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Presynaptic tyrosine availability in the phenylketonuric brain: a hypothetical evaluation
Brain Research, 272 (1983) 189-193 Elsevier
189
Presynaptic tyrosine availability in the phenylketonuric brain: a hypothetical evaluation N. A. PETERSON, S. N. SHAH, E. RAGHUPATHY and D. E, RHOADS Department of Psychiatry, University of California, San Francisco and Brain-Behavior Research Center, Sonoma Developmental Center, Eldridge, CA 95431 (U.S.A.)
(Accepted March 29th, 1983) Key words: phenylketonuria - - synaptosomal tyrosine uptake - - hyperphenylalaninemia- brain catecholamines- - amino acid inhibition
From measured effects of amino acids on synaptosomal tyrosine uptake and from published data on human CNS levels of amino acids hypothetical calculations were made to compare CNS tyrosine availability for catecholamine synthesis in the hyperphenylalaninemic and non-hyperphenylalaninemic condition. These calculations indicate an approximately two-fold reduction in the availability of tyrosine in phenylketonuria that is due solely to a reduction in CNS tyrosine alone. Sometime ago McKean 6 showed that CNS levels of catecholamines are reduced in phenylketonuria, and suggested that such reductions are due in part to competitive phenylalanine inhibition of the transport of the precursor amino acid, tyrosine, into presynaptic terminals. Recent studies on synaptosomal tyrosine transport have provided evidence 1 that phenylalanine and tyrosine do, in fact, share a common transport system at presynaptic terminals; and further 2, that CNS phenylalanine concentrations typical of phenylketonuria would significantly inhibit the uptake of tyrosine via this system. This present communication reports that several amino acids besides phenylalanine strongly inhibit synaptosomal tyrosine uptake, and attempts a quantitative comparison of hypothetical synaptic tyrosine uptake in the hyperphenylalaninemic and non-hyperphenylalaninemic conditions. The calculations given take into account the more generalized amino acid inhibition of the synaptosomal tyrosine uptake system, and argue for an approximately two-fold reduction in the availability of tyrosine in phenylketonuria that is due almost solely to a reduction in CNS tyrosine alone. Uniformly labeled L-[14C]tyrosine was obtained from New England Nuclear Corporation (Boston, MA) and had a specific activity of approximately 400 mCi/mmol. Cerebral cortices obtained from adult Sprague-Dawley rats were homogenized in 10 vols. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
of 0.32 M sucrose solution. Synaptosomal fractions were prepared from the brain homogenates by the method of Kurokawa et al?. Previous studies 10 indicated that synaptosomal accumulation of tyrosine proceeds optimally in the absence of Na ÷. The present studies were thus carried out as described previously 10 in incubation medium (TMSK) containing 10 mM Tris HCI (pH 7.2), 15 mM MgCI2, 0.32 M sucrose and 1 mM KC1; the incubation temperature was 25 °C. Under these conditions synaptosomal accumulation of tyrosine was linear with time up to 5 min of incubation with synaptosomal protein concentrations up to 0.2 mg/ml, and observed inhibitory effects were not time dependent or dependent on protein concentrations. Portions of synaptosomal suspensions containing 0.2 mg protein were thus incubated for 3 min with 0.1 p C of labeled tyrosine and concentrations of other amino acids ranging from 10-100 #M in a final incubation volume of 1 ml of TMSK solution. At the end of the incubation period the synaptosomal particles were collected on Millipore filters and assayed for radioactivity as described elsewheretO. Leucine, isoleucine, histidine, serine and threonine inhibited synaptosomal tyrosine uptake with I50 values of similar magnitude as that for phenylalanine inhibition (Table I). A kinetic analysis was performed on the inhibitory data for the purpose of ob-
190 taining estimates of values for inhibitory constants. The inhibitory data were arranged into Hill-type plots in which the velocity function, log [(Vo--V)/V], was plotted against log I (V o and v are, respectively, the uninhibited and inhibited rates of labeled tyrosine uptake, and I is the inhibitor concentration). In the case of each inhibitor amino acid the resulting Hill plots were empirically linear within the range of inhibitor concentrations used (10-100/~M), and had correlation coefficients of greater than 0.98. All slope values (Table I) for the plots were less than unity (within the range of 0.52--0.81), and hence the plots conform to the existence of two inhibitor constants corresponding to simultaneous inhibition of two distinct saturable tyrosine uptake systems (cf. ref. 9). Values for inhibitor constants were thus estimated from the isotope uptake data obtained in the experiments using the method previously proposed 9 for calculating kinetic constants representing dual transport systems (Appendix A). The resulting values are summarized in Table I. Since the value for both K~ and K~ is of essentially the same magnitude for all of the inhibiting amino acids, these amino acids can be considered a single inhibitor with a mean value for each of the constants (6.3 pM for K~ and 94 pM for K~), If the inhibitory effects of the amino acids on tyrosine uptake are of the simple competitive type, then the rate equation that applies to the inhibition is3 Wmax
v= Km
1+--~
,
(1)
I
(1 + - K T )
and the rates of tyrosine uptake in the hyperphenylalaninemic condition, Vp, and in the non-hyperphenylalaninemic condition, vn, can be expressed as the ratio, Vp/Vn, to obtain for the high and the low affinity system, respectively, K1 In 1 + - ~ - ( 1 + K--~) vo/vn =
and 1 + K~ ( 1 +
Ip__)
K2 In 1 + ~ - a ( 1 + K~-) vr,/vn =
;
1 + S~- ( 1 + K~)
(2)
where Sp and Sn are the respective tyrosine concentrations, and Ip and I n a r e the respective total concentrations of inhibitory amino acids, in the hyperphenylalaninemic and non-hyperphenylalaninemic brain. Values for Ip and I n were approximated from data on humanCNS tissues reported by McKean and PetersonS and from data on experimental hyperphenylalaninemia in the rat, reported by McKean et al. 7. These values, along with the corresponding tyrosine concentrations, are summarized in Table II. Since values for Ip and In are large relative to those for the constants, K~ and K~; and the inhibitor constants are essentially equal to the K m terms, K 1 and K2, for the substrate tyrosine (Table I), the value for the ratio, Vp/Vn, given by equation 2, approximates to vp/v, =
In Sp
I~,Sn
(3)
Although there is almost a 10-fold increase in CNS phenylalanine in the hyperphenylalaninemic condition, the net change in the total concentration of inhibiting amino acids is small, due in large part to the high brain levels of two of the inhibitors, threonine and serine. Consequently, In/Ip ~ 1 in equation 3, and any change in the rate of tyrosine uptake in the hyperphenylalaninemic brain is due essentially to changes in tyrosine levels. The values for vp/vn summarized in Table II, calculated from equation 2, predict a hypothetical rate of tyrosine uptake in synaptic terminals of the phenylketonuric brain that is less than one-half of normal. Brain tyrosine concentrations in the hyperphenylalaninemic rodent are elevated because of the activity of phenylalanine hydroxylase; the hypothetical rate of synaptic tyrosine uptake in this case was more than 1.5 times higher in the hyperphenylalaninemic than in the non-hyperphenylalaninemic condition. The values calculated for Ip and I n representing human brain (Table II) exclude tryptophan, which is reduced by a factor of two in human phenylketonuric brain6; and also exclude leucine, isoleucine, valine and methionine, for which data are not available, but which are apparently similarly reducedT. A maximum contribution of depleted levels of these amino acids to an increase in tyrosine availability in the phenylketonuric CNS can be approximated by assuming that each of these amino acids is depleted by a fac-
191 TABLE I Inhibitory constants for amino acid inhibition of synaptosomal [l~C]tyrosine uptake Synaptosomal fractions were incubated, as described in the text with 0.1/~Ci (0.25 x 10-3/~M) of labeled tyrosine in the absence of inhibitor and in the presence of 4 different concentrations (10, 30, 60 and 100 #M) of inhibitor amino acids. K' values were calculated from Hill-type plots of labeled tyrosine uptake, as described in the text. Values are the mean values obtained from the number of plots given in parenthesis. Inhibitor Amino Acid
I5o
Phenylalanine (5) Leucine (5) Isoleucine (4) Histidine(4) Serine(2) Threonine(2) Tyrosine (4)
7 11 12 15 15 13 10
+ + + + + + +
0.5 2 3 5 2 3 2
K't (I~M)
Kj (I~M)
4.4 5.3 5.7 7.1 5.0 5.4 5.6
82 102 86 74 130 99 90
+ 0.3 --- 0.7 + 1.0 + 1.0 + 0.9 + 1.3 + 0.9*
+ + + + -+ + +
Hill-plot Slope 5 10 6 5 7 1 11"
0.67 0.63 0.68 0.75 0.56 0.63 0.77
+ + + + + + +
0.02 0.03 0.02 0.03 0.03 0.03 0.03
* Values represent K mterms for synaptosomal tyrosine uptake. affinity tyrosine uptake system is in g o o d a g r e e m e n t with the value of 6/~M r e p o r t e d by A r a g 6 n et al.l, but there is almost a factor of 10 difference b e t w e e n our value of 90/~M for a low affinity system and their value of 830/zM. It is unlikely that this latter difference is due to the two different methods used for calculating the constants, since b o t h m e t h o d s yielded the same value for the high affinity K m term. M o r e o v e r , a dual u p t a k e system with K m values of 6 and 830/~M would yield a Hill plot slope value o f a p p r o x i m a t e l y 0.4, which is only one-half of the value, 0.77, that we obtained, and hence is well outside the limits of o u r isotope uptake data. W e think it likely, therefore, that this difference in the low affinity K m value reflects a fundamental difference in the kinetics of tyrosine uptake by the m e m b r a n e vesicle p r e p a r a t i o n used in their study and the synaptosomal p r e p a r a t i o n that we used. The application of equation 2 requires that the hy-
tor of two in p h e n y l k e t o n u r i a 6, and that t r y p t o p h a n , methionine and valine have inhibitory effects on synaptosomal tyrosine uptake of similar magnitude as the amino acids given in Table I. O n the basis of the values given by McKean6 for t r y p t o p h a n , and those given by R o b i n s o n and Williams 11 for leucine plus isoleucine and for valine plus methionine, these 5 depleted amino acids would contribute 900 and 450/zM to the respective values for I n and Ip given in Table II; and thus, would change the ratio, In/I p in equation 3 from 0.88 to 1.07. The contribution of depletions in these amino acids to tyrosine availability would therefore be small. Equations 2 A and 3 A do not distinguish between simple competitive and non-competitive inhibition, but inhibitory effects on tyrosine u p t a k e of the noncompetitive type lead to the same conclusions ( A p pendix B). O u r K m value of 5.6~tM (K~ in Table II) for a high TABLE II
Substrate and inhibitor concentrations and relative tyrosine uptake rates in hyperphenylalaninemic and non-hyperphenylalaninemic conditions The values for Sp and Sn are tyrosine concentrations. Concentrations are in/xM/kg brain tissue, vp/vn values were calculated as described in the text for the high affinity (H) and low affinity (L) tyrosine uptake systems.
Human brain* Rat brain**
lp
In
Sp
Sn
vp/v,(H)
vp/v,(L)
2244 1345
1978 1111
116 171
254 82
0.41 1.6
0.44 1.7
* Obtained from the data of McKean and Petersons. The Ip and I~ values are the sum of the concentrations of phenylalanine, histidine, serine and threonine. ** Obtained from the data of McKean et al. 7. The Ip and I n values are the sum of the concentrations of phenylalanine, histidine, leucine, isoleucine, valine and threonine.
192 perphenylalaninemic conditions lead to no fundamental alterations in physicochemical properties (K m and Vmax) of the tyrosine transport carrier. Although there is no evidence for such alterations or a priori reason for believing that they might occur, this possibility cannot unequivocally be ruled out. Additionally, the use of total brain amino acid concentrations in equation 2 requires the usual assumption that such concentrations represent amino acid concentrations which interact at the cellular and molecular level. This assumption remains at risk in view of the uncertainty in localized amino acid concentrations within specific cellular and subcellular compartments, and also in view of the possibility that the concentration of amino acids may vary from one brain region to an-
other. Notwithstanding these caveats, the more generalized amino acid inhibitory effects on synaptosomal tyrosine uptake, in particular those of threonine and serine, have - - hypothetically - - an interesting functional significance inasmuch as, in the absence of these effects, the inhibition of tyrosine uptake by phenylalanine alone would cause an 8--10-fold decrease in tyrosine availability for catecholamine synthesis. Of additional interest is the fact that, according to the calculations presented, tyrosine availability for cerebral catecholamine synthesis in untreated phenylketonuric individuals would be normalized by a relatively modest therapeutically induced augmentation of brain tyrosine levels.
APPENDIX A
yields values for two inhibitory constants, K~ and K~, in place of values for two Michaelis constants, K l and K~. The mean values for a' and fl', the respective fractional contributions of a high and a low affinity tyrosine uptake system to Vo, were 0.68 _+ 0.07 (n = 30) and 0.32, respectively.
The equation s representing simple competitive inhibition which is applicable to the inhibitory data given in this present report is, Km
log [(Vo--V)/V] = n log I + log Ki (Km + S') '
(1A)
where Ki and Kmhave their usual meanings and n is unity. Since the value of S', the substrate concentration associated with the isotope, was negligible in these experiments, equation IA simplifies under these conditions to, log [(Vo--v)/v] = log I - - log K i.
(2A)
APPENDIX B Equation 2A applies to non-competitive inhibition 5 and the values derived for the two inhibitory constants, K~ and K~, are applicable to the equation, analogous to equation 1, that represents non-competitive inhibition31 The relative tyrosine uptake, vp/vn, becomes in this case
Sp(K~ + I.)
Sp(K~ + I.)
%/v, - S.(K[ + Ip) and %/v, = Sn(K~+ Ip) Following the same reasoning given previously9, the equation representing simultaneous inhibition of two independent saturable substrate uptake systems by an inhibitor concentration, I, is,
log [(Vo--V~)/vl]= log [(Vo--v)/v] = log I + log (I + a'K~ + fl'K~)--log [I(ct'K~ + fl'K~) + K~K~],
(3A)
and the procedure proposed for carrying out the calculations
for the high and low affinity system, respectively, and this value reduces to equation 3 in the hyperphenylalaninemie/non-hyperphenylalaninemie condition.
Supported in part by National Institutes of Health Grants NS-15659 and NS-14938.
193 1 Arag6n, M. C., Gim6nez, C., Mayor, Jr., F., Marvizon, J. G. and Valdivieso, F., Tyrosine transport by membrane vesicles isolated from rat brain, Biochim. biophys. Acta, 646 (1981) 465-470. 2 Arag6n, M. C., Gim6nez, C. and Valdivieso, F., Inhibition by L-phenylalanine of tyrosine transport by synaptosomal plasma membrane vesicles: implications in the pathogenesis of phenylketonuria, J. Neurochem., 39 (1982) 1185-1187. 3 Dixon, M. and Webb, E. C., Enzymes, Academic Press, New York, 1979, pp. 334-337. 4 Kurokawa, M., Sakamoto, T. and Kato, M., Distribution of sodium-plus-potassium-stimulatedadenosine-triphosphatase activity in isolated nerve ending particles, Biochem. J., 97 (1965) 833-844. 5 Loftfield, R. B. and Eigner, E. A., Molecular order of participation of inhibitors (or activators) in biological systems, Science, 164 (1969) 305-308.
6 McKean, C. M., The effectsof high phenylalanine concentrations on serotonin and catecholamine metabolism in the human brain, Brain Res., 47 (1972) 469--476. 7 McKean, C. M., Boggs, D. E. and Peterson, N. A., The influence of high phenylalanine and tyrosine on the concentrationsof essentialamino acids in brain,J. Neurochem., 15 (1968) 235-241. 8 McKean, C. M. and Peterson, N. A., Glutamine in the phenylketonuric central nervous system, New Eng. J. Med., 283 (1970) 1364--1367. 9 Peterson, N. A., Analysis of dual transport systems by means of Hill plots,Anal. Biochem., 114 (1981) 322-329. I0 Peterson, N. A. and Raghupathy, E., Characteristics of amino acid accumulation by synaptosomal particlesisolated from rat brain,J. Neurochem., 19 (1972) 1423-1438. II Robinson, N. and Williams, C. B., Amino acids in human brain, Clin. Chim. Acta, 12 (1965) 311-317.
189
Presynaptic tyrosine availability in the phenylketonuric brain: a hypothetical evaluation N. A. PETERSON, S. N. SHAH, E. RAGHUPATHY and D. E, RHOADS Department of Psychiatry, University of California, San Francisco and Brain-Behavior Research Center, Sonoma Developmental Center, Eldridge, CA 95431 (U.S.A.)
(Accepted March 29th, 1983) Key words: phenylketonuria - - synaptosomal tyrosine uptake - - hyperphenylalaninemia- brain catecholamines- - amino acid inhibition
From measured effects of amino acids on synaptosomal tyrosine uptake and from published data on human CNS levels of amino acids hypothetical calculations were made to compare CNS tyrosine availability for catecholamine synthesis in the hyperphenylalaninemic and non-hyperphenylalaninemic condition. These calculations indicate an approximately two-fold reduction in the availability of tyrosine in phenylketonuria that is due solely to a reduction in CNS tyrosine alone. Sometime ago McKean 6 showed that CNS levels of catecholamines are reduced in phenylketonuria, and suggested that such reductions are due in part to competitive phenylalanine inhibition of the transport of the precursor amino acid, tyrosine, into presynaptic terminals. Recent studies on synaptosomal tyrosine transport have provided evidence 1 that phenylalanine and tyrosine do, in fact, share a common transport system at presynaptic terminals; and further 2, that CNS phenylalanine concentrations typical of phenylketonuria would significantly inhibit the uptake of tyrosine via this system. This present communication reports that several amino acids besides phenylalanine strongly inhibit synaptosomal tyrosine uptake, and attempts a quantitative comparison of hypothetical synaptic tyrosine uptake in the hyperphenylalaninemic and non-hyperphenylalaninemic conditions. The calculations given take into account the more generalized amino acid inhibition of the synaptosomal tyrosine uptake system, and argue for an approximately two-fold reduction in the availability of tyrosine in phenylketonuria that is due almost solely to a reduction in CNS tyrosine alone. Uniformly labeled L-[14C]tyrosine was obtained from New England Nuclear Corporation (Boston, MA) and had a specific activity of approximately 400 mCi/mmol. Cerebral cortices obtained from adult Sprague-Dawley rats were homogenized in 10 vols. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
of 0.32 M sucrose solution. Synaptosomal fractions were prepared from the brain homogenates by the method of Kurokawa et al?. Previous studies 10 indicated that synaptosomal accumulation of tyrosine proceeds optimally in the absence of Na ÷. The present studies were thus carried out as described previously 10 in incubation medium (TMSK) containing 10 mM Tris HCI (pH 7.2), 15 mM MgCI2, 0.32 M sucrose and 1 mM KC1; the incubation temperature was 25 °C. Under these conditions synaptosomal accumulation of tyrosine was linear with time up to 5 min of incubation with synaptosomal protein concentrations up to 0.2 mg/ml, and observed inhibitory effects were not time dependent or dependent on protein concentrations. Portions of synaptosomal suspensions containing 0.2 mg protein were thus incubated for 3 min with 0.1 p C of labeled tyrosine and concentrations of other amino acids ranging from 10-100 #M in a final incubation volume of 1 ml of TMSK solution. At the end of the incubation period the synaptosomal particles were collected on Millipore filters and assayed for radioactivity as described elsewheretO. Leucine, isoleucine, histidine, serine and threonine inhibited synaptosomal tyrosine uptake with I50 values of similar magnitude as that for phenylalanine inhibition (Table I). A kinetic analysis was performed on the inhibitory data for the purpose of ob-
190 taining estimates of values for inhibitory constants. The inhibitory data were arranged into Hill-type plots in which the velocity function, log [(Vo--V)/V], was plotted against log I (V o and v are, respectively, the uninhibited and inhibited rates of labeled tyrosine uptake, and I is the inhibitor concentration). In the case of each inhibitor amino acid the resulting Hill plots were empirically linear within the range of inhibitor concentrations used (10-100/~M), and had correlation coefficients of greater than 0.98. All slope values (Table I) for the plots were less than unity (within the range of 0.52--0.81), and hence the plots conform to the existence of two inhibitor constants corresponding to simultaneous inhibition of two distinct saturable tyrosine uptake systems (cf. ref. 9). Values for inhibitor constants were thus estimated from the isotope uptake data obtained in the experiments using the method previously proposed 9 for calculating kinetic constants representing dual transport systems (Appendix A). The resulting values are summarized in Table I. Since the value for both K~ and K~ is of essentially the same magnitude for all of the inhibiting amino acids, these amino acids can be considered a single inhibitor with a mean value for each of the constants (6.3 pM for K~ and 94 pM for K~), If the inhibitory effects of the amino acids on tyrosine uptake are of the simple competitive type, then the rate equation that applies to the inhibition is3 Wmax
v= Km
1+--~
,
(1)
I
(1 + - K T )
and the rates of tyrosine uptake in the hyperphenylalaninemic condition, Vp, and in the non-hyperphenylalaninemic condition, vn, can be expressed as the ratio, Vp/Vn, to obtain for the high and the low affinity system, respectively, K1 In 1 + - ~ - ( 1 + K--~) vo/vn =
and 1 + K~ ( 1 +
Ip__)
K2 In 1 + ~ - a ( 1 + K~-) vr,/vn =
;
1 + S~- ( 1 + K~)
(2)
where Sp and Sn are the respective tyrosine concentrations, and Ip and I n a r e the respective total concentrations of inhibitory amino acids, in the hyperphenylalaninemic and non-hyperphenylalaninemic brain. Values for Ip and I n were approximated from data on humanCNS tissues reported by McKean and PetersonS and from data on experimental hyperphenylalaninemia in the rat, reported by McKean et al. 7. These values, along with the corresponding tyrosine concentrations, are summarized in Table II. Since values for Ip and In are large relative to those for the constants, K~ and K~; and the inhibitor constants are essentially equal to the K m terms, K 1 and K2, for the substrate tyrosine (Table I), the value for the ratio, Vp/Vn, given by equation 2, approximates to vp/v, =
In Sp
I~,Sn
(3)
Although there is almost a 10-fold increase in CNS phenylalanine in the hyperphenylalaninemic condition, the net change in the total concentration of inhibiting amino acids is small, due in large part to the high brain levels of two of the inhibitors, threonine and serine. Consequently, In/Ip ~ 1 in equation 3, and any change in the rate of tyrosine uptake in the hyperphenylalaninemic brain is due essentially to changes in tyrosine levels. The values for vp/vn summarized in Table II, calculated from equation 2, predict a hypothetical rate of tyrosine uptake in synaptic terminals of the phenylketonuric brain that is less than one-half of normal. Brain tyrosine concentrations in the hyperphenylalaninemic rodent are elevated because of the activity of phenylalanine hydroxylase; the hypothetical rate of synaptic tyrosine uptake in this case was more than 1.5 times higher in the hyperphenylalaninemic than in the non-hyperphenylalaninemic condition. The values calculated for Ip and I n representing human brain (Table II) exclude tryptophan, which is reduced by a factor of two in human phenylketonuric brain6; and also exclude leucine, isoleucine, valine and methionine, for which data are not available, but which are apparently similarly reducedT. A maximum contribution of depleted levels of these amino acids to an increase in tyrosine availability in the phenylketonuric CNS can be approximated by assuming that each of these amino acids is depleted by a fac-
191 TABLE I Inhibitory constants for amino acid inhibition of synaptosomal [l~C]tyrosine uptake Synaptosomal fractions were incubated, as described in the text with 0.1/~Ci (0.25 x 10-3/~M) of labeled tyrosine in the absence of inhibitor and in the presence of 4 different concentrations (10, 30, 60 and 100 #M) of inhibitor amino acids. K' values were calculated from Hill-type plots of labeled tyrosine uptake, as described in the text. Values are the mean values obtained from the number of plots given in parenthesis. Inhibitor Amino Acid
I5o
Phenylalanine (5) Leucine (5) Isoleucine (4) Histidine(4) Serine(2) Threonine(2) Tyrosine (4)
7 11 12 15 15 13 10
+ + + + + + +
0.5 2 3 5 2 3 2
K't (I~M)
Kj (I~M)
4.4 5.3 5.7 7.1 5.0 5.4 5.6
82 102 86 74 130 99 90
+ 0.3 --- 0.7 + 1.0 + 1.0 + 0.9 + 1.3 + 0.9*
+ + + + -+ + +
Hill-plot Slope 5 10 6 5 7 1 11"
0.67 0.63 0.68 0.75 0.56 0.63 0.77
+ + + + + + +
0.02 0.03 0.02 0.03 0.03 0.03 0.03
* Values represent K mterms for synaptosomal tyrosine uptake. affinity tyrosine uptake system is in g o o d a g r e e m e n t with the value of 6/~M r e p o r t e d by A r a g 6 n et al.l, but there is almost a factor of 10 difference b e t w e e n our value of 90/~M for a low affinity system and their value of 830/zM. It is unlikely that this latter difference is due to the two different methods used for calculating the constants, since b o t h m e t h o d s yielded the same value for the high affinity K m term. M o r e o v e r , a dual u p t a k e system with K m values of 6 and 830/~M would yield a Hill plot slope value o f a p p r o x i m a t e l y 0.4, which is only one-half of the value, 0.77, that we obtained, and hence is well outside the limits of o u r isotope uptake data. W e think it likely, therefore, that this difference in the low affinity K m value reflects a fundamental difference in the kinetics of tyrosine uptake by the m e m b r a n e vesicle p r e p a r a t i o n used in their study and the synaptosomal p r e p a r a t i o n that we used. The application of equation 2 requires that the hy-
tor of two in p h e n y l k e t o n u r i a 6, and that t r y p t o p h a n , methionine and valine have inhibitory effects on synaptosomal tyrosine uptake of similar magnitude as the amino acids given in Table I. O n the basis of the values given by McKean6 for t r y p t o p h a n , and those given by R o b i n s o n and Williams 11 for leucine plus isoleucine and for valine plus methionine, these 5 depleted amino acids would contribute 900 and 450/zM to the respective values for I n and Ip given in Table II; and thus, would change the ratio, In/I p in equation 3 from 0.88 to 1.07. The contribution of depletions in these amino acids to tyrosine availability would therefore be small. Equations 2 A and 3 A do not distinguish between simple competitive and non-competitive inhibition, but inhibitory effects on tyrosine u p t a k e of the noncompetitive type lead to the same conclusions ( A p pendix B). O u r K m value of 5.6~tM (K~ in Table II) for a high TABLE II
Substrate and inhibitor concentrations and relative tyrosine uptake rates in hyperphenylalaninemic and non-hyperphenylalaninemic conditions The values for Sp and Sn are tyrosine concentrations. Concentrations are in/xM/kg brain tissue, vp/vn values were calculated as described in the text for the high affinity (H) and low affinity (L) tyrosine uptake systems.
Human brain* Rat brain**
lp
In
Sp
Sn
vp/v,(H)
vp/v,(L)
2244 1345
1978 1111
116 171
254 82
0.41 1.6
0.44 1.7
* Obtained from the data of McKean and Petersons. The Ip and I~ values are the sum of the concentrations of phenylalanine, histidine, serine and threonine. ** Obtained from the data of McKean et al. 7. The Ip and I n values are the sum of the concentrations of phenylalanine, histidine, leucine, isoleucine, valine and threonine.
192 perphenylalaninemic conditions lead to no fundamental alterations in physicochemical properties (K m and Vmax) of the tyrosine transport carrier. Although there is no evidence for such alterations or a priori reason for believing that they might occur, this possibility cannot unequivocally be ruled out. Additionally, the use of total brain amino acid concentrations in equation 2 requires the usual assumption that such concentrations represent amino acid concentrations which interact at the cellular and molecular level. This assumption remains at risk in view of the uncertainty in localized amino acid concentrations within specific cellular and subcellular compartments, and also in view of the possibility that the concentration of amino acids may vary from one brain region to an-
other. Notwithstanding these caveats, the more generalized amino acid inhibitory effects on synaptosomal tyrosine uptake, in particular those of threonine and serine, have - - hypothetically - - an interesting functional significance inasmuch as, in the absence of these effects, the inhibition of tyrosine uptake by phenylalanine alone would cause an 8--10-fold decrease in tyrosine availability for catecholamine synthesis. Of additional interest is the fact that, according to the calculations presented, tyrosine availability for cerebral catecholamine synthesis in untreated phenylketonuric individuals would be normalized by a relatively modest therapeutically induced augmentation of brain tyrosine levels.
APPENDIX A
yields values for two inhibitory constants, K~ and K~, in place of values for two Michaelis constants, K l and K~. The mean values for a' and fl', the respective fractional contributions of a high and a low affinity tyrosine uptake system to Vo, were 0.68 _+ 0.07 (n = 30) and 0.32, respectively.
The equation s representing simple competitive inhibition which is applicable to the inhibitory data given in this present report is, Km
log [(Vo--V)/V] = n log I + log Ki (Km + S') '
(1A)
where Ki and Kmhave their usual meanings and n is unity. Since the value of S', the substrate concentration associated with the isotope, was negligible in these experiments, equation IA simplifies under these conditions to, log [(Vo--v)/v] = log I - - log K i.
(2A)
APPENDIX B Equation 2A applies to non-competitive inhibition 5 and the values derived for the two inhibitory constants, K~ and K~, are applicable to the equation, analogous to equation 1, that represents non-competitive inhibition31 The relative tyrosine uptake, vp/vn, becomes in this case
Sp(K~ + I.)
Sp(K~ + I.)
%/v, - S.(K[ + Ip) and %/v, = Sn(K~+ Ip) Following the same reasoning given previously9, the equation representing simultaneous inhibition of two independent saturable substrate uptake systems by an inhibitor concentration, I, is,
log [(Vo--V~)/vl]= log [(Vo--v)/v] = log I + log (I + a'K~ + fl'K~)--log [I(ct'K~ + fl'K~) + K~K~],
(3A)
and the procedure proposed for carrying out the calculations
for the high and low affinity system, respectively, and this value reduces to equation 3 in the hyperphenylalaninemie/non-hyperphenylalaninemie condition.
Supported in part by National Institutes of Health Grants NS-15659 and NS-14938.
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