Inhibition of tyrosine aminotransferase activity by low concentrations of urea

BIOCHIMICA ET BIOPHYSICA ACTA BBA

35 1

65399

INHIBITION OF TYROSINE AMINOTRANSFERASE ACTIVITY BY LOW CONCENTRATIONS OF UREA

GERALD LITWACK, MARY LOU SEARS-GESSEL

AND

ILGA WINICOV

Fels Research Institute and the Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pa. (U.S.A.) (Received October 8th, 1965)

SUMMARY

Urea and guanidine hydrochloride in relatively low concentrations can completely inhibit tyrosine aminotransferase (r.-tyrosine .z-oxoglutarate aminotransferase, EC 2.6.1.5) activity by direct addition of the inhibitor to the assay system or prior incubation systems. Guanidine hydrochloride is more effective on a molar basis than urea, however, the action of these reagents is qualitatively similar. The kinetic mode of urea inhibition in the assay is reversible with respect to enzyme and is noncompetitive with respect to each reactant of the system. Simultaneous addition of about 10-4 M pyridoxal phosphate or pyridoxamine phosphate to the prior incubation system affords nearly complete protection from concentrations of urea or guanidine hydrochloride which would produce 50% inhibition in the subsequent assay. L- Tyrosine, a-ketoglutarate, pyridoxal hydrochloride, or pyridoxamine hydrochloride do not protect from urea inhibition under these conditions. The fact that protection from urea inactivation requires concentrations of coenzyme of about 100 X K m together with the noncompetitive mode of inhibition suggests that urea induces structural changes. The inhibition resulting from the presence of urea with enzyme in prior incubation systems is more extensive when Tris buffer is used in prior incubation than when phosphate buffer is used, although Tris buffer does not significantly inhibit enzymatic activity compared to phosphate buffer in the assay systems. This protective effect of phosphate ion can be imitated by arsenate ion in comparison to Tris buffer in the prior incubation system. Anionic surface active agents such as sodium dodecylsulfate or sodium deoxycholate are also very effective inhibitors of enzymatic activity, whereas a nonionic detergent was not inhibitory. The inhibition by anionic surface active agents is not eliminated by coenzyme in the prior incubation system and therefore appears to be qualitatively different from the action of urea or guanidine hydrochloride.

INTRODUCTION

Although many reports are contained in the literature which describe denaturation of enzymes by urea, little emphasis, until recently}, has been placed upon Biochim, Biopbys, Acta, lIS (Ig66) 35I-362

35 2

G. LITWACK, M. L. SEARS- GESSEL, I WINICOV

studies of activity and kinetics of enzymes inhibited by relatively small concentrations of urea. The finding, in this laboratory, that the tyrosine aminotransferase (t-tyrosine .z-oxoglutarate aminotransferase, EC 2.6.1.5) system is inhibited by relatively low concentrations of urea is of importance because relatively little is known concerning the molecular weight, heterogeneity, or aggregation of this enzyme. Direct information on changes in tertiary or quaternary structure by physicochemical techniques is hampered by the great lability of the purified enzyme, therefore, as an initial starting point, the effects of disrupting agents have been studied with regard to effects upon enzymatic activity. A preliminary report of this work has been mades. EXPERIMENTAL PROCEDURE

Biochemicals and reagents Pyridoxal phosphate and pyridoxamine phosphate were obtained from the Nutritional Biochemicals Corporation. The spectra of these compounds from 240500 m{-t indicated very high purification. There was no obvious contamination of one by the other. Urea was obtained from the Fisher Scientific Company and was used directly. Experiments performed with urea recrystallized from 95% ethanol and treated with Norit in the process indicated that routine purification of urea was not necessary. Guanidine hydrochloride was obtained from Distillation Products Industries. Enzyme grade ammonium sulfate (Sigma Chemical Company) was used in prior incubation studies where indicated and in all purification procedures. The other chemicals mentioned were of reagent grade and were obtained from commercial sources. Partially purified tyrosine aminotransferase was prepared as described previously". Purification above zoo-fold was not attempted because the enzyme tends to become unstable and generally unsuitable to studies involving inhibition and especially, prior incubation. The partially purified enzyme was stable for many weeks at 4°. Prior inc-ubation system and enzyme assay Prior incubation refers to a z.o-ml volume (sometimes 3.0 ml to 12.5 ml where indicated) containing 0.05 ml of partially purified enzyme, 0.2 M phosphate buffer at pH 7.6 (unless indicated to the contrary), and inhibitor with or without protecting agent. Prior incubation was carried out for 10 min at 37° with gyratory shaking. Longer prior incubations were used for serial sampling in progress experiments. The enzymatic assay was of 10 min duration and has been described previously-, When prior incubation was involved a o.y-ml aliquot was transferred to the temperature-equilibrated assay system containing all the reactants except enzyme. The transfer resulted in a 6-fold dilution of the prior incubation system. Enzymatic activity was measured by the appearance of p-hydroxyphenylpyruvate 5 • Activity or velocity expressions are based upon the change of absorbance at 850 m,u resulting from a ro-min reaction time per ml of assay system. These values may be converted to ftmole p-hydroxyphenylpyruvate' 10 min' 3.0 ml systemr" by multiplying by 0.7. Biochim, Biopby», Acta, lIB (1966) 35 1-362

TYROSI NE AMIN OT RANSFE RASE AN D URE A EFFE CT

353

1.8

8

Urea

Guanidln e'Hel

v

\

1.2

\'( \

ClB

4

\

\

\

'I -, o

3

t

""'" 1

0.1

0.3

It

COf'lcn. In assay system 1M)

Fig. 1. Concent ration-depe nde nce curves of ur ea and guanidine hyd rochloride on inhibition of t yr osine aminotran sferase activit y in th e assay syste m. Each point r epresents a separat e measurement. These studies were p erformed with three different enzyme p reparations.

RESULTS

Effects of protein structural reagents in the assay system En zym atic acti vity is ma rkedly inhibited by relat ively low concentrations of ur ea or guanidine hydrochloride in the assay system as shown in Fig. 1. 50 % inhibition by urea is obtained at a concentration of about 2 M in the assay system, whereas 50 % inhibit ion is obtained at a concentration of about 0 .1 M guanidin e hydrochloride in the assay system. The more potent effect of guanidine hydrochloride is consistent throughout all exp eriments performed. It should be noted that effecti ve inhibitory concentrations of urea and guanidine hydrochloride are much lower than thos e usually used t o achieve disaggregation, dissociation or unfolding 6. From Fi g. 2 (insert), it can be seen that the inhibition by urea and guanidine h ydrochloride is linear with the logarithm of concent ration. Howev er, when inhibition data ar e formulate d in a DIXO N plot ? as shown in Fig. 2 , it is clear that curves are

,

~---1

'--

~

Innibitor concn. 1M)

Fi g. z. DIXON pl ots of inhibition of tyrosine aminotransferase activ it y by urea and guanidine hydrochloride in the assay . The insert shows the log co ncent r at ion- de pendence cu r ves for urea and guanidine hydrochl orid e in the assay.

Bi ochi m, B iopby s. Ac ta, 1I 8 (1966) 351-362

354

G. LITWACK, M. L. SEARS-GESSEL, 1. WINICOV

v

Fig. 3. ACKERMANN-POTTER plot of inhibition by urea in the assay system showing reversibility with respect to enzyme.

obtained instead of straight lines. A deviation of this type may be associated with a reaction between inhibitor and substrate, which seems unlikely in this case, or when more than one molecule of inhibitor reacts with one molecule of enzymes.

Kinetics of inhibition in the assay system Fig. 3 shows the results of inhibition using the ACKERMANN-POTTER procedure 9. Inhibition by urea is reversible with respect to enzyme. In Fig. 4 is shown the results of kinetic studies of inhibition by urea in the assay system with respect to each reactant. The data are plotted as described by HUNTER AND DOWNS 1D .These plots show that the kinetic mode of inhibition is of non-competitive character with respect to each reactant. In addition to studying

s-

~ s-

2.)I

/s 10i&R 1~5""

>:;' 10- 4 3

2

(')

~

4 3 0 0'.:16 I~ 21 3

'-' .

o

L,~,_,_,_ L,_,_,_,_,_,_ 20 400 4 8 12 0

o

[Tyrosine] x 10

4M

4

86

00

0 5 r,.

'6

0

1

0

0

2

1-1-1-1-1-'-1-1-1-1-11-1-

8 12 16 20100 [a-ketoglutarate] x 10- 4 M

.
[pyridoxal phosphate]x10·

Rote-limiting substrate concentration Fig. 4. Mode of inhibition by urea of tyrosine aminotransferase activity with respect to each reactant in the system using the method of HUNTER AND DOWNS 10. Several concentrations of urea were used from La to 3.52 M. Temperature equilibration was carried out for I min at 37° prior to initiating the reaction by addition of a-ketoglutarate. Incubation was for 10 min at 37°. Each point is the mean of a number of experiments indicated by the number on the chart. Standard deviations were calculated for means of 4 or more observations and were usually not more than ± 25 % of the mean value. [IJ, molar concentration of urea; v and Vi refer to the initial velocity of the reaction in the absence and presence of the inhibitor, respectively.

Biochim, Biopliys. Acta, IIS (Ig66) 35I-362

TYROSINE AMINOTRANSFERASE AND UREA EFFECT

355

wide ranges of reactant concentrations, some attention was directed to the effect of varying the temperature equilibration period prior to assay. This was studied in the greatest detail when kinetics were run with respect to a-ketoglutarate. Temperature equilibration periods of from I to 10 min did not alter the resulting non-competitive mode of inhibition. Prior incubatio« systems A prior incubation time of 10 min was selected for these experiments. The usefulness of this choice was evident from progress curves of urea inhibition. In order to learn the effects of this prior incubation time upon enzymatic activity in the subsequent assay, prior incubations were run without urea but in the presence of each reactant of the tyrosine aminotransferase system. These results are presented in Fig. 5. At least 3 different preparations of enzyme were used in each case accounting PALP

L -tyrosine

a - ketoglutarat e

v

0.3

Conlroor level o 10"6 10-3 0'0-0 10- 3 0 1cr~ 10- 3 Concn in prior incubati on system (M)

Fig. 5. Activities of tyrosine aminotransferase after a to-min prior incubation in the absence or presence of various concentrations of reactants of the system. Each point represents a separate determination. PALP, pyridoxal phosphate. The "control level" refers to the average activity in the assay after the ro-min prior incubation of enzyme and buffer only as shown by the averaged point on the ordinate at a zero value of the abscissa.

for the scatter of points at each locus. The resulting curves are actually quite smooth for a given enzyme preparation. Each component of the reaction renders slight protection from loss of activity of the enzyme during the prior incubation. That afforded by pyridoxal phosphate is slightly greater, perhaps, than tyrosine or aketoglutarate. The decrease of activity below that of the control when 10-3 and IO-2 M pyridoxal phosphate is in the prior incubation system is due to the presence of inhibitory levels of coenzyme after dilution into the assay. The large drop in activity when 10- 2 M tyrosine is present in the prior incubation is due to the insolubility of the tyrosine even after dilution into the assay; this undoubtedly causes co-precipitation of some of the enzyme from solution. In Fig. 6 are shown the results of adding urea, guanidine hydrochloride, or sodium deoxycholate to the prior incubation system followed by 6-fold dilution and assay of enzymatic activity. 50% inhibition occurs with levels in the prior incubation system of 2.5 M urea, about La M guanidine hydrochloride, or with about 0.2% (5' 10-3 M) sodium deoxycholate. These results may be compared to results obtained Bioohim, Biopbys. Acta, IrS (I966) 35I-362

35 6

G. LITWACK, M. L. SEARS-GESSEL,

Ur~a

1.1 1.2

GuarOoine+Cl SOd:um " d eoxycnolol e

'-t.,

,

r. WINICOV

\

.

1.0 0.8 0.6 0.1

\

1

0.2 0

01

3

5 OQ3 09 1.5 0

04 OS

M M·'. CCl1CI\ in prior incutlation syslem

Fig. 6. Effects of urea, guanidine hydrochloride, or sodium deoxycholate added to a r o-rnin prior incubation system. Each point represents one observation. At least 3 different enzyme preparations were used in each experiment; this accounts for the scatter of points at each locus on a curve. Relation of v to units of enzymatic activity is described in EXPERIMENTAL PROCEDURE.

by adding urea or guanidine hydrochloride directly to the assay system (Fig. I). The concentrations of urea required to produce 50 % inhibition either in the assay system or in the prior incubation system aTe quite comparable. However, this is not the case when guanidine hydrochloride is considered. The concentration of guanidine hydrochloride needed to produce 50% inhibition in the assay system was 0.1 lVI, however, about 1.0 M is required in the prior incubation system to achieve the same effect. After dilution into the assay system from the prior incubation system, the concentration in the assay would be 0.17 M. This suggests that little difference obtains in the type of incubation chosen for the study of guanidine hydrochloride. On the other hand, urea inhibition is not linear with dilution. 2 M is required in the assay system to achieve 50 % inhibition, whereas 2.5 M is required in the prior incubation system. After dilution from the prior incubation system, the concentration of urea in the assay would be 0.4 M; this level would produce only 10% inhibition if added directly to the assay system. It thus appears that urea is more effective if it is incubated with enzyme prior to assay, whereas, guanidine hydrochloride is equally effective in either case. The action of sodium deoxycholate is also pronounced. 50% inhibition is achieved by having a concentration of 0.2 % (5' 10-3 M) in the prior incubation system. This level aligns well with the inhibition by sodium dodecylsulfate, discussed below. As in other studies, the scatter of points at each locus results from the use of 2 or more enzyme preparations in each experiment. The next investigation was directed at attempts to determine whether reactants of the enzymatic system and related compounds could protect from or reverse the effect of urea added to the prior incubation system. The results of such a study are presented in Table 1. It was possible to devise a "corrected control" in these studies because urea has about the same quantitative effect when added to the prior incubation system or directly to the assay system. The "corrected control", therefore, Biochim, Biophys. Acta, lIS (1966) 351- 362

357

TYROSI NE AMINOTRA NSFERASE AND UREA EFFECT

TABLE! E F FECTS OF REACTANTS AND RELAT ED CO MP OU N D S A DD ED WITH UREA IN TH E IO -MIN PRIOR INCUB ATION SYSTEM AT p H 7.6'

Addition.

Number of

experiments None (control) II Corrected for urea in assay . 0' 41\1, 90% 2.5 M urea 22 10- 0 M p yr id ox a l phosphat e 7 10-· M pyri dox al phosphate 17 + 2 ' 10- " M p yridoxal ph osphate 8 + 10-4 ~I p yridoxal phosphate 10 4 + 2 ' 10- M pyridoxal phosphate 5 + 10-3 M pyridoxal phosphate 7

+ +

+ + + + + + +

+ + + + + + + +

+ + + + + + +

10- ' lVI p yri dox a l p hosphate

5

10- 3 M a-ketoglutarate 12 10- 2 M a-ketoglutarate 5 10-1 M a-ketoglutarate 5 10 - " M t -tyrosinc 4 3 10- M L-t yrosi ne II 10- 2 M L-tyrosine -I 10- 3 M a -ketoglu t arate } .'i 10- 3 MI.-tyrosine 10-6 M p yridoxal phosphate 6 10- 3 M L-tyrosine 10- 6 M p yridoxal phosphate 4 ro - 3 M a -k etoglutarate 5 . ro -· M p yrid ox al phosphat e 10- 3 M p y ri dox al p hosp hate 10-' M pyridoxal phosphate 10- 3 M pyridoxamine phosphate 10- 2 M pyridoxamine phosphate 10-3 M pyridoxamine' HCl 10- 2 !VI pyridoxamine ' HCl 10- 3 M pyridoxal' HC l 10-' M p yridoxal ' H'Cl

J

Enzymatic activity 0.75 ± 0.01 (S.E.) 0.67 0.29 ± 0.01 0.33 ± 0.001 0.49 ± 0.02 0.57 ± 0.03 0.61 ± 0.03 0.48 ± 0.02 0.42 ± 0.02 (inhibitory level in assay) 0. 22 ± 0. 01 (inh ibi t ory le v el in as sa y) 0.32 ± 0.02 0.33 ± 0.01 o..p ± 0.01 0.27 ± 0.03 0·32 ± 0.02 0.38 ± 0.02

± 0.03 0.42 ± 0.02 0.58 ± 0 .02 0.32

0·57 0·53 0.5 0 0.69 0.56 0.25 0·33 0·35 0.26

Percent of corrected control

100 43 49 73 85 91

72 63 33 48 49 61 40 48 57 48 63 87 79 74 69 96

78 35 46 49 36

• 6-fold dilution to assay.

refers to a correction made for the 1 0 % inhibiti on resulting in the assay syste m from the 6-fold dilution of the ur ea in t he prior incubation system . A depend en ce upon the concentration of pyridoxal phosphat e for protection from the urea effect in t he pri or in cubation system is immediately appa rent . Maximal protection from the urea effect is achieved by 2 ' 10-5 to 10-4 M pyridoxal phosphate in the prior incubatio n system. Levels of coenzyme in excess of IO - 4 result in less complete protection from the urea effect and the last 2 levels present ed result in levels of coenzyme, after dilution, which ar e inhibitory in t he assay. Large concentr at ions of a-ketoglutarate or tyrosine have only very slight ability to protect from the effect of urea. One-t enth M a-ketoglutarate has a slight enhancing effect upon activity. When comb inations of the reactants are used, prot ect ion is not ed only when pyridoxal phosphate is part of the additi on to the prior incub at ion system. Coenzyme plHS a-k et oglut ara t e seems more effect ive than coenzyme plus tyrosine. P yridoxamin e phos phate as well as B iochim , B iophy s. Acta. lIB (r966) 351-362

G. LITWACK, JIif. L. SEARS-GESSEL, I. WINICOV

TABLE II EFFECTS OF REACTANTS AND RELATED COMPOUNDS ADDED WITH GUANIDINE HYDROCHLORIDE IN THE IO-MJN PRIOR INCUBATION SYSTEM AT pH 7.6'

Add-ition

Ntimber of experiments

Enzymatic activity

Percent of control

None (control) 1.2 M guanidine hydrochloride + 10- 6 M pyridoxal phosphate 10- 5 M pyridoxal phosphate + 2' 10- 5 M pyridoxal phosphate + ro- 4 M pyridoxal phosphate 10-' M pyridoxal phosphate

4 4 3 4 4 4 4

0.67 0.26 0.4 6 0·59 0.62 0·55 0·53

roo 39 69 88 93 82 79

None (control) I.2 M guanidine hydrochloride + 5 . ro- 5 M pyridoxamine' Hel + 5 . 10-' M pyridoxal' Hel 5 . 10-5 M pyridoxamine phosphate + 10-' M pyridoxamine phosphate + 5 . ro- 5 M pyridoxal phosphate 5 . IQ-5 M a-ketoglutarate + 5 . 10-5 M t.-tyrosine

3 3 3 3 3

r .71 0.24 0.26 0.29

2

0.3 0 0.5 6 0.27 0.23

+ +

+

+

3 2

2

0.21

(0.6J-o.7I) range (0.24-0. 29) (0.44-0.5 1 ) (0.57-0.62) (0-49- 0.7 1 ) (0.45-0.63) (0·3 8-0. 60)

JOO

34 37 41 30 85 79 38 32

• 6-fold dilution to assay.

pyridoxal phosphate eliminates the inhibition by urea, except that the effective concentration is somewhat higher. However, pyridoxamine hydrochloride, or pyridoxal hydrochloride, the non-phosphorylated forms, are ineffective in protecting from inhibition by urea. In Table II, similar results are presented for the case of inhibition by guanidine hydrochloride added to the prior incubation system. Pyridoxal phosphate most effectively protects from the effect of guanidine hydrochloride at a level of z . 10-5 M and the effective range of concentration of the coenzyme is from 10-5 to 10-3 M, the range being somewhat broader than that for the protection from the effect of urea. Pyridoxamine phosphate is also active in protecting from the inhibition due to guanidine hydrochloride, and, as observed before with urea inhibition, the nonphosphorylated forms, pyridoxamine hydrochloride and pyridoxal hydrochloride, are inactive in this respect. Tyrosine and a-ketoglutarate do not protect from the inhibition produced by guanidine hydrochloride. In Table III are shown the results of varying the buffer anion used in the prior incubation experiment upon enzymatic activity in the absence or presence of 2.5 M urea. While prior incubation and assay of enzymatic activity in Tris buffer compared to phosphate does not statistically affect the level of enzymatic activity, there is a marked effect when urea is present in the prior incubation system. Thus, it is clear in Expt. I that phosphate ion reduces the level of inhibition produced by urea compared to Tris buffer in the prior incubation system. The beneficial effect of phosphate is roughly proportional to its concentration when the level exceeds 0.1 mmole in the prior incubation system. In Expt. II, it is shown that phosphate and arsenate behave similarly and this similarity is expressed in the data of Expt. III showing that Biochim. Biophys. Acta, IrB (1966) 35J-362

359

TYROSINE AMINOTRANSFERASE AND UREA EFFECT TABLE III

EFFECTS OF BUFFER ANION ON ENZYMATIC ACTIVITY OF TYROSINE AMINOTRANSFERASE AND ON INHIBITION BY UREA IN THE IO-MIN PRIOR INCUBATION SYSTEM AT pH 7.6

Components of the prior incubation system are enzyme and buffer

±

urea as described in

EXPERI-

MENTAL PROCEDURE. E;~pt.

No.

Components oj prior incubation system Addition to system

Number of experiments

Concentration of buffer anion Phosphate (mmoles)

Arsenate Tris (mmoles) (mmoles)

none 0.20 0.3 0 0·39 none 0.10

0·39 0.19 0.09 none 0·39 0.29 0.19 0.09 none

Enzymatic clctivity IOOO X (umotes HPP'jIo min per 3.0 ml system)

Expt. Ib 2 3 4 5 6 7 8 9

none none none none 2.5 M urea 2.5 M urea 2.5 M urea 2.5 M urea 2.5 Murea

Expt. lIe 10 11 12 13 14 15 r6 17 18

none none none none 2.5 M urea 2.5 M urea 2.5 M urea 2.5 M urea 2.5 M urea

Expt. IIIb 19 20 21 22 23 24 25 26 27

none none none none 2.5 M urea 2.5 Murea 2.5 Murea 2.5 Iv! urea 2.5 M urea

I

0.20

0.3 0 0·39

none 0.20 0.3 0 0·39 none

o.ro o.sc 0.3 0 0·39 0·39 0.19 0.0C) none 0·39 0.29 0.19 0.09 none

± 28· ± 31

5 6 6 6 6 6 6 6 6

308 350 33 6 336 25 :Z4 106 155 174

0·39 0.19 0.09 none 0·39 0.29 0.19 0.09 none

7 7 7 7 7 7 7 7 7

650 ± 50 530 ± 50 570 ± 60 600 ± 60 220 ± 50 170 ± IO 270 ± 40 245 ± 30 200 ± 30

none 0.20 0.3 0 0·39 none o.ro 0.20 0.3 0 0·39

6 6 6 6 6 6 6 6 6

602 ± 20 553 ± 35 525 ± 49 560 ± 28 21 ± 3 56 ± 35 d 91 ± 7 77±I4 9 8 ± 35

± 39 ± 35 ± 11 ±3 ± IS ± 26 ± 30

HPP. p-hydroxyphenylpyruvate. b Enzyme assay determined using 0.2 M Tris buffer (pH 7.6). c Enzyme assay determined using 0.2 M arsenate buffer (pH 7.6). d All values were very close with the exception of one value which accounts for the high

a

S.E. e Standard error of the mean; different preparations of enzyme were used in the 3 experiments,

arsenate ion can imitate phosphate ion compared to Tris buffer in the prior incubation system. Again relatively little difference can be seen when enzymatic activity is assayed in arsenate or Tris, paralleling the case of phosphate and Tris buffer (Expt. I). Table IV presents information on the inhibition of enzymatic activity by surface active agents. Sodium dodecylsulfate inhibits enzymatic activity when it is Biochim. Biophys, Acta. JI8 (1966) 351-362

G. LITWACK , 1\1. L. SEARS-GESSEL, 1. WINICOV TABLE1V EFFECTS OF SURFACE ACTIVE AGENTS AND AMMONIUM SUL FATE ADDED TO THE J 0 - MIN PRIOR INCUBATION SYSTEM AT

pn 7.6*.

Addition

None (control) 10- 4 M so dium 2.5 ' 10-' M sod ium 5 ' 10- * 1'1 sodium 7.5 ' IO-'.M so dium 10 - ·1 .M so dium

dodecylsulfate'" dod ecylsu lfa tc ** d odecylsulfate** dodecylsu lfate"

dcdecy lsulfate'"

N one (control) 0.4 % sod iu m deoxycholate (5 . 10- 5 M} ro- G M pyridoxal ph osphate IO- 5 M pyridoxal phosp hate 2' 10- 6 M pyridoxal p hosphate 10-4 M p yridoxa l ph osp h at e ro- s M pyridoxal phosphate -I-

+ + + +

Number of ex periments

Enzymatic actiuiiy

Percent of control

4 4 4 4 4 4

0 .78 0·93 I .03 0.94 0.61 0 .3 I

(0.70-o.9I) range (0.85-1.06) (0.9 7-1.14) (0.8 6- 1.0 2) (0.49- 0.75) (0.18-0.48)

100 IIg 13 2 121 78 40

3

0.5 8 0 .2I 0 .15 0 .21 0 .2 I 0.24 0 .23

(0.5 1- 0.7 1) (0.I7-o.·z6) (0.12-0.17) (0.1 8- 0.23l (0.19-0.23 ) (0..17-0.3 I) (0. 20-0.26)

100 36 26 36 36 41 40

3 2 2

3 3 3

1. 0 7

Non e (control) 0.2 to r .0 % " cuts cu m " (di isob u tylphe no xyp olyeth oxy ethano l)

6

0 .98 t o

1.1 6

92 t o 108

None (control) 2' 10- 8 to 2 ' 10-' M ammonium sulfate

I

6

1. 0 9 0.g2 to 1.16

100 84 t o 106

100

• 6-fold dilution to assay. •• So di um dodecy Isulfat e prior in cub ation experiment s in 0.05 M pot assium phosphat e b uffer (pH 7.6) .

present in the prior incubation system at 7.6. ro-4 M and above . Sodium deoxycholat e at a level of 5' 10- 3 M in the prior incubation system inhibit ed 50% (Fig. 6). Shown here is the ineffecti veness of a wide range of concentrations of py ridoxa l phosphate in protecting from t he effect of the surface active agent. Enzymatic activity is sensitive t o anionic surface active agents, but not to nonionic surface active agents as demonstrated by the ineffectiveness of diisobutylphenoxypolyethoxyethanol in con cent rations up to I % added to the prior incubation system. Addit ional dat a presented in this t able show t hat ammonium sulfat e used in the purification is not inhi bitory up to 0.02 M. Several experiments suggest that the action of urea takes place immediately and if an appropriate level of coenzyme is added at a time prior to the maximal effect of urea, the coenzyme can prevent the further act ion of the inhibitor but does not reverse the established inhibition. DISCUSSION

It is interesting that the great instability of highly purified tyrosine aminotransferase may be related to t he high sensitivity of the partly purified, stable enzyme to disrup t ing reagents. It is also quite clear that t he action of urea and guanid ine h ydrochl oride is qualitatively different from the inhibiti on produced by B iochim. B iop hys. A cta. II8 (196 6) 351-362

TYROSINE AMINOTRANSFERASE AND UREA EHEeT

anionic surface active agents since the effects of the latter are not reversed by levels of coenzyme which when added to the prior incubation system nearly completely protect from the action of urea or guanidine hydrochloride. The activity of pyridoxamine phosphate in protecting from the inhibition by either urea or guanidine hydrochloride raises some obvious questions. Pyridoxamine phosphate is not only active in this capacity, but, surprisingly, functions also as a coenzyme in fully resolved preparations of tyrosine aminotransferase 2. The recent finding of contaminants in guanidine hydrochloride preparations which are effective as competitive inhibitors of xanthine oxidase activity and are related in structure to the substrate of this enzyme-" does not appear to satisfy the difference of potency with regard to the tyrosine aminotransferase system. Specific cases are known, such as the dissociation of human CO-hemoglobin where guanidine hydrochloride is much more potent than urea 1 ,14 . From the results of several experiments on the progress of urea inhibition in the prior incubation system it appears that the coenzyme, in concentrations of about roo X K m , protects from rather than reverses the effects of the denaturing agents. The participation of large quantities of coenzyme in this function could mean that alteration of the tertiary or quaternary structure of the enzyme involves a pyridoxal phosphate binding site other than the site of attachment of coenzyme to the active site. A precedent of this type is the case with phosphorylase 15. This interpretation is strengthened by the noncompetitive kinetics with respect to coenzyme as well as the existence of multiple forms of the liver enzyme of molecular weights in excess of 200000 which have similar amino acid substrate specificities (G. LITWACK AND 1. WINICOV, unpublished experiments). The effect of coenzyme in protecting from urea inhibition does not appear to be a function of its phosphate content alone, for by comparison to the amount of phosphate in the buffer, it is rather small. It may be surmised, therefore, that the coenzyme protects from inhibition by urea at one site, possibly a structural site, and the effect of phosphate (or arsenate) ion may take place at another site which involves, in addition, the structure of the enzyme. The remarkable effect of phosphate ion upon unfolding of ribonuclease by urea may serve in support of this concept-s,

ACKNOWLEDGEMENTS

The authors thank Miss ]. M. BIRKENHEUER for helpful technical assistance in certain experiments. This research was supported by Research Grants AM-08350 from the National Institute of Arthritis and Metabolic Diseases and C-07174 from the National Cancer Institute, U.S. Public Health Service. G. 1. is a Research Career Development Awardee, 3-K3-AM-r6, 568, from the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service. REFERENCES

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