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Suppression of tyrosine transaminase activity by rat liver ribosomes
Lüe Sciences Vol . 12, Part II, pp. 49-58, 1973 Printed in Great Britain
Pergamon Press
SûPPRESSION OF TYROSINE TRANSAMINASE ACTIVITY BY RAT LIVER RIBOS~S George P . Tryfiatee+ , Edward F . Ploy* sad Mark H . Hafner Department of Biochemistry Went Virginia ûaiveraity Medical Center Morgaatown, West Virginia 26506
(Received 29 November 1971 ; in final form 4 December 1972) Tha activity of tyrosine tranaaminase ie suppreaeed if rat liver ribosomes are present is the enzyme assay system . Thin observation is true whether highly purified rat liver enzyme or crude pH 5 .1 enzyme fraction traasaminase is emplo~ed . Ribosomal tuppresaion of enzymatic activity is not due to Mg and/or to limited substrate or cofactor sad occurs whey catalysis is linear . Suppression of enzyme activity was demonstrated independently by two different assay methods . The data are interpreted to suggest possible ribosomal involvement in the regulation of tyrosine traaaaminaae . During the course of cell-free protein synthesis ezperimeats (1,2) it was obsarvad that the presence of liver ribosomes in the enzyme assay system suppressed the tyrosine traaeaminase (L-tyrosine :
2-ozoglutarate amiaotranaferasq
E .G .2 .6 .1 .5) activity of the pH 5 .1 aazyma fraction .
Since tyrosine traas-
aminase is known to be associated with ribosomaa (3,1) ribosomal suppression of its activity aeemad paradozical and warranted further verification .
This com-
munication reporta on data regarding the suppression of the activity of highly purified tyrosine traneaminase by rat liver ribosomes . Materials and Methods Wistar strain male rata, weighing approzimately 350 g ware injected intraperitoaeally with triamcinolonn acetonida (500 mg/100 g body wt .) four hours before decapitation sad removal of the liver .
Tyrosine traasamiasee wan puri
fied from a post-~mitochondrial liver supernatant according to the procedure of
{To whoa reprint requests should be addressed . *Present address : Department of Clinical Pathology, Scripps Clinic and Research Foundation, 476 Prospect Street, LaJolla, California 92037 .
49
50
Suppression ad Tyrosine Transaminase Activity
Sayashi, nt al . (4) .
Vol. 12, No. 8
Ribosomal were prepared by the method of Wettstein, et al.
(5), ae described (6-8) from post-mitochoadrial rat or rabbit liver suparnateats .
The source of the ribosomal preparation had no influence oa the results .
During the purification of the easyma, the assay method of Dianondstone (9) was used .
However, in tests requiring the use of radioisotope the Diamondatona
assay (9) was slightly modified is that, in addition to cold L-tyrosine, 3 uCi of L-[3H]-tyrosine (sp . act . 33 .5 uCi/pmole; ~i Corp .) were included, as suggested by Weinstein, et al .
(10) .
Further, 0.58 mgs ribosome C8 OD260) per ml
assay were added, where indicated (11) .
The enzyme unit is defined ae that
amount catalyzing the formation of 1 pmole of p-hydrozybeazaldehyde in 10 minutes at 37'C
(9,12) .
Aaaaye were rue with 0 .1 ml enzyme in 4 .2 ml final volume.
After incubation for 10 minutes at 37'C the reaction was stopped by adding 0 .2 ml of cold 5x trichloroacetic acid .
The assay tubes were then allowed to stand
for 10 minutes sad the supernatant obtained by centrifugation was directly applied onto a 0.8 a 14 cm Dowea AG 50W - %8, hydrogen, (Calbiochem) column (10) .
The traasamination product [ 3H]-p-hydroayphenylpyruvate ([ 3H]p HPP) was
separated from L-[ 3H]-tyrosine as described (10) .
During standard roan, we
repeatedly recovered over 90Z of both p HPP and amino acid with the pHPP eluting in the first 55 ml . HCi .
The amino acid (L-tyrosine) was eluted with 4N
Radioactivity in the eluted fractions was measured is a Packard Tri-Garb
liquid scintillation spectrometer by adding 100 plitnre of each fraction to 10 ml of Bray's solution as previously described (1,7,8,19,20) .
Since the enzyme
preparation was entirely depleted, tyrosine transaminase was reieolated as described above (4) .
In attempts to further demonstrate ribosoml suppression
of enzymatic activity the enzyme was assayed by yet a different procedure.
In
these latter teats the assay method of Cannellakie and Cohéa (13) was employed se previously described (2) .
Under the coaditioas described, nonspecific
traasamination i.a . is the absence of enzyme or ribosomas, did not occur . Results and Discussion The results reported here were obtained using highly purified enzyme
Vol . 12, No . 2
51
Suppression od Tyrosine Transa.minase Activity
r
W C
É ô ô
Eluilon Yolu" o ( " 1)
Llutlon Yolur ("1)
Figure 1. Elution of purified Rat Liver Tyrosine Transaminase from DEAE celluloae . A. Elution vas effected using a linear gradient of decreasing pH attd increseiag salt concnatration. Other details is Results and Discussion aectioa. B. The enzyme peak in A vas chro>aatographed again by pooling the indicated fractions and applying to a second DEAF-cellulose cola®, as euggeetnd (4) . Ona optical density unit at 331 mu equals 50 .2 umoles of p-hydroxybeazaldnityda formed under the conditions of the assay (9) . preparations, i.e . specific activity of more than 10,000 (sp . act . ~ ymoles product formed per mg enzyme protein) . Hayashi, et al ., (4) .
The enzyme vas prepared according to
The preparation eluted ea a single peak from a 2 .0 cm x
30 cm DEAF-cellulose column using a linear gradient of increaeiag salt concen
tration and decreasing pH . phate pH 7 .6 buffer .
The mixing chamber contained 0 .2 M potassium phos-
The linear gradient vas formed by diluting the buffer in
the mixing chamber with 0 .2 ä potassium phosphate buffer, pH 6 .0 containing 0 .3 K xCl .
The enzyme vas eluted from the DEAF-cellulose column by directly apply-
ing the formed lunar gradient onto the column, as described (4,6,22,23) . tion vas carried at 4 " C .
Elu-
Fractions of 10 ml were collected at the rate of 1.0
52
Suppression of Z~rosine Transaminase Activity
OA
0.4
0 .1 ENZYME
OR
Vol. 12, No . 2
0.7
RI6050ME5
(M L)
Figure 2. Ribosomal Suppression of Tyrosine Traneaminase Activity . Tyrosine traasamiaase vas purified from rat liver (4) and assayed in the absence and presence of ribosomea (Materials and Methods) according to the method of Canallakis and Cohen (13,2) . A ~ Activity curve using purified enzyme ; C Activity curve using liver ribosomee as the source of ~zyme ; B ~ Curve A repeated but in the presence of 0 .13 ml ribosomes par assay; Al ~ Eapnctad Instead, the theoretical activity curve for purified enzyme plus ribosomee. result vas curve B . 0.1 ml ribosomee ~ 49 optical density units at 260 mu "3 .55 mgs (11) . 0 .1 ml enzyme ~ 60 ug Lovry protein (21) . Thn total assay volume vas 4 .2 ml . All assays were performed in duplicate . Hach point is the average of five different datnrminatioas euept for curve C which consisted of 3 determinations . The highest standard deviation observed vas t 0 .6 a 10-5 p HPP . ml in 1 .2 minutes .
The elution of tyrosine traneaminase from DEAR-cellulose is
ahovn is Figure 1 . The effect of ribosomes on the activity of purified liver tyrosine transamiaase ie shown in Figure 2 .
A curve of a sigmoid nature vas obtained with
increasing enzyme concentration (curve A) .
This observation ie in accord with
reports by different workers which either suggested or demonstrated that the enzyme is composed of four similar or identical subuaitsQ,14,4,15-17) .
Curve C
Vol . 12, No . 2
Suppression od Tyrosine Transamina.se Activity
was obtained when liver ribosomee ware used as the source of enzyme .
53 However,
if curve A was repeated is the presence of a constant amount of ribosomes (=0 .13 ml ribosomes which catalyzed the formation of 1 .125 ymoles of p HPP) curve B vas obtained rather than the theoretical expected curve A1 .
Further,
if curve C vas repeated in the presence of a constant amount of enzyme the activity of the ribosomal eazyma was similarly suppressed .
The curve obtained
vas similar to C but of lesser elope (Results sot ahwn) . The results obtained with radioactively labeled L-[3H]-tyrosine (Materials sad Methods) era summarized is Table 1 . activity is present in ribosomee,
Despite the fact that transaminase
the activity of the purified enzyme vas lees
when ribosomaa were present in the assay system .
The two enzyme activities,
i.e . activity of the purified enzyme and that of the ribosomal enzyme were definitely not additive when assayed together .
As a matter of fact, the
amount of product ([ 3H] p HPP) formed is less than nzpected by either the action of the purified enzyme or by the action of the ribosomal enzyme .
This
paradozical finding vas repeatedly verified not only with the purified enzyme, but also with liver pH 5.1 fraction traasaminase (1) .
Ia the latter case
suppression of enzymatic activity was observed with over 60 different prepare" bona and without failure. The ribosomal suppressioa of tyrosine traasaminase activity dcea not seem to be due to limited substrate or coenzyme concentration. Hayaahi, et al . (4), reported Michaelis' conataate of 1 .7 a 10-3 , 7 z 10
-4
sad 1.7 z 10 g for tyrosine, a-ketoglutarate and pyridozal phosphate, respectively .
The coacantratioaa of the amino sad keto acids in the assay system (9)
ware 6 : 10-3M and 9.4 z 10-3li, respectively . al phosphate inhibits tyrosine traneaminase
At high concentrations, pyridoz-
[Ki~l .l a 10-3 , (4)] .
tration of the coaazyme in the assay vas 4 .25 z 10 -5M .
Tha coacan-
Further, under the
sway conditions, the reaction vas linear with approzimately up to 0 .20 ml of easysia par assay.
This observation lends credence to ribosomal inhibition of
traaaamiaase activity which is not a slight 4x as may ba inferred from Table I
Suppression of Tyrosine Tra,nsaminase Activity
54
Vol. 12, No . 2
TABLE I Effect of Liver Ribosomes on the Activity of Purified Rat Liver Tyrosine Transaminase . Variable
Counts per Minute [3H]pHPP Peak (product)
L-[ 3H]-tyrosine Peak (eubetrate)
Total Counts Recovered
Per Cent Comrersion
Purified Enzyme (Coatrol)
172,000
233,000
405,000
42 .5
Liver Ribosomes
176,000
220,000
396,000
44 .4
Control + Liver Ribosomes
145,000
227,000
372,000
38 .6
Highly purified tyrosine traaaaminaea vas assayed is the absence and presence of rat liver ribosomee (0 .6 mg/ml assay) using 0 .1 ml enzyme . The reacThe reaction vas tion vas linear with up to 0.20 ml of enzyme per assay. The clear supernatant stopped by the addition of 0.2 ml of cold 5X TCA . obtained by centrifugation vas directly applied on Doves 50W-8S, hydrogen to separate the product from the eubetrate (10) . Coatrol tests shoved no transamiaation occurring and the counts under the [3H] p HPP peak were recovered as tyrosine is the tyrosine peak . but 38 .6 from an eapected theoretical value of 44 .4 + 42 .5 (i .e . .38 .6/86 .9 44 .42X) . Although Mg++ inhibits activity (18) the 3nhibitory .effect of ribosomes does sot appear to be due to the presence of Mgt .
Results obtained from assays
performed is the presence of ribosomes after treatmaat with EDTA or after dielyeie to remove Mgt (11) were practically identical as reported above (Table I) . The paradoxical inhibitory effect of liver ribosomal on the activity of purified or crude tyrosine traneaminase (1) ie difficult to interpret .
It
would appear that is the codez nukaryotic cells ribosomes in addition to their role in protein synthesis, may also participate in the regulation of enzymatic activity .
Although a macromolecule such se albumin is kaova to sta-
bilize traasaminaaa activity enzyme is sot understood .
(4) how ribosomes suppress the activity of the
To our knowledge ribosomal suppression of enzymatic
activity has not been reported and further teats are mended to rigorously
Vol . 12, No. 2
Suppression ad Tyrosine Tra.nsaminase Activity
55
demonstrate whether ribosomes am indeed involved in the regulation of eaaymatit activity as suggsated by our results. Acknowledgement This study van initially supported by general research support awards from the Schools of Dentistry, Medicine and the local branch of the American Ceacer Society and later by research Grant 1801 CA 13759-01 NTN . Rnfarencas 1.
G. P . TRYFIATSS, Biochim. Biophys . Acta, 174,
779(1969) .
2.
G. P . TRYFLATSS aad G . LITWACK, Biochem., 3, 1483(1964) .
3.
G. LITWACIC, M. L . SEARS aad T. I . DL9MONDSTONE, J . Biol . Chem ., 238, 302 (1963) .
4.
S . HAYASHI, D. H. GBAlOiER and G . M. TOMICINS, J. Biol . Chem ., 242, 3998(1967~
5.
F. 0 . WSTTSTSIN, T . STAHASLIN and H . NOLI, Nature, 197, 430(1963) .
6.
G. P . TRYFIATES, Proc . Soc . Ezptl . Biol . Mnd ., 134, 644(1970) .
7.
G. P . TRYFIATES, Biocham. Pharmacol ., 20, 1669(1971) .
8.
G. P . TRYFIATES and R. F. RRAÜSS, Proc . Soc . S:ptl . Biol . Med ., 136, (1971) .
9.
Y. i . DLAMONDSTONE, Anal . Biodran., 16, 395(1966) .
10 .
A. WSINSTEIN, G. MEDSS and G. LITWAC~, Anal . Biochem., 21, 86(1967) .
11 .
Y. TASHIAO and T . li0SII~60'lb, Biothun. Biophys . Acta ., 123, 523(1966) .
12 .
G. P . TSYFIlITSS, Life Scieacas,. (Part II), 10, 1147(1971) .
13 .
Z . N. r~rrßrs.~ATS aad P P. COHBN, J. Biol . Cham ., 222, 52(1956) .
14 .
F.T . YBNNEY, J . Biol . Chen ., 237, 1605(1962) .
15 .
P .G . HOLT and LT. OLIVER, FSBS Letters, 5, 89(1969) .
16 .
F. ADRICCHIO, F. VALHRIOTE, G. TOM&INS aad W. D . RILSY, Biocrim. Biophys . Acta ., 221, 307(1970) .
946
17 .
Y. Tv~c~YT and H. C . PITOT, Lifa Sciences, 10, (Part II), 1071(1971) .
18 .
G. P. TRYFIATES and S. PLOW, 156th Nat'1 Meeting Amer . Cheer. Society, (1!6 ") . Aba . 75 .
19 .
G. P . TRYFIATES and R. F . ESAÛBS, Life Sciencna,
20 .
G. P. TRYFIATES
Sept .
(Part II), 10, 1092(1971) .
aß~ J. LA82Z0, Natura, 213, 1025(1967) .
58
Suppresaion ad T`yroaine Z`ransaminase Acüvity
Vol. 12, No . 2
21 .
0 . H . LOÜRY, N . J . ROSEBROUGH, A . L . FARB aad R . J . RANDALL, J . Biol . Chem. 193, 265(1951) .
22 .
G . P . TRTFLATBS, Prepay . Biochea ., 2(4), 355(1972) .
23 .
G . P . TRYFLATES aad N . H . CHOÛLIS, J . Pharmac . Sci ., 61, 465(1972) .
Pergamon Press
SûPPRESSION OF TYROSINE TRANSAMINASE ACTIVITY BY RAT LIVER RIBOS~S George P . Tryfiatee+ , Edward F . Ploy* sad Mark H . Hafner Department of Biochemistry Went Virginia ûaiveraity Medical Center Morgaatown, West Virginia 26506
(Received 29 November 1971 ; in final form 4 December 1972) Tha activity of tyrosine tranaaminase ie suppreaeed if rat liver ribosomes are present is the enzyme assay system . Thin observation is true whether highly purified rat liver enzyme or crude pH 5 .1 enzyme fraction traasaminase is emplo~ed . Ribosomal tuppresaion of enzymatic activity is not due to Mg and/or to limited substrate or cofactor sad occurs whey catalysis is linear . Suppression of enzyme activity was demonstrated independently by two different assay methods . The data are interpreted to suggest possible ribosomal involvement in the regulation of tyrosine traaaaminaae . During the course of cell-free protein synthesis ezperimeats (1,2) it was obsarvad that the presence of liver ribosomes in the enzyme assay system suppressed the tyrosine traaeaminase (L-tyrosine :
2-ozoglutarate amiaotranaferasq
E .G .2 .6 .1 .5) activity of the pH 5 .1 aazyma fraction .
Since tyrosine traas-
aminase is known to be associated with ribosomaa (3,1) ribosomal suppression of its activity aeemad paradozical and warranted further verification .
This com-
munication reporta on data regarding the suppression of the activity of highly purified tyrosine traneaminase by rat liver ribosomes . Materials and Methods Wistar strain male rata, weighing approzimately 350 g ware injected intraperitoaeally with triamcinolonn acetonida (500 mg/100 g body wt .) four hours before decapitation sad removal of the liver .
Tyrosine traasamiasee wan puri
fied from a post-~mitochondrial liver supernatant according to the procedure of
{To whoa reprint requests should be addressed . *Present address : Department of Clinical Pathology, Scripps Clinic and Research Foundation, 476 Prospect Street, LaJolla, California 92037 .
49
50
Suppression ad Tyrosine Transaminase Activity
Sayashi, nt al . (4) .
Vol. 12, No. 8
Ribosomal were prepared by the method of Wettstein, et al.
(5), ae described (6-8) from post-mitochoadrial rat or rabbit liver suparnateats .
The source of the ribosomal preparation had no influence oa the results .
During the purification of the easyma, the assay method of Dianondstone (9) was used .
However, in tests requiring the use of radioisotope the Diamondatona
assay (9) was slightly modified is that, in addition to cold L-tyrosine, 3 uCi of L-[3H]-tyrosine (sp . act . 33 .5 uCi/pmole; ~i Corp .) were included, as suggested by Weinstein, et al .
(10) .
Further, 0.58 mgs ribosome C8 OD260) per ml
assay were added, where indicated (11) .
The enzyme unit is defined ae that
amount catalyzing the formation of 1 pmole of p-hydrozybeazaldehyde in 10 minutes at 37'C
(9,12) .
Aaaaye were rue with 0 .1 ml enzyme in 4 .2 ml final volume.
After incubation for 10 minutes at 37'C the reaction was stopped by adding 0 .2 ml of cold 5x trichloroacetic acid .
The assay tubes were then allowed to stand
for 10 minutes sad the supernatant obtained by centrifugation was directly applied onto a 0.8 a 14 cm Dowea AG 50W - %8, hydrogen, (Calbiochem) column (10) .
The traasamination product [ 3H]-p-hydroayphenylpyruvate ([ 3H]p HPP) was
separated from L-[ 3H]-tyrosine as described (10) .
During standard roan, we
repeatedly recovered over 90Z of both p HPP and amino acid with the pHPP eluting in the first 55 ml . HCi .
The amino acid (L-tyrosine) was eluted with 4N
Radioactivity in the eluted fractions was measured is a Packard Tri-Garb
liquid scintillation spectrometer by adding 100 plitnre of each fraction to 10 ml of Bray's solution as previously described (1,7,8,19,20) .
Since the enzyme
preparation was entirely depleted, tyrosine transaminase was reieolated as described above (4) .
In attempts to further demonstrate ribosoml suppression
of enzymatic activity the enzyme was assayed by yet a different procedure.
In
these latter teats the assay method of Cannellakie and Cohéa (13) was employed se previously described (2) .
Under the coaditioas described, nonspecific
traasamination i.a . is the absence of enzyme or ribosomas, did not occur . Results and Discussion The results reported here were obtained using highly purified enzyme
Vol . 12, No . 2
51
Suppression od Tyrosine Transa.minase Activity
r
W C
É ô ô
Eluilon Yolu" o ( " 1)
Llutlon Yolur ("1)
Figure 1. Elution of purified Rat Liver Tyrosine Transaminase from DEAE celluloae . A. Elution vas effected using a linear gradient of decreasing pH attd increseiag salt concnatration. Other details is Results and Discussion aectioa. B. The enzyme peak in A vas chro>aatographed again by pooling the indicated fractions and applying to a second DEAF-cellulose cola®, as euggeetnd (4) . Ona optical density unit at 331 mu equals 50 .2 umoles of p-hydroxybeazaldnityda formed under the conditions of the assay (9) . preparations, i.e . specific activity of more than 10,000 (sp . act . ~ ymoles product formed per mg enzyme protein) . Hayashi, et al ., (4) .
The enzyme vas prepared according to
The preparation eluted ea a single peak from a 2 .0 cm x
30 cm DEAF-cellulose column using a linear gradient of increaeiag salt concen
tration and decreasing pH . phate pH 7 .6 buffer .
The mixing chamber contained 0 .2 M potassium phos-
The linear gradient vas formed by diluting the buffer in
the mixing chamber with 0 .2 ä potassium phosphate buffer, pH 6 .0 containing 0 .3 K xCl .
The enzyme vas eluted from the DEAF-cellulose column by directly apply-
ing the formed lunar gradient onto the column, as described (4,6,22,23) . tion vas carried at 4 " C .
Elu-
Fractions of 10 ml were collected at the rate of 1.0
52
Suppression of Z~rosine Transaminase Activity
OA
0.4
0 .1 ENZYME
OR
Vol. 12, No . 2
0.7
RI6050ME5
(M L)
Figure 2. Ribosomal Suppression of Tyrosine Traneaminase Activity . Tyrosine traasamiaase vas purified from rat liver (4) and assayed in the absence and presence of ribosomea (Materials and Methods) according to the method of Canallakis and Cohen (13,2) . A ~ Activity curve using purified enzyme ; C Activity curve using liver ribosomee as the source of ~zyme ; B ~ Curve A repeated but in the presence of 0 .13 ml ribosomes par assay; Al ~ Eapnctad Instead, the theoretical activity curve for purified enzyme plus ribosomee. result vas curve B . 0.1 ml ribosomee ~ 49 optical density units at 260 mu "3 .55 mgs (11) . 0 .1 ml enzyme ~ 60 ug Lovry protein (21) . Thn total assay volume vas 4 .2 ml . All assays were performed in duplicate . Hach point is the average of five different datnrminatioas euept for curve C which consisted of 3 determinations . The highest standard deviation observed vas t 0 .6 a 10-5 p HPP . ml in 1 .2 minutes .
The elution of tyrosine traneaminase from DEAR-cellulose is
ahovn is Figure 1 . The effect of ribosomes on the activity of purified liver tyrosine transamiaase ie shown in Figure 2 .
A curve of a sigmoid nature vas obtained with
increasing enzyme concentration (curve A) .
This observation ie in accord with
reports by different workers which either suggested or demonstrated that the enzyme is composed of four similar or identical subuaitsQ,14,4,15-17) .
Curve C
Vol . 12, No . 2
Suppression od Tyrosine Transamina.se Activity
was obtained when liver ribosomee ware used as the source of enzyme .
53 However,
if curve A was repeated is the presence of a constant amount of ribosomes (=0 .13 ml ribosomes which catalyzed the formation of 1 .125 ymoles of p HPP) curve B vas obtained rather than the theoretical expected curve A1 .
Further,
if curve C vas repeated in the presence of a constant amount of enzyme the activity of the ribosomal eazyma was similarly suppressed .
The curve obtained
vas similar to C but of lesser elope (Results sot ahwn) . The results obtained with radioactively labeled L-[3H]-tyrosine (Materials sad Methods) era summarized is Table 1 . activity is present in ribosomee,
Despite the fact that transaminase
the activity of the purified enzyme vas lees
when ribosomaa were present in the assay system .
The two enzyme activities,
i.e . activity of the purified enzyme and that of the ribosomal enzyme were definitely not additive when assayed together .
As a matter of fact, the
amount of product ([ 3H] p HPP) formed is less than nzpected by either the action of the purified enzyme or by the action of the ribosomal enzyme .
This
paradozical finding vas repeatedly verified not only with the purified enzyme, but also with liver pH 5.1 fraction traasaminase (1) .
Ia the latter case
suppression of enzymatic activity was observed with over 60 different prepare" bona and without failure. The ribosomal suppressioa of tyrosine traasaminase activity dcea not seem to be due to limited substrate or coenzyme concentration. Hayaahi, et al . (4), reported Michaelis' conataate of 1 .7 a 10-3 , 7 z 10
-4
sad 1.7 z 10 g for tyrosine, a-ketoglutarate and pyridozal phosphate, respectively .
The coacantratioaa of the amino sad keto acids in the assay system (9)
ware 6 : 10-3M and 9.4 z 10-3li, respectively . al phosphate inhibits tyrosine traneaminase
At high concentrations, pyridoz-
[Ki~l .l a 10-3 , (4)] .
tration of the coaazyme in the assay vas 4 .25 z 10 -5M .
Tha coacan-
Further, under the
sway conditions, the reaction vas linear with approzimately up to 0 .20 ml of easysia par assay.
This observation lends credence to ribosomal inhibition of
traaaamiaase activity which is not a slight 4x as may ba inferred from Table I
Suppression of Tyrosine Tra,nsaminase Activity
54
Vol. 12, No . 2
TABLE I Effect of Liver Ribosomes on the Activity of Purified Rat Liver Tyrosine Transaminase . Variable
Counts per Minute [3H]pHPP Peak (product)
L-[ 3H]-tyrosine Peak (eubetrate)
Total Counts Recovered
Per Cent Comrersion
Purified Enzyme (Coatrol)
172,000
233,000
405,000
42 .5
Liver Ribosomes
176,000
220,000
396,000
44 .4
Control + Liver Ribosomes
145,000
227,000
372,000
38 .6
Highly purified tyrosine traaaaminaea vas assayed is the absence and presence of rat liver ribosomee (0 .6 mg/ml assay) using 0 .1 ml enzyme . The reacThe reaction vas tion vas linear with up to 0.20 ml of enzyme per assay. The clear supernatant stopped by the addition of 0.2 ml of cold 5X TCA . obtained by centrifugation vas directly applied on Doves 50W-8S, hydrogen to separate the product from the eubetrate (10) . Coatrol tests shoved no transamiaation occurring and the counts under the [3H] p HPP peak were recovered as tyrosine is the tyrosine peak . but 38 .6 from an eapected theoretical value of 44 .4 + 42 .5 (i .e . .38 .6/86 .9 44 .42X) . Although Mg++ inhibits activity (18) the 3nhibitory .effect of ribosomes does sot appear to be due to the presence of Mgt .
Results obtained from assays
performed is the presence of ribosomes after treatmaat with EDTA or after dielyeie to remove Mgt (11) were practically identical as reported above (Table I) . The paradoxical inhibitory effect of liver ribosomal on the activity of purified or crude tyrosine traneaminase (1) ie difficult to interpret .
It
would appear that is the codez nukaryotic cells ribosomes in addition to their role in protein synthesis, may also participate in the regulation of enzymatic activity .
Although a macromolecule such se albumin is kaova to sta-
bilize traasaminaaa activity enzyme is sot understood .
(4) how ribosomes suppress the activity of the
To our knowledge ribosomal suppression of enzymatic
activity has not been reported and further teats are mended to rigorously
Vol . 12, No. 2
Suppression ad Tyrosine Tra.nsaminase Activity
55
demonstrate whether ribosomes am indeed involved in the regulation of eaaymatit activity as suggsated by our results. Acknowledgement This study van initially supported by general research support awards from the Schools of Dentistry, Medicine and the local branch of the American Ceacer Society and later by research Grant 1801 CA 13759-01 NTN . Rnfarencas 1.
G. P . TRYFIATSS, Biochim. Biophys . Acta, 174,
779(1969) .
2.
G. P . TRYFLATSS aad G . LITWACK, Biochem., 3, 1483(1964) .
3.
G. LITWACIC, M. L . SEARS aad T. I . DL9MONDSTONE, J . Biol . Chem ., 238, 302 (1963) .
4.
S . HAYASHI, D. H. GBAlOiER and G . M. TOMICINS, J. Biol . Chem ., 242, 3998(1967~
5.
F. 0 . WSTTSTSIN, T . STAHASLIN and H . NOLI, Nature, 197, 430(1963) .
6.
G. P . TRYFIATES, Proc . Soc . Ezptl . Biol . Mnd ., 134, 644(1970) .
7.
G. P . TRYFIATES, Biocham. Pharmacol ., 20, 1669(1971) .
8.
G. P . TRYFIATES and R. F. RRAÜSS, Proc . Soc . S:ptl . Biol . Med ., 136, (1971) .
9.
Y. i . DLAMONDSTONE, Anal . Biodran., 16, 395(1966) .
10 .
A. WSINSTEIN, G. MEDSS and G. LITWAC~, Anal . Biochem., 21, 86(1967) .
11 .
Y. TASHIAO and T . li0SII~60'lb, Biothun. Biophys . Acta ., 123, 523(1966) .
12 .
G. P . TSYFIlITSS, Life Scieacas,. (Part II), 10, 1147(1971) .
13 .
Z . N. r~rrßrs.~ATS aad P P. COHBN, J. Biol . Cham ., 222, 52(1956) .
14 .
F.T . YBNNEY, J . Biol . Chen ., 237, 1605(1962) .
15 .
P .G . HOLT and LT. OLIVER, FSBS Letters, 5, 89(1969) .
16 .
F. ADRICCHIO, F. VALHRIOTE, G. TOM&INS aad W. D . RILSY, Biocrim. Biophys . Acta ., 221, 307(1970) .
946
17 .
Y. Tv~c~YT and H. C . PITOT, Lifa Sciences, 10, (Part II), 1071(1971) .
18 .
G. P. TRYFIATES and S. PLOW, 156th Nat'1 Meeting Amer . Cheer. Society, (1!6 ") . Aba . 75 .
19 .
G. P . TRYFIATES and R. F . ESAÛBS, Life Sciencna,
20 .
G. P. TRYFIATES
Sept .
(Part II), 10, 1092(1971) .
aß~ J. LA82Z0, Natura, 213, 1025(1967) .
58
Suppresaion ad T`yroaine Z`ransaminase Acüvity
Vol. 12, No . 2
21 .
0 . H . LOÜRY, N . J . ROSEBROUGH, A . L . FARB aad R . J . RANDALL, J . Biol . Chem. 193, 265(1951) .
22 .
G . P . TRTFLATBS, Prepay . Biochea ., 2(4), 355(1972) .
23 .
G . P . TRYFLATES aad N . H . CHOÛLIS, J . Pharmac . Sci ., 61, 465(1972) .