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Heterogeneous nature of certain ribonucleases. Human urine and sperm ribonucleases
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
83,
39@407
(19%)
Heterogeneous Nature of Certain Ribonucleases. Human Urine and Sperm Ribonucleases’ Anwar From
A. Hakim
the Biochemistry Research Laboratories, Miami Heart Institute, Miami Florida; and the Department of Biochemistry, School of Medicine, University of Miami, Coral Gables. Florida Received
November
Beach,
28, 1958
There is an increasing interest in ribonucleases from biological sources, their specific activities, and their mode of action. Previous studies on crystalline ribonuclease (l-3) established the enzymic and electrophoretic heterogeneity of crystalline ribonuclease (RNase). The pancreas ribonuclease, despite its homogeneity as observed by sedimentation studies (4), has been separated into two fractions by partition (5) and ion-exchange chromatography (6), and has been resolved into four enzymically active fractions by paper electrophoresis (7). There is increasing evidence for the presence of different specific ribonucleases in certain biological materials. The behavior of the acid and alkaline ribonucleases under various conditions (8-10) has led to attempts at characterization of ribonuclease in normal human urine and in human sperm and comparison with ribonuclease from calf pancreas. EXPERIMENTAL Materials
and Methods
Substrate. The substrate, ribonucleic acid (RNA), was prepared from yeast (11, 12). Uridine 2’,3’-phosphate, cytidine 2’,3’-phosphate, adenosine 2’,3’-phosphate and guanosine 2’,3’-phosphate were obtained by procedures similar to those described in an earlier communication (1). Twenty-four-hour urine samples were collected and lyophilized to dryness immediately upon collection. Sperm samples were exposed to the same treatment. Preparation of Combined Ribonuclease from Human Sperm and from Human Urine. The dried urine or sperm powder was homogenized in 9 vol. of ice-cold, sterile glassdistilled water with the use of an Elvehjem all-glass homogenizer. The homogenate was centrifuged in a Spinco model L preparative ultracentrifuge at 60,000 X 9 for 1 Presented X-18, 1958.
in part
at the
New
York
Academy 390
of Science
Conference,
October
URINE
AND
SPERM
RIBONUCLEASES
391
40 min.; the supernatant was then decanted and recentrifuged at 60,000 X g for 26 min. The supernatant fluid from the second centrifugation was used in the study of the free and combined ribonucleases. Thirty milliliters of the urine or sperm supernatant obtained as described above was treated with solid ammonium sulfate (12.54 g.) to 30% saturation. The precipitate obtained by centrifugation was discarded. The supernatant was made up to 50% ammonium sulfate saturation, and the mixture was stirred and centrifuged. The supernatant fluid was poured off, and the precipitate was dissolved in 10 ml. of distilled water. To the latter solution 1.8 g. calcium phosphate gel was added, and the mixture was thoroughly stirred, allowed to stand at 0°C. for 25 min., and then was centrifuged at 15,000 X g for 30 min. The supernatant solution contained the combined inactive ribonuclease (C-RNase). Using a procedure similar to that described for urine, a combined ribonuclease (C-S.RNase) was prepared from the dried human sperm. Preparation of Purified Alkaline Ribonuclease. The alkaline RNase was prepared by a modification of the method of Kunitz (13) as follows: Thirty g. of dried urine powder was homogenized in an equal volume of distilled water in the cold. This homogenate was made up to 0.25 M with respect to sulfuric acid. The homogenate was left at 0°C. for 24 hr. with intermittent stirring and then filtered. The residue was discarded. The filtrate was centrifuged at 2000 X g for 10 min. The supernatant liquid was brought to 0.6 saturation with ammonium sulfate, and the precipitate formed was separated by centrifugation and discarded. To the supernatant, enough ammonium sulfate was added with continuous stirring to obtain 0.8 saturation. The solution was left at 0°C. for 24 hr. The precipitate formed was collected by centrifugation and dissolved in 25 ml. of distilled water, and t,he resultant solution was dialyzed for 48 hr. at 0°C. against large volumes of distilled water. The dialyzed solution was heated for 10 min. in a boiling water bath and cooled. Ammonium sulfate treatment was then repeated, and the 0.8 saturation ammonium sulfate precipitate was collected. The latter precipitate was dissolved in distilled water, dialyzed at O”C., and lyophilized to dryness. Using a similar procedure as above, a purified alkaline RNase was prepared from dried sperm. Preparation of Acid Ribonuclease. The acid ribonuclease was prepared by the following procedure [cf. (14)]: Twenty-five grams of the dried urine or sperm was extracted with 50 ml. of 0.2 N acetate buffer, pH 6.0 (15) for 4 hr. The suspension was centrifuged. The supernatant was adjusted to pH 5.0, and ammonium sulfate was added to obtain a solution of 0.6 saturation. The pH was maintained at 5.0 during the addition of ammonium sulfate. The precipitate formed was discarded. More ammonium sulfate was added to the supernatant, to obtain 0.8 saturation, and was left for 24 hr. at 0°C. The precipitate formed was collected by centrifugation and dissolved in 15 ml. of distilled water. The solution was adjusted to pH 8.0 and maintained at this pH, while ammonium sulfate treatment was repeated as above, and the supernatant solution was left at 0°C. for 48 hr. The precipitate formed was collected by centrifugation and dissolved in distilled water, dialyzed for 48 hr. against large volumes of distilled water, and lyophilized to dryness. All operations were carried out at 0°C. Using the same procedure, an acid ribonuclease was obtained from the dried human sperm.
Analytical
Procedures
Action of Acid or Alkaline Ribonucleases on Yeast Ribonucleic Acid. The reaction mixture contained yeast ribonucleic acid (2 mg.) in 0.5 ml. of 0.2 M acetate buffer at pH 5.3, about 100 kunitz units of ribonuclease (acid or alkaline type), and a few drops
392
HAKIM
of toluene; it was incubated at 37°C. for 24 hr. Aliquots of 0.2 ml. were applied in a band form on Whatman 13 MM filter paper and allowed to dry. The chromatograms were dried and photographed. Four distinct bands were located. The material from each band was concentrated at a wick, or eluted or applied on paper electrophoresis. The chemical identity of the substances present in each of the bands was determined by the use of the following analytical procedures for each one of the four bands: (a) Electrophoresis in 0.05 M ammonium formate at pH 3.5. Separately, the spots observed on the electrogram were eluted. The eluates were concentrated to dryness, the residue was hydrolyzed with 72$& perchloric acid (HClOa), and the hydrolysis products were subjected to chromatography in an isopropyl alcohol-HCl-water system. (5) To the action of snake venom, where the reaction mixture contained the eluate, 0.05 M Verona1 buffer at pH 8.5 (0.5 ml.), 0.02 M MgSOd , and purified snake venom. (c) To the action of phosphomonoesterase in a mixture containing the eluate, 0.1 M acetate buffer at pH 5.0 and phosphomonoesterase. (d) To chromatographic separation using a solvent system consisting of saturated ammonium sulfate solution-isopropyl alcohol-l iIf acetate buffer (80:2:18) (16).
Assay of Enxymic Activity Ribonuclease activity of free and combined urine or sperm enzyme preparation, and of acid or alkaline ribonuclease was determined by the rate of hydrolysis of uridine 2’,3’-phosphate and cytidine 2’,3’-phosphate. The rate of hydrolysis of the latter two anhydrides was obtained by microtitration of the secondary phosphoryl groups that were liberated by the action of the enzymes. The procedure is as follows: 2 ml. of 0.25 N solution of sodium sulfate and 0.05 M of the anhydride was placed in a 4-ml. beaker equipped with a glass electrode (1). The pH was adjusted to 7.5 with 0.1 N NaOH, and 10 pl. of the enzyme preparations was added. The amount of 0.01 N NaOH required to keep the solution at pH 7.5 for a period of 30 min. was noted. The enzymic hydrolysis of yeast ribonucleic acid by the above enzyme preparations was carried out by mixing 0.1 ml. of 0.1 M phosphate buffer, ionic strength 0.05, and pH 6.00, containing 5 mg. ribonucleic acid, with a 25-ml. aliquot of the enzyme preparation, and incubating at 37°C. for 48 hr. Ten microliters of chloroform was added as a bacteriostatic agent. The perchloric acid-uranyl acetate reagent (17) was used to precipitate the polymerized RNA. The mononucleotides liberated were separated by paper chromatography. Chromatograms of the digests of ribonucleic acid with free or combined acid or alkaline ribonuclease obtained from urine or sperm were developed by the descending technique in two dimensions (12). Permanent records of the chromatograms were obtained by the technique of Markham and Smith (18). The located mononucleotides were cut from the paper, eluted, and quantitatively determined (1). Hydrolysis of Cytidine Benzyl Phosphate or Uridine Benzyl Phosphate. The hydrolysis of uridine or cytidine benzyl phosphate was measured chromatographically. Solutions containing 2.5 pmoles cytidine or uridine benzyl phosphate, 25 al. of either the urine or sperm enzyme preparation, and 0.3 rmole of phosphate buffer at pH 6.00, to give a total volume of 0.25 ml. and an ionic strength of 0.10, were incubated at 37°C. Before the addition of the enzyme preparation, and at intervals thereafter, 25-~1. aliquots were applied to Whatman 11 filter-paper sheets. The chromatograms were developed with the isopropyl alcohol-ammonia-water system (19). The mononucleotides were located, cut from the chromatograms, eluted, and their concentration determined (1).
URINE AND SPERM RIBONUCLEASES TABLE Effect
of Heat”
I
on Ribonuclease Activities of Preparations from Human Urine and Sperm
Per cent of original
Preparation 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
393
Sperm acid RNase Sperm alkaline RNase Sperm-free RNase Sperm-combined RNase Urine Sib acid RNase Urine S,ib alkaline RNase Urine Sz? acid RNase Urine SZib alkaline RNase Urine-free RNase Urine-combined RNase
n All the preparations were exposed to 98-100°C. 10 min. at pH 7.2, then quickly cooled and activity b Sn is the preparation obtained from 24-hr. urine day of the menstrual cycle. c Szi is the preparation obtained from 24-hr. urine first day of the menstrual cycle.
activity
pH 5.8
pH 7.8
15 82 20 18 40 92 50 10 35 20
80 95 85 50 92 96 65 64 55 65
in a water bath for a period of determined. sample collected on the eleventh sample collected on the twenty-
RESULTS
Products of Enzymic
Hydrolysis
Chemical and enzymic studies of the four bands located on chromatoof the enzymic action (enzyme preparations listed in Table I) on yeast ribonucleic acid showed the following chemical identities: Band 1 contained guanosine 2’- or 3’- or 5’-phosphate or a mixture of all the three nucleotides (step a). Action of snake venom (step 6) indicated the absence of guanosine Y-phosphate. Phosphomonoesteraseaction (step c) confirmed the presence of guanosine 2’- or 3’-phosphate, and only one spot corresponding to guanosine 3’-phosphate was observed in step d. By similar analogy, band 2 was found to contain adenosine 3’-phosphate; band 3 contained guanosine 2’,3’-phosphate (alkaline urine or sperm ribonuclease and band 4 contained the 2’ ,3’-phosphate cyclic derivatives of adenosine (alkaline urine or sperm ribonuclease), cytidine, and uridine grams
(acid urine
or sperm
ribonuclease).
The chemical identity was confirmed by the electromobility in specific buffer
systems
on paper
electrophoresis
reference compounds (Fig. 1).
using authentic
pure derivatives
as
Starti line
w
Star ting line 4
3
2 B 394
1
URINE
AND
SPERM
395
RIBONUCLEASES
sta1rting line 1
2
3
4
C FIG. 1. Action of sperm alkaline ribonuclease on synthebic substrates. Paper electrophoresis was performed in an electrophoresis apparatus as described earlier (1). Sodium tetraborate, 0.4 M, pH 13, separated the cyclic anhydride and the nucleotide. A current of 10 ma. at 1100 v., applied for 1 hr., resulted in a clear separation of the two groups. A. Action of sperm alkaline ribonuclease on uridine 2’,3’-phosphate. 1. Uridine 2’, 3’-phosphate; 2. Uridine 2’, 3’-phosphate after the action of sperm alkaline ribonuclease; 3. Uridine 3’-phosphate. B. Action of sperm alkaline ribonuclease on adenosine 2’, 3 ‘N-phosphate. 1. Adenosine 2’,3’-phosphate; 2. Adenosine 2’,3’-phosphate after the action of combined urine ribonuclease; 3. Adenosine 2’,3’-phosphate after the action of combined sperm ribonuclease; 4. Adenosine 3’.phosphate. C. Action of sperm alkaline ribonuclease on guanosine 2’,3<-phosphate. 1. Guanosine 2’,3’-phosphate after the action of combined sperm ribonuclease; 2. Guanosine 2’,3’-phosphate alone; 3. Guanosine 2’,3’-phosphate after the action of sperm acid ribonuclease; 4. Guanosine 2’,3’-phosphate after the action ~of free sperm ribonuclease. The location of guanosine 2’.phosphate and guanosine 3’-phosphate has been checked using pure isomeres.
Further Purijication of Ribonucleaseson Ion-Exdange Columns precipitating between 30 and 50 % The urine- or sperm-free ribonuclease ammonium sulfate saturation was dissolved in 0.2 M ~phosphate buffer at pH 6.2, dialyzed against the phosphate buffer, and th&n chromatographed
over IRC-50 resin (XE-64, 200-mesh) previously equilibrated with the
396
HAKIM
d d
0.700
-
0.600
-
0.500
-
0.400-
x .5 0.300 E T)
-
2=
0.200
-
0.100
-
0.050
-
‘0 0
0
4
6
li
16 .iO 24 Elution
28
volume
32 36
40 44
48
52
(ml)
Urine ribonuclease. Plots of enzymic activity elution pattern of human urine ribonuclease, at pH 7.0 assay. Optical density determined at 260 mp. a. Crystalline pancreatic ribonuclease ( . . .) ; b. Urine-free ribonuclease (+--) ; c. Urine-combined ribonuclease (- - - -). FIG.
2.
same buffer (Figs. 2 and 3). Crystalline pancreas ribonuclease (Fig. 2~) showed two major peaks, the larger peak being RNase A and the smaller peak RNase B. Urine ribonuclease (Fig. 2b) showed two larger peaks and at least four smaller peaks. Human sperm ribonuclease gave one larger peak and two smaller. The elution data showed differences in behavior between urine ribonucleases, sperm ribonucleases,and crystalline pancreatic ribonucleases. Taking crystalline pancreas RNase to consist of at least two enzymic fractions, urine RNase then consisted of at least six, and sperm RNase of at least three. The behavior of sperm-free RNase and spermcombined RNase on the ion-exchange column, IR-C50 resin (XE-64, 200mesh) presented in Figs. 36 and 3c, showed that free sperm RNase consisted of at least three major peaks (Fig. 3b) while combined sperm RNase after activation is made up of three components (Fig. 3~). Properties of Pur$ed RibonucleasePreparations from Human Sperm and Urine A. Determination of pH-Optimum Curves for the Urine and Sperm Ribonucleases.Aliquots of urine, free and combined ribonucleases, and of free
URINE
AND
SPERM
397
RIBONUCLEASES
0.000 I-
d d
0.700
-
0.600
-
(? I ‘r . :
i : ! i : I : I
-
0.500
3.
0
4
8
12
16 20 Elution
FIG. 3. Human sperm ribonuclease. Plots human sperm ribonuclease, at pH 7.0 assay. a. Cryst,alline pancreatic ribonuclease (---.-) c. Sperm-combined ribonuclease (- - - -).
\ I : I I : I \
24
28 32
volume
36 40
44
48
52
(ml)
of enzymic activity elution pattern of Opt,ical density determined at 260 rnp. ; b. Sperm-free ribonuclease (--) ;
and combined sperm ribonucleases were assayed for ribonuclease enzymic activity in the presence of phosphate and acetate buffers of different pH values, but with the same ionic strength (0.15). The enzymic activity of the combined urine RNase (CU RNase), and of the combined sperm RNase (CS RNase) was assayed after activation with p-chloromercuribenzoic acid (1 X 10e6 M). The results are shown in Figs. 4a and 4b. Both CU RNase and CS RNase showed a pH optimum at about 8.00, progressive increase in activity occurring on the acid side, and a sharp decrease on the alkaline side. Both urine-free ribonuclease (FU RNase) and sperm-free ribonuclease (FS RNase) had two pH optima: at pH 5.70 and at pH 8.50. Urine alkaline or acid RNase is characterized by a pH optimum at 5.70 or 8.50, respectively. The pH-activity curves of acid and alkaline ribonuclease preparations are given in Fig. 5. A sharp optimum is obtained for acid ribonuclease from both human sperm and urine. Free ribonuclease either from human sperm or urine showed a broad optimum activity. The optimum pH of human urine RNases were at pH 5.5-5.7 and 7.8-8.0. Sperm ribonucleases showed
IIAKIM
IOOIOOLago.;t 00O 70E z 60.i 50-
L3.00
Fro. 4. Human b. Human
--1
5.00
4.00
6.00
7.00
0)
3.00
I
4.00
9.00
10.00
PH
urine ribonuelease. a. Human ribonuclease (- - - -).
urine-free
0.00
.
urine-combined
I
5.00
1
6.00
7.00
0.00
ribonuclease
I
9.00
(-);
1
10.00
PH
FIG. 5. Human sperm ribonuclease. a. Human b. Human sperm alkaline ribonuclease (-O-O-); (-.-.-) ; d. Human sperm-combined ribonuclease
sperm acid ribonuclease (- - - -); c. Human sperm-free ribonuclease (--).
optimum activity at pH 5.8 (acid enzyme), pH 8.0 (alkaline enzyme), and pH’s 5.8 and 8.0 for the free enzyme. The curves of Fig. 4a show interesting points which indicate differences between the pH-optimum curves of the acid or alkaline RNases, but certain similarity to the curves for RU RNase or FS RNase. The latter curves could be considered the sum of the acid and alkaline curves of the urine or sperm. Although these differences are
URINE
AND
SPERM
RIBONUCLEASES
399
lOOr 90
-
0 5
IO 20 30 40 50 60 70 00 90 100 Temperature
*C.
FIG. 6. Effect
of heat on human urine ribonuclease. Aliquots of the solution of each enzyme were heated separately for 5 min. at 30,40,50,60,70,80,90,95, and 100°C. and cooled directly on ice. Heating was performed after adjusting the pH of each solution to 7.2. a. Human urine alkaline ribonuclease (---); b. Human urine acid ribonuclease (- - - -); c. Human urine-free ribonuclease (-.-.-).
very preliminary, they do suggest that alkaline RNase and acid RNase fractions are different enzymes. B. E$ect of Heating on Acid, Alkaline, Free and Combined Ribonucleases from (Jrine and Sperm. Heat-stability studies were made on purified acid, alkaline, or free ribonuclease solutions. Similar studies were carried out with the use of combined ribonucleases (urine or sperm) which had been activated. Aliquots of the solution of each enzyme were heated separately for 5 min. at 30, 40, 50, 60, 70, 80, 90, 95, and lOO”C., and cooled directly on ice. Heating was performed after adjustment of the pH of each solution to pH 7.2. The results of typical assaysare shown in Fig. 6. In a seriesof experiments, the heating effect was studied on the enzyme solutions preadjusted to pH 7.2, then heated to 98-100°C. in a water bath for a period of 10 min. following which ribonuclease activity was assayed at pH’s 5.8 and 7.8. Sperm or urine alkaline ribonuclease showed 82 and 92% activity recovery, respectively, when determined at pH 7.8. All the other enzyme preparations showed 50-85 % loss in acidic (pH 5.8) activity. Other variations in heat stability of the alkaline enzymic activity are shown in Table I. The per cent recovery for free and combined ribonuclease activity was 35 and 20 for urine, and 18 for sperm. The corresponding figures for the alkaline ribonuclease activity of the same enzyme preparation were 55 and 65 for urine, and 85 and 50 for sperm. In general, the sperm enzymes showed greater stability than the urine preparations.
400
HAKIM
XpeciJic Activity A. Action on Synthetic Substrates. The synthetic substrates cytidine 2’, 3’-phosphate, uridine 2’, 3’-phosphate (UZZBP), adenosine 2’) 3’-phosphate (Az:aP), and guanosine 2’, 3’-phosphate were hydrolyzed to the extent of 25, 20, 15, and 18% by sperm alkaline ribonuclease; 30, 22, 12, and 14% by urine alkaline ribonuclease. Sperm acid ribonuclease hydrolyzed 85, 82, 98, and 95% of the above four synthetic substrates, respectively. Uridine alkaline ribonuclease hydrolyzed cytidine 2’) 3’-phosphate (65 %), urine 2’,3’-phosphate (74 %), adenosine 2’,3’-phosphate (25 %), and guanosine 2’) 3’-phosphate (20 %), while acid ribonuclease obtained from the same urine sample hydrolyzed 81, 80, 98, and 96% of the synthetic substrates, respectively. Free ribonuclease from sperm or from urine acted on 98 and 92 % of cytidine 2’,3’-phosphate; both enzyme preparations hydrolyzed 98 % of uridine 2’,3’-phosphate, 55 and 35 % of adenosine 2’,3’-phosphate, and 75 and 25 % of guanosine 2’,3’-phosphate. Free sperm ribonuclease showed greater activity on the guanosine cyclic compound than did free urine ribonuclease. Although the combined urine ribonuclease showed greater activity on the pyrimidine synthetic compounds (85 and 82 % of cytidine 2’,3’-phosphate and uridine 2’,3’-phosphate as compared to 65 and 70 % for the combined sperm ribonuclease), combined sperm ribonuclease action on the purine cyclic compounds (88 and 95% of adenosine 2’, 3’-phosphate and guanosine 2’) 3’-phosphate) was more efficient than the action of combined urine ribonuclease. All the enzyme preparations studied differed in action on the synthetic substrates from crystalline calf pancreas ribonuclease (cf. Table II). Table III presents the effect of heating on the different ribonuclease preparations. The results show that after 17 hr. of incubation, acid ribonuclease splits Az:sP to give AZP as indicated by the Rj values. No A3P could be detected. After 40 hr. incubation only one spot was detected with Rf value of 0.28, indicating a complete breakdown of Az:~P; illustrating the fact that the activity of acid ribonuclease was destroyed by heating. Acid ribonuclease acted on UzZtP to give uridylic acid; the reaction was slower than with Az:~P;~ Uz:aP could still be demonstrated after 40 hr. of incubation, Alkaline ribonuclease and heated acid ribonuclease showed no effect on Uz:fP. These results show important differences between the acid and the alkaline ribonucleases. They also show that sperm or urine alkaline ribonuclease could be different from the pancreatic ribonuclease which split pyrimidine cyclic mononucleotides whereas the sperm or urine enzyme did not. * The rate of activity scribed earlier (1).
on the cyclic
anhydrides
was carried
out by a technique
de-
a
cycle.
on the
Alk. RNase AC. RNase Alk. RNase AC. RNase Alk. RNasea AC. RNase* free RNase comb. RNase free RNase comb. RNase pancreas RNase (HzO)
Preparation
Samples collected
Sperm Sperm Urine Urine Urine Urine Sperm Sperm Urine Urine Cryst. Control
the menstrual
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Enzyme
-
20 82 22 79 74 80 98 70 98 82 92 12
day
of the menstrual
CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’(2’) CMP-2’(3’) CMP-3’(2’) CMP-3’(2’) CMP-3’(2’) CMP-3’(2’)
II or Urine
cycle;
the
UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’(2’) UMP-2’(3’) UMP-3’(2’) UMP-3’(2’) UMP-3’(2’) UMP-3’(2’) other
Reaction products
Uridine 2’, 3’phosphate Extent of hydrolysis
25 85 30 80 65 81 98 65 92 85 99 10
-
Sperm
TABLE Human
%
Reaction products
from
%
Extent of hydrolysis
-
Cytidine 2’, 3’phosphate
of Ribonucleases
twenty-first
-
Action
Substrates
-
.-
sample
15 98 12 96 25 98 55 88 12 35 12 10
%
was collected
AMP-3’ AMP-2’ AMP-3’ AMP-2’ AMP-3’ AMP-2’ AMP-3’(2’) AMP-2’(3’) AMP-3’(2’) AMP-3’(2’) AMP-3’ AMP-3’(2’)
Reaction products
Adenosine 2’, 3’phosphate
Synthetic
Extent of hy,drolysis
urine
-
--
--
- or -
day
GMP-3’ GMP-3’ GMP3’ GMP-3’ GMP-3’ GMP-3’ GMP-3’(2’) GMP-2’(3’) GMP-3’(2’) GMP-3’(2’) GMP-3’ GMP-3’(2’)
Reaction products
on the eleventh
18 95 14 97 20 96 75 95 25 48 23 12
%
Extent of hydrolysis
Guanosine 2’, 3’phosphate
of
the
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Preparation
a Samples menstrual
collected cycle.
-
Ribonucleases
25 80 23 65 35 60 55 60 80 70 92 10
%
Extent of hydrolysis
day
-
22 75 18 58 30 55 60 60 85 68 88 12
%
Sperm
III
cycle;
the
UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’(2’) UMP-3’ UMP-3’(2’) UMP-3’(2’) UMP-3’(2’)
Substrates
sample
2 0 2 0 5 0 3 70 2 20 0 10
%
-
was collected
AMP-3’(2’)
AMP-3’ AMP-3’(2’) AMP-3’ AMP-3’ -
AMP-3’
AMP-3’ -
AMP-3’ -
Reaction products
Adenosine 2’, 3’phosphate
on Synthetic
Extent of hy,drolysis
urine
-
-
or Urine
other
Reaction products
Uridine 2’, 3’phosphate
Extent of hy,drolysis
__
-
TABLE
from Human
of the menstrual
CMP3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’(2’) CMP-3’ CMP-3’(2’) CMP-3’(2’) CMP-3’(2’)
Reaction products
Cytidine 2’, 3’phosphate
of Heated
on the twenty-first
Sperm Alk. RNase Sperm AC. RNase Urine Alk. RNase Urine AC. RNase Urine Alk. RNasea Urine AC. RNase” Sperm free RNase Sperm comb. RNase Urine free RNase Urine comb. RNase Cryst. pancreas RNase Control (HZO)
Enzyme
Action
-
-
day
GMP-3’(2’)
GMP-3’ GMP-3’(2’) GMP-3’ GMP-3’ -
GMP-3’ -
GMP-3’ -
GMPS
Reaction products
on the eleventh
10 0 8 00 6 0 5 80 5 15 0 12
%
Extent of hydrolysis
Guanosine 2’, 3’phosphate
of
URINE
AND
SPERM
RIBONUCLEASES
403
B. Action on Yeast Ribonucleic Acid. Incubation of yeast ribonucleic acids with sperm-free ribonuclease for 24 hr. produced 7.5 and 13.5% of adenylic acid and guanylic acid, respectively. Urine-free ribonuclease produced 8.5 and 10.5%. Sperm-combined ribonuclease when incubated with yeast ribonucleic acid resulted in 22.5 and 28.5% of cytidylic acid and guanylic acid, respectively. Urine-combined ribonuclease liberated only 8.9 and 14.8% of the mononucleotides, respectively. Although slight differences are noted between the action of the free enzymes on yeast ribonucleic acid, the combined enzymes differed in the amount of adenylic acid and guanylic acid produced by their action. Urine-combined ribonuclease produced approximately twice the amount of cytidylic acid that is produced by sperm-combined ribonuclease. It is suggested from these data that sperm- or urine-combined ribonuclease differed in their specific activity on yeast ribonucleic acid. Although no adenylic acid and only 1.2 % guanylic acid resulted from the action of urine (SJ alkaline ribonuclease on yeast ribonucleic acid and guanylic acid, respectively, the amount of pyrimidine nucleotides produced by the action of urine (Sn) alkaline ribonuclease is relatively more than those liberated by alkaline ribonuclease obtained from urine sample (&I). Urine (Sn) acid ribonuclease liberated 16.0 and 11.2 % of adenylic acid and guanylic acid. Urine (&I) acid ribonuclease produced 7.2 and 14.5 % of the purine mononucleotides, respectively. The acid or the alkaline ribonucleases present in urine samples (FL) and (SZl) appear to differ in their action on yeast ribonucleic acid. Sperm alkaline ribonuclease produced 3.5, 5.5, 6.5, and 7.8%, while sperm acid ribonuclease resulted in 20.8, 25.4, 15.0, and 17.5 % of adenylic acid, guanylic acid, cytidylic acid, and uridylic acid, respectively. These data indicate either that sperm alkaline ribonuclease is less active than sperm acid ribonuclease on yeast ribonucleic acid, or sperm alkaline ribonuclease possesses a secondary specific activity (i.e., a synthetic activity). Further studies are in progress to define the specificity of these two sperm enzymes on yeast ribonucleic acid. Although all the enzymes studied in this report (except urine E&l alkaline ribonuclease) produced adenylic and guanylic acid, crystalline pancreas ribonuclease did not produce the purine mononucleotides. On the basis of these data the enzymes presented in Table IV differed in their specific activit,y on yeast ribonucleic acid from that of crystalline pancreas ribonuclease. DISCUSSION
Ribonucleases are enzymes that degrade ribonucleic acids so that they no longer show their typical macromolecular properties. They have been
HAKIM
404
TABLE Mononucleotides
Liberated Sperm
IV
by the Action of Ribonuclease and Urine on Yeast Ribonucleic
Preparations Acid
moles 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Alkaline hydrolysis” Crystalline RNase* Free Sperm RNase Combined Sperm RNase Sperm Alkaline RNase Sperm Acid RNase Urine Sllc Alkaline RNase Urine Siic Acid RNase Urine Szi~ Alkaline RNase Urine S~,C Acid RNase Free Urine RNase Combined Urine RNase
0
5
IO 20
30
per 100 moles
24.8 -
31.6 -
7.5 22.5 3.5 20.8 0.0 16.0 1.2 7.2 8.5 8.9
13.5 28.5 5.5 25.4 1.2 11.2 12.2 14.5 10.5 14.8
a One normal NaOH at 37°C. for 24 hr. * Crystalline ribonuclease (Armour). The enzyme before use. 0 Urine samples collected on the eleventh day (Sn) of the menstrual cycle.
40
Temperature
50
60
23.8 15.7 15.5 7.8 6.5 15.0 14.5 13.6 11.5 10.5 14.8 14.5
was
crystallized
and the twenty-first
70
Human
from
00
90
18.7 18.1 18.1 10.5 7.8 17.5 18.0 14.0 12.5 11.5 16.5 16.9
three day
times (Ssi)
100
‘C
FIQ. 7. Effect of heat on human sperm ribonucleases. Aliquots of the solution of each enzyme were heated separately for 5 min. at 30, 40, 50, 60, 70, 80, 90, 95, and 100°C. and cooled directly on ice. Heating was performed after adjusting the pH of each solution to 7.2. a. Human sperm acid ribonuclease (-.-.-); b. Human sperm alkaline ribonuclease (--) ; c. Human sperm-free ribonuclease (-O-O-) ; d. Human combined ribonuclease (- - - -).
URINE
AND
SPERM
RIBONUCLEASES
405
found and studied in the pancreas (13, 1,6), spleen (14), and liver (20,21), and in leaves, shoots, and seeds of certain plants (22, 23). In the present studies, purified ribonuclease preparations are obtained from human sperm and urine. The enzymes are prepared by procedures which result in the separation of a free and combined ribonuclease, as well as acid and alkaline ribonucleases. Plots of the enzymic activities of the individual components collected from column chromatography of each of the eight preparations of ribonuclease (FS RNase, CS RNase; S. Alk. RNase; S. Acid RNase; RU RNase; CU RNase; U. Alk. RNase, and U. Acid RNase) indicated that each of the ribonuclease preparations contained many enzymically active components separable on column chromatography. The curves revealed the presence of variations in the number, proportion, and rate of elution of the ribonuclease active components in each of the ribonuclease preparations. Urine-free ribonuclease showed the presence of two major and four minor components, while sperm ribonuclease exhibited only three major components. Urine RNase major components differed from both RNase A or RNase B in their elution, while certain similarities occurred with sperm ribonuclease (free or combined). Combined ribonuclease from either urine or sperm resembled alkaline ribonuclease in their optimum pH, whereas two pH optima (pH’s 5.70 and 8.50) were found for free urine or sperm ribonuclease. Urine alkaline RNase and sperm-free ribonuclease are very unstable, 50 % of their ribonuclease activity being lost when exposed to 90°C. Sperm and urine acid ribonucleases and free and combined sperm ribonucleases act on the four cyclic anhydrides (uridine 2’,3’-phosphate, cytidine 2’) 3’-phosphate, adenosine 2’) 3’-phosphate, and guanosine 2’) 3’phosphate) the 3’-phosphate derivatives comprising the major end products, whereas free or combined urine ribonuclease acts only on the pyrimidine 2’,3’-phosphate and thus is similar to pancreatic ribonuclease (i.e., ribonuclease A) in activity. Adenosine 2’-phosphate results from the action of sperm or urine acid ribonuclease on adenosine 2’ ,3’-phosphate, while both adenosine 2’- (and 3’-)phosphate are produced from the action of free or combined sperm ribonuclease, and of free or combined urine ribonuclease. Although these results demonstrate differences between the various ribonuclease preparations obtained from human sperm and urine, they do not indicate that the enzymically active components are different. The nucleotides in ribonucleic acid are connected by phosphate bridges connecting position 3 in the ribose of one nucleotide to positions in the ribose of the next (24,25). This structural consideration gives ample chance
406
HAKIM
for specificity in ribonuclease activity since possibilities exist for some ribonuclease preparations to break the link connecting phosphate and position 5 in a ribose; while others break it on the other side between the phosphate and position 3. The susceptibility of an internucleotide link to attack depends on which bases are present in adjacent nucleotides and, in certain cases, on the length of the polynucleotide chain. The results reported here confirm the differential specificity of ribonuclease preparations and confirm the existence of separate active centers for each of the enzyme activities of ribonuclease. ACKNOWLEDGMENT this
The author manuscript.
acknowledges
the kind
help
of Professor
G. T. Lewis
in the revision
of
SUMMARY
Purified ribonuclease preparations are obtained from human sperm and urine. The enzymes are prepared by procedures which result in the separation of a free and combined ribonuclease, as well as acid and alkaline ribonuclease. Differences in the action of these various enzyme preparations on yeast ribonucleic acid and on certain synthetic substrates are reported. Differences between these ribonuclease preparations as to heat stability and optimum pH, and elution from an ion-exchange resin column are described. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
HAKIM, A. A., Arch. Biochem. Biophys. 70, 591 (1957). HABIM, A. A., Enzymologia 18, 252 (1957). HAKIM, A. A., J. Biol. Chem. 228,459 (1957). ROTHEN, A., J. Gen. Physiol. 24,203 (1940). MARTIN, A. J. P., AND PORTER, R. R., Biochem. J. 49,215 (1951). HIRS, C. H. W., STEIN, W. H., AND MOORE, S., J. Biol. Chem. 200, 493 (1953). HAKIM, A. A., Anal. Chim. Acta 17, 439 (1957). ALLARD, C., DE LAMIRANDE, G., AND CANTERO, A., Exptl. Cell Research 13, 69 (1957). ALLARD, C., DE LAMIRANDE, G., AND CANTERO, A., Can. J. Biochem. and Physiol. 84, 170 (1956). ALLARD, C., DE LAMIRANDE, G., AND CANTERO, A., Cancer Research 17,862 (1957). HAKIM, A. A., Proc. Intern. Congr. Biochem., 3rd Congr., Brussels, 1966, Sec. 8, p. 70, No. 1. HAKIM, A. A., J. Biol. Chem. 226,609 (1957). KUNITZ, M., J. Gen. Physiol. 24,15 (1940). HEPPEL, L. A., MARKHAM, R., AND HILMOE, R. J., Nature 171, 1152 (1953). HAKIM, A. A., Enzymologia 17, 383 (1956). KAPLAN, N. O., AND LIPMANN, F., J. Biol. Chem. 174.37 (1948). ANFINSEN, C. B., REDFIELD, R. R., CHORATE, W. L., PAGE, J., AND CARROLL, W. R., J. Biol. Chem. 207, 201 (1954).
URINE
18. 19. 20. 21. 22. 23. 24. 25.
AND
SPERM
RIBONUCLEASES
MARKHAM, R., AND SMITH, J. D., Biochem. J. 62,552 (1952). BROWN, D. M., AND TODD, A. R., J. Chem. Sot. 1963,204O. ROTH, J. S., J. Biol. Chem. 231, 1097 (1958). ZYTKO, J., DE LAMIRANDE, G., ALLARD, C., AND CANTERO, A., Biochim. phys. Acta 27, 495 (1958). JONC), Y., Acta Schol. Med. Univ. Koo 13, 162,182 (1930). HOLDEN, M., AND PIRIE, N. W., Biochem. J. 60,39 (1955). BROWN, D. M., HEPPEL, L. A., AND HILMOE, R. J., J. Chem. Sot. 1964,40. MARKHAM, R., AND SMITH, J. D., in “The Proteins” (Neurath, H. and K., eds.), Vol. II, p. 1. Academic Press, New York, 1954.
407
et Bio-
Bailey,
OF
BIOCHEMISTRY
AND
BIOPHYSICS
83,
39@407
(19%)
Heterogeneous Nature of Certain Ribonucleases. Human Urine and Sperm Ribonucleases’ Anwar From
A. Hakim
the Biochemistry Research Laboratories, Miami Heart Institute, Miami Florida; and the Department of Biochemistry, School of Medicine, University of Miami, Coral Gables. Florida Received
November
Beach,
28, 1958
There is an increasing interest in ribonucleases from biological sources, their specific activities, and their mode of action. Previous studies on crystalline ribonuclease (l-3) established the enzymic and electrophoretic heterogeneity of crystalline ribonuclease (RNase). The pancreas ribonuclease, despite its homogeneity as observed by sedimentation studies (4), has been separated into two fractions by partition (5) and ion-exchange chromatography (6), and has been resolved into four enzymically active fractions by paper electrophoresis (7). There is increasing evidence for the presence of different specific ribonucleases in certain biological materials. The behavior of the acid and alkaline ribonucleases under various conditions (8-10) has led to attempts at characterization of ribonuclease in normal human urine and in human sperm and comparison with ribonuclease from calf pancreas. EXPERIMENTAL Materials
and Methods
Substrate. The substrate, ribonucleic acid (RNA), was prepared from yeast (11, 12). Uridine 2’,3’-phosphate, cytidine 2’,3’-phosphate, adenosine 2’,3’-phosphate and guanosine 2’,3’-phosphate were obtained by procedures similar to those described in an earlier communication (1). Twenty-four-hour urine samples were collected and lyophilized to dryness immediately upon collection. Sperm samples were exposed to the same treatment. Preparation of Combined Ribonuclease from Human Sperm and from Human Urine. The dried urine or sperm powder was homogenized in 9 vol. of ice-cold, sterile glassdistilled water with the use of an Elvehjem all-glass homogenizer. The homogenate was centrifuged in a Spinco model L preparative ultracentrifuge at 60,000 X 9 for 1 Presented X-18, 1958.
in part
at the
New
York
Academy 390
of Science
Conference,
October
URINE
AND
SPERM
RIBONUCLEASES
391
40 min.; the supernatant was then decanted and recentrifuged at 60,000 X g for 26 min. The supernatant fluid from the second centrifugation was used in the study of the free and combined ribonucleases. Thirty milliliters of the urine or sperm supernatant obtained as described above was treated with solid ammonium sulfate (12.54 g.) to 30% saturation. The precipitate obtained by centrifugation was discarded. The supernatant was made up to 50% ammonium sulfate saturation, and the mixture was stirred and centrifuged. The supernatant fluid was poured off, and the precipitate was dissolved in 10 ml. of distilled water. To the latter solution 1.8 g. calcium phosphate gel was added, and the mixture was thoroughly stirred, allowed to stand at 0°C. for 25 min., and then was centrifuged at 15,000 X g for 30 min. The supernatant solution contained the combined inactive ribonuclease (C-RNase). Using a procedure similar to that described for urine, a combined ribonuclease (C-S.RNase) was prepared from the dried human sperm. Preparation of Purified Alkaline Ribonuclease. The alkaline RNase was prepared by a modification of the method of Kunitz (13) as follows: Thirty g. of dried urine powder was homogenized in an equal volume of distilled water in the cold. This homogenate was made up to 0.25 M with respect to sulfuric acid. The homogenate was left at 0°C. for 24 hr. with intermittent stirring and then filtered. The residue was discarded. The filtrate was centrifuged at 2000 X g for 10 min. The supernatant liquid was brought to 0.6 saturation with ammonium sulfate, and the precipitate formed was separated by centrifugation and discarded. To the supernatant, enough ammonium sulfate was added with continuous stirring to obtain 0.8 saturation. The solution was left at 0°C. for 24 hr. The precipitate formed was collected by centrifugation and dissolved in 25 ml. of distilled water, and t,he resultant solution was dialyzed for 48 hr. at 0°C. against large volumes of distilled water. The dialyzed solution was heated for 10 min. in a boiling water bath and cooled. Ammonium sulfate treatment was then repeated, and the 0.8 saturation ammonium sulfate precipitate was collected. The latter precipitate was dissolved in distilled water, dialyzed at O”C., and lyophilized to dryness. Using a similar procedure as above, a purified alkaline RNase was prepared from dried sperm. Preparation of Acid Ribonuclease. The acid ribonuclease was prepared by the following procedure [cf. (14)]: Twenty-five grams of the dried urine or sperm was extracted with 50 ml. of 0.2 N acetate buffer, pH 6.0 (15) for 4 hr. The suspension was centrifuged. The supernatant was adjusted to pH 5.0, and ammonium sulfate was added to obtain a solution of 0.6 saturation. The pH was maintained at 5.0 during the addition of ammonium sulfate. The precipitate formed was discarded. More ammonium sulfate was added to the supernatant, to obtain 0.8 saturation, and was left for 24 hr. at 0°C. The precipitate formed was collected by centrifugation and dissolved in 15 ml. of distilled water. The solution was adjusted to pH 8.0 and maintained at this pH, while ammonium sulfate treatment was repeated as above, and the supernatant solution was left at 0°C. for 48 hr. The precipitate formed was collected by centrifugation and dissolved in distilled water, dialyzed for 48 hr. against large volumes of distilled water, and lyophilized to dryness. All operations were carried out at 0°C. Using the same procedure, an acid ribonuclease was obtained from the dried human sperm.
Analytical
Procedures
Action of Acid or Alkaline Ribonucleases on Yeast Ribonucleic Acid. The reaction mixture contained yeast ribonucleic acid (2 mg.) in 0.5 ml. of 0.2 M acetate buffer at pH 5.3, about 100 kunitz units of ribonuclease (acid or alkaline type), and a few drops
392
HAKIM
of toluene; it was incubated at 37°C. for 24 hr. Aliquots of 0.2 ml. were applied in a band form on Whatman 13 MM filter paper and allowed to dry. The chromatograms were dried and photographed. Four distinct bands were located. The material from each band was concentrated at a wick, or eluted or applied on paper electrophoresis. The chemical identity of the substances present in each of the bands was determined by the use of the following analytical procedures for each one of the four bands: (a) Electrophoresis in 0.05 M ammonium formate at pH 3.5. Separately, the spots observed on the electrogram were eluted. The eluates were concentrated to dryness, the residue was hydrolyzed with 72$& perchloric acid (HClOa), and the hydrolysis products were subjected to chromatography in an isopropyl alcohol-HCl-water system. (5) To the action of snake venom, where the reaction mixture contained the eluate, 0.05 M Verona1 buffer at pH 8.5 (0.5 ml.), 0.02 M MgSOd , and purified snake venom. (c) To the action of phosphomonoesterase in a mixture containing the eluate, 0.1 M acetate buffer at pH 5.0 and phosphomonoesterase. (d) To chromatographic separation using a solvent system consisting of saturated ammonium sulfate solution-isopropyl alcohol-l iIf acetate buffer (80:2:18) (16).
Assay of Enxymic Activity Ribonuclease activity of free and combined urine or sperm enzyme preparation, and of acid or alkaline ribonuclease was determined by the rate of hydrolysis of uridine 2’,3’-phosphate and cytidine 2’,3’-phosphate. The rate of hydrolysis of the latter two anhydrides was obtained by microtitration of the secondary phosphoryl groups that were liberated by the action of the enzymes. The procedure is as follows: 2 ml. of 0.25 N solution of sodium sulfate and 0.05 M of the anhydride was placed in a 4-ml. beaker equipped with a glass electrode (1). The pH was adjusted to 7.5 with 0.1 N NaOH, and 10 pl. of the enzyme preparations was added. The amount of 0.01 N NaOH required to keep the solution at pH 7.5 for a period of 30 min. was noted. The enzymic hydrolysis of yeast ribonucleic acid by the above enzyme preparations was carried out by mixing 0.1 ml. of 0.1 M phosphate buffer, ionic strength 0.05, and pH 6.00, containing 5 mg. ribonucleic acid, with a 25-ml. aliquot of the enzyme preparation, and incubating at 37°C. for 48 hr. Ten microliters of chloroform was added as a bacteriostatic agent. The perchloric acid-uranyl acetate reagent (17) was used to precipitate the polymerized RNA. The mononucleotides liberated were separated by paper chromatography. Chromatograms of the digests of ribonucleic acid with free or combined acid or alkaline ribonuclease obtained from urine or sperm were developed by the descending technique in two dimensions (12). Permanent records of the chromatograms were obtained by the technique of Markham and Smith (18). The located mononucleotides were cut from the paper, eluted, and quantitatively determined (1). Hydrolysis of Cytidine Benzyl Phosphate or Uridine Benzyl Phosphate. The hydrolysis of uridine or cytidine benzyl phosphate was measured chromatographically. Solutions containing 2.5 pmoles cytidine or uridine benzyl phosphate, 25 al. of either the urine or sperm enzyme preparation, and 0.3 rmole of phosphate buffer at pH 6.00, to give a total volume of 0.25 ml. and an ionic strength of 0.10, were incubated at 37°C. Before the addition of the enzyme preparation, and at intervals thereafter, 25-~1. aliquots were applied to Whatman 11 filter-paper sheets. The chromatograms were developed with the isopropyl alcohol-ammonia-water system (19). The mononucleotides were located, cut from the chromatograms, eluted, and their concentration determined (1).
URINE AND SPERM RIBONUCLEASES TABLE Effect
of Heat”
I
on Ribonuclease Activities of Preparations from Human Urine and Sperm
Per cent of original
Preparation 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
393
Sperm acid RNase Sperm alkaline RNase Sperm-free RNase Sperm-combined RNase Urine Sib acid RNase Urine S,ib alkaline RNase Urine Sz? acid RNase Urine SZib alkaline RNase Urine-free RNase Urine-combined RNase
n All the preparations were exposed to 98-100°C. 10 min. at pH 7.2, then quickly cooled and activity b Sn is the preparation obtained from 24-hr. urine day of the menstrual cycle. c Szi is the preparation obtained from 24-hr. urine first day of the menstrual cycle.
activity
pH 5.8
pH 7.8
15 82 20 18 40 92 50 10 35 20
80 95 85 50 92 96 65 64 55 65
in a water bath for a period of determined. sample collected on the eleventh sample collected on the twenty-
RESULTS
Products of Enzymic
Hydrolysis
Chemical and enzymic studies of the four bands located on chromatoof the enzymic action (enzyme preparations listed in Table I) on yeast ribonucleic acid showed the following chemical identities: Band 1 contained guanosine 2’- or 3’- or 5’-phosphate or a mixture of all the three nucleotides (step a). Action of snake venom (step 6) indicated the absence of guanosine Y-phosphate. Phosphomonoesteraseaction (step c) confirmed the presence of guanosine 2’- or 3’-phosphate, and only one spot corresponding to guanosine 3’-phosphate was observed in step d. By similar analogy, band 2 was found to contain adenosine 3’-phosphate; band 3 contained guanosine 2’,3’-phosphate (alkaline urine or sperm ribonuclease and band 4 contained the 2’ ,3’-phosphate cyclic derivatives of adenosine (alkaline urine or sperm ribonuclease), cytidine, and uridine grams
(acid urine
or sperm
ribonuclease).
The chemical identity was confirmed by the electromobility in specific buffer
systems
on paper
electrophoresis
reference compounds (Fig. 1).
using authentic
pure derivatives
as
Starti line
w
Star ting line 4
3
2 B 394
1
URINE
AND
SPERM
395
RIBONUCLEASES
sta1rting line 1
2
3
4
C FIG. 1. Action of sperm alkaline ribonuclease on synthebic substrates. Paper electrophoresis was performed in an electrophoresis apparatus as described earlier (1). Sodium tetraborate, 0.4 M, pH 13, separated the cyclic anhydride and the nucleotide. A current of 10 ma. at 1100 v., applied for 1 hr., resulted in a clear separation of the two groups. A. Action of sperm alkaline ribonuclease on uridine 2’,3’-phosphate. 1. Uridine 2’, 3’-phosphate; 2. Uridine 2’, 3’-phosphate after the action of sperm alkaline ribonuclease; 3. Uridine 3’-phosphate. B. Action of sperm alkaline ribonuclease on adenosine 2’, 3 ‘N-phosphate. 1. Adenosine 2’,3’-phosphate; 2. Adenosine 2’,3’-phosphate after the action of combined urine ribonuclease; 3. Adenosine 2’,3’-phosphate after the action of combined sperm ribonuclease; 4. Adenosine 3’.phosphate. C. Action of sperm alkaline ribonuclease on guanosine 2’,3<-phosphate. 1. Guanosine 2’,3’-phosphate after the action of combined sperm ribonuclease; 2. Guanosine 2’,3’-phosphate alone; 3. Guanosine 2’,3’-phosphate after the action of sperm acid ribonuclease; 4. Guanosine 2’,3’-phosphate after the action ~of free sperm ribonuclease. The location of guanosine 2’.phosphate and guanosine 3’-phosphate has been checked using pure isomeres.
Further Purijication of Ribonucleaseson Ion-Exdange Columns precipitating between 30 and 50 % The urine- or sperm-free ribonuclease ammonium sulfate saturation was dissolved in 0.2 M ~phosphate buffer at pH 6.2, dialyzed against the phosphate buffer, and th&n chromatographed
over IRC-50 resin (XE-64, 200-mesh) previously equilibrated with the
396
HAKIM
d d
0.700
-
0.600
-
0.500
-
0.400-
x .5 0.300 E T)
-
2=
0.200
-
0.100
-
0.050
-
‘0 0
0
4
6
li
16 .iO 24 Elution
28
volume
32 36
40 44
48
52
(ml)
Urine ribonuclease. Plots of enzymic activity elution pattern of human urine ribonuclease, at pH 7.0 assay. Optical density determined at 260 mp. a. Crystalline pancreatic ribonuclease ( . . .) ; b. Urine-free ribonuclease (+--) ; c. Urine-combined ribonuclease (- - - -). FIG.
2.
same buffer (Figs. 2 and 3). Crystalline pancreas ribonuclease (Fig. 2~) showed two major peaks, the larger peak being RNase A and the smaller peak RNase B. Urine ribonuclease (Fig. 2b) showed two larger peaks and at least four smaller peaks. Human sperm ribonuclease gave one larger peak and two smaller. The elution data showed differences in behavior between urine ribonucleases, sperm ribonucleases,and crystalline pancreatic ribonucleases. Taking crystalline pancreas RNase to consist of at least two enzymic fractions, urine RNase then consisted of at least six, and sperm RNase of at least three. The behavior of sperm-free RNase and spermcombined RNase on the ion-exchange column, IR-C50 resin (XE-64, 200mesh) presented in Figs. 36 and 3c, showed that free sperm RNase consisted of at least three major peaks (Fig. 3b) while combined sperm RNase after activation is made up of three components (Fig. 3~). Properties of Pur$ed RibonucleasePreparations from Human Sperm and Urine A. Determination of pH-Optimum Curves for the Urine and Sperm Ribonucleases.Aliquots of urine, free and combined ribonucleases, and of free
URINE
AND
SPERM
397
RIBONUCLEASES
0.000 I-
d d
0.700
-
0.600
-
(? I ‘r . :
i : ! i : I : I
-
0.500
3.
0
4
8
12
16 20 Elution
FIG. 3. Human sperm ribonuclease. Plots human sperm ribonuclease, at pH 7.0 assay. a. Cryst,alline pancreatic ribonuclease (---.-) c. Sperm-combined ribonuclease (- - - -).
\ I : I I : I \
24
28 32
volume
36 40
44
48
52
(ml)
of enzymic activity elution pattern of Opt,ical density determined at 260 rnp. ; b. Sperm-free ribonuclease (--) ;
and combined sperm ribonucleases were assayed for ribonuclease enzymic activity in the presence of phosphate and acetate buffers of different pH values, but with the same ionic strength (0.15). The enzymic activity of the combined urine RNase (CU RNase), and of the combined sperm RNase (CS RNase) was assayed after activation with p-chloromercuribenzoic acid (1 X 10e6 M). The results are shown in Figs. 4a and 4b. Both CU RNase and CS RNase showed a pH optimum at about 8.00, progressive increase in activity occurring on the acid side, and a sharp decrease on the alkaline side. Both urine-free ribonuclease (FU RNase) and sperm-free ribonuclease (FS RNase) had two pH optima: at pH 5.70 and at pH 8.50. Urine alkaline or acid RNase is characterized by a pH optimum at 5.70 or 8.50, respectively. The pH-activity curves of acid and alkaline ribonuclease preparations are given in Fig. 5. A sharp optimum is obtained for acid ribonuclease from both human sperm and urine. Free ribonuclease either from human sperm or urine showed a broad optimum activity. The optimum pH of human urine RNases were at pH 5.5-5.7 and 7.8-8.0. Sperm ribonucleases showed
IIAKIM
IOOIOOLago.;t 00O 70E z 60.i 50-
L3.00
Fro. 4. Human b. Human
--1
5.00
4.00
6.00
7.00
0)
3.00
I
4.00
9.00
10.00
PH
urine ribonuelease. a. Human ribonuclease (- - - -).
urine-free
0.00
.
urine-combined
I
5.00
1
6.00
7.00
0.00
ribonuclease
I
9.00
(-);
1
10.00
PH
FIG. 5. Human sperm ribonuclease. a. Human b. Human sperm alkaline ribonuclease (-O-O-); (-.-.-) ; d. Human sperm-combined ribonuclease
sperm acid ribonuclease (- - - -); c. Human sperm-free ribonuclease (--).
optimum activity at pH 5.8 (acid enzyme), pH 8.0 (alkaline enzyme), and pH’s 5.8 and 8.0 for the free enzyme. The curves of Fig. 4a show interesting points which indicate differences between the pH-optimum curves of the acid or alkaline RNases, but certain similarity to the curves for RU RNase or FS RNase. The latter curves could be considered the sum of the acid and alkaline curves of the urine or sperm. Although these differences are
URINE
AND
SPERM
RIBONUCLEASES
399
lOOr 90
-
0 5
IO 20 30 40 50 60 70 00 90 100 Temperature
*C.
FIG. 6. Effect
of heat on human urine ribonuclease. Aliquots of the solution of each enzyme were heated separately for 5 min. at 30,40,50,60,70,80,90,95, and 100°C. and cooled directly on ice. Heating was performed after adjusting the pH of each solution to 7.2. a. Human urine alkaline ribonuclease (---); b. Human urine acid ribonuclease (- - - -); c. Human urine-free ribonuclease (-.-.-).
very preliminary, they do suggest that alkaline RNase and acid RNase fractions are different enzymes. B. E$ect of Heating on Acid, Alkaline, Free and Combined Ribonucleases from (Jrine and Sperm. Heat-stability studies were made on purified acid, alkaline, or free ribonuclease solutions. Similar studies were carried out with the use of combined ribonucleases (urine or sperm) which had been activated. Aliquots of the solution of each enzyme were heated separately for 5 min. at 30, 40, 50, 60, 70, 80, 90, 95, and lOO”C., and cooled directly on ice. Heating was performed after adjustment of the pH of each solution to pH 7.2. The results of typical assaysare shown in Fig. 6. In a seriesof experiments, the heating effect was studied on the enzyme solutions preadjusted to pH 7.2, then heated to 98-100°C. in a water bath for a period of 10 min. following which ribonuclease activity was assayed at pH’s 5.8 and 7.8. Sperm or urine alkaline ribonuclease showed 82 and 92% activity recovery, respectively, when determined at pH 7.8. All the other enzyme preparations showed 50-85 % loss in acidic (pH 5.8) activity. Other variations in heat stability of the alkaline enzymic activity are shown in Table I. The per cent recovery for free and combined ribonuclease activity was 35 and 20 for urine, and 18 for sperm. The corresponding figures for the alkaline ribonuclease activity of the same enzyme preparation were 55 and 65 for urine, and 85 and 50 for sperm. In general, the sperm enzymes showed greater stability than the urine preparations.
400
HAKIM
XpeciJic Activity A. Action on Synthetic Substrates. The synthetic substrates cytidine 2’, 3’-phosphate, uridine 2’, 3’-phosphate (UZZBP), adenosine 2’) 3’-phosphate (Az:aP), and guanosine 2’, 3’-phosphate were hydrolyzed to the extent of 25, 20, 15, and 18% by sperm alkaline ribonuclease; 30, 22, 12, and 14% by urine alkaline ribonuclease. Sperm acid ribonuclease hydrolyzed 85, 82, 98, and 95% of the above four synthetic substrates, respectively. Uridine alkaline ribonuclease hydrolyzed cytidine 2’) 3’-phosphate (65 %), urine 2’,3’-phosphate (74 %), adenosine 2’,3’-phosphate (25 %), and guanosine 2’) 3’-phosphate (20 %), while acid ribonuclease obtained from the same urine sample hydrolyzed 81, 80, 98, and 96% of the synthetic substrates, respectively. Free ribonuclease from sperm or from urine acted on 98 and 92 % of cytidine 2’,3’-phosphate; both enzyme preparations hydrolyzed 98 % of uridine 2’,3’-phosphate, 55 and 35 % of adenosine 2’,3’-phosphate, and 75 and 25 % of guanosine 2’,3’-phosphate. Free sperm ribonuclease showed greater activity on the guanosine cyclic compound than did free urine ribonuclease. Although the combined urine ribonuclease showed greater activity on the pyrimidine synthetic compounds (85 and 82 % of cytidine 2’,3’-phosphate and uridine 2’,3’-phosphate as compared to 65 and 70 % for the combined sperm ribonuclease), combined sperm ribonuclease action on the purine cyclic compounds (88 and 95% of adenosine 2’, 3’-phosphate and guanosine 2’) 3’-phosphate) was more efficient than the action of combined urine ribonuclease. All the enzyme preparations studied differed in action on the synthetic substrates from crystalline calf pancreas ribonuclease (cf. Table II). Table III presents the effect of heating on the different ribonuclease preparations. The results show that after 17 hr. of incubation, acid ribonuclease splits Az:sP to give AZP as indicated by the Rj values. No A3P could be detected. After 40 hr. incubation only one spot was detected with Rf value of 0.28, indicating a complete breakdown of Az:~P; illustrating the fact that the activity of acid ribonuclease was destroyed by heating. Acid ribonuclease acted on UzZtP to give uridylic acid; the reaction was slower than with Az:~P;~ Uz:aP could still be demonstrated after 40 hr. of incubation, Alkaline ribonuclease and heated acid ribonuclease showed no effect on Uz:fP. These results show important differences between the acid and the alkaline ribonucleases. They also show that sperm or urine alkaline ribonuclease could be different from the pancreatic ribonuclease which split pyrimidine cyclic mononucleotides whereas the sperm or urine enzyme did not. * The rate of activity scribed earlier (1).
on the cyclic
anhydrides
was carried
out by a technique
de-
a
cycle.
on the
Alk. RNase AC. RNase Alk. RNase AC. RNase Alk. RNasea AC. RNase* free RNase comb. RNase free RNase comb. RNase pancreas RNase (HzO)
Preparation
Samples collected
Sperm Sperm Urine Urine Urine Urine Sperm Sperm Urine Urine Cryst. Control
the menstrual
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Enzyme
-
20 82 22 79 74 80 98 70 98 82 92 12
day
of the menstrual
CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’(2’) CMP-2’(3’) CMP-3’(2’) CMP-3’(2’) CMP-3’(2’) CMP-3’(2’)
II or Urine
cycle;
the
UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’(2’) UMP-2’(3’) UMP-3’(2’) UMP-3’(2’) UMP-3’(2’) UMP-3’(2’) other
Reaction products
Uridine 2’, 3’phosphate Extent of hydrolysis
25 85 30 80 65 81 98 65 92 85 99 10
-
Sperm
TABLE Human
%
Reaction products
from
%
Extent of hydrolysis
-
Cytidine 2’, 3’phosphate
of Ribonucleases
twenty-first
-
Action
Substrates
-
.-
sample
15 98 12 96 25 98 55 88 12 35 12 10
%
was collected
AMP-3’ AMP-2’ AMP-3’ AMP-2’ AMP-3’ AMP-2’ AMP-3’(2’) AMP-2’(3’) AMP-3’(2’) AMP-3’(2’) AMP-3’ AMP-3’(2’)
Reaction products
Adenosine 2’, 3’phosphate
Synthetic
Extent of hy,drolysis
urine
-
--
--
- or -
day
GMP-3’ GMP-3’ GMP3’ GMP-3’ GMP-3’ GMP-3’ GMP-3’(2’) GMP-2’(3’) GMP-3’(2’) GMP-3’(2’) GMP-3’ GMP-3’(2’)
Reaction products
on the eleventh
18 95 14 97 20 96 75 95 25 48 23 12
%
Extent of hydrolysis
Guanosine 2’, 3’phosphate
of
the
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Preparation
a Samples menstrual
collected cycle.
-
Ribonucleases
25 80 23 65 35 60 55 60 80 70 92 10
%
Extent of hydrolysis
day
-
22 75 18 58 30 55 60 60 85 68 88 12
%
Sperm
III
cycle;
the
UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’ UMP-3’(2’) UMP-3’ UMP-3’(2’) UMP-3’(2’) UMP-3’(2’)
Substrates
sample
2 0 2 0 5 0 3 70 2 20 0 10
%
-
was collected
AMP-3’(2’)
AMP-3’ AMP-3’(2’) AMP-3’ AMP-3’ -
AMP-3’
AMP-3’ -
AMP-3’ -
Reaction products
Adenosine 2’, 3’phosphate
on Synthetic
Extent of hy,drolysis
urine
-
-
or Urine
other
Reaction products
Uridine 2’, 3’phosphate
Extent of hy,drolysis
__
-
TABLE
from Human
of the menstrual
CMP3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’ CMP-3’(2’) CMP-3’ CMP-3’(2’) CMP-3’(2’) CMP-3’(2’)
Reaction products
Cytidine 2’, 3’phosphate
of Heated
on the twenty-first
Sperm Alk. RNase Sperm AC. RNase Urine Alk. RNase Urine AC. RNase Urine Alk. RNasea Urine AC. RNase” Sperm free RNase Sperm comb. RNase Urine free RNase Urine comb. RNase Cryst. pancreas RNase Control (HZO)
Enzyme
Action
-
-
day
GMP-3’(2’)
GMP-3’ GMP-3’(2’) GMP-3’ GMP-3’ -
GMP-3’ -
GMP-3’ -
GMPS
Reaction products
on the eleventh
10 0 8 00 6 0 5 80 5 15 0 12
%
Extent of hydrolysis
Guanosine 2’, 3’phosphate
of
URINE
AND
SPERM
RIBONUCLEASES
403
B. Action on Yeast Ribonucleic Acid. Incubation of yeast ribonucleic acids with sperm-free ribonuclease for 24 hr. produced 7.5 and 13.5% of adenylic acid and guanylic acid, respectively. Urine-free ribonuclease produced 8.5 and 10.5%. Sperm-combined ribonuclease when incubated with yeast ribonucleic acid resulted in 22.5 and 28.5% of cytidylic acid and guanylic acid, respectively. Urine-combined ribonuclease liberated only 8.9 and 14.8% of the mononucleotides, respectively. Although slight differences are noted between the action of the free enzymes on yeast ribonucleic acid, the combined enzymes differed in the amount of adenylic acid and guanylic acid produced by their action. Urine-combined ribonuclease produced approximately twice the amount of cytidylic acid that is produced by sperm-combined ribonuclease. It is suggested from these data that sperm- or urine-combined ribonuclease differed in their specific activity on yeast ribonucleic acid. Although no adenylic acid and only 1.2 % guanylic acid resulted from the action of urine (SJ alkaline ribonuclease on yeast ribonucleic acid and guanylic acid, respectively, the amount of pyrimidine nucleotides produced by the action of urine (Sn) alkaline ribonuclease is relatively more than those liberated by alkaline ribonuclease obtained from urine sample (&I). Urine (Sn) acid ribonuclease liberated 16.0 and 11.2 % of adenylic acid and guanylic acid. Urine (&I) acid ribonuclease produced 7.2 and 14.5 % of the purine mononucleotides, respectively. The acid or the alkaline ribonucleases present in urine samples (FL) and (SZl) appear to differ in their action on yeast ribonucleic acid. Sperm alkaline ribonuclease produced 3.5, 5.5, 6.5, and 7.8%, while sperm acid ribonuclease resulted in 20.8, 25.4, 15.0, and 17.5 % of adenylic acid, guanylic acid, cytidylic acid, and uridylic acid, respectively. These data indicate either that sperm alkaline ribonuclease is less active than sperm acid ribonuclease on yeast ribonucleic acid, or sperm alkaline ribonuclease possesses a secondary specific activity (i.e., a synthetic activity). Further studies are in progress to define the specificity of these two sperm enzymes on yeast ribonucleic acid. Although all the enzymes studied in this report (except urine E&l alkaline ribonuclease) produced adenylic and guanylic acid, crystalline pancreas ribonuclease did not produce the purine mononucleotides. On the basis of these data the enzymes presented in Table IV differed in their specific activit,y on yeast ribonucleic acid from that of crystalline pancreas ribonuclease. DISCUSSION
Ribonucleases are enzymes that degrade ribonucleic acids so that they no longer show their typical macromolecular properties. They have been
HAKIM
404
TABLE Mononucleotides
Liberated Sperm
IV
by the Action of Ribonuclease and Urine on Yeast Ribonucleic
Preparations Acid
moles 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Alkaline hydrolysis” Crystalline RNase* Free Sperm RNase Combined Sperm RNase Sperm Alkaline RNase Sperm Acid RNase Urine Sllc Alkaline RNase Urine Siic Acid RNase Urine Szi~ Alkaline RNase Urine S~,C Acid RNase Free Urine RNase Combined Urine RNase
0
5
IO 20
30
per 100 moles
24.8 -
31.6 -
7.5 22.5 3.5 20.8 0.0 16.0 1.2 7.2 8.5 8.9
13.5 28.5 5.5 25.4 1.2 11.2 12.2 14.5 10.5 14.8
a One normal NaOH at 37°C. for 24 hr. * Crystalline ribonuclease (Armour). The enzyme before use. 0 Urine samples collected on the eleventh day (Sn) of the menstrual cycle.
40
Temperature
50
60
23.8 15.7 15.5 7.8 6.5 15.0 14.5 13.6 11.5 10.5 14.8 14.5
was
crystallized
and the twenty-first
70
Human
from
00
90
18.7 18.1 18.1 10.5 7.8 17.5 18.0 14.0 12.5 11.5 16.5 16.9
three day
times (Ssi)
100
‘C
FIQ. 7. Effect of heat on human sperm ribonucleases. Aliquots of the solution of each enzyme were heated separately for 5 min. at 30, 40, 50, 60, 70, 80, 90, 95, and 100°C. and cooled directly on ice. Heating was performed after adjusting the pH of each solution to 7.2. a. Human sperm acid ribonuclease (-.-.-); b. Human sperm alkaline ribonuclease (--) ; c. Human sperm-free ribonuclease (-O-O-) ; d. Human combined ribonuclease (- - - -).
URINE
AND
SPERM
RIBONUCLEASES
405
found and studied in the pancreas (13, 1,6), spleen (14), and liver (20,21), and in leaves, shoots, and seeds of certain plants (22, 23). In the present studies, purified ribonuclease preparations are obtained from human sperm and urine. The enzymes are prepared by procedures which result in the separation of a free and combined ribonuclease, as well as acid and alkaline ribonucleases. Plots of the enzymic activities of the individual components collected from column chromatography of each of the eight preparations of ribonuclease (FS RNase, CS RNase; S. Alk. RNase; S. Acid RNase; RU RNase; CU RNase; U. Alk. RNase, and U. Acid RNase) indicated that each of the ribonuclease preparations contained many enzymically active components separable on column chromatography. The curves revealed the presence of variations in the number, proportion, and rate of elution of the ribonuclease active components in each of the ribonuclease preparations. Urine-free ribonuclease showed the presence of two major and four minor components, while sperm ribonuclease exhibited only three major components. Urine RNase major components differed from both RNase A or RNase B in their elution, while certain similarities occurred with sperm ribonuclease (free or combined). Combined ribonuclease from either urine or sperm resembled alkaline ribonuclease in their optimum pH, whereas two pH optima (pH’s 5.70 and 8.50) were found for free urine or sperm ribonuclease. Urine alkaline RNase and sperm-free ribonuclease are very unstable, 50 % of their ribonuclease activity being lost when exposed to 90°C. Sperm and urine acid ribonucleases and free and combined sperm ribonucleases act on the four cyclic anhydrides (uridine 2’,3’-phosphate, cytidine 2’) 3’-phosphate, adenosine 2’) 3’-phosphate, and guanosine 2’) 3’phosphate) the 3’-phosphate derivatives comprising the major end products, whereas free or combined urine ribonuclease acts only on the pyrimidine 2’,3’-phosphate and thus is similar to pancreatic ribonuclease (i.e., ribonuclease A) in activity. Adenosine 2’-phosphate results from the action of sperm or urine acid ribonuclease on adenosine 2’ ,3’-phosphate, while both adenosine 2’- (and 3’-)phosphate are produced from the action of free or combined sperm ribonuclease, and of free or combined urine ribonuclease. Although these results demonstrate differences between the various ribonuclease preparations obtained from human sperm and urine, they do not indicate that the enzymically active components are different. The nucleotides in ribonucleic acid are connected by phosphate bridges connecting position 3 in the ribose of one nucleotide to positions in the ribose of the next (24,25). This structural consideration gives ample chance
406
HAKIM
for specificity in ribonuclease activity since possibilities exist for some ribonuclease preparations to break the link connecting phosphate and position 5 in a ribose; while others break it on the other side between the phosphate and position 3. The susceptibility of an internucleotide link to attack depends on which bases are present in adjacent nucleotides and, in certain cases, on the length of the polynucleotide chain. The results reported here confirm the differential specificity of ribonuclease preparations and confirm the existence of separate active centers for each of the enzyme activities of ribonuclease. ACKNOWLEDGMENT this
The author manuscript.
acknowledges
the kind
help
of Professor
G. T. Lewis
in the revision
of
SUMMARY
Purified ribonuclease preparations are obtained from human sperm and urine. The enzymes are prepared by procedures which result in the separation of a free and combined ribonuclease, as well as acid and alkaline ribonuclease. Differences in the action of these various enzyme preparations on yeast ribonucleic acid and on certain synthetic substrates are reported. Differences between these ribonuclease preparations as to heat stability and optimum pH, and elution from an ion-exchange resin column are described. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
HAKIM, A. A., Arch. Biochem. Biophys. 70, 591 (1957). HABIM, A. A., Enzymologia 18, 252 (1957). HAKIM, A. A., J. Biol. Chem. 228,459 (1957). ROTHEN, A., J. Gen. Physiol. 24,203 (1940). MARTIN, A. J. P., AND PORTER, R. R., Biochem. J. 49,215 (1951). HIRS, C. H. W., STEIN, W. H., AND MOORE, S., J. Biol. Chem. 200, 493 (1953). HAKIM, A. A., Anal. Chim. Acta 17, 439 (1957). ALLARD, C., DE LAMIRANDE, G., AND CANTERO, A., Exptl. Cell Research 13, 69 (1957). ALLARD, C., DE LAMIRANDE, G., AND CANTERO, A., Can. J. Biochem. and Physiol. 84, 170 (1956). ALLARD, C., DE LAMIRANDE, G., AND CANTERO, A., Cancer Research 17,862 (1957). HAKIM, A. A., Proc. Intern. Congr. Biochem., 3rd Congr., Brussels, 1966, Sec. 8, p. 70, No. 1. HAKIM, A. A., J. Biol. Chem. 226,609 (1957). KUNITZ, M., J. Gen. Physiol. 24,15 (1940). HEPPEL, L. A., MARKHAM, R., AND HILMOE, R. J., Nature 171, 1152 (1953). HAKIM, A. A., Enzymologia 17, 383 (1956). KAPLAN, N. O., AND LIPMANN, F., J. Biol. Chem. 174.37 (1948). ANFINSEN, C. B., REDFIELD, R. R., CHORATE, W. L., PAGE, J., AND CARROLL, W. R., J. Biol. Chem. 207, 201 (1954).
URINE
18. 19. 20. 21. 22. 23. 24. 25.
AND
SPERM
RIBONUCLEASES
MARKHAM, R., AND SMITH, J. D., Biochem. J. 62,552 (1952). BROWN, D. M., AND TODD, A. R., J. Chem. Sot. 1963,204O. ROTH, J. S., J. Biol. Chem. 231, 1097 (1958). ZYTKO, J., DE LAMIRANDE, G., ALLARD, C., AND CANTERO, A., Biochim. phys. Acta 27, 495 (1958). JONC), Y., Acta Schol. Med. Univ. Koo 13, 162,182 (1930). HOLDEN, M., AND PIRIE, N. W., Biochem. J. 60,39 (1955). BROWN, D. M., HEPPEL, L. A., AND HILMOE, R. J., J. Chem. Sot. 1964,40. MARKHAM, R., AND SMITH, J. D., in “The Proteins” (Neurath, H. and K., eds.), Vol. II, p. 1. Academic Press, New York, 1954.
407
et Bio-
Bailey,