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Non-enzymatic hydrolysis of transfer RNA by bull semen
Life Sciences Vol . 10, Part II, pp. 919-925, 1971 . Printed in Great Britain
Pergamon Press
NON-ENZYMATIC HYDROLYSIS OF TRANSFER RNA BY BU..L SEMEN Bettrum Sheid and Susan M . Wilson Department of Pharmacology, State University of New York Downetate Medical Center, Brooklyn, New York 11203
(Received S1 March 1971; in final form 6 July 1971) su®a ry Dilutions of deproteinised seminal plasma ae lw as 1 :5000 were ahovn to contain sufficient amounts of a compounds) which nonensysntically hydrolysed O .l mg tRNA into acid soluble fragments . Other evidence is presented to eliminate the possibility of the active seminal plasma substance (s) being ribonuclease contaminaat(s) . PREVIOUS ezperiaents have daaonstrated the existence of a very potent inhibitor s) of tRNA methylation in bull semen (1,2) .
The active subetance(s) was
not affected by daneturing the semen proteins with trichloracetic acid or chloroform :isoanyl alcohol .
The semeu compouad(s) ws also unaffected by 100°-
C heat, trypsia and prooase digestion .
Other experiments eliminated the poss-
ibility of the coo~pouad(s) being a lipid, nucleic acid or a small molecule such as salts or amino acids (2) .
Periodate oxidation destroyed the tRNA
methy L se inhibitory activity, thus suggesting a polysaccharide as the active component in semen . In the present report, data are presented to ahoy that bull semen contains a caozpou~(s) which praventa tRMA methylation by degrading the tRNA substrate in a non-ensymatic manner .
Other evidence is presented to further
eliminate the possibility of the degradation of tRNA being specifically due to contaminating ribonucleasea . Materials and Methods Bull seism was collected over liquid nitrogen without preservatives or antibiotics added to the final pooled sample .
919
The product was provided by the
920
Non-Enzymatic Hydrolysis ad tRNA
Vol, 30, No. 16
Eastern Artificial Insemination Corporation, Ithaca, New York . Escherichia coliß t$NA was purchased from the General Biochemical Corporation . 8amiaal plasma was separated from sperm cells after centrifugation at 850 x g for 10 min in a modal PR-2 Iateraational centrifuge .
Although the ac-
tive component was present is both of the snman fractioae (2), only the seminal plasma was used for these studies .
The seminal plasma was deproteinized by
incubating 1 ml of a 1 :400 dilution (300 Wg protein) with 100 W g of trypain or LOO~,g of chymotrypsin for 2 .5 hr at 37 ° C .
These enzymes were subeequently
dastroged without affecting the active component(a) in seminal plasma by heating at 80 ° C for 20 min .
Thn seminal p L ema was also deproteinizad by extract-
ing 1 ml of uadilutad seminal pLss~a with 3 volumes of alcohol ;ether (3 ;1) at 4°C, or 1 volume of phenol at 4 ° C .
Another method employed to denature seminal
plasma proteins was by adding perchloric acid to a 1 :75 dilution of seminal pLsan until the solution was 6x with respect to the acid .
This particular sol-
ution was neutralised with NaOH and then dialyzed against water .
The appro-
priate dilutiona of the deproteiniaed extracts were made with distilled water . The assay mixture used to determine the non-enzymatic hydrolysis of tRNA coasiatad of 100y~moles of Tris buffer pfl 8 .4, 0 .1 mg tRNA and 0 .2 ml of varying dilutiow of the deproteiaised seminal plasma extracts .
After 1 hr at 37°C
the reactions were terminated with trichloroacetic acid (final concentration 6Z acid) .
The 260 nm aborbance of the supernatant fluid was then read in a Beck-
man spectrophotometer to determine the percent hydrolysis of tRNA . wsahed Sephadex G-100 was loaded into a column 10 x 140 cm .
One-ml sam-
plea containing 0 .25 mg of ta1~A praincubated at 37°C, with and without 1 ;1000 diluted and daproteinizad extract of seminal p Lama (pronase and trypain di gestion, followed by trichloroacetic acid precipitation ; then followed by neutralisation with NaON), ware dialyzed against water for 24 hr before being added to the column .
NaCl (1M) waa employed to elute the samples with 1-ml
aliquots being collected .
The tRNA in each sample was determined by measuring
the 260 nm absorbance in a Beckman spectrophotamcter .
Vol. 10, No. 18
Non-Ensymatic Hydrolysis of tRNA
921
Besulte Deproteiniaation of seminal plasma with either trypain, chymotrypsin, perchloric acid or phenol had no effect on the ability of seminal plasma to hydrolyze tRNA .
Perchloric acid could only be used on seminal plasma diluted
at least 1 :75 .
If undilutefl seminal plasma was used, the active aubetance(s)
was co-precipitated loan with the proteins . undiluted seminal plasma .
Phenol was used successfully with
Deproteinisatioa of seminal plasma by a combination
of phenol and chlorotorm :isoamyl alcohol extractions (2x each) showed no detectable protein .
Despite these various treatments to denature the proteins,
seminal plasma dilutione as low as 1 ;5000 were able to totally hydrolyze 0 .1 mg of tRNA in 1 hr at 37 ° C . Figure 1 depicts a Sephadax G-100 profile of tRNA and tRNA pre incubated with a 1 :1000 dilution of deproteinised seminal plasma (pronase treatment follaaed by trichloroacetic acid precipitation) .
Both tRNA samples were
dialysed for 24 hr against water at 4°C prior to being placed oa the column . The results (F ig . 1) show that after the tRNA was incubated with the deproteinised seminal plasma there was a shift is the 260 nm abeorbance of the 5s peak (~6 tubes ; 6 ml), and a shift in the 4a peak (22 tubes ; 22 ml) .
The shift
shows a degradation of the tRNA into shorter polynucleotide chains .
The tRNA,
after incubation with the deproteinised seminal plasma did not lone anq 260 nm absorbing material after dialysis .
Also, the area under the curves for tR2iA
and tRNA plus deproteinised seminal plasma were approximately the same .
These
latter two facts indicate that mono or diaucleotidea were not produced by the reaction .
The production of mono and dinucleotides is usually associated with
a ribonuclease .
When tR2iA was incubated with seminal plasma which was ant de-
proteinized, there wen a SX lose of 260 nm absorbing material after dialysis, thus indicating the presence of a contaminating nuclease in the untreated seminal plasma .
922
Non-Enzymatic Hydrolysis ad tRNA
Vol. 10, No. 18
E c O c0
N m C O aD
a
FIG . i Sephadex G-100 profile of tRDiA iacui~ated with deproteinised aemiael plasma, and tRNA without any prior treatment, eluant .
Each test tube represents 1 ml of
Tha methods ara described in the text .
Sucrose gradients of tRNA and tENA plus deproteiaized seminal plasma showed similar results for the 4s peak as the sephadex gel data .
The 5a
peak could not be separated with the use of the sucrose gradients .
The
diluted seminal plasma extract, by itself, had no 260 or 280 nm absorption after gel chromatography or sucrose gradient assays,
Increased abeorp~ion
(hyperchramicity) at 260 om was also observed when similar dilutions of the deprotainizad seminal plasma extract were incubated with tRNA .
This data
further subatantiatea the hydrolysis of the tRNA aubatrate by the seminal plasma extract . Since it waa demonstrated that incubation of deprotainized extracts of aeminel plasma with tRNA results in acid-soluble tRK9 fragments is the absence of protein, it was possible to determine the kinetics of tRirA hydrolysis ôy measuring the acid solubility of tRNA at 260 nm .
This type of asFay wns em-
ployed to ahw that the active rubatance(a) in seminal plasma reacted in a non-enzymatic manner with the aubatrate tR~IA, and was dependent oa the time of
voi .
io,
No. 16
Noa-Enzymaüc Hydrolysis ad tRNA
923
inuubation (Fig, 2) " The reactions were completed in 70-80 min at 37°C, after which constant levels of tRNA acid solubility were maintaiand .
A 1 :5000 dilu-
tion of seminal plasma completely hydrolysed 0 .1 mg of tRNA while 1 ;10000 and 1 :30000 dilutions hydrolysed tRNA SOx and 32x, respectively .
There were no
effects on the hydrolysis of tRNA if the deprotefniaed extracts of seminal plasma and tRNA were preincubated alone for 100 min at 37°C prior to being mixed together .
100
Q 2
80
20
MINUTES FIG . 2 The effect of time of incubation and concentration of seminal plasma on tRNA solubility .
One-tenth mg of tRNA was incubated with varying concentrations
of deproteinised seminal plasma .
Details of the assay are described in the
section on methods . Discussion
924
Non-$nzymaüc i~ydrolysis od tRNA
Vol. 10, No. 1 8
The rasulta obtained with the Sephadex G-100 chromatography, sucrose gradients sad spectral studies indice n that very dilute amounts of deproteiniaad saotiaal plasma hewn sufficient amouata of a aube nace(a) which cas hydrolyae CßD1A .
The activa aubannce(s) was shown to react in a aon-ensymatic
manner with the tRNA (Fig . 2) .
These results, plue the data showing that
mono and dinucleotodes are sot products of the reaction and the fact that various proteases, inorganic and organic protein precipitants failed to destroy the active substancea(s), indicate that a ribonuclaase was not responeible for the hydrolysis of taNA .
However, the potnacy of minute amounts
sad resistance to degradation of ribonucleases, in general, is wall recognised . Therefore, the possibility of the active compouad(a) being ribonuclease contamiaaats can not be completely eliminated .
This would still be true even
after an apparent isolation of the active substancn(s) .
However, if a ribo-
nuclease ie involved in the hydrolysis of tßPiA, it must possess an unusually large polysaccharide component nncesaary to nxplaia
the acquired da n . Other
nucleases, such ae ribonuclease B, which coataine a polysaccharide component, are deasturad by away of the techniques which had no nffecta on the bull semen compouad(s) . Ia an initial study (2), extensive hydrolysis of tRNA was not observed after being incubated with seminal plasma .
This was due to the co-precipita-
tion of the resulnat tRIiA fragments with the large amounts of liver protein present is the assay mixture for hepatic tRNA methylase activity .
The prea-
seta of those fragments was verified by Sephadex C-100 column chromatography after their extraction by phenol from the liver protein .
Their elution pro-
file wets similar to that produced by tRNA which had been incubated with seminal plasma in the absence of large amounts of liver protein . the resulting t&NA fragments were acid soluble .
Ia the latter case,
None of the isolated tRNA
fragments were able to fuactioa ae a substrate for tRNA methylaees .
It is non
avldent that the inhibition of tBNA mathylation by seminal plaams as reported in a previous publication (2), was due to the presence of hydrolytic subetance(s)
Vol. 10, No. 18
Non-Enzymatic Hydrolysis ~ tRNA
92S
in the bull semen which degraded both, the tBNA substrate, sad any of the methylatad tBNA product which may 4ave been formed . It is difficult to envisage a polysaccharide hydrolysing tRNA .
Roaevar,
the possibility of the polysaccharide chemically binding to tRNA does exist . Initial experiments were unsuccessful in demonstrating the existence of a polysaccharide-tRNA complex .
Ha+aver, identification of such a complex may
prove to be a formidable task, since degradation of the tRNA may occur imsediately after binding .
If the polysacchar üe does bind to tRNA, it sry
conceivably produce a molecular strain on the tRNA, resulting in the observed degradation . Ackaovledßement This study was supported by Grant No . 12-711äA from the New York State Research Foundation . References 1.
B . Shaid and S .M . Wilson, Federation Proc . 29 : 3883 (1970) .
2.
B . 5heid and S .M . Wilson, Siochim . Bioohys . Acu 224 :382 (1970) .
3.
O .H . Loary, N .J . Rosebrough, A .L . Farr and R .J . Bandall, J . Biol . Chem . 1 94 : 265 (1951) .
Pergamon Press
NON-ENZYMATIC HYDROLYSIS OF TRANSFER RNA BY BU..L SEMEN Bettrum Sheid and Susan M . Wilson Department of Pharmacology, State University of New York Downetate Medical Center, Brooklyn, New York 11203
(Received S1 March 1971; in final form 6 July 1971) su®a ry Dilutions of deproteinised seminal plasma ae lw as 1 :5000 were ahovn to contain sufficient amounts of a compounds) which nonensysntically hydrolysed O .l mg tRNA into acid soluble fragments . Other evidence is presented to eliminate the possibility of the active seminal plasma substance (s) being ribonuclease contaminaat(s) . PREVIOUS ezperiaents have daaonstrated the existence of a very potent inhibitor s) of tRNA methylation in bull semen (1,2) .
The active subetance(s) was
not affected by daneturing the semen proteins with trichloracetic acid or chloroform :isoanyl alcohol .
The semeu compouad(s) ws also unaffected by 100°-
C heat, trypsia and prooase digestion .
Other experiments eliminated the poss-
ibility of the coo~pouad(s) being a lipid, nucleic acid or a small molecule such as salts or amino acids (2) .
Periodate oxidation destroyed the tRNA
methy L se inhibitory activity, thus suggesting a polysaccharide as the active component in semen . In the present report, data are presented to ahoy that bull semen contains a caozpou~(s) which praventa tRMA methylation by degrading the tRNA substrate in a non-ensymatic manner .
Other evidence is presented to further
eliminate the possibility of the degradation of tRNA being specifically due to contaminating ribonucleasea . Materials and Methods Bull seism was collected over liquid nitrogen without preservatives or antibiotics added to the final pooled sample .
919
The product was provided by the
920
Non-Enzymatic Hydrolysis ad tRNA
Vol, 30, No. 16
Eastern Artificial Insemination Corporation, Ithaca, New York . Escherichia coliß t$NA was purchased from the General Biochemical Corporation . 8amiaal plasma was separated from sperm cells after centrifugation at 850 x g for 10 min in a modal PR-2 Iateraational centrifuge .
Although the ac-
tive component was present is both of the snman fractioae (2), only the seminal plasma was used for these studies .
The seminal plasma was deproteinized by
incubating 1 ml of a 1 :400 dilution (300 Wg protein) with 100 W g of trypain or LOO~,g of chymotrypsin for 2 .5 hr at 37 ° C .
These enzymes were subeequently
dastroged without affecting the active component(a) in seminal plasma by heating at 80 ° C for 20 min .
Thn seminal p L ema was also deproteinizad by extract-
ing 1 ml of uadilutad seminal pLss~a with 3 volumes of alcohol ;ether (3 ;1) at 4°C, or 1 volume of phenol at 4 ° C .
Another method employed to denature seminal
plasma proteins was by adding perchloric acid to a 1 :75 dilution of seminal pLsan until the solution was 6x with respect to the acid .
This particular sol-
ution was neutralised with NaOH and then dialyzed against water .
The appro-
priate dilutiona of the deproteiniaed extracts were made with distilled water . The assay mixture used to determine the non-enzymatic hydrolysis of tRNA coasiatad of 100y~moles of Tris buffer pfl 8 .4, 0 .1 mg tRNA and 0 .2 ml of varying dilutiow of the deproteiaised seminal plasma extracts .
After 1 hr at 37°C
the reactions were terminated with trichloroacetic acid (final concentration 6Z acid) .
The 260 nm aborbance of the supernatant fluid was then read in a Beck-
man spectrophotometer to determine the percent hydrolysis of tRNA . wsahed Sephadex G-100 was loaded into a column 10 x 140 cm .
One-ml sam-
plea containing 0 .25 mg of ta1~A praincubated at 37°C, with and without 1 ;1000 diluted and daproteinizad extract of seminal p Lama (pronase and trypain di gestion, followed by trichloroacetic acid precipitation ; then followed by neutralisation with NaON), ware dialyzed against water for 24 hr before being added to the column .
NaCl (1M) waa employed to elute the samples with 1-ml
aliquots being collected .
The tRNA in each sample was determined by measuring
the 260 nm absorbance in a Beckman spectrophotamcter .
Vol. 10, No. 18
Non-Ensymatic Hydrolysis of tRNA
921
Besulte Deproteiniaation of seminal plasma with either trypain, chymotrypsin, perchloric acid or phenol had no effect on the ability of seminal plasma to hydrolyze tRNA .
Perchloric acid could only be used on seminal plasma diluted
at least 1 :75 .
If undilutefl seminal plasma was used, the active aubetance(s)
was co-precipitated loan with the proteins . undiluted seminal plasma .
Phenol was used successfully with
Deproteinisatioa of seminal plasma by a combination
of phenol and chlorotorm :isoamyl alcohol extractions (2x each) showed no detectable protein .
Despite these various treatments to denature the proteins,
seminal plasma dilutione as low as 1 ;5000 were able to totally hydrolyze 0 .1 mg of tRNA in 1 hr at 37 ° C . Figure 1 depicts a Sephadax G-100 profile of tRNA and tRNA pre incubated with a 1 :1000 dilution of deproteinised seminal plasma (pronase treatment follaaed by trichloroacetic acid precipitation) .
Both tRNA samples were
dialysed for 24 hr against water at 4°C prior to being placed oa the column . The results (F ig . 1) show that after the tRNA was incubated with the deproteinised seminal plasma there was a shift is the 260 nm abeorbance of the 5s peak (~6 tubes ; 6 ml), and a shift in the 4a peak (22 tubes ; 22 ml) .
The shift
shows a degradation of the tRNA into shorter polynucleotide chains .
The tRNA,
after incubation with the deproteinised seminal plasma did not lone anq 260 nm absorbing material after dialysis .
Also, the area under the curves for tR2iA
and tRNA plus deproteinised seminal plasma were approximately the same .
These
latter two facts indicate that mono or diaucleotidea were not produced by the reaction .
The production of mono and dinucleotides is usually associated with
a ribonuclease .
When tR2iA was incubated with seminal plasma which was ant de-
proteinized, there wen a SX lose of 260 nm absorbing material after dialysis, thus indicating the presence of a contaminating nuclease in the untreated seminal plasma .
922
Non-Enzymatic Hydrolysis ad tRNA
Vol. 10, No. 18
E c O c0
N m C O aD
a
FIG . i Sephadex G-100 profile of tRDiA iacui~ated with deproteinised aemiael plasma, and tRNA without any prior treatment, eluant .
Each test tube represents 1 ml of
Tha methods ara described in the text .
Sucrose gradients of tRNA and tENA plus deproteiaized seminal plasma showed similar results for the 4s peak as the sephadex gel data .
The 5a
peak could not be separated with the use of the sucrose gradients .
The
diluted seminal plasma extract, by itself, had no 260 or 280 nm absorption after gel chromatography or sucrose gradient assays,
Increased abeorp~ion
(hyperchramicity) at 260 om was also observed when similar dilutions of the deprotainizad seminal plasma extract were incubated with tRNA .
This data
further subatantiatea the hydrolysis of the tRNA aubatrate by the seminal plasma extract . Since it waa demonstrated that incubation of deprotainized extracts of aeminel plasma with tRNA results in acid-soluble tRK9 fragments is the absence of protein, it was possible to determine the kinetics of tRirA hydrolysis ôy measuring the acid solubility of tRNA at 260 nm .
This type of asFay wns em-
ployed to ahw that the active rubatance(a) in seminal plasma reacted in a non-enzymatic manner with the aubatrate tR~IA, and was dependent oa the time of
voi .
io,
No. 16
Noa-Enzymaüc Hydrolysis ad tRNA
923
inuubation (Fig, 2) " The reactions were completed in 70-80 min at 37°C, after which constant levels of tRNA acid solubility were maintaiand .
A 1 :5000 dilu-
tion of seminal plasma completely hydrolysed 0 .1 mg of tRNA while 1 ;10000 and 1 :30000 dilutions hydrolysed tRNA SOx and 32x, respectively .
There were no
effects on the hydrolysis of tRNA if the deprotefniaed extracts of seminal plasma and tRNA were preincubated alone for 100 min at 37°C prior to being mixed together .
100
Q 2
80
20
MINUTES FIG . 2 The effect of time of incubation and concentration of seminal plasma on tRNA solubility .
One-tenth mg of tRNA was incubated with varying concentrations
of deproteinised seminal plasma .
Details of the assay are described in the
section on methods . Discussion
924
Non-$nzymaüc i~ydrolysis od tRNA
Vol. 10, No. 1 8
The rasulta obtained with the Sephadex G-100 chromatography, sucrose gradients sad spectral studies indice n that very dilute amounts of deproteiniaad saotiaal plasma hewn sufficient amouata of a aube nace(a) which cas hydrolyae CßD1A .
The activa aubannce(s) was shown to react in a aon-ensymatic
manner with the tRNA (Fig . 2) .
These results, plue the data showing that
mono and dinucleotodes are sot products of the reaction and the fact that various proteases, inorganic and organic protein precipitants failed to destroy the active substancea(s), indicate that a ribonuclaase was not responeible for the hydrolysis of taNA .
However, the potnacy of minute amounts
sad resistance to degradation of ribonucleases, in general, is wall recognised . Therefore, the possibility of the active compouad(a) being ribonuclease contamiaaats can not be completely eliminated .
This would still be true even
after an apparent isolation of the active substancn(s) .
However, if a ribo-
nuclease ie involved in the hydrolysis of tßPiA, it must possess an unusually large polysaccharide component nncesaary to nxplaia
the acquired da n . Other
nucleases, such ae ribonuclease B, which coataine a polysaccharide component, are deasturad by away of the techniques which had no nffecta on the bull semen compouad(s) . Ia an initial study (2), extensive hydrolysis of tRNA was not observed after being incubated with seminal plasma .
This was due to the co-precipita-
tion of the resulnat tRIiA fragments with the large amounts of liver protein present is the assay mixture for hepatic tRNA methylase activity .
The prea-
seta of those fragments was verified by Sephadex C-100 column chromatography after their extraction by phenol from the liver protein .
Their elution pro-
file wets similar to that produced by tRNA which had been incubated with seminal plasma in the absence of large amounts of liver protein . the resulting t&NA fragments were acid soluble .
Ia the latter case,
None of the isolated tRNA
fragments were able to fuactioa ae a substrate for tRNA methylaees .
It is non
avldent that the inhibition of tBNA mathylation by seminal plaams as reported in a previous publication (2), was due to the presence of hydrolytic subetance(s)
Vol. 10, No. 18
Non-Enzymatic Hydrolysis ~ tRNA
92S
in the bull semen which degraded both, the tBNA substrate, sad any of the methylatad tBNA product which may 4ave been formed . It is difficult to envisage a polysaccharide hydrolysing tRNA .
Roaevar,
the possibility of the polysaccharide chemically binding to tRNA does exist . Initial experiments were unsuccessful in demonstrating the existence of a polysaccharide-tRNA complex .
Ha+aver, identification of such a complex may
prove to be a formidable task, since degradation of the tRNA may occur imsediately after binding .
If the polysacchar üe does bind to tRNA, it sry
conceivably produce a molecular strain on the tRNA, resulting in the observed degradation . Ackaovledßement This study was supported by Grant No . 12-711äA from the New York State Research Foundation . References 1.
B . Shaid and S .M . Wilson, Federation Proc . 29 : 3883 (1970) .
2.
B . 5heid and S .M . Wilson, Siochim . Bioohys . Acu 224 :382 (1970) .
3.
O .H . Loary, N .J . Rosebrough, A .L . Farr and R .J . Bandall, J . Biol . Chem . 1 94 : 265 (1951) .