complete structural analysis of
a nucleic acid, Holley and coworkers' (3, u) elucidation of

the primary structure of an alanine transfer RNA, was done

on a molecule of merely 25,000 molecular weight and 77

2

nucleotides in length. The task becomes quite formidable
for DNA molecules even from the viruses, because these have
molecular wehghts which are several million in magnitude.
Let us examine some of the problems associated with deter-
mining the primary sequence of a transfer RNA. Holley (3, 5)
made good use of the chemical property of RNA which makes
it susceptible to partial or total hydrolysis by base.
Even with this tool, and others which made it possible to
separate the products of hydrolysis, without the help of
some enzymes of rather high specificity the task would have
been much more difficult. For instance, his use of venom
phosphodiesterase to degrade oligonucleotides (5) of a
specific length to a mixture of products varying from one
another by one nucleotide, greatly facilitated the deter-
mination of their primary sequence. Initially, the oligo-
nucleotides with which Holley worked were formed by specif—
ically acting endonucleases, enzymes that cleave nucleic
acids preferentially at positions other than the termini.
These endonucleases have been used for a great variety of
structure determinations of RNA molecules. One of the
biggest problems in the field of nucleic acid chemistry
today is the fact that there are no known.endonucleases
which will degrade DNA with as precise a specificity as
such endonucleases as ribonuclease T1 (6).

The search for new and more useful tools of nucleic
acid degradation has produced a multitude of various kinds

of deoxyribonucleases. Thus a need for a system of classi-

 

 

fication of
Specificity
the f‘ollowirI

1..
2.

3

fication of these various enzyme types with regard to their

Specificity has prompted a worker (7) in the field to devise

the following:

1.
2.

9.

Specificity toward the sugar moiety.

Exo- versus endonucleolytic mode of
action.

Cleaving the internucleotide bond on
the 3'AP versus the P-5' side, and
forming products bearing either 5' or
3' monophosphate.

Nature of bases adjacent to the suscep-
tible linkage.

Specificity toward secondary structure
of the DNA; native (double-stranded)
versus the denatured (monostranded) DNA.

Inability to attack the DNA from the
same species.

Nucleases incapable of hydrolyzing
dinucleotides.

Exonucleases incapable of attacking
either native or denatured DNA but
capable of hydrolyzing oligonucleotides.

Ability of some endonucleases to hydro-
lyze both strands of the native DNA
simultaneously at the same locus.

Classifications like the preceding must be out of necessity

in a constant state of revision.because new nucleases are

being reported that have all possible combinations of

properties.

The only classification which has remained

absolute is number 3.

It would be a pointless and rather lengthy task to

attempt to review all of the outstanding nucleases that

have been reported over the past few years. Suffice to say

u
that extremely unusual enzymes showing much promise as poten-
tial tools have been isolated from almost every living organ-
ism. For more information.regarding nucleases from.various
sources the reader is referred to the following recent
reviews (7, 8, 9).

This work has centered on enzymes derived from plants.
Plants have been used for many years as potential sources
for the purification of both RNases and DNases. Workers
looking for either one or the other have, in the course of
their work, discovered that a rather unusual combination of
nuclease and nucleotidase activities purify as one species
from a variety of plant sources. Generally speaking, these
nucleases (attacking both.RNA and DNA) appear to prefer the
single stranded form of the substrate. Optimal activity is
observed at pH's between 5 and 7. An additional activity,
3'-nucleotidase (often reported as 3'-AMPase) has been found
to accompany many of the previously mentioned nuclease
activities. Molecular weights have usually been observed
to be in the 15-30,000 range. Plants which have been found
to contain these nuclease: 3'-nucleotidase combinations
are barley (10. 11), ryegrass (12), rice bran (13). mung
bean (1h-18), wheat (19), corn (20, 21), Penicillium
citrinum (22, 23, 21+) , and 211225 cucurbita-cearum (25). The
enzymes best characterized from this series are those from
mung bean and wheat. No one has stated that the associated
RNasezDNase:3'-nucleotidase activities are part of the same

protein molecule; in.fact, Walters and Loring have concluded

5
that the mung bean DNase and RNasez3'-nucleotidase reside in
different proteins. This view has been challenged by Johnson
and Laskowski (26). The final conclusion.regarding this
dilemma awaits more purified preparations of enzyme. What-
ever conclusions appear, the fact remains that a number of
these enzymes appear to exhibit some preference for certain
nucleotide bases early in the stage of hydrolysis. It was
for this reason that a number of other plants were surveyed
for their content of DNase activity.

The association of deoxyribonuclease, ribonuclease,
and 3'-nucleotidase seems common throughout all levels of
complexity in the plant kingdom. The fact that these associ-
ated activities have been found in dormant seeds as well as
in rapidly growing seedlings suggests a possible role in the
process of DNA repair or replication.

This thesis reports work done originally on the puri-
fication of a DNase from the seeds of muskmelon. As will
become apparent, RNase and 3'-nucleotidase activities were
found to be closely associated with the DNase activity.

Some of the properties and specificities of this association

have been studied and are described.

EXPERIMENTAI.PROCEDURE
Materials

Enzyme Source
Muskmelon seeds (Cucumis melo), Honey Rock variety,
were purchased in a 100 pound quantity from Farm.Bureau

Services, Lansing, Michigan.

Nucleic Acids and Derivatives

DNA1 from salmon.8perm (Type III), DNA from calf
thymus (Type I), various nucleotide and nucleoside deriva-
tives, imidazole (Grade I), and glycine were purchased from
Sigma Chemical Company. Exceptions to this were 3'-GMP1,
3'-UMP, and 3'-CMP which were obtained from P-L Biochemicals,
Inc. ‘Polyadenylic acid (Poly A), polycytidylic acid.(Poly C),
and polyuridylic acid.(Poly U), each containing 2.5 umoles
of polynucleotide phosphorus per mg of nominal polymer weight
(apparent average molecular weight 105 to 106) were purchased
from Schwarz Bioresearch, Inc. PolyU,C1 was a generous gift
from Dr. F. M. Rottman. The nucleotide derivatives 5'727
adenylate, 5'-p-nitr0phenyl cytidylate, 5'-p-nitr0pheny1-2'-
deoxyadenylate, and 3'gp-nitropheny1 thymidylate were prepared

 

1The abbreviations are those specified by the Journal
of Biological Chemistry. .Also see the appendix. Reference
to this footnote will be made frequently throughout the body
of this thesis. Whenever it appears the Appendix should be
consulted for an appropriate explanation.

6

7
and donated generously by Mr. R. E. Jagger. p-Nitrophenyl
phosphate (disodium.hexahydrate), Grade A: bis-(p-nitro-
phenyl) phosphoric acid; and sodiumfip-nitrophenyl thymidine-
5'-phosphate, Grade B were purchased from.Calbiochem.

Enzymes
5'-Nucleotidase, Grade II was purchased from Sigma

Chemical Company. Pancreatic RNase (5x crystallized) was
purchased from Calbiochem. Snake venom phosphodiesterase
(Crotalus adamanteus, VPH); phosphodiesterase II (Bovine
spleen, SPH) and alkaline phosphatase (E. 2211. BAPC) were
purchased from Worthington Biochemical Corporation.

Resins

DEAR-cellulose (Cellex-D), 0.99 meQ/g and.P-100
(50-150 mesh) were purchased from Bio-Rad Laboratories.
CM-cellulose (course mesh). 0.60 meq/g was purchased from
Sigma Chemical Company. c-25 (100-270 mesh), G-100 (#0-
120 u). and LEAR-Sephadex A-25. 3.5 t 0.5 meq/g (ho-120 u)

were obtained from Pharmacia Fine Chemicals Inc.

Reagents

All chemicals were reagent grade materials purchased
from either J. T. Baker Chemical Company or Mallinckrodt
Chemical Works unless otherwise specified. Lanthanum
nitrate was purchased from E. H. Sargent and Company.
Ammonium formats and Buffalo Black NBR were obtained from

Allied Chemical Company. Tris(hydroxymethyl)aminomethane

8
(Trizma Base, reagent grade) was purchased from Sigma Chomp
ical.Company. Bromphenol.B1ue was purchased from Fisher
Scientific Company. Acrylamide, N,N,N',N'-tetramethyl-
ethylenediamine (Temed), and N,N'-methy1enebisacrylamide

(Bis) were obtained from Canalco.
M13932

Preparation of Buffers

Two buffers were used during the course of the puri-
fication procedure. Buffer A was an ammonium acetate solu-
tion, 0.1 mM in zinc acetate, adjusted to pH 8.0 with 28.9
percent ammonium hydroxide. Buffer B was an ammonium acetate
solution, 0.1 mM in zinc acetate, adjusted to pH 5.5 with
glacial acetic acid. All designations of concentration
refer to the concentration of ammonium acetate before pH

adjustment was made.2

.All other buffers used in.this study
were made in a similar manner, e.g. 0.2 M Tris-H01, pH 8.2
refers to a 0.2 M solution of tris(hydroxymethyl)aminomethane
adjusted to pH 8.2 with 36 percent HCl. Triethylammonium
bicarbonate (TEAB) was made from triethylamine (TEA) by the
following procedure: A 520 ml quantity of cold} deion-
ized water was mixed with 280 ml of coldl, twice redistilled
TEA. Carbon dioxide from a dry ice generator was bubbled

through the solution which was stirred constantly in an ice

 

2The change in ammonium acetate concentration.after

the pH was adjusted was less than 0.1 percent.

9

bath. The rate of 002 evolution.was increased after an hour
and continued until the pH of a ten-fold diluted sample of
solution was in the range of 7.8 t 0.1. A 200 ml quantity

of cold1 deionized water was then.added (total volume equaled
950 ml after the addition). A 2 M TEAB stock solution was
obtained which, if stored at #9, was stable for about two

weeks e

Preparation of RNA from.Yeast

All.RNm used in this study, unless otherwise speci-
fied, was prepared by the method of Crestfield gt 2.1.. (27).
The yeast used was bought fresh for each preparation.

The final nucleic acid product was stored at -10°.

Preparation of Dialysis Tubing

All dialysis tubing used in the enzyme purification
procedure was routinely boiled for 1 hour in 0.2 M NaZCOB,
0.01 M EDTA solution with frequent stirring. This treatment
was followed by washing with deionized water. The boiling
procedure was repeated until the resulting wash liquid was
colorless. At this point the tubing was washed extensively
with deionized water to remove all Na2C03 and EDTA, and
stored in 50 percent ethanol at 4°.

Preparation of Ion Exchange Resins
DEAE-cellulose: A 40 g quantity of dry resin
(Cellex-D, Bio-Rad) was suSpended in 2 liters of deionized

10

water stirred mechanically in a 2 liter beaker. Fines were
removed by allowing the resin to settle for 20 minutes,
decanting the suspension.above the settled resin and resus-
pending in.2 liters of deionized water. Three to four such
cycles were sufficient to remove most of the smallest par-
ticles. (All of the following washings were done with the
aid of a sintered glass filter (2 liter capacity) fitted to
a suction flask and water aspirator. The resin was suspended
on the filter in two 2 liter batches of 0.1 N NaOH. Follow-
ing the base wash, the resin was washed with 2 liters of
deionized water. The base wash was repeated if the previous
water filtrate exhibited any color. After washing the resin
with water, 2 liters of 0.1 N HCl were added, the resin was
rapidly suspended, and the HCl removed immediately. The
resin was washed with 2 liter portions of deionized water
until the pH of the filtrate was about 5 (using pH paper).
A 2 liter portion of 0.1 N neon was then added and the resin
was washed with deionized water until the filtrate pH equaled
5. The washed, settled resin usually amounted to about 500
m1. This resin was then.washed with two 500 ml portions of
0.12 H Buffer A (see Preparation of Buffers) and stored at
4° in the same buffer prior to loading into a column.

CM-cellulose: A 25 g quantity of dry resin was sus-
pended in 2 liters of deionized water in a 2 liter beaker
and fines were removed by a procedure similar to that used
with DEAE-cellulose, except that with.CM-cellulose the
settling time was 7 minutes. The settled resin was then

11

resuspended and washed on a sintered glass filter, using
suction, with 1 liter of 0.5 M NaCl, 0.5 N NaOH solution.
Two liters of deionized water were used to wash the resin.
An.additional washuwith.NaCl-NaOH solution usually was
required to remove the last traces of color appearing in
the filtrate. A rapid washing cycle was then carried out
with 1 liter of 0.1 N HCl followed immediately by 2 liters
of deionized water. Finally, the 1 liter NaCl-NaOH wash
was done and followed by washing with 6 liters of deionized
water. If the resin was to be stored for a long period of
time before use it was washed with 1 liter of 95 percent
ethanol, dried at room temperature for 2 days, and stored
in a closed container. Just prior to use in a column, the
resin was suspended in 1 liter of 0.01 M Buffer B.

IEAE-SephadexlA-25: A 50 g quantity was allowed to
swell with constant stirring in 1 liter of deionized water
at b0 for 15 hours. Fines were removed by a method similar
to that used with DEAE-cellulose except that the settling
time for IEAE-Sephadex was 15 minutes. The resin was washed
with 0.1 N NaOH (2 liters per wash cycle) until the acidified
filtrate gave a negative test for chloride with silver
nitrate solution (usually 5-6 wash cycles were sufficient
for 50 g of resin). Base was removed by three 2 liter washes
with deionized water. If the resin was to be used with
ammonium formats, it was washed with 2 liters of 0.1 N formic
acid followed by removal of the excess acid with two 1 liter

washes of 1.0 M ammonium formats, pH 7.5. The resin.was

12
then equilibrated with 0.01 M ammonium formats, pH 7.5 by
repeated washing. If the resin was to be used with tri-
ethylammonium bicarbonate (TEAB) it was washed, following
the last base and water wash, with 2 liters of 0.5 M TEAB,
pH 7.8, followed by equilibration with 0.01 M TEAB. pH 7.8.

Thin-Layer Chromatography

For the rapid separation of individual deoxy- or
ribomononucleotides the method of Handerath (28) was used.
Essentially the procedure consisted of ascending thin-layer
chromatography on.polyethylenimine (PEI) cellulose sheets
using acetic acidpldCl solvents.

Paper Chromatography

The two-dimensional paper chromatographic method of
Felix‘gtwgl. (29) was used for the separation of nucleosides,
nucleotides, and nucleoside diphosphates of the deoxy series.
Chromatography in.the first direction was accomplished by use
of a solvent (solvent I) which was prepared by mixing 75
parts (by volume) of 95 percent ethanol and 30 parts (by
volume) of 1 M ammonium acetate adjusted to pH 7.5. This
solvent system.separated three groups of compounds the migra-
tion rates of which decrease in the following order:
nucleosides > nucleotides) nuc leoside diphoephates. The
four components of each group were separated in the second
direction by use of a solvent (solvent II) composed of iso-
propanol, water, and a saturated solution of ammonium sulfate

(2:18:80 by volume, respectively). Whatman No. 3 MM paper

13
(57 x 46 cm) was used for all separations. Further experi-
mental details are given by Laskowski (30) concerning this
method.

Polyacrylamide Disc Electrophoresis

The method of Ornstein (31) and Davis (32) was used
for polyacrylamide gel electrophoresis. Columns, 0.5 x 11.5
cm, were filled to a height of 5 cm with separating gel con-
taining acrylamide at 7 percent (w/v) concentration. The
other components in the separating gel were Tris-H01 buffer,
0.370 M, pH 8.9; N,N,N',N'-tetramethylethylenediamine (Tamed),
0.03 percent (v/v); ammonium persulfate, 0.07 percent (w/v);
and N,N'-methylenebisacrylamide (Bis), 0.181+ percent (w/v).
This gel ran at pH 9.5. The stacking gel contained Tris-H01
buffer, 0.062 M, pH 6.8; Temed, 0.0575 percent (v/v);
acrylamide, 2.5 percent (w/v), Bis, 0.625 percent (w/v);
riboflavin, 0.5 m8 percent and sucrose, 20 percent (w/v). The
buffer used in the electrode compartments was 0.025 M in
Tris and 0.192 M in glycine. Samples (0.5 ml) to be analyzed
were made 10 percent (w/v) in sucrose and 0.001 percent (w/v)
in Bromphenol Blue as tracking dye. Such samples were
routinely layered onto the stacking gel by diSplacement of
electrode buffer. A potential of 350-400 volts and a current
of 5 MA per column was maintained for a period of approxi-
mately 2 hours, or until the tracking dye band was observed
to reach the end of the gel. Immediately after turning off

the current, the gels were removed from the columns and

in
either sliced and eluted for subsequent enzyme assays, or
stained. The staining procedure consisted of soaking the
gels in a 0.5 Percent (w/v) solution of Buffalo Black in
7 percent (v/v) acetic acid for 1 hour. Destaining was
accomplished by gentle stirring in several 500 ml portions
of 7 percent acetic acid over a 2# hour period. Subsequently,
the destained gels were stored in closed tubes in 7 percent

acetic acid.

DNase Assays

When it was discovered that the hydrolysis of dena-
tured DNA exhibited two separate pH optima, assay methods
were developed to measure activity at both pH 8.0 (termed
DNase B) and pH 5.7 (termed DNase A).

DNase B: This assay measures the depolymerization
of heat-denatured DNA to products which are soluble in
lanthanum nitrate-H01 reagent. The reaction mixture con-
tained, in a total volume of 1.2 ml: 0.6 ml of 0.2 M
tris(hydroxymethyl)aminomethane, pH 8.23, 0.5 ml of dena-
tured DNA (15 minutes at 100°. followed by quick cooling to
0°) at a concentration of #0.0 (final conc. = 1.92 mg/ml)
5260 units1 per ml (determined following the heating proce-
dure), and 0.1 ml of enzyme diluted sufficiently with
0.12 H Buffer.A so as to obtain 5260 values between 1.0 and
2.0. The combination of DNA and Tris buffer was brought to

 

3Final pH at 37° with all components = 8.0.

15
37° and the reaction initiated by the addition of enzyme.
Incubation of the mixture was carried out for 20 minutes
at the end of which a 1.0 ml quantity of cold1 lanthanum
nitrate-HCl reagent (0.02 M La(N03)3, 0.2 N HCl) was added.
The mixture was stirred on.a vortex mixer and maintained at
4° for 15 minutes. Centrifugation at 1,100 x g for 20
minutes at u° was then.used to separate the precipitate
which had formed. The 5260 of the supernatant solution was
then determined in a Beckman DB Spectrophotometer. .Absor-
bance readings above 1.0 were determined by diluting the
solutions with water.

A unit of DNase B activity is defined as that amount
of enzyme which in 20 minutes causes the formation of
lanthanum nitrate-H01 soluble material amounting to 1.0 5260
unit1 per ml of reaction mixture.

DNase A: This assay measures the same type of
parameter as that in the DNase B assay except that the reac-
tion mixture is maintained at pH 5.7. The only difference
1n.reaction components is the substitution of 0.2 M imidazole,
pH 5.9“ for Tris. All other conditions as outlined under the
DNase B assay are maintained in the assay for DNase A. A
unit of DNase A activity is defined in the same terms as a
unit of DNase B with the implied restriction of pH.

RNase Assay
This assay measures the depolymerization of yeast RNA
to products which are soluble in lanthanum nitrate-H01 reagent.

 

*Final pH at 37° = 5.7.

16
The reaction mixture contained, in.a total volume of 1.2 ml,
0.6 ml of 0.2 M imidazole, pH 5.9“. 0.5 ml of yeast RNA
(see Methods for preparation) at a concentration of 90.0
A260 units1 per ml, and 0.1 ml of enzyme diluted sufficiently
with 0.12 M Buffer A so as to obtain.A260 values between
1.0 and 2.0. The procedures employed were essentially idsnp
tival to those described under the assay for DNase B. Solu-
tions of RNA were made fresh for each assay by dissolving
the nucleic acid (stored at -10°) in.watsr at a concentra-
tion of 2.0-2.2 mg per ml and then diluting the mixture to
a concentration of #0.0 A260 units1 per ml.

A unit of RNase activity is defined in the same terms
as a unit of DNase activity, i.e. that amount of enzyme which
in 20 minutes causes the formation of lanthanum nitrate-H01
soluble material amounting to 1.0 A260 unit1 per ml of

reaction mixture.

Q'qAMPase Assay

This assay measures the conversion of adenosine-3'-
monophOSphoric acid (3'-AMP) to free adenosine and inorganic
phosphate. The extent of reaction is determined by measuring
inorganic phosphate [by a modification of the method of
Dreisbach (33)] after a suitable incubation of enzyme and
3'-AMP. The reaction mixture contained, in,a total volume
of 2.0 ml: 1.8 ml of 0.11 M tris(hydroxymethyl)aminomethane,
pH 8.2. 0.10 ml of 0.04 M 3'-AMP, and 0.1 ml of enzyme
diluted sufficiently with 0.12 M Buffer A so as to obtain

1?
A310 values between 0 and 1.0. The combination of 3'-AMP
and Tris was brought to 37° and the reaction initiated by
addition of enzyme. A 0.5 ml aliquot of reaction mixture
was removed immediately after the addition of enzyme and
mixed with an equal volume of water-saturated liquid
phenol.5 Another 0.5 ml portion was removed after 15
minutes and treated in the sane manner. The aqueous and
phenol phases were separated by centrifugation at 1,100 x g
for 5 minutes at room temperature. A determination of inor-
ganic phosphate was then carried out on the aqueous (upper)
phase according to the following procedure. A 0.1 ml
aliquot of aqueous phase was added to 1.5 ml of a solution6
composed of 2 N sulfuric acid: 8 percent ammonium molybdate
(2:1, v/v) in screw-cap culture tubes. Two ml of xylene-
isobutanol (65:35, v/v) were then added. The tubes were
capped immediately and shaken 15 seconds, and centrifuged
for 5 minutes at 1,100 x g at room temperature. The A310 of
the upper (organic) phase was then determined in a Beckman
DB spectrophotometer in capped cuvettes. The xylene-iso-
butanol solution described above was used in the reference
cell. A standard curve relating A310 to inorganic phosphate
was constructed for each assay. The inorganic phosphate
standards were taken through the same extraction procedures

as the aliquots of reaction mixtures containing enzyme and

 

5rhis treatment eliminated the nonspecific binding of
inorganic phosphate to protein.

6Made fresh daily.

 

I"!

I'd

(D

l0~o

I“

I II

In

18
and 3'qAMP. A unit of 3'nAMPase activity is defined as
that amount of enzyme which causes the release of 0.1 umole
of inorganic phosphate per ml of reaction mixture in 15

minutes.

Protein Determination

Protein concentratiom were determined by the method
of Lowry 23 2;. (3“) using bovine serum albumen.as a refer-
ence standard. Colorimetric readings were made at 750 mu
on a Beckman DB Spectrophotometer.

Determination of Average Chain Length of Mixed Oligonucleotides

A.method for the determination of average chain length
of a mixture of oligonucleotides was provided by the determin-
ation of the ratio of total to terminal phoSphate. The
terminal phosphate was removed by the action of alkaline
phoSphatase (35). The total phosphate was obtained in inor-
ganic form by aching the sample with sulfuric acid. Inor-
ganic phoSphate was then measured in.both samples using the
same assay procedure described previously for 3'-AMPase.

Assay for total phosphate: A modification of the
method of Fiske and SubbaRow (36) was used for ashing the
sample. A 0.5 ml sample of oligonucleotide solution having
an.A260 of 2-3 was heated over a flame with 1.0 ml of 5 N
sulfuric acid in a 25 ml Folianu tube until heavy white
vapors appeared. After cooling, 2 drops of 2 N nitric acid
were added and heating was continued until white vapors

again appeared. If the sample still remained brown in color,

19

another drop of nitric acid was added. After cooling, 0.5
m1 of H20 was added to the colorless solution and the tube
was placed in a boiling water bath for 5 minutes. A 0.10
ml aliquot of this mixture was then added directly to 1.5
ml of the ammonium molybdate-sulfuric acid solution previ-
ously described under 3'-AMPase assay. The same hydrolysis
procedures were employed with inorganic phoSphate standards
ranging in concentration from 0.1-1.0 umole per ml, and a
standard curve relating A310 to phosphate concentration was
constructed.7

Assay for terminal phoSphate: The reaction mixture
contained 1.0 ml of 0.2 M Tris, pH 8.2, 0.10 ml alkaline
phosphatase (diluted to 20 units8 per ml with 0.1 M Tris,
pH 8.2), 0.25-0.50 ml of oligonucleotide solution, 40 A260
units1 per ml, and water up to a final volume of 2.0 m1.
This mixture was incubated at 37° for L: hours. A o. 5 ml
aliquot was then removed and mixed with 0.5 ml of water-
saturated liquid phenol. After separation of the aqueous
and organic phases by centrifugation (1100 x g for 5 minutes),
a 0.1 ml aliquot of aqueous phase was assayed for inorganic
phoSphate as described previously under 3'qAMPase assay.
The apprOpriate blanks lacking enzyme were included in order
to compensate for any free phosphate present in the oligonuc-

 

7It was found that this standard curve differed some-
what in slaps from that obtained from the 3'-AMPase assay
because of slight volume differences.

8As defined by Carin and Ievinthal (35).

20
leotide mixture. Average chain length was then determined
from the ratio of total phosphate to terminal phosphate.

EXPERIMENTAL.RESUITS
Purification of Enzyme

The term "enzyme," as used throughout this descrip-
tion, refers to all associated nuclease and 3'-nucleotidase
activities which subsequently will be shown to be insepar-
able.

Preparation of Muskmelon Seeds

It has been found that the purification procedure is
most conveniently carried out on not more than 1 kilogram of
seeds at a time.

One kilogram of dry seeds was washed in a 12 liter
chromatography jar with distilled water over a. one half
hour period with frequent stirring. The wet seeds were
then spread on a double layer of cheesecloth and dried in an

oven overnight at a temperature of 35-“00.

Homogenization of Seeds

Unless otherwise specified all procedures were carried
out at 0-h°. The washed, dried seeds were broken.up into a
meal using a Hobart (Model #612) meat grinder with strainer
fitting containing h mm diameter openings. The meal was
then divided into two equal parts and each was homogenized
with 1 liter of cold1 deionized water for 2 minutes in a

21

22
commercial Waring Blendor (1 gallon capacity). Following
homogenization, the suspension was centrifuged at 13,200 x g
for 30 minutes and the supernatant solution was filtered
through glass wool to separate unwanted bits of floating

material.

Ammonium Sulfate Fractionation

The filtered crude extract was brought to 87 percent
of saturation by the addition of solid ammonium sulfate
(previously passed through a standard 40 mesh screen) in the
amount of 575 g per liter of extract. Addition of the
ammonium sulfate was done slowly over a 1 hour period while
the solution was being stirred mechanically. After stirring
for 20 minutes the solution was centrifuged at 13,200 x g
for 20 minutes. Following centrifugation the supernatant
fluid was discarded and the precipitate was dispersed into
#00 ml of 55 percent ammonium sulfate solution (made by
combining 220 ml of saturated solution with 180 ml of deion-
ized water). The suspension was stirred mechanically for
30 minutes after breaking up any lumps of precipitate result-
ing from the transfer procedure. The suspension was then
centrifuged at 13,200 x g for #5 minutes. The supernatant
solution, after being separated from the precipitate, was
brought to 90 percent of saturation by the addition of solid
ammonium sulfate in the amount of 253 g per liter of solution.
After stirring the suspension for 15 minutes it was centri-

fuged at 27,000 x g for 30 minutes. The supernatant fluid

‘ A

81:13

23
was discarded and the precipitate dissolved in 100 ml of
0.06 M Buffer A. This solution.was stored at 4° for approx—
imately 12-15 hours before proceeding with dialysis.
Dialysis was carried out in two 2.3 cm diameter cellulose
bags (see Methods for washing procedure) for a total of 3
hours against three 2 liter portions of 0.12 M Buffer A.
The contents of both bags were combined and centrifuged at
27,000 x g for 15 minutes in order to remove a small amount

of precipitate which had formed during the dialysis.

DEAF-Cellulose Chromatography

The supernatant solution from the previous centrifuga-
tion.was brought to room temperature1 and diluted with an
equal volume of deionized water, also at room temperature.
The remaining manipulations concerned with DEAE-cellulose
chromatography were carried out at room temperature. Follow-
ing dilution the dialyzed ammonium sulfate fraction was
applied to a DEAR-cellulose column (3.0 x 20.0 cm) previously
equilibrated with 1 liter of 0.12 M Buffer A at a flow rate
of 2.0 ml per minute regulated with a peristaltic pump.
Following application of the ammonium sulfate fraction, the
column.was washed with the same buffer with which it was
equilibrated until the optical density at 280 mu of column
effluent came down to 0.10 (time required 4 hours at a flow
rate of 2.0 ml per minute). A gradient prepared from 500 ml
each of 0.25 M Buffer A and 0.75 M Buffer A was applied to
the column at a flow rate of 1.5 ml per minute. The collec-

tion of 10 minute fractions was begun immediately after

 

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(‘3

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"1

24
starting the Buffer A gradient.

The elution pattern of protein and enzyme activity is
shown in Figure 1. Enzyme activity was located by assaying
0.10 ml portions of alternating fractions. The bulk of
activity was consistently eluted between 0.30 and 0.40 M
ammonium acetate. Fractions containing at least 0.50 units
of DNase per ml (see DNase B assay under Methods) were
pooled. About 60 percent of the activity applied to the
column was found in the pooled material whereas less than
2 percent of the applied protein was present in this frac-

tion.

CM-Cellulose Chromatography

The pooled fraction from the DEAR-cellulose column
was cooled to 4° and adjusted to pH 5.5 with 1 N acetic acid.
Dialysis was then performed for a total of 6 hours against
three 2 liter portions of 0.01 M Buffer B at 4°. The refrac-
tive index was routinely determined to provide a measure of
the salt concentration9 before applying the material to the
CM-cellulose column. The dialyzed DEAE-cellulose fraction
was then applied to a CM-cellulose column (2.2 x 30.0 cm),
previously equilibrated with 1 liter of 0.01 M Buffer B at a
regulated flow rate of 2.0 ml per minute. The column was
washed with the same buffer for 1 hour at a flow rate of

2.0 ml per minute. A gradient prepared from 200 ml each of

 

9It was previously found that if the initial salt
concentration was above 0.07 M the enzyme would pass through
the column.

25

 

 

 

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27
0.01 M and 0.40 M Buffer B was used at a flow rate of 1.0 ml
per minute to elute the enzyme. Fractions were collected at
10 minute intervals. Figure 2 shows the elution pattern for
protein and enzyme activity. The peak of activity was
located in a manner similar to that used on the DEAR-cellulose
column. .All fractions containing any measurable DNase activ-
ity were pooled. The pooled fraction was adjusted to pH
9.010 with 1 N ammonium hydroxide before storage at 4°.

Chromatography on P-100 (RAB-Cellulose)
The following procedure was carried out at room tem-

1 A Bio-Gel.P-100 column (2.0 x 70 cm) was OOH!

perature.
structed. A pad of glass wool 3-4 mm thick was placed on
the resin and a 2.0 x 10 cm bed of DEAE-cellulose was poured
on top of the glass wool. The column was equilibrated with
1 liter of 0.12 M Buffer A at a flow rate of 1.0 ml per
minute. The CM-cellulose enzyme fraction, previously
brought to room temperature after storage at 4° for 12-15
hours, was applied to the column at a flow rate of 1.0 ml
per minute. Following application of the sample, the column
was washed with 0.12 M Buffer A for 2 hours at a flow rate
of 1.0 ml per minute. No enzyme activity could be detected
in the pass-through or wash fractions. A 30 m1 quantity of

0.75 M Buffer A was then applied to the column followed by

 

1°11: has been found that extensive loss of enzyme
activity occurred if the CM-cellulose pooled material was
stored at pH 5.5 for any amount of time: therefore, assays
and pH adjustment were done as soon as possible.

28

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30
the previously used 0.12 M Buffer A. Beginning at the time
when the 0.75 M buffer was applied, five minute fractions
were collected at a flow rate of 1.0 ml per minute. Figure
311 indicates the relative positions of elution of the
enzyme activity and protein. The central six fractions
from the enzyme peak were pooled and stored at 4°. This
purification step routinely gave 80-90 percent recovery of
enzyme activity originally applied to the column, an
increase in specific activity to a value 5-6 times that of
the CM-cellulose enzyme fraction, and a reduction in volume
to less than one tenth of that of the CM-cellulose fraction.
The enzyme preparation in this form was stored at -20° over
a period of 6 months without any detectable loss of activ-
ity.

In order to have a sufficient quantity of enzyme
preparation with which to do both characterization and
specificity studies six 1 kilogram batches were purified up
to and through the DEAE-cellulose step. The DEAR-cellulose
fractions from three of these were combined and carried
through the remaining procedures. This same treatment was
repeated for the other three DEAR-cellulose fractions. At
the conclusion of the purification, both batches (represent-
ing a total of 6 kilograms of dry seeds originally) were
combined. This enzyme preparation.was placed in storage in

1.0 ml quantities at -20°. Unless otherwise specified all

 

11
enzyme.

Figure 3 summarizes the results of a 3 Kg. batch of

31

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as on 4 .ouoh scam came on» no meson N you sasaoo on» now: on come on:

d nomusm z NH.0 one .oeasaa sea as 0.a no open scam o no oodaaao mos

noauomnh sauce omoafiaaeouzo 0:» .4 Mahmum : NH.0 ho Honda H Spa: Sade—”00

on» so 3395338 mosaics .so 3 no senses a 8 Enos 87m one

go no» so pounce not con names onoHsHHooumdmn 4 .pwoe on» ad pondaonoo
no oopospphsoo no: .80 0.05 H 0.m .sasaoo 00H:m_Hooaodm a

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essences. cocaina one Joe: .8on .58on so .338 soap
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32

(lw/suun) KHAHQV

 

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33
results given in this thesis have been derived from the use
of the 6 kilogram preparation of enzyme. Table I gives the
results of a typical 1 kilogram preparation carried through
all previously described manipulations.

Further Attempts to Separate the Four Activities

Isoelectric Focusing

Even though the four activities appeared to purify
together as shown in the previous discussion it was desirable
to have additional evidence bearing on the question of their
inseparability. Thus, electrofocusing was used in an attempt
to separate the activities. .An.LKB Model 8101 electrofocus-
ing column (total capacity a 106 ml) was loaded with ampho-
lyte solutions to give a final linear pH gradient of 3.0-10.0.
A 1.0 ml sample of P-100 enzyme (200 DNase B units) was
added after one half of the total ampholyte solution had
been layered in the apparatus. A potential of 300 volts was
applied to the column for a period of 48 hours using a
Krohn-Hite Model UHR-240 constant voltage power supply. The
column was emptied from the bottom by pumping water into the
top at a flow rate of 1.0 ml per minute while collecting
1.0 minute fractions. During loading, electrofocusing, and
unloading the apparatus was maintained at a temperature of
o.5°. The pH of every fifth fraction was determined while
nuclease and 3'-AMPase were measured in alternate fractions.
Data from the above determinations are summarized in.Figure 4.

It was evident that all activities were found in the same

34

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as ooa.c no.0 oms.~ scans
ms oao.a aa.n oo~.m oeoHsHHoonzo
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mm sa.s mmo.~ o~0.m oneness asascssa
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m onozo ws\nsass “wee em onozo
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H wands

35

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one: omomzdiR use .335 .omozn not mashed .msogoonm speeds 0.H was

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s so.“ 00.30am no: n30» 00m mo asapsopoa 4 .msfisfioaao on» ad coached

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mo 0:30on me Home: H.930 m 93% on muoapsHom 3.43293 suds pocooa no:
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36

(Iw/suun) Mmuov

emnErz 00:00am

 

 

 

 

 

 

O: OO_ 00 00 O 00 00 CV Om ON 0_
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37

peak at a position correSponding to an isoelectric point of
4.4. The ratios of the various activities, however, did not
conform to those previously observed in the latter stages of
the purification scheme. This discrepancy was probably due
to loss of activity by the enzyme preparation, a condition
not surprising in light of previous observations of greatly
decreased stability at pH values below 6.0. The RNase and
3'-AMPase determinations were performed 15 and 18 hours
respectively later than the DNase measurements. .An attempt
was made to reactivate the enzyme by storing the peak tubes
at 4° after adjusting the pH to 8.0 (with and without added

zinc acetate, 0.1 mM), but this attempt proved unsuccessful.

Polyacrylamide Disc ElectrOphoresis

Two 0.5 ml quantities of P-100 enzyme each containing
22 ug of protein (100 DNase B units) were subjected to elec-
trophoresis in separate columns using a 7 percent polyacryl-
amide gel at pH 9.5 (as described in Methods). Bromphenol
blue was present in both samples to act as a tracking dye.
When the dye band reached the end of the gel, the current
was turned off. Both gels were removed, one was stained with
Buffalo Black (as described in Methods), and the other was
out into sections each 2 mm thick. Each section was placed
in 1.0 m1 of 0.1 M Tris buffer, pH 8.2, and allowed to stand
at 4° for 15 hours. At the end of the 15 hour period the
gel slices were discarded and the eluates assayed for DNase,

RNase, and 3'-AMPase (as described in Methods).

38

Figure 5 indicates the staining pattern for protein
and the relative position of the enzyme activities found in
the slices. It is evident that even at this degree of
purity the enzyme preparation contained a number of differ-
ent proteins. The four enzyme activities, however, were
found to be associated with one protein band (the slowest
moving one) and were evidently distributed through this
band in a constant ratio to one another. Subsequent experi-
ments utilizing up to 100 ug of protein per sample gave
essentially the same staining pattern as that observed in
Figure 5.

PrOperties of the P-100 Enzyme Preparation

Contaminating Enzyme Activities

In order to determine whether or not significant
amounts of contaminating phosphomono- or phOSphodiesterase
activities were present in the enzyme preparation following
the P-100 purification step, aliquots of the P-100 fraction,
each containing 2.0 units of DNase B activity (specific
activity of 4,550), were tested with various substrates at
pH's 4.5, 7.0, and 9.0 for 24 hours at 37°. No detectable
hydrolysis was observed with bicep—nitrophenyl phoSphate,
S'gpenitrOphenyl uridylate, S'fip-nitrophenyl thymidylate,
5'-penitrophenyl-2'-deoxyadenylate, or 3'ep-nitrophenyl
thymidylate. The four common 2'-deoxynucleoside-5'-mono-
phOSphates (dAMP, dGMP, dCMP and TMP) were also not hydro-
lyzed under the above conditions. A slight amount of

 

39

Figure 5: Staining Pattern of Protein and Distribution of
Enzyme Activities in a Polyacrylamide Gel.After
Electrophoresis

Two 22 ug quantities of P-100 enzyme (each containing

100 units of DNase B) were subjected to electrophoresis in

7% polyacrylamide gels at pH 9.5 (as described in Methods).

One gel was stained for the presence of protein and the other

out into sections each 2 mm thick. Each section, after soak-

ing in 0.1 M Tris buffer, pH 8.2, for 15 hours, was assayed
for DNase, RNase, and 3'-AMPase (as described in Methods).

Units/ml Units/ml Units/ml

Units/ml

9[ Ill llllll G
9991!
“zin'ifi'htfifl“3.3..

30,. DNase A

60b-

40-

20—

30_ DNase B

60—

40r-

20+-

RNase

.40- 32mm

I20-

80%

4O

4O

 

 

 

 

 

 

 

 

 

 

 

   

        

41
hydrolysis of p—nitrophenyl phosphate was observed. Further
discussion concerning other phosphomonoesterase activity is
found under Specificity Studies. During the course of
extensive hydrolysis of denatured DNA no nucleosides nor
inorganic phosphate could be found. Thus, it has been con.
cluded that 5'-nucleotidase and phosphodiesterase activities
were absent from the enzyme preparation following chroma-

tography on P-100.

pH thima of DNase Activity

Figure 6 indicates the relationship between pH and
DNase activity as measured by the standard lanthanum nitrate-
HCI assay described under DNase Assays in Methods. Each
reaction contained 0.65 ml of 0.2 M buffer, 0.5 m1 denatured
DNA (as described in Methods), and 0.05 ml of P-100 enzyme
preparation previously diluted to a concentration of 20 DNase
B units per ml with 0.12 M Buffer A. Measurements of pH
were made on the substrate—buffer mixture at 37°. Reactions
showing the highest activity underwent a pH change of less
than 0.1 over the 20 minute incubation.period. The existence
of two separate pH optima, first observed with cacodylate and
Tris buffers, was substantiated when reactions were carried
out in imidazols and histidine buffers in the pH 5 to 7
:range. Another important result was the great difference
between the activities of the enzyme in the presence of
ihistidine and imidazols buffers at pH 8.0-8.5. A similar
relationship has also been observed with the 3'-AMPase

42

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a DIIID .33 “III- .opoamooooo nollno .ouoeooo ”I .3933

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some ca 2 moa.0 no: soapoausoosoo acumen Honda .noospo: ca confluence no
poaaomnoa one: 020 possessoonpoos means hedbapoo omozm no“ whomn<

onozn you obnso mpabapodima ”0 onwmam

 

43

 

 

 

 

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44
activity (see Figure 10). In order to rule out buffer effects
(note in Figure 6 the shift of the optimum to a lower pH and
the stimulation of activity with imidazole and histidine as
compared to cacodylate), DNase activity was measured in the
absence of buffers by use of a pH stat (E. H. Sargent and
Co.). The instrument was used principally to maintain the
pH and temperature of a reaction. Aliquots were withdrawn
and assayed at the beginning and end of the incubation
period. In a final volume of 6.0 ml, a typical reaction con-
tained: 5.0 ml denatured DNA at a concentration of 1 mg per
ml in 0.12 M NaCl (18.0 A260 units1 per ml after denatura-
tion), 0.1 ml of P-100 enzyme previously diluted to a con-
centration of 40 DNase B units per ml with 0.10 M NaCl, 0.1
to 0.2 m1 of 0.01 N HCl or 0.01 N NaOH to bring the pH to an
appropriate value, and water to 6.0 ml. All reactions were
incubated at 37° for 20 minutes after adding enzyme. Aliquots
of 1.2 ml were removed immediately after adding enzyme and
at the end of 20 minutes. These aliquots were added to 1.0
ml of cold1 lanthanum nitrate-H01 reagent and treated as
described under DNase Assays in Methods. Figure 7 indicates
the relative activities obtained at various pHs. It is
evident that the enzyme does exhibit DNase activity over a
rather wide range of hydrogen ion concentration. The
results shown in.Figure 7 are qualitatively similar to the
cacodylate-Tris buffer curves presented in Figure 6.

45

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nu masons ooozn noose oondnoooo no anowoon vauouospas sanonusoa pace
so as o.a seas soaeocaasooea so posses sonssssosa one so one one so
one oahuno «o nodpdooo one Hopes maouoaoosaa oohoooo oaos Ha N.H mo
oposodad .onm no oopsnaa 0N you 950 coannoo onoz oshuno mo soapauoo
one wedsoaaom uncaponsonH. .Hs 0.0 no ossaob Honda o op nopos one
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ionoo as 0.m "wadsoaaoh one weanaonsoooonspwas nodpooon o no oposoaao
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46

 

 

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47

pH Optimum of RNase Activity

The relationship between.RNase activity and pH for
various buffers is shown in Figures 8 and 9. Conditions
used are described in Methods under RNase Assay. One mod-
ification made, however, was that the enzyme was diluted
to a concentration one fourth that used in the DNase pH
optima study. The absolute activities at the maxima in
Tris and imidazols buffers were equal. As with the DNase,
the RNase exhibits activity over a rather wide pH range.
As with the DNase activity, the pH Optimum of RNase activ-
ity shifts from about 6.5 to 5.5 when imidazols or histidine
is substituted for cacodylate buffer.

pH Optimum of 3'-AMPase.Activity

Figure 10 shows the effect of varying pH on 3'-AMPase
activity. The assays were done in accordance with the
details given.under 3'-AMPase Assay. Enzyme concentrations
were equal to those used in the determination of DNase pH

optima (Figure 6).

Effect of Metal Ions

In order to investigate the relative effects of vari-
ous metals on DNase,.RNase, and 3'eAMPase activities a
series of experiments were carried out using the assay
conditions outlined in Methods for the appropriate activity.
.All metals were added as the chloride salts to the incuba-
tion mixtures just before the addition of enzyme. The final

concentration was 10'3 M (except for Na, Li, and K ions

48

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aonwdn 0.3 .00 0080009 000 09 809.348 .908 09‘88 0 00020 0.m «0 80990.99
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89 009980000 00 0089098908 00: 8099099800800 800.880 009.302 89 00004

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49

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IO

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i. : Figure 9: pH-Activity Curve for RNase
l Assays for RNase activity were performed as described

in Methods. All other conditions were as described in the

legend for Figure 8. Buffers: imidazole, A—A;

histidine , 0—4.

51

 

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53

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5“
which were 10'2 M). The appropriate controls containing
metal ions but without enzyme and/or substrate were included
in order to detect invalid A260 or A310 readings. The P-iOO
enzyme preparation used was diluted to a concentration of
10 DNase B units per ml with 0.12 M Buffer A. The final
concentration of zinc in the DNase and RNase assays. there-
fore, was h x 10'7 H, and in the 3'-AMPase assay, 2.4 x
10"7 M. These levels were assumed to be insignificant com-
pared to the levels of added metals. Table II summarizes
the results obtained. None of the metal ions led to major
increases in.anzyme activity. although the divalent cations,
Mg“. Ba‘H', Ca‘H', and 30‘” stimulated DNase B and RNase
activity 7-20 percent. and similar increases in.aNase and
3'-AMPase activity were found with Li”. Na+, and K+. The
other divalent metal ions tested were generally inhibitory
to all activities of the preparation.

It must be pointed out, however, that these changes
in enzymatic activity brought about by the addition of mono-
or divalent cations could reflect interactions of the metals
with substrates, especially in the case of polynucleotides
(9), rather than direct effects on the enzyme involved.

Ratios. of Dflase AJ RNase, and 'qAMPase to Dnase B
Through Purification

In order to ascertain whether or not the four activ-
ities mentioned above purify together, measurements of each
activity were made following each step in the purification
jprccedure. Table III indicates the relationships, expressed

97.7.7.“
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55
TABLE II
Effect of Cations on.Activity

 

 

Percent of Activity in
Absence of Cation

 

Addition to Assayb DNase Ba DNase Aa RNase“ 3'-AMPasea

 

pH 8.0 pH 5.75 PH 5.7 pH 8.0
None° 100 100 100 100
Mgc12 112 1ou 107 97
BaClz 11b 98 107 102
SrClz 120 ion 113 98
oac12 123 91 11t 127
MnClz 63 77 57 25
ch:12 3a 66 37 6
coci2 52 78 51 77
Fe012 18 35 12 72
NiClz 37 62 #5 83
CdClz 36 56 29 #9
CuClZ 82 #3 27 115
1401 98 90 117 114
xc1 94 84 123 125
Neal 96 92 122 123

 

gAJJ.reactions were performed at the pH optima values indi-
cated for each type of activity.

‘firne final concentration of the additives were 0.001 M
except for LiCl. KCl, and NaCl which were 0.01 M.

9A1J.reactions contained 0.01 M ammonium acetate (except
:3'-AMPase in.which the ammonium acetate concentration was
().006 M) because of the enzyme addition.

56

 

 

 

 

 

 

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HHH Manda

57
as the ratio of each specific activity to that of DNase B,
obtained from the same preparation as that used for the data
in Table I. As is evident, the ratios involving DNase A
and 3'qAMPase change in the same relative prOportions
throughout most of the purification scheme. The ratio
involving RNase, however, does not stabilize in the last
step of the purification. This could reflect the presence
of some RNase activity not associated with the other

nuclease activities in the preparation.

Molecular Weight Determination

In order to estimate the molecular weight of the P-1OO
enzyme preparation. the gel filtration method of Andrews
(37, 38) was used. A 2.5 x #6 cm column of Sephadex G-100
(ho-120 u bead size) was equilibrated with 0.12 M Buffer A
at a flow rate of 0.5 ml per minute at 4°. All samples were
applied to the column in.a volume of 2.0 m1 of equilibration
buffer containing 10 mg of sucrose. The material loaded on
the column was layered beneath the equilibration buffer
above the resin bed. Buffer flow was maintained at a con-
stant rate by the use of a peristaltic pump; fractions were
collected at 10 minute intervals. ‘Various combinations of
standard proteins were chromatographed to give a calibration
curve relating elution volume to the known molecular weight
of each standard. The variation of elution.volume of consecu-
'tive passes of the same protein through the column was in the
order of 3 percent. Table IV lists the various standard

58

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59

proteins used in constructing the calibration curve shown in
Figure 11. As can be seen in.Figure 11 the extrapolated
value for the molecular weight of the P-100 purified musk—

melon nuclease is about 50.000.

Stability of P-100 Enzzge Preparation

It has been previously shown (see Purification) that
the P-100 enzyme preparation is unusually stable to storage
at -100 for relatively long periods of time. In addition,
less than 15 percent of the DNase activity was lost in 6
months when P-100 enzyme preparations were stored at 4° or
lyophilized and kept at -1o°. An experiment designed to
find a way to abolish DNase activity indicated that at pH
7.5 in the presence of 1 mM magnesium chloride and sub-
strate levels of dDNA the P-100 enzyme [Nase activity was
not affected by eXposure to temperatures of 100° for up to
15 minutes. At pH 10, however, the DNase activity was
destroyed. Preliminary work on the purification scheme
also showed that following the ammonium sulfate fractiona-
tion the DNase activity was particularly stable when heated
to a temperature of 80° for 20 minutes if two Specific con-
ditions were maintained. These conditions were the presence
of 1 mM zinc chloride at pH 9.0. Maximum stability was
observed only when both.parameters of pH and zinc concentra-
tion were maintained. This heat treatment would have been
retained as part of the purification procedure were it not

for the fact that this procedure appeared to significantly

60

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61

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62
reduce the yield of DNase activity eluted from the DEAE
cellulose column in the subsequent step as compared to
preparations not heated. Dialysis for an extended period
of time also appeared to decrease DNase activity. When a
h ml sample of P-100 enzyme was dialyzed against 2 liters
of 0.10 M sodium chloride at pH 8.0 for 27 hours a 50 per-
cent decrease in total DNase activity was observed as com-
pared to an undialyzed control. Complete loss of enzyme
activity was also observed when.a 1.0 m1 sample of P-100
enzyme was passed through a 1.0 x 15.0 cm Bio-Gel P-Z
column previously equilibrated with 0.01 M sodium chloride
at pH 5.7. Attempts to reactivate the enzyme by addition
of zinc acetate (0.1 mM final concentration) and/or adjust-

ment of the pH to 8.0 were unsuccessful.
Studies on.Enzyme Specificity

Relative 3'-Nuoleotidase Activities

Since nuclease preparations from other plant sources
have been shown.to exhibit phosphatase activity toward not
only 3'~AMP but 3'-UMP. 3'-GHP, and 3'-CMP as well. it was
desirable to see whether or not the muskmelon nuclease also
demonstrated such a range of specificity. The four 3'-phos-
phoryl mononucleotides were used as substrates in a reaction
containing components as described in Methods for the 3'-AMPase
Assay. Each of the four substrates was present at a final
concentration of 2 mm in the assay. Equal amounts of P-100

enzyme were added to each reaction. Aliquots were removed

63

at two incubation times, 15 and 30 minutes, in.arder to
check for linearity of inorganic phosphate production with
time. Under the conditions where half of the 3'-AMP had
been hydrolyzed in 30 minutes no hydrolysis of 3'-CMP could
be detected. The relative rates of hydrolysis, expressed
as percent of maximum activity with 3'-AMP equal to 100.
for all four mononucleotides tested is given.in Table V.

The next question to be considered was whether or
not the correSponding 2'-deoxynucleoside-3'-monophoSphates
were hydrolyzed by the muskmelon nuclease preparation.
Work with the nuclease from mung bean (26) indicated that
the 3'-nuc1eotidase of that enzyme was sugar-specific in
that only 3'-ribonucleotides were hydrolyzed. Preliminary
eXperiments with the muskmelon nuclease using enzyme and
substrate concentrations comparable to those used in the
study summarized in.Tab1e V indicated that no breakdown of
2'-decxyribonucleoside-3'-monophoSphates occurred. However,
in order to be certain, the levels of enzyme were increased
forty-fold, and incubation times were maintained at 15 hours.
Reaction mixtures of 0.10 ml total volume contained either
imidazole, 0.10 M, pH 5.9 or Tris-HCl, 0.10 M, pH 8.2 plus
mononucleotide at 1 mM final concentration” After the incu-
bation period, all mixtures were frozen in a dry ice-ethanol
bath and lyophilized to dryness. The dry residue in.aach
case was dissolved in 0.04 ml of H20, spotted an.uhatman No. 1
paper, and chromatographed in solvent I for 16 hours. Mix-

tures without enzyme were included as controls in the incuba-

64

TABLE'V

Relative 3'-Nucleotidase Activities Toward
Ribonucleoside-3'-Monophosphates

 

 

 

Mononucleotide Percent of
Assayeda Maximum.Aotivity
3'-AMP 1oo
3'-GMP 73
3'-UMP 34
3'-CMP 0

 

QAssays were carried out as described in Methods for 3'-AMPase.
Incubation times of 15 and 30 minutes were used. Liberation
of inorganic phosphate was linear with time up to 30 minutes.
Activities were calculated from the data obtained in the 15
minute incubation.

65
tion and chromatographic procedures. The four 2'-deoxynuc-
leosides were also chromatographed in lanes adjacent to each
corresponding nucleotide. Table VI summarizes the results
found. At pH 8.0 there definitely was hydrolysis of 3'-dAMP.
3'-dGMP, and 3'-THP but not of 3'-dCMP. The only nucleotide
dephosphorylated at pH 5.7 was 3'-dAHP, and even that was
not completely hydrolyzed under the conditions used.

Activity Toward Cyglic Mononucleotides 5

RNases from various sources (9) have been shown to y

 

catalyze the hydrolysis of nucleoside-2',3'-cyclic monophos-
phates or the corresponding 3',5'-cyclic derivatives.

Because a number of these enzymes have been found in plant
tissues, it was necessary to determine whether or not the
muskmelon nuclease preparation exhibited such activity.

Since it was already known that the muskmelon nuclease
formed 5'-phosphoryl terminated products upon hydrolysis of
both.RNA and DNA (see later sections on the determination of
phosphate position in mononucleotides from hydrolyzates of
RNA and dDNA), the presence of cyclic phosphodiesterase
activity would indicate that the enzyme preparation was con-
taminated by at least one other RNase. Reactions containing
the following substrates - adenosine-3'.5'-monophosphate,
adenosine-2',3'-monophosphate, guanosine-B',5'-monophosphate,
cytidine-Z',3'-monophosphate, and uridine-Z',3'-mcncphosphate -
were incubated 15 hours at 37° under the same conditions of

substrate and enzyme concentration as those involving the

66

TABLE VI

Relative 3'-Nucleotidase Activities Toward
2'-Deoxyribonucleoside-3'-Monophosphates

 

 

 

Mononucleotide pH 5.7 pH 8.0
Testedb
3'-dAMP ta +
3'-dGMP - :
3'-TMP_ - +'
3'-dCMP - -

 

+ signifies complete conversion to the Zi-deoxynucleoside

l+

signifies partial conversion to the 2'-deoxynuoleoside

- signifies no detectable conversion to the 2'-deoxynuoleo-
side.

bAll substrates were present at a final concentration of 1 mM.
Each reaction contained 2.0 units of DNase (8.2 units of

3'-AMPase) in a final volume of 0.10 ml. Reactions were
incubated at 37° for 15 hours.

6?
2i-deoxyribonucleoside-B'-monophosphates. Chromatography
of the reactions in Solvent I12 for 15 hours indicated that
no hydrolysis of the cyclic mononucleotides had occurred as
a result of prolonged exposure to the muskmelon nuclease

preparation.

Relative Hydrolysis Rates of Native DNA.
Denatured DNA. and RNA

The earliest work done on the purification of DNase
activity from muskmelon seeds involved the use of native
DNA exclusively (39). When it became apparent that heat-
denatured DNA and RNA were hydrolyzed more rapidly than
native DNA, an experiment was conducted to demonstrate the
relative rates of hydrolysis of all three substrates. Since
activity toward denatured DNA exhibited two separate pH
optima, rates of hydrolysis were measured at both pHs for
an additional means of comparison. Reactions containing
native DNA13, denatured DNA or RNA at concentrations speci-
fied in Methods under the appropriate assay, but ten-fold
greater in volume, were brought to 37°. A 1.0 m1 aliquot
was removed immediately, and at various time intervals,
after adding 1.0 m1 of P-100 enzyme (previously diluted to
a concentration of 20 DNase B units per ml with 0.12 M
Buffer A). One ml of cold1 La(N03)3-HCl reagent was mixed

 

12Cyclic mononucleotides migrate in Solvent I with
Rf values midway between the normal phosphomonoesters and
their corresponding nucleosides.

13The concentration of native DNA was adjusted to
equal that of denatured DNA on a weight per volume basis,

68
immediately with each reaction aliquot and the mixture treated
as described for the appropriate assay in Methods. ApprOp-
riate blanks not containing enzyme were included and incu-
bated in a parallel fashion. Figure 12 summarizes the data
obtained. An additional experiment using calf thymus DNA
instead of DNA from salmon sperm gave almost identical results

to those shown in.Figure 12.

Mode of Action on RNA

The method of Birnboim (40) was used to characterize
hydrolysis products in order to determine whether the mode
of action of the P—100 enzyme preparation was endo- or exo-
nuoleolytic in nature. Essentially, the method consisted of
separating the products of early stages of hydrolysis on a
gel filtration column. Exonucleases characteristically
degrade polynucleotides by stepwise removal of mononucleo-
tides from one end of the chain. Endonucleases, on the
other hand, make scissions in the polynucleotide chain at
points other than the ends thus giving mainly oligonucleotide
products of decreasing chain length as hydrolysis proceeds.
In this experiment it was desirable to test the method with
enzymes which were known to be either exo- or endonucleases.
Figures 13a-13c summarize the results obtained from three
separate reactions containing the components described in
the legends. For those reactions containing enzymes (see
Figures 13b and 13c), 0.5 ml aliquots were removed at various

times after addition of the enzyme. The aliquot was pipetted

69

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"all. .o.m mo1§oe “mile {..m mousse “41114 4.6 mousse

”ma1opoapmnsm .Aommo oceanaoaaae on» you scones: a« confluence

no psomeoa Hom1mfimozvoq mo as 0.6 spas pounce» one: mposdaao omega

.Ad Hommsm z NH.o and: as you mean: m onezn om mo soapenpsoosoo

m on passage mamooa>onav manage ooa1m do as o.H ho sodpauoe 0:» Hopes

mead» msoaaob so poboaon one: He o.H no nposodad .muonpez_sa whence

opeaaaoaaae on» nous: confluence coon» soap Hopeoaw mead» ca one:

moasao>_soapooom .moospoz ad confluence escapenusooaoo no 42m no .420
anode soeaon consensoo no spans: accede oosampsoo macapoeom

38 one .22qu SS
eossoesoo . 2755 58 33oz do ooeom 392823 ohaoaom .3 353a

7O

0 —

 

 

 

 

 

CON

J On:

01 lo). 00.

1.1. .1. on
all

\ 0 \O \O\ \
_ \4
q l

 

092W

 

71

Figure 13a: Chromatography on Sephadex G-25 of a Mixture of
RNA and 5'-AMP

Figure 13b: Chromatography on Sephadex G-25 of a Reaction
Containing RNA and Venom.Ph03phodiesterase, a
Known.Exonuclease, at Various Stages in the
Hydrolysis

The reaction contained 8.0 ml RNA at a concentration
of 20.0 A260 units per ml in 0.1 M Tris-HCl, pH 8.8, 0.8 ml
of 0.36 M magnesium chloride, and 0.8 ml of venom phosPho-
diesterase, 2.3 mg per ml. Each time a 0.5 ml aliquot was
removed for chromatography on Sephadex G-25, a 1.0 ml aliquot
was simultaneously removed and treated with 1.0 m1 of cold
lanthanum nitrate-RC1 reagent as described in Methods under
RNase Assay. The various elution profiles are thus expressed
in terms of the percent of total nucleic acid converted to
lanthanum nitrate-RC1 soluble products as determined by the
A260- a) 0%: b) “7%: c) 71%.

Figure 130: Chromatography on Sephadex 6-25 of a Reaction
Containing.RNA and Pancreatic RNase, a Known
Endonuclease, at Various Stages in the Hydrolysis

The reaction contained 8.0 ml RNA at a concentration
of 20.0 A260 units per ml in 0.1 M Tris-HCl, pH 7.5, 0.8 ml
water, and 0.8 ml pancreatic RNase, 0.2 ug per ml. Aliquots
of 0.5 and 1.0 ml were removed at various times after the
addition of enzyme and treated as described in the legend for
Figure 13b. The elution profiles represent the following
stages of hydrolysis: a) 0%; b) 32%; c) 63%; d) 77%.

Figure 13d: Chromatography on Sephadex 0-25 of a Reaction
Containing.RNA and P-100 Purified Muskmelon
Nuclease at Various Stages in the Hydrolysis

The reaction contained components as described in
Methods under RNase Assay except that the volume of each
component was increased ten-fold. Aliquots were removed at
various times and treated as described in the previous figure
legends. The elution.profiles represent the following stages
in the hydrolysis: a) 0%; b) 35%; c) 72%,

A254

A254

A254

A 254

72

 

0.4 —-
0.3 '-
02 "
0. I "

 

AMP

 

 

 

 

0.4 -
0.3 -
0.2 -

0.| -

 

 

 

0.4 "
0.3 -
0.2 -
0. I '-

 

 

 

 

 

 

0.4 -
0.3 -
0.2 -
0.2 -

 

 

 

 

0

 

If F28

 

Volume (ml)

 

73
into a test tube containing 0.05 ml of glacial acetic acid
to terminate the reaction. A half ml of 0.1 M sodium
acetate, pH 4.5, was then added and 0.5 ml of this mixture
was applied to a 1.2 x 13 cm.Sephadex G-25 column.previously
equilibrated with 0.1 M sodium acetate, pH 4.5, at a regu-
lated flow rate of 1.0 ml per minute. As can be seen in
Figures 13b the typical elution.pattern for an exonuclease
was obtained from.venom phosphodiesterase, an enzyme previ-
ously shown (41) to be an exonuclease. Using Figure 13a as
a reference, it can be seen that the only products eluting
from the column (in Figure 13b) were mononucleotides. In
Figure 13c, however, the elution profile indicates that as
hydrolysis proceeds products are formed which vary in size
from mononucleotides up to the large polynucleotides in the
unhydrolyzed, excluded peak. This is a typical elution
profile for an endonuclease (pancreatic RNase). Figure 13d
summarizes the results obtained when the muskmelon nuclease
(P-100 fraction) was tested. Conditions used were identical
to those used in the assay for RNase (see Methods) except
that the volume of each reaction component was increased
tenpfold. The P-100 enzyme preparation was diluted to a
concentration of 4.0 DNase B units per ml before use. As
is evident from the results of Figure 13d, the muskmelon
nuclease appeared to degrade RNA in an endonucleolytic

fashion.

74
Mode of Action on Denatured DNA (dDNA)

The same method (40) as that described directly above
was used to determine the mode of action of the P-100 enzyme
preparation on heat denatured DNA. Because of the presence
of two DNase pH optima, it was desirable to test the enzyme
at both pH 5.7 and 8.0. Reactions containing dDNA, P-100
enzyme, (20 DNase B units per ml), and either Tris or
imidazole buffer in the amounts described in.Methods under
assays for DNase B and DNase A, respectively (except that
the volume of each component was ten-fold greater) were
incubated at 37°. Aliquots of o. 5 and 1.0 ml were removed
at various times and treated as described in the previous
section.(see Mode of Action on RNA). ‘Figures 14b and 14c
summarize the results obtained from.reactions at pH 5.7 and
8.0 respectively. The enzyme appeared to act endonucleo-

lytically on dDNA at both pH optima.

Mode of Action on Native DNA (nDNA)

Since the P-100 enzyme preparation exhibited signifi-
cant activity on native DNA, cepecially at pH 5.7, (see
Relative Hydrolysis Rates of Native DNA, Denatured DNA, and
lRNA and.Figure 12) it was thought important to characterize
the mode of action on this substrate as well. Therefore,
reactions containing native DNA (at concentrations comparable
to those used in the previous study on dDNA), P-100 enzyme
(20 DNase B units per ml), and either Tris or imidazole

buffer in amounts described in Methods under assays for

 

75

Figure 14a: Chromatography on Sephadex G-25 of a Mixture of
dDNA and dAW?

Figure 14b: Chromatography on Sephadex 6-25 of a Reaction,
at pH 5.7, Containing dDNA and P-100 Enzyme at
Various Stages in the Hydrolysis

The reaction contained components as described in
Methods under DNase A assay except that the volume of each
component was increased ten-fold. Aliquots were removed at
various times and treated as described in the text (see Mode
of Action on.RNA). The elution profiles re resent the fol-
lowing stages in the hydrolysis: a) 0%: b 54%; c) 91%.

Figure 140: Chromatography on Sephadex G-25 of a Reaction,
at pH 8.0, Containing dDNA and P-100 Enzyme at
Various Stages in the Hydrolysis

The reaction contained components as described in
Methods under the assay for DNase B except that the volume
of each component was increased ten-fold. Aliquots were
removed at various times and treated as described in the
text (see Mode of Action on.RNA). The elution profiles
represent the following stages in the hydrolysis: a) 0%:
b) 45%: c) 87%.

76

 

dDNA
Cl

A254

 

 

 

 

 

 

 

A254

 

 

 

 

 

 

 

 

 

 

A254

 

 

 

 

 

 

 

 

 

 

Volume (ml)

77

DNase B and DNase A, respectively (except that the volumes
were tenpfold greater) were incubated at 37°. Aliquots were
removed at various times and treated as described in the
discussion titled Mode of Action of RNA except that each
aliquot to be chromatographed was brought to 100° for 10
minutes and quickly cooled in ice in order to denature the
DNAlu. Figures 15b and 15c indicate that at pH 5.7 and 8.0,
respectively, the enzyme degrades nDNA in an endonucleolytic

manner.

Determination.of‘Phosphate Position in Mononucleotides
Obtained From a Hydrolyzate of RNA

Preliminary experiments with the products of hydrolysis
of RNA by the P-100 enzyme preparation indicated that mono-
nucleotides as well as oligonucleotides were formed upon
extensive15 degradation. Treatment of these products with
Sl-nucleotidase (42) (which dephosphorylates only nucleoside-
5'-monoph08phates) produced significant amounts of inorganic
phosphate and nucleosides. Therefore, it was desirable to
determine whether or not all mononucleotides produced in a
hydrolysis of RNA were the 5'-phosphoryl derivatives.

A reaction containing 25 ml of yeast RNA (see Methods:
Preparation of RNA from Yeast) at a concentration of 2 mg
per ml; 30 ml of 0.2 M imidazole, pH 5.9: and 5.0 ml of P-100

enzyme previously diluted to a concentration of 20 DNase B

 

1”Native DNA was found to decrease the flow rate of
the Sephadex G-25 column.

15Complete conversion of substrate to lanthanum
nitrate-H01 soluble products when measured by the RNase
assay as described in Methods.

 

78

Figure 15a: Chromatography on Sephadex 0-25 of a Mixture of
nDNA and dAMP

Figure 15b: Chromatography on Sephadex G-25 of a Reaction,
at pH 5.7. Containing nDNA and P-100 Enzyme at
Various Stages in the Hydrolysis

The reaction contained components as described in
Methods under DNase A assay except that the DNA was not
denatured and the volume of each component was increased ten-
fold. Aliquots were removed at various times and treated as
described in the text. The elution.profiles represent the
following stages in the hydrolysis: a) 0%; b) 55%; c) 83%.

Figure 150: Chromatography on Sephadex 0-25 of a Reaction,
at pH 8.0, Containing nDNA and P-100 Enzyme at
Various Stages in the Hydrolysis

The reaction contained components as described in
Methods under DNase B assay except that the DNA was not
denatured, and the volume of each component was increased
ten-fold. Aliquots were removed at various times and treated
as described in the text. The elution profiles represent the
following stages in the hydrolysis: a) 0%; b) 21%: c) 43%.

79

 

nDNA

.0 .0
01 b
I I
(—

CD

dAMP

A254
0
I“

 

 

 

 

 

A254
0 .0
0° 0‘

 

.0
1

 

 

 

 

 

 

 

.0
.b
I

 

A254
0
1?

 

 

 

 

 

 

Volume (ml)

80
units per ml with 0.12 M ammonium acetate, pH 8.0: was incu-
bated at 37° for 90 minutes. At the end of the incubation
period a 0.6 ml aliquot was removed, added to 0.5 ml of cold1
lanthanum nitrate-RC1 reagent, and treated as described in
Methods under RNase assay. No visible precipitate formed
following centrifugation. The A260 of the mixture was 12.4.
These results indicated that all substrate in the reaction
had been converted to lanthanum nitrate-RC1 soluble products.
The hydrolyzate from which the aliquot was taken was adjusted
to pH 8.0 with NaOH and diluted to 120 ml with deionized
water in order to prepare it for chromatography on.DEAE-
Sephadex. The diluted hydrolyzate was applied to a 2.2 x 14
cm DEAR-Sephadex A-25 column (see Methods: Preparation of
Ion Exchange Resins) previously equilibrated with 2 liters
of 0.05 M triethylammonium bicarbonate (TEAB), pH 8.0, (see
Methods: iPreparation.of Ion Exchange Resins) at a flow rate
of 1.0 ml per minute. The collection of 5 minute fractions
was begun immediately. After all the sample had been
applied, the column was washed with 0.05 M TEAB, pH 8.0,
until the A260 of the effluent was 0.025. The column was
then treated with a gradient prepared from 500 ml each of
0.05 M and 0.30 M TEAB, both at pH 8.0, at a flow rate of
1.0 ml per minute. The 5260 elution profile determined dur-
ing application of the sample, washing, and gradient elution,
is shown in.Figure 16. The center three fractions from each
peak, A-D in Figure 16, were pooled. TEAB was removed from

each pooled fraction.by repeated evaporation to dryness on

81

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.o.m me so soon .mema a om.o use a no.0 so sees as oom sosa assessed assesses d can:

season» soap or: sssaoo one .mmo.o mes ososfleao on» do come one Hausa .o.m ma

.meaa z no.0 some serum: as: sasaoo one .eosaaas soon so: season on» ads soeee

.haopcaeoaaa games no: maoapomam enemas n no soapooaaoo one .opsaaa pea Ha o.H mo

opus scan a pm Amsanom owsmao m 20H no sodpenmaonm "apogee: oomv o.m ma .m4me

z no.0 mo unopda m and: oopmnnaadsoo anmsoabona Amsamom owsenowm.soH ho sodomy

1maehm ”apogee: oomv sasHoo mm1< Noodaaom1m4mq Bo ed M ~.N m on ooaaaae no: open

neaoness ecosaso one .nopd: eonasosoe eons as one on ooosaso use mosz can: o.m no

on denounce no: camshaoneh: use .maoapdusoo o>ono on» House mposooaa cansaom

Hom1opuapa: asamzuamd op oopaobsoo no: opmnpmnsm Ham pus» poop was» scum mason

no: pH .pnop one an confluence no pscwmoa Hom1oponpd: sasmzpsea oaoo suds condone

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m ommzn om no acapmapsoosoo m on oopsado hamsoabona ceases ooa1m go as o.m use

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82

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md

5952 cocooi

 

 

 

 

 

 

 

 

 

 

 

mum OmN 0mm CON L. Mb 001% O
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83
a Buchler flash-evaporator at 40°. Material from each peak
amounting to 0.2 A260 unit was spotted on a polyethylenimine
(PEI) cellulose coated sheet (9 x 15 cm) adjacent to the
four known ribonucleoside-Sl-monophosphates. Thin layer
chromatography was performed as described in Methods. The
material from each peak in Figure 16 gave one spot which
migrated with an.Rf value identical to the following stan-
dard mononucleotides: Peak A, CMP: Peak B, UMP: Peak C,
AMP: and Peak D, GMP. The spectral characteristics of
material from each peak, measured at pH 2, supported the
identification made using thin layer chromatography. With
the above evidence proving that peaks A-D (Figure 16) con-
tained only mononucleotides, the pooled, desalted fractions
from all four peaks were themselves pooled in a total volume
of 2.0 ml giving what was termed a "mixed-mononucleotide"
fraction. This mixed-mononucleotide fraction was then sub-
jected to hydrolysis by alkaline phOSphatase and 5'-nucleo-
tidase under the following conditions. Each reaction con-
tained 1.0 ml of 0.2 M Tris-H01 buffer, pH 8.2, 0.2 ml of
mixed-mononucleotide at a concentration of 61.0 A260 units
per ml, 0.2 ml of either 5'-nucleotidase (Sigma, Grade II),
60 us per ml or alkaline phosphatase (Worthington, BAPC),
2.1 mg per ml and water to a final volume of 2.0 ml. The
appropriate control reactions lacking enzymes or substrate
were included as well as were reactions containing 5'-AMP.
All reactions were incubated at 37° for one hour. At the

end of the incubation.period a 0.5 ml aliquot was removed,

84
treated with 0.5 ml of phenol, and assayed for inorganic
phosphate by the procedure described in.Methods under'B'qAMPase
assay. Virtually 100 percent of the inorganic phOSphate
released from the mixed-mononucleotide by alkaline phOSpha-
tase was released by 5'-nucleotidase. The same relative
amounts of inorganic phosphate were found in those reactions
containing 5'-AMP. Thus, all mononucleotides isolated from
a hydrolyzate of RNA by the P-100 enzyme were the 5'-phos-
phoryl derivatives. The assumption can also be made that
the oligonucleotide products were terminated by a 5'-phosphate.

Determination.of Phosphate Position in Mononucleotides
Obtained From a Hydrolyzate of Denatured DNA at pH 5.7

In order to determine the nature of the phosphate
position in mononucleotides from a hydrolyzate of dDNA at
pH 5.7 an eXperiment similar to that utilized for the mono-
nucleotides fromeNA was performed.

A reaction containing 25 ml of denatured DNA (heated
at 100° for 15 minutes, quickly cooled to 4° in an ice-water
bath) from salmon sperm (Sigma, Type III) at a concentration
of 2.0 mg per ml: 30 ml of 0.2 M imidazole, pH 5.9: and 5.0
ml of P-100 enzyme previously diluted to a concentration of
20 DNase B units per ml with 0.12 M ammonium acetate, pH 8.0:
was incubated at 37° for 2.5 hours. At the end of the incu-
bation period 0.6 ml of reaction mixture was assayed as
described in Methods for DNase A. It was found that all
substrate was converted to lanthanum nitrate-RC1 soluble

products under the above conditions. The hydrolyzate was

85
then adjusted to pH 8.0 with NaOH and diluted to 120 ml with
deionized water. This diluted hydrolyzate was applied to a
2.2 x 15 cm DEAR-Sephadex A-25 column (see Methods: Prepara-
tion of Ion Exchange Resins) previously equilibrated with 2
liters of 0.05 M TEAB, pH 8.0, (see Methods: Preparation of
Ion Exchange Resins) at a flow rate of 1.0 ml per minute.
The collection of 5 minute fractions was begun immediately.
After all the sample had been applied, the column was washed
with 0.05 M TEAB, pH 8.0. until the A260 of the effluent was
0.025. The column was then.treated with a gradient prepared
from 500 ml each of 0.05 M and 0.30 M TEAB, both at pH 8.0,
at a flow rate of 1.0 ml per minute. The A260 elution pro-
file determined during application of the sample, washing,
and gradient elution, is shown in.Figure 17. Previous
chromatography of the four common 5'-phosphoryl-2l-deoxymono-
nucleosides under the same conditions as used in this experi-
ment, showed that 5'-dCMP and 5l-TMP were eluted together in
the first peak, while 5l-dAMP and 5'-dGMP, respectively, were
eluted immediately after the pyrimidines (dCMP and TMP) in
two well separated peaks, all with 95-100 percent recoveries.
The center three fractions from each.peak (A-B, C, and D in
Figure 17) were pooled and TEAB was removed by flash-evapora-
tion as in the previous eXperiment utilizing the mononucleo-
tides from.RNA. Thin layer chromatography (see Methods) of
the material from each peak along with the determination of
spectral characteristics, at pH 2, indicated that peak A-B
contained mostly C>75 percent) TMP, peak C contained only

86

.aopoaoposaoapooau

mm smexoom o ad oosaahopoo no: soapooam some mo come one .opsaaa pea

as o.« mo open Sofia o no .o.w we as anon .mdme z om.o one 2 mo.o mo some

as com acne oonmaoaa psoaooaw m and: condos» some new sasaoo 0:9 .mmo.o

no: paosammo one mo owmw on» Hausa .o.w ma .mwma : mo.o spas cosmos mos

cadaoo one .ooaaaao some can oaaaom oz» Ham amped .aaopoapoeaa sswon

mos ncoapooaw enemas m ho soapooaaoo mas .opssaa sea as o.H mo open

30am 6 no Amsamom owsesoam_soH mo scapegoaoam “apogee: oomv menace

mm1d Hoomzaom1m4ma ao ma N N.N o co oodaaao mos Ada owav ondwhaoaoaz

popsaao one .msoapaosoo obono one hopes oposooaa oHQSHom Hom1opoapa:

assmzpsmfl op oopaossoo no: opoapmpsm Ham pomp venom no: pa use 4 omozm

you mooapoz ad confluence we commune mos posoaao Ha w.o o ooaaoa soap

1onaoca one no use one am .mHSOS m.m you can no oopopsoad mos ”o.w ma

.opmpooo adasoaso z NH.o spas as yea means m onmza om no soapoapsoosoo

s on eoesase aamsoasoaa osanso ooaua no as o.m use “o.m ma .oaondesaa

: m.o mo as on "Ha pea we o.N mo soapehuaooaoo a pm AHHH mama .mawdmv

saoan soaaom scam Aspen nopoz1ooa as ea c: on ooaooo aonasd .moussaa ma
Mom OOOH no oopoozv 42m consumsop go as mN wadsaopaoo soapomon <

a.m ma as seesaw scale as 426 eonsossoo
do oosnaaoseam e do mule woossaomumeua so asassmoosaonso use chewed

87

(W) ”OllDJlUGOUOO EVELL

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88
dAMP, and peak D contained only dGMP. The material from the
central portions of peaks A-B, C, and D were combined in a
total.volume of 2.0 ml and termed mixed-mononucleotide.
This mixed-mononucleotide fraction was then subjected to
hydrolysis by alkaline phosphatase and 5'-nucleotidase under
the same conditions as those used previously with the mixed-
mononucleotide fraction obtained from.RNA. The 5260 of
the mixed-mononucleotide fraction from dDNA was 37.0. It
was found that 5l-nucleotidase released 96 percent of the
inorganic phoSphate, from the mixed-mononucleotide fraction,
that was released by alkaline phoSphatase. This result
indicated that the mononucleotides released from dDNA at
pH 5.7 by the P-100 enzyme were the 5'-phOSphoryl deriva-

tives,

Determination of Phosphate Position in Oligonucleotides
Obtained From a Hydrolyzate of Polyadepylic Acid by P-100

Enzyge
In a hydrolyzate of poly-2l-O-methyladenylic acid

(poly Am) produced by the P-100 enZyme preparation (see
Hydrolysis of Poly-2'-O-methyladenylic Acid for further
details) a significant amount of 2'-0-methyladenosine and
oligonucleotides lacking phosphate at either terminus was
present. One or both of the following may have been the
cause of the presence of dephoSphorylated products in the
hydrolyzate: a nuclease producing 3'-ph08phoryl terminated
products which possibly would have in turn been dephoSphory-

89
lated by the 3'-nucleotidase activity16 or a separate phos-
phatase capable of removing 5'-phosphates from mono- and
oligonucleotides. Because of the extreme conditions of
both incubation time and amount of enzyme needed to give
significant degradation of the poly Am, the effect of any
nonspecific phosphomonoesterases or other nucleases present
would be greatly magnified. For this reason it was desir-
able to characterize the shorter oligonucleotides from a
partial hydrolysis of polyadenylic acid (poly A) to see
whether or not they contained dephosphorylated products.
Reaction mixtures composed of 0.60 ml poly A, 1.0 mg per ml
(15.8 A260 per ml): 0.30 ml of 0.20 M Tris-H01, pH 8.2;
0.060 ml of P-iOO enzyme previously diluted to concentra-
tions of 20 and 40 DNase B units per ml with 0.12 M Buffer
A; and 0.24 ml of water were incubated at 37°. Aliquots of
0.40 ml were removed after i and 2 hours of incubation.
These aliquots, as well as the remaining 0.40 ml which was
incubated for a total of 3 hours, were frozen immediately
upon removal, and lyophilized. Each residue was dissolved
in 0.04 ml of water and Spotted on Whatman 3MM paper for
chromatography in.a-prOpanol:29 percent ammonium hydroxide:

water solvent system (55:10:35. respectively. V/Vll7- The

 

16

Dr. M. Laskowski, Sr. has, by personal communication,

stated that the 3'-nucleotidase from mung bean is capable of
dephosphorylating 3'-phosphory1 terminated dinucleotides.

17This solvent system is capable of resolving small
oligonucleotides of homOpolymers from 1-5 in length when
used with Whatman No. i or 3MM paper.

90
solvent was permitted to migrate for a period of 18 hours
after which the paper was dried. It was evident from the
distribution of U}V. absorbing spots observed that 2 or 3
hours incubation at an enzyme concentration of 40 DNase B
units per ml, and 3 hours incubation at an enzyme concentra-
tion of 20 DNase B units per ml gave the most desirable
degree of hydrolysis, i.e. all of the substrate was con-
verted to mono- and oligonucleotides up to tetranucleotide
in size. The above conditions outlined as optimal for the
production of di-, tri-, and tetranucleotides were then
used with three separate reaction mixtures. Chromatography
on Whatman BMM paper in the solvent system described previ-
ously (pppropanolzammonium hydroxidexwater) was again used
to separate the products of hydrolysis. A reaction lacking
enzyme and containing 5'qAMP instead of poly A was also
chromatographed in order to have a marker for mononucleo-
tide. A series of four to five spots was observed on the
chromatograms from each.reaction that contained enzyme.
This series corresponded to the following products18 of
hydrolysis, arranged in order of decreasing Rf: mononucleo-
tide (pA)>dinucleotide (pA2)>trinucleotide (pA3)>tetra-
nucleotide (pAu)>»pentanucleotide (pAs). All spots contain.
ing di-, tri-, and tetranucleotide were eluted from the
paper by the method of Davis, gg.‘gl. (43). The eluants

from Spots of correspondingin's were combined in a total

 

18Dr. F. M. Rottman, personal communication.

91
volume of 2.0 ml. The following amounts of each type of
oligonucleotide were recovered: pAz. 5.41 A260 unitslg
pA3. 5.71 5260 units: pAu, 4.82 5260 units. Each oligo-
nucleotide solution was lyophilized and the residue dis-
solved in a sufficient amount of water to give a concentra-
tion of 20.0 ‘260 units per ml. In order to determine
whether or not the isolated oligonucleotides contained 3'-
phosphoryl termini or, alternatively, no phosphoryl termini,
each was completely hydrolyzed by snake venom phosphodiester-
ase under the following conditions: a typical reaction mix-
ture contained 0.025 ml of Tris-succinate, 0.15 M, pH 6.0:
0.025 ml of magnesium acetate, 0.012 M; and 0.020 ml of
venom phosphodiesterase (Worthingtonw VPH), 0.5 mg per ml,
potency of 0.3. These reactions were incubated at 37° for
3 hours. (The above condition of pH has been described by
Richards and Laskowski (44) to be optimal for the hydrolysis
of 3l-phosphoryl terminated oligonucleotides by venom phos-
phodiesterase.) Aliquots of 0.010 ml were removed before
adding enzyme and at the end of the incubation period. In
preparation for thin layer chromatography each aliquot was
spotted in a separate lane along the longer side of a 20 x
10 cm sheet of PEI-cellulose (MNePolygram.CEL 300 PEI,
Brinkman Instruments, Inc.). Preliminary experiments using
0.75 M lithium chloride (45) as solvent showed that a mix-
ture of adenosine, 5'-AMP. and pAz, pA3, or pAu were
separated by ascending thin layer chromatography on PEI-
cellulose with the following Rf values: adenosine, 0.53:

92
5'-AMP, 0.32; pAz. 0.18: pA3. 0.15: and 954' 0.13. Experi-
ments designed to determine the lowest level of adenosine
that could be detected on a PEI-cellulose sheet after thin
layer chromatography in 0.75 M lithium chloride indicated
that this value was 0.003 5260 unit. All reactions tested
by the chromatographic method indicated that the three
types of oligonucleotides were completely degraded to a
compound giving a single spot with an Rf equal to that of
5'-AMP. No Spots with RfISO.2, indicative of unhydrolyzed
oligonucleotide or nucleoside diphosphates, could be
detected, nor could spots having an.Rf equal to that of
adenosine be seen. Taking into account that a 0.010 ml
aliquot of reaction mixture contained 0.06? 5260 unit it is
evident that approximately 5 percent (0.003 A260 unit) of
this amount could have been detected as adenosine, if this
nucleoside had been produced in the course of hydrolysis by
venom phOSphodiesterase. These results indicate two facts.
First, the oligonucleotide products of hydrolysis of poly A
were terminated on the 5' end by a phoSphate and on the 3'
end by a free hydroxyl group. Second, if there were dephos-
phorylated oligonucleotides present, they were present in
amounts less than 10 percent of the total dinucleotide,
15 percent of the total trinucleotide, and 20 percent of the

total tetranucleotide.

93

Susceptibility of Polyadenylic Acid, Polyuridylic AcidJ
and'Polycytidylic Acid to Hydrolysis by P-100 Enzyge
When it was found that the P-100 enzyme preparation

 

exhibited great differences in its ability to dephoSphory-
late the various nucleoside-3'-monophosphates (see Relative
3'-Nuc1eotidase Activities) it was reasoned that this dif-
ference might have some bearing on the ability of the enzyme
to hydrolyze polyribonucleotides containing only one type
of purine or pyrimidine base. Walters and Loring (46) have
suggested that the differences observed in 3'-nucleotidase
activities of a partially purified nuclease from mung bean
might have some bearing on the specificity of the accompany-
ing RNase activity. With this reasoning in mind the follow-
ing eXperiment was conducted. Reaction mixtures were pre-
pared to contain 110 microliters of 0.20 M Tris—RC1 buffer,
pH 8.2: 10 microliters of P-100 enzyme (200 DNase B units
per ml): and 100 microliters of either polyadenylic acid
(poly A), polyuridylic acid (poly U), or polycytidylic acid
(poly C), each at a concentration of 1.0 mg per ml in water.
These mixtures were incubated at 37° for 1 hour. The
appropriate controls lacking enzyme were included in the
incubation procedure. At the end of the incubation period
.10 microliter aliquots of each reaction were spotted 2 cm
from the shorter side of a 14 x 11 cm piece of MN-Polygram
CEL BOO/W254 (Brinkman Instruments, Inc.). The three cor-
reSponding nucleoside-5'-mon0phosphates (AMP. UMP, and. CMP)

were Spotted in positions adjacent to appropriate reactions

94
containing polynucleotide and enzyme. The sheet was devel-
oped in a closed tank for 3 hours by ascending thin layer
chromatography using the solvent, gyprOpanol:29 percent
ammonium hydroxidezwater (55:10:35. rSSpectively, v/v).
The solvent migrated to a distance of 11 cm from the origin
in the 3 hour period. After drying the chromatogram the
U.V. absorbing Spots were marked. The following results
were observed. Each standard mononucleotide migrated with
an Rf offi*0.h5. The positions Spotted with reactions con-
taining polynucleotide but lacking enzyme each exhibited
one Spot which had not migrated from the origin (indicating
no degradation19 of the polynucleotide). The positions
Spotted with reactions containing poly A plus P-iOO enzyme
and poly U plus P-iOO enzyme each exhibited one Spot which
had migrated to a position adjacent to the standard mono-
nucleotide Spots. The position Spotted with the reaction
containing poly C plus P-ioo enzyme, however, diSplayed one
Spot which had not migrated from the origin. These results
indicate that the P-iOO enzyme preparation was capable of
hydrolyzing poly A and poly U completely to mononucleotides,
but was not capable of hydrolyzing poly C.

 

1'9On thin-layer plates with this solvent partial
degradation of a polynucleotide results in streaking of
U.V. absorbing material from the origin to the mononucleo-
tide position.

95

Characterization of the Products of Hydrolysis of
Polyuridylic-Cygidylic Acid.(Poly U,C) Produced by
P-iOO Enzyme

The preceding section along with the one describing
differences in 3'-nucleotidase activities have suggested
that the 3' oxygen to phoSphorus bond of cytidylic acid is
not hydrolyzed by the P-iOO enzyme preparation. In order
to test this hypothesis more rigorously, a ribonucleotide
copolymer containing uracil and cytosine (base ratio of
2:1, reSpectively) was subjected to extensive hydrolysis
by P-iOO enzyme under the following conditions. Poly U,020
(3.“ m1 at a concentration of 82.1 5260 units1 per ml) was
thoroughly mixed with 3.4 ml of redistilled, water-saturated
phenol. The phases were separated by centrifugation at
1100 x g for 10 minutes at room temperature. Three ml of
aqueous phase was recovered and made 0.2 M in potassium
acetate by the addition of 0.33 ml of the 2 M salt solution.
The polynucleotide was precipitated at 4° by the addition
of 6.0 ml of 95 percent ethanol. Centrifugation at 27,000
x g for 10 minutes was used to separate the precipitate.
This was subsequently dissolved in 2.0 m1 of water after
discarding the supernatant solution. The ethanol precipi—
tation was repeated and the precipitate dissolved in 2.0 ml
of water. This solution was then lyophilized to dryness to
remove the last traces of ethanol. The white, fibrous

 

20A generous gift from Dr. F. M. Rottman.

96
residue was dissolved in 3.0 ml of water. It diSplayed an
A260 of 67.0. A reaction mixture containing 0.747 ml of
poly U,C (67.0 A260 units per ml); 1.25 ml of 0.20 M Tris-
HCl, pH 8.2; 0.25 ml of water (pentachlorophenol, 0.5 pg
per ml); and 0.25 ml of undiluted P-100 enzyme was incu-
bated in a closed container at 37° for 16 hours.21 The
reaction mixture was diluted with 2.5 m1 of water before
applying it to a 1.2 x 32 cm column of DEAE-Sephadex A-25
at a flow rate of 0.8 ml per minute. The tota1.A260 in
the sample was 49.2 unitsl. The resin was prepared in the
formats form.(see Methods: Preparation of Ion.Exchange
Resins) and equilibrated with 0.05 M ammonium formats,
pH 7.5. A 100 ml quantity of 0.05 M ammonium formate,
pH 7.5, was used to wash the column. Column effluent was
continuously monitored using an Isco Model UA ultraviolet
optical unit recording A254. Fractions of 5 ml each were
collected throughout the application of the sample, wash-
ing, and gradient elution. Following the washing procedure,
the column was treated with a gradient prepared from 500 ml
each of 0.05 M and 0.50 M ammonium formate, both at pH 7.5
at a flow rate of 0.8 ml per minute. The elution profile
determined from the A250 of each fraction during the course
of the gradient elution is Shown in Figure 18. At the
termination.of the 0.05-0.5 M gradient, the column was

 

21Preliminary experiments using thin layer chromatog-
raphy on.PEI-oellulose indicated that the hydrolysis had
reached a limit under these conditions.

97

 

.nopoaoposaonpoomm mm seafloom d ca dosaanopoo we: Sodpooau Some no oomd one
.codpaflo anoaooyw use .ws«:md3 .uodpmoaaaam madame no omnsoo as» madame
oopooaaoo one: some as n no mnoapomnm .n.e mm .opdanoh adanoaad z o.a

do HS com spa: wanna: was nasaoo on» .peodemnw on» go monumedanop as» »4
.opzsaa non as 0.0 00 open 30am a pm .m.e me am npon .opmanom azdaoaad

z m.o one no.0 go some as 00m Soup oonmaohm pcoaomnw a and: condohp

zone was menace one .m.e ma .opmanom asacoaso z no.0 no as 00H spas dogmas
we: nasaoo on» oaaamm on» wsdoom nopm< .m.m ma .opoanoh adenoaam z no.0

mo whopaa m spas oopennaadsvo mm: Sasaoo on» .oaspwaa Sodpoooh on» no soap
nmoaaaam onomom .opzcaa Hon as 0.0 mo open loan a pm maid Noomsaomlmdun no
menace 80 mm M N.a m on pa weakened muomon Hopes no He m.N and: oopnaao

mos endpwaa Soauooon one .tho: 0H you own no nozdapnoo oomoao a ma
oopmnso:« was oshuno ooanm copsaaoss mo HS mm.o use “AHS you w: m.o .Hoaosa

-oeoaeompcooo “spa: do Ha m~.o “m.m mg .Humumdna 2 o~.o ea Ha m~.H “Aaa non

moans oomd o.eov 0.0 neon no as mem.o wcdaampmoo oHSpNaa noduomoa 4

H

,msausm ooaam an 0.: aaom mo mdmeaoueam mo
mposoonm esp mo nasaoo mmn4 Noomsaomnm4mm a 8099 shopped Soapsam ”ma onswam

 

98

(w) uououueouog momma wnguowwv

 

X

\

to. v. m. N
O O O O
I I I
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50

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Fraction Number

50

 

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x
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092v

99
washed with 200 ml of 1.0 M ammonium formats, pH 7.5. This

washing step resulted in the elution of a small peak as
indicated by the A25“ monitor. The fractions containing
this material were pooled and termed "1.0 M Wash" (Fraction
F). The following fractions were pooled and each pooled
group given.a letter designation as shown in.Figure 18;
Peak A, fractions 17-38; Peak B, 47-54; Peak C, 87-94;

Peak D, 116-123; and Peak E, 140-147. .Ammonium.formate was
removed from each fraction (A-F) by repeated lyophilization:
the residue was dissolved in approximately 5 ml of water
for each cycle. After all ammonium formats was removed,
the remaining residue in each fraction was dissolved in
water and brought to 1.0 ml in volume. The 5260 of each
fraction was determined. A percentage yield was calculated
for each fraction from this value and the total of 49.2
A260 unitsl applied to the DEAE-Sephadex A-25 column.

These results are Shown in Table VII. In order to charac-
terize each fraction.with regard to 5' and 3' terminal
nucleotides, each fraction.(A-E) was dephoSphorylated using
alkaline phoSphatase. Separate reaction mixtures for each
fraction were incubated at 37° for 3 hours. Each reaction
mixture contained 1-4 A26o units1 from a Specific fraction.
0.11 ml of alkaline phOSphatase (Worthington, BAPC) at a

concentration of 0.92 mg per ml (20 units8

per mg) in 0.2 M
Tris-HCl, pH 8.2, and water up to 1.0 ml. Each reaction
was lyophilized following the incubation period. The

residue was dissolved in 0.04 ml of water and the entire

100

.nasaoo Hoodsnomnmdmn op ooaannd mean: oomd «.039
.ma onswam some

 

 

 

oonobooom
Hauoe 0.Hm
Hm.e -.~ Anna: 2 o.flo a
oo.e e~.~ m
0.HH 0:.m Q
«.mm 0.0H o
e.mm 3.5H m
I?
a¢.m mm.” 4
new 00 maid op oodaaa¢ oonobooom
apes: 0N<.pampoe no usoonom means 00m<.adpoe escapomnh

 

 

nausea oo~nm.ap in.a aaomv ence chasedpaouodaaedhshaom
mo mamhaonohm no oposoonm on» mo acapmwdnopomndso

HH> mumde

101
amount was Spotted on Whatman No. 1 paper. Chromatography
for 14.5 hours in Solvent I (see Methods: Paper Chroma-
tography) along with standard UMP, CMP, uridine and cytidine
gave the following results. The reaction mixture containing
fraction.A gave one very light Spot under U.V. which
migrated near the solvent front faster than either
standard mononucleotides or nucleosides. Fraction B
material showed one Spot withRf equal to that of uridine.
Fraction C reaction mixture exhibited one Spot with Rf of
0.17, i.e. significantly less than either standard mononuc-
leotide. Material from.Fraction D migrated just off the
origin, while material from Fraction.E appeared not to have
moved. Chromatography in Solvent I was used for two
reasons: first, to get some idea of the nature (mono- or
oligonucleotide) of the material in each fraction; and
second, to separate each dephoSphorylated compound from
the alkaline phoSphatase (which remains at the origin)17.
The Spots containing dephoSphorylated material from Frac-
tions C and I>were eluted from the paper with water. The
eluant from each was divided into two equal parts, one
half to be completely hydrolyzed with venom phOSphodiester-
ass and the other to be hydrolyzed with potassium hydroxide.
The residues from C and D to be hydrolyzed with KOH were
dissolved in 0.050 ml of water. An equal volume of 0.2 N
KOH was added to each and the reactions were heated in a
boiling water bath for 20 minutes. The salt concentration

was reduced by perchloric acid treatment as described by

102
Eavidson.and Smellie (47). Thin.layer chromatography of C
on.PEI-cellulose (see Methods: Thianayer Chromatography)
revealed one mononucleotide22 Spot migrating like 3'-CMP.
The only nucleoside component in base-hydrolyzed C was
Shown by paper chromatography in Solvent I to be uridine.
The material resulting from KOH hydrolysis of D appeared
to contain one mononucleotide, 3'-CMP, when chromatographed
on PEI-cellulose. Again, the only nucleoside component, as
Shown by paper chromatography in Solvent I, in base-hydro-
lyzed D was uridine. In order to verify the possible
structures for C and D suggested by the above results, the
other halves of dephoSphorylated C and D fractions were
subjected to complete hydrolysis by venom phoSphodiesterase.
Venom phoSphodiesterase is known to hydrolyze oligonucleo-
tides by stepwise liberation of nucleoside-5'-monophoSphates
from the 3'-hydroxyl terminus. If the oligonucleotide is
dephOSphorylated (both termini), the nucleotide residue on
the 5' terminus appears as a nucleoside. Reaction mixtures
containing 0.05 ml of either dephoSphorylated C or D
(2.0 A260 units); 0.01 ml of Tris-acetate, 0.3 M, pH 8.8;
0.01 ml of 0.3 M magnesium.acetate; 0.01 ml of venom phos-
phodiesterase (Worthington, VPH), 0.5 mg per ml at a potency
of 0.3; and 0.02 ml of water were incubated for 21 hours at
37°. Identical reaction mixtures lacking oligonucleotide
but containing instead either 5'-CMP and 5'-UMP or the

nucleosides, cytidine and uridine, were incubated under the

 

22Nucleosides migrate with the solvent front.

103
same conditions. Chromatography of the reaction mixtures
after the incubation period on paper in Solvent I, and also
on.PEI-cellulose, gave the following results: the reaction
mixture containing hydrolysis products from dephoSphorylated
C contained only 5'-UMP and cytidine, whereas the D reaction
mixture exhibited Spots correSponding to 5'-UMP, 5'-CMP, and
cytidine, but no uridine. Because at least two possible
oligonucleotide structures, differing in chain length by at
least one nucleotide, were possible for D, the Spots on the
paper chromatograph of base-hydrolyzed E>were removed and
eluted, each with 1.5 ml of 0.01 N HCl. Since the 3'
terminus was Shown to be uridine, and the only other com-
ponent from base-hydrolysis was 3'-CMP, the molar ratio of
uridine to 3'-CMP would indicate the chain length and; the
most plausible structure of D. The ratio of uridine to
3'-CMP was found to be 1:2.7. This indicated that peak D
(Figure 18) was a mixture of the tri- and tetranucleotides
pCpCpU and pCpCpCpU. A summary of the characterization of
peaks C and D is Shown in Table VIII. Peak B was rather
conclusively shown both by thin layer chromatography on
PEI-cellulose and by the determination of Spectral charac-
teristics to contain only 5'-UMP. Peaks E and F, because
of the limited amount of material available, were not
characterized beyond the point of determining Spectral
characteristics. Evidently, E and F contained a high
proportion of cytidylic acid judging from the position of
their maximum absorption in the U.V. The 280:260 ratios

104

TABLE VIII

Summary of the Characterization of Peaks "C" and "D"
From DEAE-Sephadex A-25 Column.(Figure 18)

 

DephOSphorylated Products from Products from PrOposed
Oligonucleotide KOH Hydrolysis VPH Hydrolysis Structure

 

 

C [XpY] Cp + Ur Cr + pU CpU
D [(Xp)nl] Cp + Ur Cr + pC + pU CpCpCpU
+. a
CpCpU }

 

Xp = 3'-mononucleotide
pX = 5'-mononucleotide
Xr = nucleoside

8Proposed to be the mixture since the molar ratio of Ur to
Cp was found to be 1:2.7.

105
observed for peaks B-F increased steadily from 0.48 (pH 7.0)
for B to 1.06 for F, another indication that the proportion
of cytidylic acid to uridylic acid was increasing. Thus,
the tacit assumption is made that these later peaks are of

the same general structure as C and D, i.e. (pC)npU.

Characterization of the Products of Hydrolysis of
Denatured DNA'Produced‘by'P-ioo Enzyme atng 8.0

Because of the apparent degree of Specificity exhibited
by the P-100 enzyme preparation with reSpect to the various
polyribonucleotides tested it appeared desirable to determine
whether or not polydeoxyribonucleotides were hydrolyzed in a
similar way. For this reason, the following experiment was
performed. A 100 mg quantity of DNA from salmon Sperm was
dissolved in water at a concentration of 2.5 mg per ml by
gentle stirring at 4° over a 15 hour period. The DNA was
denatured by heating at 100° for 30 minutes followed by
rapid cooling in an ice-water bath. The volume was then
brought to 50.0 ml with water. This 50 ml quantity of dDNA
was combined with 60 ml of 0.2 M Tris-H01, pH 8.2, and 10 m1
of P-100 enzyme (previously diluted to a concentration of 20
DNase B units per ml with 0.12 M ammonium acetate, pH 8.0).
The reaction.mixture was incubated at 37° for 9 hours.
.Aliquots of 0.5 ml were removed at 20 minute intervals and
treated with 0.5 ml of cold lanthanum nitrateeHCl reagent in
order to follow the progress of the reaction. The production
of lanthanum nitrate-H01 soluble, UV-absorbing materials was
found to reach a limit, indicating the lack of susceptibility

106
of the degradation products to further hydrolysis, at about
180 minutes. This time was arbitrarily tripled in deciding
on a total incubation time of 9 hours. The reaction mix-
ture (117 ml) was diluted to 336 ml with water after the
incubation period. This diluted sample, containing a total
of 1880 A260 units, was applied to a 2.5 x 48 cm DEAE-
Sephadex Aa25 column previously equilibrated with 0.01 M
ammonium formate, pH 7.5. at a flow rate of 1.5 ml per
minute. Following application of the sample, the column
was washed with 1200 ml of 0.01 M ammonium formate, pH 7.5.
A gradient prepared from 2000 ml.each of 0.01 M and 1.0 M
ammonium formate, both at pH 7.5, was used as the eluting
agent at a regulated flow rate of 1.3 ml per minute. Frac-
tions were collected at 10 minute intervals. The elution
pattern, determined by A260; is shown in Figure 19. After
the 0.01 M-1.0 M gradient had gone to completion, a second
gradient prepared from 1000 ml each of 1.0 M and 2.0 M
ammonium formate, both at pH 7.5. was established. No A260
above a base line value of 0.01 could be detected in frac-
tions collected during the course of elution by the 1.0-2.0
M gradient. Previous experiments in which hydrolyzates of
dDNA were fractionated on DEAE-Sephadex showed that mono-
nucleotides were eluted in the first three peaks; i.e. the
first peak contained a mixture of the two pyrimidine mono-
nucleotides, the second was dAMP, and the third was dGMP.
Material from the center of each of the first three peaks

(See Figure 19) was chromatographed on.PEI-cellulose with

107

.paoaocnw z 0.Nio.a on» up sodasao mo consoo as» waanso
eopooaaoo maoapomnm ad 00900900 on dance ”0.0 mo ozfiob mafia omen o obonc coma oz
.paomc wadpsao on» ma poms we: .m.n we no anon .opmanom asaaoaac z o.m use : o.H go
some Ha oooH Scam ocnmaoha unoaomam daooom a .aoapoflgaoo op oaow om: paoaomnw pace
2 o.aiao.o can hoped .mambpopaa spends 0a pm oouocaaoo one; maoapochm .opsada
you as m.H no open 30am oopcaswoa c pd enema waduseo on» we come no: .m.n we as
soon .opmanoh addaoaad z 0.H use 2 ao.o.mo some as ooom Bonn oohcaona paoaooaw 4
.m.a ma .opesaoe asaaoaao z Ho.o co as coma spa: corner on: asoaoo or» .oaason or»
no Scapcodaaam wsdzoaaom .opsaaa Hon Ha m.a mo ouch 30am a no .m.e ma .opmanou
Bdaaoaac z «0.0 and: oopmnpaaasoo mamsoaboha cadaoo n~i< Noodsaomimdmn So m: H m.m
a on ocaaaac was .mpaa: oomd ommfl mo Hmuop d waaaampsoo .maaamm oopsaao mane
.Hmumz ooNdQOAoo spas Ha 0mm 0» oouzado mm: oefipfida moapomoe 0:» oodhom noapmnfioca
can Hopm< .maSo: 0 Mom can no condensed was Ao.m ma cpcpooo Seacoaam a NH.o and:
as non means m omczm om mo soapmauacoaoo c on oopsaao adenoaboaav caenao ooalm
co as ea one .m.m ma .Homuaaae z «.0 co as oo .Aoe op waaaooo eaaoa an eozoaaoo
mopzaaa on How oooH pm oopconv «290 no HE om wadaampaoo ondpwaa soapomon Q

o.m ma an neaaam ooana an oooaooaa «zoo oo nanaaoaeam
co naoooona or» co aaaaoo mmie woooraomumemn a song aaoppomxaoapoam “ma enemas

 

 

IV

 

I<——l——->l<——II—>l<—|||—>le

108

(W) UOIIDJIUGOUOO GIDUJJOj UJI’IIUOLUUJV
0. oo. o <r. N.
_ O O O O
I I I I 7

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II

 

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x W“?

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30"
2.0-
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no

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<

240

|80

|20

60

Fraction Number

109
the four common 2'-decxynucleoside-5'-mon0phOSphates as
reference standards. The second and third peaks were found
to contain MM? and dGMP, reSpectively. The first peak
contained only TMP. Because these three peaks were not
well separated from one another, they were pooled and
termed Fraction I (as shown in Figure 19). The remaining
tubes were pooled at points in the elution profile shown in
Figure 19 to give Fractions II, III, and IV. All fractions
collected during the washing step were pooled and termed
"WashpThru." The A260 of Fractions I-IV and the WashJThru
was determined. These values are shown in.Table Ix. In
order to remove the volatile ammonium formate, each frac-
tion was flashpevaporated to a volume of approximately 50
ml. Repeated cycles of lyophilization were then used to
remove the remainder of the salt. Each.residue was then
dissolved in a 1-2 ml of water at a concentration which
ranged from 138-220 A260 units per ml. These concentrated
fractions were stored at -10° when not in use. The first
type of characterization to be done was a determination of
the relative molar proportions of mononucleotides present
in Fraction I. Two dimensional paper chromatography on
Whatman 3MM sheets in Solvents I and II (see Methods: Paper
Chromatography) was used to separate the various mononuc-
leotides. The four common 2'-deoxynucleoside-5'-monophos-
phates and 2'-deoxynucleosides were also chromatographed as I
reference standards. All.U.V. absorbing Spots were cut

from the chromatographs and the compounds eluted with 0.01 N

TABLE IX

Relative Amounts of Products of Hydrolysis of dDNA
Recovered From.a.DEAE-Sephadex A-25 Column

 

 

 

Fraction ‘Volume A260 Total
(m1) A260 Units
WashpThru 1580 0.023 36.3
I 560 0.650 364
II 370 1.42 525
III 325 1.38 448
IV 1070 0.423 453

 

1826.3 Total
Recovered

 

1826.3 Recovered
1880 Applied

 

x 100 = 97.2 Percent Recovery

Material eluted from a IEAE-Sephadex A-25 column was
pooled into four fractions (Fractions I-IV, Figure 19) plus
a WashpThru fraction consisting of all eluate collected from
the beginning of the application of hydrolyzate to the
beginning of the 0.01-1.0 M gradient elution. The A260 of
each fraction.was determined on a Beckman DB Spectrophotometer.

111
HCl. The recovery of standard mononucleotides was found to
be in the range of 92-110 percent. Table X summarizes the
relative amounts of mononucleotides found in Fraction I
(Figure 19). In order to determine the average Size of
the products in each of Fractions I-IV, the procedure in
Methods was used (see Determination of Average Chain Length
of Mixed Oligonucleotides). The values obtained by this
procedure are shown inITable XI along with data pertaining
to the percent of the total hydrolyzate recovered in each
fraction. In order to verify that oligonucleotides pro-
duced from dDNA at pH 8.0 by P-100 enzyme were, in fact,
5'-phoSphoryl terminated, the following digestion with
venom phOSphodiesterase was performed. Reaction mixtures
consisting of 0.02 ml of either Fraction II (220 A260 units
per ml) or Fraction III (182 A260 units per ml); 0.01 ml of
0.3 M Tris-acetate, pH 8.8; 0.01 ml of 0.3 M magnesium
acetate; 0.03 ml of venom phoSphodiesterase, 0.5 m8 Per ml
in water, potency 0.3; and 0.03 ml of water were incubated
at 37° for 14.5 hours. Reaction mixtures lacking oligonuc-
leotide but containing instead 2'—deoxynucleoside-5'-mono-
phOSphateS and 2'-deoxynucleosides were treated in the same
way. Following the incubation period all reactions were
lyophilized and chromatographed on.Whatman 3MM paper using
Solvent I. All Spots absorbing under U.V. light were
removed, and the substances eluted with 0.01 N H01. The
A260 and A270 values of the eluates were determined, lead—
ing to the following result. It was found that 3.73 percent

112

TABLE X

Relative Amounts of Mononucleotides in Fraction I
(Figure 19)

 

 

Percent of

 

Mononucleotide Total Mononucleotides
dAMP 28.5
dCMP 31.4
TMP 40.1
dCMP Less than 2 percent

 

A 0.058 ml quantity of Fraction I (138 A2 0 units per
ml) was chromatographed on.Whatman.3MM paper in olvents I
and II as described in Methods. The four common 2'-deoxy-
nucleoside-5'-monophoSphates were also chromatographed as
reference standards. All U.V. absorbing Spots were removed
from the chromaotgraphs and eluted with 0. 01 N HCl. The
recovery of standard mononucleotides was found to be in the
range of 92-110 percent.

113

TABLE XI
Distribution of Products in a Hydrolyzate of dDNA

 

 

Fractiona Average Percent of Total
Chain Length Hydrolyzate Recovered

 

I 1.04 20.4
II 2.08 29.4
III 2.96 25.0
IV 3.98 25.2

 

a'Fraction numbers as given in Figure 19.

Material from each of Fractions I-IV was assayed for
the ratio of total to terminal phoSphate as described in
Methods (see Determination of Average Chain Length of
Mixed Oligonucleotides). The percent of the total hydrol-
yzate in each fraction was calculated from data Shown in
Table IX.

114
(measured by A270) and 4.15 percent (measured by 5260) of the
total venom phOSphodiesterase hydrolyzate of Fraction II
appeared as nucleoside. Likewise, 2.74 percent (A270) and
3.18 percent (A260) of the total hydrolyzate of Fraction III
appeared as nucleoside. Taking into account the average
chain length of each fraction these figures indicate that
7.5-8.3 percent of Fraction II was dinucleoside monophos-
phates (XpX), and 8.2-9.5 percent of Fraction III was tri-
nucleoside diphoSphates (prpX). Even with these results,
the experiment still shows that principally 5'-phOSphoryl
terminated oligonucleotides were produced from dDNA at

pH 8.0 by the P-100 enzyme.

The Hydrolysis of Poly-2'-O-Methyladegylic Acid by
P-100 Enzyme

When it was found that the P-100 enzyme preparation
acted endonucleolytioally on both RNA and DNA to give 5'-
phOSphoryl terminated products, it was thought desirable to
attempt to hydrolyze the polynucleotide poly-2'-0-methyl-
adenylic acid (Poly Am). Because this polymer exhibited
unusually high viscositiss at pH values below 6.0. it was
decided to use pH 8.0 as one of the conditions for hydrolysis
(since the P-100 enzyme exhibited significant RNase and DNase
activities at this pH). A reaction containing 0.04 ml
poly.Am, 27.5 A257 units per ml in water; 0.05 ml‘rris-HCI,
0.2 M, pH 8.2; and 0.01 ml of undiluted P-100 enzyme was
incubated at 37°. Aliquots of 0.01 ml were removed at

various times and Spotted on a 14 x 20 cm MN 300 cellulose

115
thin layer sheet for chromatography in the n—propanol:29
percent ammonium hydroxidezwater solvent system described
in the section, Susceptibility of Poly A, Poly U, and Poly
C to Hydrolysis by P-100 Enzyme. The mononucleotide 2'-0-
methyladenosine-S'-mon0phoSphate (5'-AmMP) and the corres-
ponding nucleoside 2'-0-methyladenosine (Am) were also
included on the chromatogram as reference standards. Their
Rf values were 0.57 and 0.86 reSpectively. It was evident
by the appearance of the chromatogram after chromatography
that even at 0.5 hour incubation a significant amount of
the polymer had been degraded by the P-100 enzyme. By 2.0
hours products of hydrolysis as small as the mononucleotide
(5'-AmMP) were being formed. At 4.0 hours incubation, most
of the U.V. absorbing material was adjacent to and just
behind the mononucleotide area, however, it also was evi-
dent that larger products still remained in the hydrolyzate
because U.V. material was still visible all the way to the
origin. By 13 hours of incubation a single streaked Spot
was observed to extend from about half the distance between
5'-AmNP and the origin to a position adjacent to the 5'-AmMP
Spot. NoAm (nucleoside) was observed in the hydrolyzate,
but there was a Spot, which became visible at about 2.0
hours incubation, that migrated approximately half way
between the nucleoside and mononucleotide. The intensity
of this Spot increased with longer incubation times indi-
cating that greater amounts of this product were being
formed with time.

_116

Two separate methods were then employed for the separa-
tion of oligonucleotides of adenylic acid up to a chain
length of four so that a more accurate indication of the
amounts of smaller products being formed could be made.
These methods were ascending thin layer chromatography on
PEI-cellulose using 0.8 M Tris-RC1, pH 8.4, as solvent and
paper electrophoresis (48) on DEAE-cellulose strips in 0.1 M
Tris-phOSphate, pH 6.6. In both of these systems migration
rates of the individual compounds decreased in the order
mononuc le otide > dinuc leotide > tr inucleot ids > tetranuc le ot ide .

It was apparent after using the previously described
separation techniques that much larger amounts of P-100
enzyme along with longer incubation times than those used
with poly A were necessary to achieve the same stage of
hydrolysis with poly.Am.

In order to prepare 10-20 5260 units of the 5‘-phos—
phoryl terminated tri- and tetranucleotides of 2'-0-methyl-
adenylic acid (49) the following experiment was performed.

A reaction mixture consisting of 6.90 ml polyAm (20.0 A260
units per ml): 3.45 ml of 0.10 M Tris-H01, pH 8.2; 3.45 ml
of undiluted P-100 (200 DNase B units per ml); and 3.45 ml
of water containing pentachlorophenol at a concentration of
0.5 ug per ml was incubated at 37° for 47 hours. Another
1.72 ml of P-100 enzyme was added and the reaction mixture
was incubated for another 21 hours at 47°. The reaction
mixture was then diluted to 100 ml with deionized water and
applied to a 1.5 x 33 cm column of Whatman Advanced DE-23

117
which had previously been converted to the formats form with
ammonium formats. The total 5260 units applied to the
column was 195.4. Following application of the sample the
column was washed with 300 ml of 0.01 M ammonium formate,
pH 7.5. The column was then treated with a gradient pre-
pared from 800 ml each of 0.01 M and 1.0 M ammonium formate,
both at pH 7.5. Fractions of 400 drops each were collected
throughout the washing and gradient elution procedures.
After the 0.01 M-1.0 M salt gradient had gone to completion,
the column.was washed with 100 ml of 2 M ammonium formats.
Figure 20 shows the A260 elution pattern obtained during
the 0.01-1.0 M salt gradient elution. No significant
increase was observed in the 5260 of fractions collected
during either the application of the sample or the washing
procedure. The fractions containing the highest concentra-
tion of products, as indicated by A260, were pooled as
separate peaks (see Figure 20 and Table XII). The A260
observed in each of these pooled peaks is Shown in Table
XII. Electrophoresis on DEAE-cellulose in 0.1 M Tris-phos-
phate buffer, pH 6.6 of material from peaks II-V indicated
that each peak contained at least two components. These
components were separated by paper electrophoresis on
Whatman No. 40 using 0.1 M ammonium bicarbonate, pH 7.8.
The characterization of each component separated by electro-
phoresis was then carried out by a determination of the ratio
of nucleoside to mononucleotide produced by venom phoSphodi-

esterase after dephoSphorylation by alkaline phOSphatase.

118

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no «a 00« ap«s oozed: mos nasaoo cap .:o«pc«aaoo op doom do: p:o«oonm pawn : o.«
i: «0.0 can Hopu< .moasooooaa ao«p:«o p:o«oonw one wc«smo3 can psoawzonzu oopooa
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one 2 «0.0 no some as 000 Scum oonoaopa pao«ooaw o £p«3 condone none as: asoaoo
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«upon one .mmuoo ooosueoq scares: co ass«oo so mm a m.« m on oo««aao one acre:
oou«ao«oo :p«s «a 00« on oops««o soap mos on:pH«a :o«poooa use .05: no masos
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119

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TABLE XII

Recovery From a Whatman Advanced III-23 Column of
the Products of Hydrolysis of Poly‘Am
as Determined by 5260

 

 

 

Peak Numbera Fractions Pooled Total.A260

I 5-14 12.?

II 17-18 15.2
III 21-23 17.8
IV 25-26 11.1
V-A 30 3.6
v-3 31-33 26.2

VI 37-40 28.0
VII 42-44 18.7
VIII 47-50 23.3
2 M Wash (all fractions) 11.0

 

167.6 Total
Recovered

 

8See Figure 20.

121
As can be seen by the data in Table X11123 a significant
percentage of the total hydrolyzate consisted of oligonuc-
leotides lacking a terminal phoSphate group.

 

23The author thanks Dr. F. M. Rottman for generously
providing this data.

122

TABLE XIII

Characterization of the Products of Hydrolysis of
Polyh2'-0-Methyladenylic Acid Eluted from a
Whatman Advanced DE-23 Column

W

 

Total
Peak Number Identity A260 Recovered
I AgAm-+ Am N.D.a
II pAm + Am N.D.
III .Agegam 16.53
IV pAg‘A‘“ 8.78
V-A pApApAm m m 0.812
m m
(Ap)3A 3.43
V-B pAgAgAm 19.22
m m
(Ap)3A 10.90
(A§)2Am 2.74
(A:)gAm 14.29
VII (Mill’s 13.33
VIII (pAm)6 16.12
(11:56“ 4.47
2 M Wash N.D.

 

aNot Determined.

The material in each.peak was resolved by electro-
phoresis on Whatman No. 40 paper using 0.1 M ammonium bicar-
bonate, pH 7.8. The characterization of each component was
performed by a determination of the ratio of nucleoside to
mononucleotide produced by venom phOSphodiesterase after
dephoSphorylation by alkaline phoSphatase.

DISCUSSION

The nuclease from muskmelon seeds described hereto-
fore has been purified about 2900 X. Recoveries of total
DNase activity have ranged between 10 and 15 percent of
that measured in crude extracts. Some problems have been
encountered when nuclease activities were measured in crude
homogenates because there was enough.material absorbing
strongly at 260 mu to give relatively high control values
in the lanthanum nitrate—H01 assay. Assays for activity
in crude homogenates were always done within 24-48 hours
after preparation since noticable changes in the solution,
such as precipitation, took place on prolonged storage at 4°.

Fractionation with ammonium sulfate was attempted in
the normal way by adding increasing amounts of the salt
and separating the various fractions precipitated by cenp
trifugation. A much better separation of DNase from non-
Specific phOSphodiesterase [as determined by the liberation
of p-nitrophenol from bis-(p-nitrophenyl) phosphate] occurred,
however, when the ammonium sulfate fractionation was done in
the reversed procedure: i.e. precipitating first with a
high concentration of the salt, and then dissolving the
precipitate in a solution of lower ammonium sulfate concen-

tration.2u The final solution in the ammonium sulfate

 

2“Dr. A. B. Adams, personal communication.

123

124

fractionation before dialysis could be stored as long as a
week at 4° without detectable losses in DNase activity. The
dialysis preceding DEAF-cellulose chromatography consis-
tently resulted in about a 10 percent decrease in total
activity which could not be accounted for in terms of the
small amount of precipitate formed during this procedure.

Chromatography on DEAE-cellulose represented the
best step in the purification procedure as far as overall
purification was concerned. The enzyme activities were
consistently eluted from the resin between 0.3 and 0.4 M
ammonium acetate, indicating a strong affinity of the
enzyme for the resin at pH 8.0. The presence of a Second
peak of RNase activity preceding the nuclease peak indicates
that at this point in the purification the nuclease prepara-
tion is still contaminated with some other RNase not associ-
ated with the nuclease;3'-nucleotidase complex.

SuSpicions regarding the presence of contaminating
RNase following the DEAE—cellulose chromatography were con-
firmed when significant amounts of RNase activity could be
measured in the CM-cellulose pass-through fraction (before
gradient elution of the nuclease) . No detectable DNase
(A or B) nor 3'-AMPase was present in this same pass-through
solution. This apparent separation of contaminating RNase
activity, however, was not reflected in a Significant change
in the RNase to DNase B Specific activity ratio. The
nuclease appears to have a relatively low affinity for CM-
cellulose at pH 5.5 as judged by the low salt concentration

 

 

125
required to elute it from the resin. This treatment, how-
ever, provided a rather good purification even though only
about 50 percent of the total activity applied to the
column was recovered. The loss in activities evidently
was not due to the separation of nuclease activities since
no other nuclease nor 3'-AMPase could be detected in the
pass-through volume or at higher salt elutions up to 1.0 M.
The other possibility remains, however, that separation
could have occurred but the separated nuclease may have
been inactive and hence, not detected. Instability to
storage at low pH (4-6) has been consistently observed with
the muskmelon nuclease, and has also been reported in the
case of the mung bean nuclease (18).

Chromatography on the Bio-Gel.P-100 DEAE-cellulose
combination column resulted in two beneficial accomplish-
ments: i.e. the volume following chromatography on CM-
cslluloss was decreased to about one tenth and a five- to
six-fold increase in Specific activity over that of the
CM-cellulose fraction was obtained. Most of the ammonium
acetate (0.75 M) used to elute the enzyme from the DEAE-
cellulose was removed during the subsequent pass through
the P-100 resin. This method utilizing a small pore resin
such as P-2 could find great use in the concentration.of
large volumes of solutions containing enzymes or nucleic
acids provided an appropriate ion exchange resin could be
found. Enzyme preparations following P-100 chromatography

could be stored at -10° or 4° over a period of several

m“...

126
months without more than 10-15 percent loss of DNase activ-
ity. These same preparations appear to survive lyophiliza-
tion and storage at -10° in the anhydrous state.

The enZyme preparation following P—100 chromatography
exhibited no activity when a variety of penitrophenyl nucleo-
tide derivatives were tested at pH 4.5, 7.0, and 9.0. The
enzyme also appeared to be free of 5'-nucleotidase activity
as indicated by the absence of inorganic phoSphate in 24
hour incubations with the four 2'-deoxynucleoside-5'-mono-
phOSphateS. A slight but significant hydrolysis of p-nitro-
phenyl phoSphate at the higher pH range (7-9) was observed,
however. The significance of this finding will become
apparent later in the discussion.regarding the characteriza-
tion of products of hydrolysis of dDNA and poly.A“.

Initially, work with.extracts of muskmelon seeds
centered on the purification of DNase activity assayed at
pH 7.5. It became evident, however, that RNase and 3'-nuc1eo-
tidase activities were closely associated with the DNase
activity. This close association of these three types of
activities has also been observed in mung bean Sprouts (14-
18), rice bran (13), ryegrass (12), barley (10, 11), the
fungus Penicillium citrinum (22-24), wheat seedlings (19),
and corn (20, 21). When DNase, RNase, and 3'-AMPase were
assayed at pH 7.5 the ratios of the Specific activities of
RNase and 3'-AMPase remained constant throughout the latter
three steps of the purification. A thorough study of the

12?
activities as a function of pH, however, revealed an addi-
tional DNase activity.

The first series of experiments testing the relation-
ship of pH and DNase activity, using dDNA, revealed what
appeared to be two pH optima (using Tris-H01, cacodylate,
and acetate buffers). These occurred at pH 6.0 and pH 8-9.
When imidazole and histidine buffers were substituted for
cacodylate and Tris, a striking difference in the activity
at the higher pH was noted. A reasonable explanation for
this effect might be that at higher pH a metal ion was
required for activity and histidine, rather than imidazole,
could have removed this metal from the enzyme by chelation.
In the presence of histidine and imidazole the pH optimum
occurred at about pH 5.7, and was comparable in magnitude
to the optimum at pH 8-9 in Tris. PhoSphate and citrate
buffers used in the pH 5-7 range were strongly inhibitory.
The activity observed with these buffers was only 10 percent
of that observed with histidine or imidazole. When the
pH-activity relationship for DNase was investigated, using
a pH stat , with 0.10 M NaCl as the only major ionic Species
present, a curve was obtained which was very Similar in
shape to the curve resulting from the use of Tris, cacodylate
and acetate buffers. This result conclusively proved the
existence of two pH Optima with reSpect to DNase activity on
denatured DNA in the absence of buffer effects.

The RNase exhibited activity over a rather wide range

of pH values when Tris and cacodylate buffers were utilized.

128
AS in.axperiments with DNase activity the substitution of
imidazole and histidine buffers resulted in a sharp opti-
mum of activity at pH 5.7. This was the first evidence
besides the purification data that these two activities
were very intimately related.

Results from the experiments with 3'qAMPase indicated
that maximal activity occurred between pH 7.5 and 8.5. The
effects of imidazole and histidine at pH 8.0 were much like
those observed for DNase at pH 8.0, i.e. very low levels of
activity with histidine, and approximately half maximal
(activity in the presence of Tris-HCl = 100 percent) with
imidazole. A small peak of activity appeared to exist in
the pH 5.0-5.5 range with histidine.

For the sake of convenience the lower [Nose optimum
(pH 5.7) was termed DNase A (acidic) and the higher one
(pH 8.0) DNase B (basic). The RNase was assayed at pH 5.7
and the 3'-AMPase, at pH 8.0. Once the pH optima were
determined, a more valid measure of the ratios of the vari-
ous activities through the purification could be made. It
was found, for instance, that levels of RNase in crude and
ammonium sulfate fractions were much higher when assayed in
imidazole at pH 5.7 than when measured at pH 7.5 in Tris
buffer. When assays were performed at the pH optima at
each stage in the purification, it was evident that the
ratios of DNase A, RNase, and 3'-AMPase to DNase B did not
vary significantly in the last three steps of the procedure.
The degree of purification through these last three steps

 

129
was greater than forty-fold. It would seem reasonable to
assume that if there were more than one protein entity
reSponsible for the four activities present in the prepara-
tion, that a variety of separation techniques such as
chromatography on DEAE-cellulose, CM-celluloss, and Bio-
Gel.P-100 would result in appreciable variation in the
ratios of Specific activities.

Two additional experiments were performed in an
effort to separate the four activities. The isoelectric
focusing treatment appeared to confirm the conclusion
regarding the close association of all four activities,
although the apparent lack of stability at the isoelectric
point (pH 4.4) and the determination of the different
activities at different time intervals after recovery from
the column caused the ratios of activities to vary greatly
from those observed in the P-100 enzyme preparation.
.Another possible explanation for the great variation in
Specific activity ratios in this experiment would be that
RNase and 3'-nucleotidase may in part have been separated
from the DNase but, once separated, were inactive and thus
not detectable. I

The other experiment performed in an effort to effect
a separation was polyacrylamide disc gel electrophoresis.
As shown in.Experimental Results, all activities were
localized in one protein band, and the ratios of activities
through this band remained essentially constant. Since the
four activities were found to migrate more slowly than any

 

 

 

 

130

other detectable protein, the possibility exists that a
still greater purification might be feasible with the use
of preparative disc electrophoresis. EXperiments designed
to provide mg quantities of P-100 enzyme are now in progress
to further investigate the possible use of preparative
electrophoresis. In light of the results obtained from
the purification procedure and the isoelectric focusing
and electrophoresis experiments, it must be concluded that
DNase A, DNase B, RNase, and 3'qAMPase either reside in a
Single protein Species or are part of a complex which is
held together extremely tightly.

There is much evidence to indicate that zinc plays
a major role in the stabilization of these associated activ-
ities in the pH range of 4-6. This fact has been well docu-
mented for the nuclease; 3'-nucleotidase activities of
mung bean (18), P, citrinum (22, 23), and wheat seedlings
(19). Although zinc has been observed to be inhibitory at
a concentration of 10"3 M in assays it appears to greatly
stabilize the muskmelon nuclease at high temperatures at
10"3 M. It therefore is likely that zinc plays some role
in maintaining a stable conformation of the protein (or
protein complex).

Another rather interesting difference between the
muskmelon nuclease and many of the other plant nucleases
was the finding that the molecular weight, determined on
Sephadex G-100, was about 50,000. Most of the plant

nuclease; 3'-nucleotidase enzymes cited previously have

131
been observed to be smaller Species migrating on gel filtra-
tion columns like proteins with molecular weights of 15-
30.000. It is intriguing to Speculate on the possibility
that the additional DNase pH Optimum (at pH 8.0) might
represent an additional protein subunit amounting to a
molecular weight integral of 15-20.000. The possibility
exists that methods of isolation may play a large part in
determining the nature of the final product, eSpecially if
this product consists of a complex of protein subunits. For
example, consider the hypothetical situation where a
nuclease:3'-nucleotidase complex dissociates at pH values
below 6.0 to give inactive subunits. This dissociation at
low pH is blocked, or retarded by the presence of zinc. If
this hypothetical situation were true, then it is possible
that some of the differences observed among the nuclease:
3'-nucleotidase enzymes from different plant sources might
simply be caused by the pH at which the initial extraction
procedure was done.

The effects of cations on.the muskmelon nuclease and
3'-nucleotidase activities appear to be complex. Certainly
no cation affected all four activities in the same way.

Some trends, however, are significant. The divalent cations
of the alkaline earth series led to minor increases in

DNase B and RNase activities. Calcium appeared to stimu-
late 3'-AMPase to some extent as well. The monovalent
cations, sodium, lithium, and potassium brought about slight

increases in RNase and 3'-AMPaSe, but were without much

132
effect on DNase A or B. The other divalent cations tested
seemed to cause inhibition of all four activities. The
relative magnitude of inhibition by these divalent cations
seems to be similar for the DNase B and RNase activities
(see Table II for summary).

It is rather difficult to draw conclusions regarding
the nature of the interaction of metals in the various reac-
tions. The experiment was originally done to see if any of
the more common cations functioned to bring about major
increases in any or all of the enzyme activities. Apparent
stimulation of nuclease activities by metal ions is often
brought about by effects of the metal on the conformation
of the polynucleotide substrate (9) rather than by direct
effects on the enzyme itself. One metal effect on the musk-
melon nuclease which may have importance regarding the
Specificity of the endonuclease is that of zinc. As indi-
cated in.Tabls II moderate inhibition of the nuclease
activities by zinc was contrasted with almost complete
inhibition of the 3'-AMPase. In a previous discussion (see
Susceptibility of Poly A, Poly U, and Poly C to Hydrolysis
by P-100 Enzyme) the relative 3'-nucleotidase activities
were implicated as possibly eXpressing the Specificity of
bond cleavage in the polynucleotide. A possibly related
observation is that the presence of zinc (10"+ - 10-3 M)
during the course of hydrolysis of high molecular weight
RNA by both mung bean (46) and wheat seedling (19) nuclease:

3'-nucleotidases appeared to greatly increase the percentage

 

 

.IDEMN.

a.

133
of mononucleotides in the hydrolyzate over hydrolyzates not
containing zinc. These two results seem to indicate that
zinc has the ability to change the Specificity of the
nuclease:3'-nucleotidass to one involving fewer endo- and
more exonucleolytic kind of scissions. Alternatively, zinc
may function in some way to facilitate the hydrolysis of
di- and trinucleotides, thus giving a higher percentage of
mononucleotides in the hydrolyzate compared to hydrolyzates
lacking zinc.

Certainly, with an enzyme system as complex as this
one, much work on the properties of the various activities
remains to be done. The main thrust, however, of this
author's work has been toward an elucidation of the possible
Specificities of the RNase and DNases.

When it became evident that the muskmelon nuclease
exhibited 3'-nucleotidase activity, a study designed to com-
pare the activity on the four ribonucleoside-3'-monophos-
phates was undertaken. The pH optimum of 3'-AMPase (pH 8.0)
was chosen.as a standard condition under which the other
three nucleotides would be tested. A striking difference
in relative rates was observed as shown in.Tabls V. This
same order of reactivity (A> G>U>C) has been observed with
the mung bean 3'-nucleotidase (18). Under the conditions
used in the muskmelon 3'-nucleotidase assay, no detectable
hydrolysis of 3'-CMP occurred (as little as 2 percent
hydrolysis could have been detected with the phoSphate assay
employed). This result along with later ones indicating

134

that CMP was either totally absent or present in only very
minor amounts in hydrolyzates of dDNA and RNA directed the
work toward elucidation of the details of enzyme Specificity.

When the 2'—deoxynucleoside-3'-monoph03phates were
tested as possible 3'-nucleotidase substrates under condi-
tions of substrate and enzyme concentrations similar to
those used with the ribonucleoside-B'-monophoSphates no
hydrolysis could be detected. When the enzyme concentration
was increased forty-fold and the incubation time was
lengthened from 0.5 hour to 15 hours, however, hydrolysis
of the compounds took place. The most important finding,
however, was that 3'-dCMP was not degraded and 3'-dGMP was
only partially degraded. This result points up another dif-
ference between the muskmelon nuclease:3'-nucleotidase and
the analogous enzyme from mung bean. Johnson and Laskowski
(26) have stated that only 3'-ribonucleotides were hydrolyzed,
thus conferring sugar Specificity at least on the nucleoti-
dase activity. The muskmelon enzyme, however, appears to
hydrolyze both ribo- and 2'-deoxyribonucleotides although
there is a vast difference in the relative rates of hydroly-
sis. The total absence of detectable hydrolysis of 2'-deoxy-
cytidine-3'-phoSphate is the most interesting result in
light of the previous findings with cytidine-3'-phOSphate.

In order to test the muskmelon nuclease preparation
for the presence of RNases or cyclic phoSphodiesterases the
enzyme preparation was exposed to various cyclic phoSphodi-

ester derivatives of the mononucleotides (both 3',5'— and

135
2*,3'-cyclic mononucleotides). Under conditions of nucleo-
tide concentration, enzyme concentration, and incubation
time identical to those used with the 2'-deoxynucleosids-3'-
monophoSphates no significant hydrolysis of the cyclic
nucleotides could be detected either at pH 5.7 or pH 8.0.
A slight amount of nucleoside formation.evident in the reac-
tions containing enzyme was attributed to dephOSphorylation
of the 3'-phOSphoryl nucleosides which were produced in low
concentrations by nonenzymatic breakdown of the cyclic
derivatives. This nonenzymatic breakdown to the phOSpho-
monoesters was detected, in reactions lacking enzyme, as a
more Slowly migrating Spot in Solvent I.

The earliest work on the purification of the muskmelon
nuclease utilized native DNA as substrate with the hope that
a DNase Specific for double stranded DNA could be isolated.
Experiments with the enzyme purified approximately 100-fold
indicated that denatured DNA was hydrolyzed much more rapidly
than was the double stranded form. When a more detailed
study was undertaken with enzyme purified through the P-100
step (A'2900-fold) it became apparent that some striking
differences existed in the hydrolysis rates of various poly-
nucleotide substrates. As the kinetic study in Figure 12
shows, RNA is hydrolyzed about twice as fast as denatured
DNA. The relative rates of hydrolysis of denatured DNA at
pH 5.7 and 8.0 appear to be equal up to about 60 percent
conversion of the substrate to lanthanum nitrate-H01 soluble

products. At this point the rate at pH 8.0 drops below that

 

136

measured at pH 5.7. This Slowing in the rate of reaction
has been described by Vanecko and Laskowski (50) as "auto-
retardation," i.e. intermediates in the reaction are pro-
gressively more resistant Substrates as their size decreases.
The rate of the reaction Slows down considerably and can be
restored only by the addition of large amounts of enzyme.
Such an observation was made with the muskmelon nuclease
while using a pH stat to follow the progress of a reaction
at pH 8.0. One explanation for the apparent difference in
rates above 60 percent conversion of substrate at pH 5.7 and
8.0 could be the charge density on a small oligonucleotide.
Since the primary phOSphate has a pKa of 0.7-1.6 and the
secondary phoSphate a pKa of 6.0-6.5 Short oligonucleotides
would have a larger charge to size ratio at pH 8.0 than at
pH 5.7. The fact that the additional charge at pH 8.0 would
be located at the terminus adds to its interfering effect
the shorter the oligonucleotide gets eSpecially in the case
of endonucleolytic type cleavages. In a long polynucleotide
an endonuclease would rarely encounter a terminal phoSphate
as far as the points of preferential cleavage are concerned.

The rate of release of lanthanum nitrate-H01 soluble
products from native DNA at pH 8.0 appears to be less than
10 percent of that of denatured DNA. ‘The ratio of these
rates at pH 5.7. however, increases quite significantly to
about 20 percent. This same difference in the reactivity of
native DNA at pH 5.7 and 8.0 was observed with DNA from calf
thymus. Such a difference could also be rationalized by the

13?
same reasoning of substrate ionization as was used for
denatured DNA.

The great difference in reactivity of denatured DNA
and native DNA is potentially useful in a variety of Situa-
tions. It is doubtful, however, that the muskmelon nuclease
exhibits a strict Specificity for single stranded DNA, since
with salmon Sperm DNA, conversions of greater than 50 percent
of the native DNA substrate to lanthanum nitrate-RC1 soluble
products were observed at pH 8.0. A strict judgment on this
question must await experiments using INA which has been
shown by other criteria to contain no single stranded breaks
or regions. Mikulski, Johnson, and Laskowski (51) have
reported that a highly purified preparation of mung bean
nuclease is capable of Specifically degrading only denatured
DNA in a mixture of denatured and native DNA.

It was encouraging from the standpoint of potential
Specificity to find that the nuclease activities on RNA at
pH 5.7, on denatured 111A at pH 5.7 and 8.0, and also on
native DNA at pH 5.7 and 8.0 exhibited an endonucleolytic
mode of action. The gel filtration method used, however,
does have one inherent disadvantage. The presence of rela-
tively large amounts of exonucleass could be masked by the
presence of small amounts of endonuclease the hydrolysis
products of which Spill over into the position at which mono-
nucleotides are eluted from the column (see Figures 13-15).
The mode of hydrolysis of poly A at pH 8.0 was also observed
to be endonucleolytic since paper chromatographic evidence

138
indicated that di-, tri-, and tetranucleotides were formed
at early stages in the reaction.

Experiments utilizing the action of alkaline phoSpha-
tase, a nonSpecific phOSphatase, and 5'-nucleotidase, a
Specific phoSphatase which removes phoSphate from only 5'-
phoSphoryl mononucleotides, conclusively demonstrated that
muskmelon nuclease forms nucleoside-5'-monoph03phates from
RNA at pH 5.7. and the correSponding 2'-deoxy derivatives
from dDNA at the same pH. Furthermore, the use of snake
venom phOSphodiesterase confirmed the presence of 5'-phos-
phoryl terminated oligonucleotides in hydrolyzates of poly
A produced by the muskmelon enzyme at pH 8.0. Because of
the presence of 3'-nucleotidase activity relatively large
amounts of nucleosides would be present in hydrolyzates of
RNA if ribonucleoside-3'-monophoSphateS were formed. This‘
was not observed. The only contradiction to this reasoning
would be the possible inhibition of 3'-nuclsotidase by the
presence of nucleoside-5'-monophoSphates. At present no
data are available to either confirm or deny this possibil-
ity.

It was surprising to find that when polyadenylic acid,
polyuridylic acid, and polycytidylic acid each were tested
as possible substrates for the muskmelon nuclease, hydrolysis
of only the first two could be detected. This finding
strengthened the suSpicion, acquired from data on the rela-
tive 3'-nucleotidase activities, that the CpY bond was par-

ticularly invulnerable to the action of muskmelon nuclease.

 

139
The reaction mixtures were buffered at pH 8.0 instead of 5.7
in order to take advantage of the Specificity observed with
the 3'—nucleotidase at pH 8.0. Other experiments also
showed that poly A could be hydrolyzed to 5'-AMP at pH 5.7
by the 9-100 enzyme preparation. '

The next logical question to ask was what would happen
if one attempted to hydrolyze a polyribonucleotide containing
cytidylic acid plus one other type of nucleotide. The hypo-
thesis regarding the CpY bond would predict that all oligo-
nucleotide products should be of the general structure
(pC)an where n is any number equal to or greater than 1.0.
Of course, no CMP should be found in.a limitzu hydrolyzate
if the CpY bond is inrulnerable. The other nucleotide is
eXpected to be present in hydrolyzates as the free mononuc-
leotide Since the occurrence of the YpY sequence should allow
for the formation of p! (it must be remembered that 5'-phos-
phoryl terminated products are formed by the muskmelon
nuclease).

The results obtained from the characterization of the
products of hydrolysis of polyuridylic-cytidylic acid (poly
U,C) support the previously described hypothesis. Chemical
and enzymatic characterization of 84 percent of the recovered
products indicated that UMP, pCpU, (pC)2pU, and (pC)3pU were

 

21”"Limit" hydrolysis is here defined as one which has
been.permittsd to proceed to the point where it is thought
ihatdall vulnerable phoSphodiester bonds have been hydro-
yze .

 

140
the major components in.the hydrolyzate. .Another 10 percent
of the recovered material, which was eluted from the DEAE-
Sephadex A—25 column, had Spectral characteristics that
indicated the presence of high proportions of cytidylic
acid. Table VIII summarizes the chemical and enzymatic
methods of characterization used on the major oligonucleo-
tide Species.

Another observation regarding recovery of the charac-
terized Species was that the amount of each obtained was in
agreement with the expected probabilities of occurrence of
those sequences in a random capolymer as calculated by the
method of Nirenberg, 23. 2}. (52), For example, a polynuc-
leotide containing uridylic acidzcytidylic acid in a molar
ratio of 2:1 reSpectively should contain 44.5 percent of its
weight as the nucleotide sequence UpU (0.666 x 0.666), 29.6
percent as UpUpU, 22.2 percent as CpU, 7.4 percent as CpCpU,
and 3.7 percent as CpCpC. As can be seen in Table VII and
VIII the major proportion of products have been recovered in
amounts approximating these predicted frequencies.

The fractionation and partial characterization of
hydrolysis products produced from denatured DNA by the musk-
melon nuclease have brought to light some important properties
of this enzyme. First, this experiment, as well as a number
of others done previously under Similar conditions, have
shown.that the nuclease converts about 20 percent of the
total substrate to mononucleotides. The composition of this

mononucleotide fraction, however, is unusual in that no dCMP

 

 

141
has been detected. Taking into account the observations
made with reSpect to the 3'-nucleotidase activities on 2'-
deoxyribonucleoside-3'-monophOSphates one might predict the
same sort of Specificity for the hydrolysis of dDNA, i.e.
inability of the enzyme to attack the dedY bond, thus pro-
ducing no free cytidylic acid except in those cases where
the DNA chains have cytidylic acid on the 3'-terminus.
Most experiments in which calculations of the relative :
amounts of each mononucleotide were done showed that the

purines were produced in about equal amounts; very often the I

 

amount of dCMP was slightly greater than that of dAMP. This
result seems to be in contrast to those found with the
nuclease:3'-nucleotidases of wheat (19) and mung bean (16)
where dAMP was always found to be the mononucleotide in
excess. The determination of average chain length of the
fractions eluted from DEAE-Sephadex A-25, immediately after
the mononucleotides together with the 5260 values for these
fractions indicates that roughly equal quantities of di-,
tri-, and tetranucleotides were formed. It is difficult to
say whether or not these products represent the extreme
limit of the reaction in terms of size since as was previ-
ously mentioned the phenomenon of autoretardation could be
operative in the latter stages of digestion.

Probably one of the most important, as well as the
most discouraging results to come out of the work on the
characterization of hydrolysis products of dDNA was the
finding that approximately 7-8 percent of the dinucleotide,

 

 

{IIIIIII III! 1‘

142

and 8-9 percent of the trinucleotide fractions appeared to
lack terminal phoSphate groups. The most plausible reason
for this result would be that the enzyme preparation must
be contaminated with a slight amount of nonSpecific phos-
phatase capable of removing phoSphates from either 5'-phos-
phoryl mononucleotides or oligonucleotides. These dephos—
phorylations would only become evident when the products of
hydrolysis (5'-phoSphoryl terminated mono- and oligonucleo-
tides) were incubated with the enzyme preparation for rela-
tively long periods of time. Reactions in which the sub-
strate was hydrolyzed rather rapidly, and in which only
small amounts of enzyme were required, were found to contain
no detectable dephoSphorylated products (see Determination
of PhoSphate Position in Oligonucleotides Obtained from a
Hydrolyzate of Polyadenylic Acid). The suSpicion concerning
nonSpecific phoSphatase contamination in.the P-100 enzyme
preparation was confirmed with assays on Slices from a poly-
acrylamide gel electrophoretic separation of the muskmelon
nuclease preparation. With_p-nitrophenyl phoSphate as sub-
strate, two or possibly three regions of phoSphatase activ-
ity were detected, regions not associated with the nuclease:
3'-nucleotidase band. ‘The major amount of p-nitrophenyl
phOSphats phoSphatase, however, was observed in the nuclease:
3'-nucleotidase band.

In the course of elucidating the Specificity of the
muskmelon nuclease it was found that the enzyme was capable

of hydrolyzing the phoSphodiester bonds of the polynucleotide

 

143
poly-2'-O-methyladenylic acid with the formation of 5'-phos-
phoryl terminated mono- and oligonucleotides ranging up to
the heptanucleotide in length. This enzyme preparation is
the first one to the author's knowledge capable of degrad-
ing this polynucleotide with the formation of 5'-ph08phoryl
terminated oligonucleotides. A report in the literature
(53) concerning a Specific RNase capable of degrading only
2'-0-methylated polynucleotides has since been retracted by
the senior author17. The potential usefulness of the musk-
melon nuclease in this latter case was complicated by the
contaminating phoSphatase activity in the preparation.
Since the polymer was degraded much more slowly than was
normal poly A, an incubation period amounting to 68 hours
was needed. AS is indicated inITable XIII, these extreme
conditions produced dephOSphorylated oligonucleotides comp
prising over 50 percent of the total oligonucleotide frac-
tion. This fact necessarily complicated the isolation of
the desired 5'-phoSphoryl terminated tri- and tetranucleo-
tides. A report of the use of these isolated tri- and
tetranucleotides is presently being published (48).

...—...;(‘L'Ibl'

 

 

 

SUMMARY

A nuclease from the seeds of muskmelon has been puri-
fied about 2900-fold with recoveries of DNase activities rang-
ing between 10 and 15 percent of that measured in crude extracts.

The purified preparation hydrolyzes native DNA, dena-
tured DNA, RNA, and the 3'-phoSphoryl bond of 3'-AMP. 3'-GMP,

 

and 3'-UMP but not of 3'-CMP.

 

Included in the purification procedure is an.ammonium
sulfate fractionation, column chromatography on DEAE- and CM-
cellulose and gel filtration on a combination column of Bio-Gel
P-ioo and DEAR-cellulose.

The DNase activity on denatured DNA exhibits two pH
cptima, one at 5.7 (DNase A) and the other at 8.0 (DNase B).
RNase activity is optimal at pH 5.7. and 3'-AMPase exhibits
optimal activity at pH 8.0.

A close association of all four activities: DNase A,
DNase B, RNase, and 3'-AMPase has been observed to exist
through a variety of treatments. The ratios of DNase A, RNase
and 3'-AMPase to mase B do not vary significantly in the
last three steps of the purification.procedure. No separa-
tion of the activities is effected by either isoelectric
focusing or polyacrylamide disc gel electrOphcresis at pH 9.5.
At this stage of purification the enzyme preparation exhibits
10 protein.bands on.pclyacrylamide disc gel electrophoresis.

144

145
The Slowest migrating band contains all four activities.

The four activities migrate together through Sephadex
G-100 like a protein Species having a molecular weight of
about 50.000.

Cations affect the nuclease and 3'-nuclectidase activ-
ities in a complex manner. No cation affects all four activ-
ities to the same degree. Cations of the alkaline earth
series cause 7-20 percent increases in DNase B and RNase
activities. The moncvalent cations, sodium, lithium, and

potassium bring about similar increases in.aNase and 3'—AMPase

 

but are without much effect on DNase A or B. All.other di-
valent cations cause varying degrees of inhibition of all
four activities. The relative magnitude of inhibition caused
by these divalent cations seems to be similar for the DNase B
and RNase activities.

At pH 8.0 the order of reactivity of the nucleoside-
3'-moncphOSphates is 3'-AMP>3'-GMP> 3'-UMP. Under the condi-
tions used in the 3'-nucleotidase assay no detectable hydroly-
sis of 3'-CMP occurs. Hydrolysis of three of the 2'-deoxy-
nucleoside-3'-monoph08phates occurs but at a significantly
slower rate. 2'-Deoxycytidine-3'-monophoSphate is not dephos-
phorylated.

When reacted with cyclic phoSphodiester derivatives of
the mononucleotides the nuclease preparation is unable to
catalyze any significant hydrolysis at either pH 5.7 or
pH 8.0 of various cyclic phoSphodiester derivatives of the

146
mononucleotides, namely the 3',5'- and 2',3'-cyclic mononuc-
leotides.

RNA is hydrolyzed by the nuclease about twice as
rapidly as denatured DNA at pH 5.7. The relative rates of
hydrolysis of denatured DNA at pH 5.7 and 8.0 are nearly
equal up to about 60 percent conversion of the substrate to
lanthanum nitrate-HCl soluble products. At this point the
rate at pH 8.0 draps below that measured at pH 5.7.

The rate of hydrolysis of native DNA at pH 8.0 appears
to be less than 10 percent of that of denatured DNA. The
ratio of these rates at pH 5.7, however, increases quite
significantly to about 20 percent.

Muskmelon nuclease appears to act endonucleolytically
on RNA at pH 5.7. on denatured DNA at both pH 5.7 and 8.0,
and on native DNA at both pH 5.7 and 8.0.

At their pH cptima, all three nuclease activities
form 5'-phOSphoryl terminated products.

At pH 8.0 the nuclease preparation hydrolyzes poly-
adenylic acid and polyuridylic acid but not polycytidylic
acid. There is strong evidence to indicate trat at pH 8.0
polyuridylic-cytidylic acid (poly U,C) is hydrolyzed to a
mixture of products containing UMP and oligonucleotides of
the following general structure: (pC)npU where n is any
number equal to or greater than 1.0.

The partial characterization of the products of
hydrolysis produced from denatured DNA by the muskmelon
nuclease at pH 8.0 has indicated that about 20 percent of

 

147

the substrate is converted to mononucleotides, 30 percent to
dinucleotides, and the remainder to equal amounts of tri-
and tetranucleotides. The mononucleotide fraction lacks
dCMP. The other three mononucleotides occur in the follow-
ing percentages (of total mononucleotides isolated): TMP,
40.1: dCMP. 31.4: and dAMP, 28.5.

The enzyme preparation is suSpected of being contam-
inated with nonSpecific phoSphomonoesterase activity because I

about 7-8 percent of the dinucleotides and 8—9 percent of

 

’-

the trinucleotides isolated from an extensively hydrolyzed
sample of denatured DNA appear to lack terminal phoSphate
groups. This same enzyme preparation, however, appears to
be free of nonSpecific phOSphodiesterase activities.

The muskmelon nuclease preparation is capable of
hydrolyzing poly-2'-O-methyladenylic acid to 5'-phOSphoryl

terminated mono- and oligonucleotides.

11.

12.

13.
14.

15.

16.

B IB LI OGRAP HY

Watson, J. D., and F. H. C. Crick, Nature, 171. 737
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Holley. R. W., Apgar, J., Everett, G. A., Madison, J. T., i,
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Holley, R. W., Apgar, J., Everett, G. A., Madison, J. T., r.
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Holley, R. W., Madison, J. T., ard A. Zamir, Biochem.
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APPENDIX

Definition of Terms

A260 unit: A solution with an.A26 of 1.0 (1.0 cm path
length) is defined to 8ontain 1.0 1260 unit
per ml.

dDNA: Denatured deoxyribonucleic acid

nDNA: Native deoxyribonucleic acid

poly.Am: poly-2'-O-methyladenylic acid

 

poly U,C: polyuridylic-cytidylic acid
cold: 0-4° centigrade

room temperature: 23° centigrade

Commercial Locations

Allied Chemical Company: Morristown, New Jersey, U.S.A.

Baker Chemical Company: Phillipsburg, New Jersey, U.S.A.

Bio-Rad Laboratories: Richmond, California, 94804, U.S.A.

Brinkman Instruments, Inc.: Westbury, New York, 11590. U.S.A.

Calbiochem: Los.Angeles, California, 90054, U.S.A.

Canalco: Rockville, Maryland, 20852, U.S.A.

Fisher Scientific Company: iFair Lawn, New Jersey, U.S.A.

Mallinckrodt, St. Louis, Mo.. U.S.A.

Pharmacia Fine Chemicals, Inc.: Uppsala, Sweden

P-I.Biochemicals, Inc.: Milwaukee, Wis. 53205, U.S.A.

Sargent and Company: Detroit, Michigan, U.S.A.

Schwarz Bioresearch, Inc.: Orangeburg, New York, 10962, U.S.A.

Sigma Chemical.Company: St. Louis, Mo.. 63178, U.S.A.

Worthington Biochemical Corporation: Freehold, New Jersey,
07728. UeSeAe

151

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