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Taenia crassiceps: Uptake of basic and aromatic amino acids and imino acids by larvae
EXPERIMENTAL PARASITOLOGY 27, 256264
Taenia
crassiceps:
Amino
Acids
(1970)
Uptake and W.
Department
of Biochemistry
Medical (Submitted
of Basic and
lmino
Acids
Aromatic
by Larvae
D. G. Haynes
and Chemistry, St. Bartholomew’s College, London, England for publication,
10 January
Hospital
1969)
Haynes, W. D. G. 1970. Taenia crassiceps: Uptake of Basic and Aromatic Amino Acids and Imino Acids by Larvae. Experimental Parasitology 27, 25&264. A study was made of the uptake of lysine, tyrosine, phenylalanine, and proline by metacestode larvae of Taeniu crassiceps (cestoda, cyclophyllidea) by observing in each case the rate of uptake, the kinetics of uptake, and the effects upon uptake of the presence of a variety of other compounds. Lysine was absorbed by a mechanism of active transport specific for molecules with a charged basic group. Tyrosine and phenylalanine were absorbed by an active mechanism specific for neutral aromatic amino acids, Proline was absorbed by a mechanism showing affinity for both imino and amino acids, but the involvement of active processes was not conclusively demonstrated for this mechanism. INDEX DESCRIPTORS: amino acids; proline; imino acids; Cestoda; lysine; tyrosine; phenylalanine; physiology; metabolism.
In a number of recent studies, tapeworms have been shown to absorb amino and imino acids by a variety of active transport mechanisms. The most detailed work has been carried out on adult Hymenolepis diminuta, and separate mechanisms have been demonstrated in this species for the active uptake of neutral aliphatic amino acids, aromatic amino acids, basic amino acids, acidic amino acids, and imino acids (Read, Rothman, and Simmons 1963, Kilejian 1966). Larvae of Taenia crassiceps have been shown to absorb L-proline, L-glutamic acid, and a number of neutral aliphatic amino acids (Haynes and Taylor 1968). In the lyresent work a study has been made of the uptake of basic amino acids, aromatic amino acids, and imino acids by the larvae, and transport mechanisms specific for each groups are described. 256
MATERIALS
Biological
Taeniu crassiceps;
AND METHODS
Materials
Larval stages of the TO1 strain of Taenia crassiceps were maintained in the peritoneal cavities of white mice (TO strain), where they multiplied rapidly by ascolar budding. Transfer from host to host was effected by intraperitoneal injection, an initial injection of about 10 larvae resulting, about 6 months later, in an infection of 1000-3000. Two main types of larvae were obtained, a solid, infective, metacestode stage about l-2- by M-mm in size, and a cysticercus stage. Because of the greater fragility of the cysticerci, only metacestodes were used in the present study. Chemicals Radiochemicals were obtained from the Radiochemical Centre, Amersham; all other
UPTAKE
OF AMINO
ACIDS BY
chemicals were of analytical grade and obtained from British Drug Houses Ltd., Poole, Dorset. Only the L-isomers of amino and imino acids were used. All solutions were made up in Hanks’s saline (in gm: NaCl 8.0; KC1 0.4; CaClz 0.14; MgSO**7 HZ0 0.10; MgC1,*6 Ha0 0.10; NaZHPO*. 12 HZ0 0.12; KH,POI 0.06. The solution was made up to 1 liter with distilled water). The pH of this saline was adjusted to 7.4 by the addition of small amounts of sodium bicarbonate, and the maintenance of this pH was checked by observation of the color of phenol red included as an indicator. Taylor (1963) has shown that the larvae can survive for several days in this saline. Uptake was studied of the basic amino acid lysine, the aromatic amino acids tyrosine and phenylalanine, and the imino acid proline. Procedure for Experiments For each experiment larvae were removed from a single host mouse and washed at least three times in saline at room temperature. The metacestodes were then separated and preincubated in a large volume of saline (pH 7.4) at 37°C for at least 15 minutes. Larvae were incubated in groups of lO15, using stainless-steel gauze baskets and 5-ml aliquots of medium. All incubations were at 37°C and pH 7.4 and at least five replicate incubations were carried out in each medium used. The compound under study was labeled either with l”Carbon at 0.1 v C./ml or tritium at 0.5-2 u C./ml. After incubation in the labeled compound, the larvae were washed three times in saline at room temperature, dried on hard filter paper, then extracted for at least 48 hours in at least 100 times their own volume (normally 2.0 ml) Of 70% ethanol or methanol. Read, Rothman, and Simmons (1963) have shown that treatment of tapeworms in this manner results
257
Taenia CrUSSiCepS
in the complete extraction of all their free amino acids. Because of the large numbers of larvae that could be obtained from a single host, it was possible to study the uptake of a compound from a number of media in a single experiment. Thus, when studying the inhibition of uptake of a compound in the presence of a wide variety of inhibitors, it was possible to test all of the inhibitors in only one or two experiments. Calculation
of Uptake
The radioactivity in the alcoholic larval extracts was measured as desoribed by Haynes and Taylor ( 1968), using a Beckman LS 100 scintillation counter with external standard. Uptake was normally related to larval dry weight, obtained by heating the extracted larvae for at least 5 hours at 105110°C. However, uptake could also be related to larval volume by means of the formula Uptake/ml
larvae = uptake/gm specific gravity
dry weight
X
wet weight/dry
weight ratio.
Since the specific gravity of the larvae is known to be 1.06 (Haynes and Taylor 1968 ) , and since the wet weight/dry weight ratio was found to be 5.25, the formula can be modified as follows: Uptake/ml
larvae = uptake/gm dry weight
Uptake/ml
larvae = uptake/gm dry weight
1.06 X 5.25 X 0.202.
Since it has been shown that metabolism of amino acids by tapeworms is minimal during short incubations (Read et al. 1963, Haynes and Taylor 1968), this value for uptake will be a fair approximation of the (absorbed) concentration of the compound achieved within the larvae. However, it
258
W.
D. G. HAYNES
will tend to be an underestimate of total internal concentration, as no account is taken of the concentration within the larvae prior to incubation.
TABLE II The Apparent Internal Concentrations of Lysine, Tyrosine, Phenylalanine, and Proline Achieved by Taenia crassiceps Larvae after 4-64-Minute Incubations in 0.1 mM Solution@ Length
REXJLTS
4
Observed Rates of Uptake Uptake was studied initially after 4-minute incubations, using 0.1 mM solutions, and the results are shown in Table I. Comparison is made in the table with the uptake of a group of nine neutral aliphatic amino acids, all of which are known to be absorbed by the larvae by active processes (Haynes and Taylor 1968, Haynes, unpublished experiments). Lysine, tyrosine, and phenylalanine appear to be absorbed at about the same rate as the neutral aliphatic amino acids, but proline is taken up much more slowly. In Table II the results of a number of experiments are shown in terms of p moles of compound absorbed/ml larvae, using the formula given in Materials and Methods. As discussed previously, these figures will be approximately values for the internal (mM) concentrations of the compounds achieved by the larvae. Results are shown for the 4-minute experiments alTABLE I The Absorption of Lysine, Tyrosine, Phenylanine, and Proline by Taenia crassiceps Larvae, from 0.1 mM Solutions in 4-Minute Incubations: a Comparison with the Uptake of Some Neutral Aliphatic Amino Acids Commund absorbed I
Neutral aliphatic amino acidsb Lysine Tyrosine Phenylalanine Proline
Uptake
( 2 SD)a
0.17 to 0.65 0.26 + 0.093 0.41 & 0.088 0.13 SI 0.024 0.027 f. 0.0045
5 Uptake is expressed as pmoles/gm dry weight larvae. t Haynes and Taylor, 1968; Haynes, unpublished.
of incubation 8
Lysine
0.053
0.051
Tyrosine Phenylalanine
0.082 0.026
0.14
Proline
0.0055
16 0.084
a All the figures refer to apparent centration within the larvae (umoles larval volume).
(minutes) 32
64
0.12
0.21
0.048
0.077
(mM) conabsorbed/ml
ready described and for a number of experiments of longer duration. The larvae appear to achieve internal concentration of lysine and tyrosine greater than the concentration in the medium: this occurs within 16 to 32 minutes of incubation for lysine and within 8 minutes for tyrosine. Proline, by contrast, is never at a greater concentration in the larvae than in the medium, even after 64 minutes of incubation. Kinetics of Uptake The kinetics of uptake were studied by the method of Lineweaver and Burk (Dixon and Webb 1965), by measuring uptake of a compound from different external concentrations in a fixed time period (4 minutes) and plotting the reciprocal of uptake against reciprocal concentration. The concentrations used were 0.1-0.4 or 0.5 mM for lysine, tyrosine, and phenylalanine, and l-5 mM for proline. For each compound a straight line graph was obtained (a Lineweaver-Burk plot), with a finite maximum rate of uptake (uptake at infinite external concentration). The lines of the graphs were calculated by the method of least mean squares and values were obtained for both the maximum uptake rate (V,,,) and transport
UPTAKE
TABLE
OF AMINO
ACIDS BY
III
The Kinetics of Uptake of Lysine, Tyrosine, Phenylalanine, and Proline by Taenia crassiceps Lafvae in 4-Minute Incubations: a Comparison with the Uptake of Some Neutral Aliphatic Amino Acids
259
Taenia CrasSiCepS
used if no inhibition was observed at the initial concentration. Four minute incubations were used throughout, and the results are shown in Tables IV-VI; only those inhibitions that are statistically significant the method of standard deviations) shown.
of up(K,) was calculated for the inhibitor, as deK, value Compound scribed by Read et al. (1963). Ki for a comabsorbed (mM) pound is a reciprocal measure of its ability Neutral to inhibit the uptake of other compounds: aliphatic” in the case of simple competition between 0.52-1.2 0.29-2.7 amino acids 0.1-0.5 two compounds for a single uptake mech0.082 0.14 0.1-0.4 Lysine anism, K, and Ki for one of the compounds 0.25 0.20 0.1-0.5 Tyrosine 0.055 0.1-0.5 0.067 Phenylalanine should be synonymous. KS values are of 0.40 5.3 Proline l-5 importance in membrane transport studies, a Maximum uptake rates are expressed as first, because they allow a direct comparison kmoles absorbed/gm dry weight of larvae/minute of a compounds properties during absorpof incubation. tion and when acting as an inhibitor, and b Haynes and Taylor, 1968, Haynes, unpubsecond, because a small difference in a lished. percentage inhibition of uptake often corresponds to a comparatively large differconstant (K,) of each compound: the Kt of ence in Ki of the inhibitor. Thus the difa compound is the concentration at which ferences in inhibitory ability within a group uptake is half maximal and is analogous to of compounds are often much more clearly the Michaelis constant of an enzymic shown by a study of Ki values than by a process. The results are shown in Table table of percentage inhibitions under any III, and comparison is again made with one set of conditions. The Ki values obresults obtained using the nine neutral tained in the present work are shown in aliphatic amino acids. Tables IV-VI. Lysine, tyrosine, and phenylalanine have Inhibition of lysine uptake (Table IV) very low values for both Kt and V,,,. only occurred in the presence of other basic Proline, by contrast, has a high Kt value, amino acids. Of these inhibitors, only and a V,,, of the same order as the lysine and arginine have Ki values of the neutral aliphatic amino acids. same order as the Kt for lysine, the other Ki values being much higher. Inhibition of Uptake Inhibition of tyrosine uptake (Table V) occurred both in the presence of neutral A number of amino acids and other compounds were tested as inhibitors of the aliphatic amino acids and in the presence uptake of lysine, tyrosine, and proline. In of the aromatic amino acids whether neutral or slightly charged. There was no these experiments the compound absorbed was at a concentration in the same range inhibition in the presence of basic amino as its Kt value, while the inhibitors were acids, imino acids or nonamino organic acids. Of those compounds which did ininitially at 10 times this concentration: higher concentrations of inhibitor were hibit tyrosine uptake, the aromatic amino Concentration range used (mM)
Whenever a significant
Maximum uptake rate (V max)a
take
occurred,
an inhibition
inhibition
(by are
constant
260
W. D. G. HAYNES TABLE
IV
TABLE
Inhibition of the Uptake of 0.1 mM Lysine by Taenia crassiceps Larvae in the Presence of 1 mM Concentrations of Some Amino Acids and Imino
Acids in
4-Minute Incubatio@ 70 Inhibitor
Inhibition observed 61 74 32 8.9b 17b
Lysine Arginine Hydroxylysine Histidine Ornithine
Ki Value of inhibitor (mM) 0.36 0.20 1.2 6.1 2.8
a No inhibition was observed in the presence of taurine, threonine, valine, serine, aspartic acid, proline, or hydroxyproline. b Calculated from the results of experiments in which 5 mM inhibitor concentrations were used.
acids, and possibly valine, have Ki values of about the same order as the K, for tyrosine, while threonine and glycine have much higher Ki values. Inhibition of proline uptake (Table VI) occurred in the presence of the neutral aliphatic and aromatic amino acids, the imino acids, and several nonamino organic TABLE
V
Inhibition of the Uptake of 0.1 mM Tyrosine by Taenia crassiceps Larvae in the Presence of 1 mM Concentrations of Amino Acids and Some Other Compounds in I-Minute Incubations
% Inhibitor
Inhibition observed
Ki Value of inhibitor (mM)
Tyrosine Phenylalanine Tryptophan Histidine
50 62 87 50
0.66 0.41 0.098 0.66
Threonine Valine Glycine Lysine
24 46 21 0
2.1 0.78 2.5 -
0
-
0
-
0
-
Proline p-Aminobenzoic acid Phenylacetic acid
VI
Inhibition of the Uptake of 1 mM Proline by Taenia crassiceps Larvae in the Presence of 10 mM Concentrations of Amino Acids and Some Other Compounds in 4-Minute Incubations % Inhibitor
Inhibition observed
& Value of inhibitor (mM)
Proline Hydroxyproline
46 26
7.1 24
Methionine Valine Glycine Tyrosine
38 32 19 11
14 18 37 67
Omithine Histidine Lysine Citrulline Hypoxanthine Thymine Cytosine Adenine
31 0 0 N.S.a 0 0 0 0
19 -
38
14
39 38
13 14
p-Aminobenzoic acid 4-Amino-n-butyric acid Succinic acid a N.S. = Inhibition
not statistically
significant.
acids. All the ZG values obtained, with the exception of that for proline, were considerably above the proline &. DISCUSSION
Lysine Uptake Taenia crassiceps larvae absorb lysine by means of a mechanism in which active processes are probably involved. The rate of uptake is similar to that of other actively transported compounds, for example the neutral aliphatic amino acids, and the kinetics of uptake resemble those of mediated processes rather than those of simple diffusion. The ability to concentrate lysine against a gradient is indicated by the apparent achievement of lysine concentrations within the larvae greater than the concentration in the medium (Table II),
UPTAKE
OF AMINO
ACIDS BY
but is not shown with complete certaintv because of the length of the incubations involved ( 32 or 64 minutes ) and the absence of detailed knowledge of amino acid metabolism in cestodes (for exallrple the presence of lysine carboxylase activity). The mechanism appears to be specific for amino acids with a side chain that contains a charged basic group at physiological pH: affinity for the mechanism, as indicated by the ability to inhibit lysine uptake (Table IV), can be related both to the nature of this basic group and to the length of the side chain. Histidine appears to have the weakest affinity among the basic amino acids for the lysine uptake mechanism; it is also the only basic amino acid that has any affinity for the mechanisms of neutral amino acid uptake ( Haynes and Taylor 1968, and present work). The basic charge on the histidine molecule is caused by the presence of an imine group in the side chain. This group has a pK value of between 6 and 7, which means that at the experimental pH (7.4) only a small fraction of the imine groups will be charged and only a small fraction of the histidine molecules will be acting as true basic amino acids. Inhibition of the uptake of neutral amino acids in the presence of histidine could be due to the fact that those histidine molecules in which the side chain is uncharged will be acting as neutral amino acids. All of the other basic amino acids possess a side chain with either an imidine group (arginine) or an amine group (lysine, hydroxylysine, omithine ) . Roth groups have a very high pK value of 10-12, which means that at the experimental pH virtually all of the molecules will have a basic charge; these amino acids can therefore bc expected to inhibit lysine uptake to a greater degree than histidine. The fact that none of these strongly basic amino acids show any aflinity for the mechanisms of neutral amino acid uptake, despite the
Taenia cmassicep
261
fact that they all possess a normal amino zwitterion, indicates that the affinity between a charged basic group and the uptake sites specific for it must be considerably stronger than the affinity between the amino zwitterion and the uptake sites specific for the neutral amino acids. This is perhaps confirmed by the fact that the K, for lysine uptake (Table III) is considerably below that of any of the neutral aliphatic amino acids, The affinity for uptake of a basic amino acid appears to be considerably decreased by the presence on the side chain of a (weakly acidic) hydroxyl group. Thus hydroxylysine inhibits lysine uptake considerably less than does lysine itself. The affinity of a basic amino acid for the lysine uptake mechanism can also be directly related to the length of its side chain. Thus the greatest inhibition of lysine uptake occurs in the presence of arginine, which has a side chain of one nitrogen and five carbon atoms; rather less inhibition occurs in the presence of lysine (a side chain of five carbon atoms only), while ornithine, with a side chain of only three carbon atoms, is a still weaker inhibitor. Since the basic group is always attached to the last carbon atom of the side chain, this effect could be simply a matter of the distance between the basic group and the amino zwitterion: however, no firm conclusions can be drawn in the absence of detailed studies of the pattern of absorption for all the basic amino acids. The very low V,,,,, for lysine uptake (Table III) could indicate that there are fewer sites available for the uptake of basic amino acids than for compounds, such as the neutral aliphatic amino acids, which have comparatively high V,,,,, values. Read et al. (1963) have described a mechanism for the uptake of basic amino acids in adults of H. climinuta which very closely resembles the mechanism described above for T. crassiceps larvae. The K, for
262
W.
D. G. HAYNES
mechanisms with overlapping affinities, lysine uptake in H. diminuta is 0.11 mu, absorbing aromatic as compared to 0.14 mM in T. crassiceps, one preferentially amino acids, the other the neutral aliphatic while lysine, arginine, ornithine, and histidine inhibit lysine uptake in the same amino acids. Uptake of a compound by means of the relative order in both species. Histidine is mechanism for the neutral aliphatic amino the only basic amino acid to show any acids has been shown to be initially specific affinity for the mechanisms for neutral amino acid uptake in H. diminuta, just as for molecules with an amino zwitterion. The inability of proline to inhibit tyrosine in T. crassiceps. The conditions of incubation used in the uptake could indicate a similar specificity in the mechanism for aromatic amino acid work on H. diminuta differed considerably uptake. The effect of the side chain on affrom those employed in the present study, finity for the latter mechanism is clearly especially as regards length of incubation much less specific since affinities for this and external concentration of the absorbed mechanism are shown by amino acids havcompounds, so that a detailed comparison of uptake rates in the two species is not ing a wide variety of aromatic side chain possible. However, lysine is certainly more groups, including compounds, such as tyrosine, in which the side chain is slightly rapidly absorbed in H. diminuta, probably about five times as rapidly as in T. was- charged. Both tyrosine and phenylalanine have siceps; a difference of this order could be very low values for Kt and V,,, (Table due simply to the greater surface area/ III). This could indicate that these comvolume ratio of the adult tapeworm form, pounds have very high afhnities for a comas discussed previously by Haynes and paratively small number of uptake sites, Taylor ( 1968). as was suggested in the case of Iysine. It Tyrosine and Phenylulanine Uptake is, however, difficult to see why the aromatic amino acids should have a much The uptake of tyrosine and phenylalanine greater affinity for uptake than the neutral by T. crassiceps larvae appears to involve aliphatic amino acids, when uptake in each active transport processes. This is indicated, as in the case of lysine uptake, by the rates case is specific for the amino zwitterion. It of uptake, the kinetics of uptake, and the can, therefore, be suggested that the low apparent ability of the mechanism to cause Kt values of tyrosine and phenylalanine concentration against a gradient. Con- merely reflect the fact that if fewer uptake sites are available for the preferential abcentration against a gradient of tyrosine appears to occur within 8 minutes of in- sorption of these molecules, then all of the cubation, as compared with 32 minutes for sites will be saturated at a lower external concentration. This, in turn, would suggest lysine (Table II ) . that the common practice of using the K, Affinity for the mechanism, as indicated by the ability to inhibit tyrosine uptake, is value as a measure of a compounds affinity for uptake has no great validity, unless shown most strongly by the aromatic amino other factors, for example the V max of the acids (Table V). The neutral aliphatic amino acids have lower affinities. The compound, are taken into consideration. The mechanism present in T. crassiceps mechanism by which the larvae preferlarvae for the uptake of aromatic amino entially absorb the neutral aliphatic amino acids is very similar to a mechanism deacids is similar in having a definite but weak affinity for the aromatic amino acids scribed in adults of H. diminuta by Read ( Haynes and Taylor 1968) ; thus, it seems et al. (1963). In H. diminutu, as in T. c~a.ssiceps, tyrosine and phenylalanine were ablikely that the larvae possess two uptake
UPTAKE
OF AMINO
ACIDS BY
sorbed at much the same rate as the other amino acids, and had very low values for Kt, (0.17 mM for tyrosine, 0.14 mA4 for phenylalanine ) : there was again a clear preference for the aromatic amino acids, although tryptophan and histidine were not tested as inhibitors of tyrosine uptake. The rate of tyrosine and phenylalanine uptake in H. diminuta appeared to be about five times that in T. crassiceps, but a direct comparison is not possible, as was discussed in the section on lysine uptake.
Proline Uptake The rate of proline uptake in T. crassiceps larvae appears to be about 10% of that of most of the amino acids (Table I). Since, in addition, the concentration of proline against a gradient was never demonstrated (Table II), it is possible that proline uptake does not involve active transport. However, the kinetics of uptake do indicate the presence of a mediated process, while the effect of other compounds on proline uptake (Table VI) indicates the presence of considerable specificity in this process. Affinity for the mechanism involved, as indicated by the ability to inhibit proline uptake, is shown by imino acids and by neutral amino acids, whether aromatic or aliphatic. The greatest affinity is shown by proline itself, while hydroxyproline has a much greater affinity for the proline uptake mechanism than for the mechanism for uptake of neutral aliphatic amino acids (Haynes and Taylor 1968); thus it seems possible that the larvae possess a mechanism preferentially absorbing the imino acids, but that this mechanism has affinities that overlap with those of the mechanisms specific for the uptake of neutral amino ac1‘d s. The imino acids are aromatic compounds that possess both an imine group and a carboxvl group attached to the same carbon atom. Since both of these groups are fully
Taenia crassiceps
263
ionized at the experimental pH, there is a close similarity in this arrangement to that of the amino zwitterion. However, the structural differences of the two types of molecule are such that the imine group of an imino acid is considerably further from the carboxyl group than is the amine group of an amino acid. It is therefore of interest that nonamino organic acids are able to inhibit the uptake of imino acids (Table VI), but not apparently that of neutral amino acids (Haynes and Taylor 1968). This suggests the possibility that the imine and carboxyl groups of an imino acid may be sufficiently far apart for the carboxyl group to have some affinity for sites specific only for this group; an investigation is at present being carried out on the effect of imino acids on the uptake of several nonamino organic acids. If proline does not have affinity both for the mechanisms for neutral amino acid uptake and for a mechanism specific for molecules with a carboxyl group, it is possible that the larvae may not in fact possess a mechanism specific for imino acids as such; the slow rate of proline uptake and the high K, value observed could be the result of the fact that proline possesses neither a true amino zwitterion nor a completely free carboxyl group. The lack of interference during uptake between proline and the basic amino acids (Table VI, and Haynes and Taylor 1968), indicates that the imine group present in proline has little or no affinity for the mechanism of basic amino acid uptake. The uptake of proline by T. crassiceps larvae, as described above, differs considerably from the pattern of proline uptake in H. diminuta adults, as described by Kilejian ( 1966). In the latter species the Kt for proline uptake was comparatively low, at about 0.3 mM, while the rate of proline uptake was greater that that of most of the amino acids. There appeared to be a similar overlap in the affinities of the mechanisms for proline and neutral amino acid uptake, but the effect of non-
264
W. D. G. HAYNES
amino organic acids on proline uptake was not tested. ACKNOWLEDGMENT I would like to thank the Science Research Council for a grant for the purchase of a Beckman LS 100 scintillation counter. REFERENCES DIXON, M., AND Longmans, HAYNES, W. D. Sstudies on
WEBB, E. C. 1965. “Enzymes.” Green, London. Pp. 63-75. G., AND TAYLOR, A. E. R. 1968. the absorption of amino acids by
larval tapeworms ( Cyclophyllidea: Taenia crassiceps) . Parasitology 58, 47-59. KILEJIAN, A. 1966. Permeation of L-proline in the cestode Hymenolepis diminuta. Journal of Parasitology 52, 1108-1115. READ, C. P., ROTHMAN, A. H., AND J. E. SIMMONS, JR. 1963. Studies on membrane transport, with special reference to hostparasite integration. Annals of the New York Academy of Sciences 113, 154-205. TAYLOR, A. E. R. 1963. Maintenance of larval Taenia crassiceps ( Cestoda: Cyclophyllidea) in a chemically defined medium. Experimental Parasitology 14, 304-310.
Taenia
crassiceps:
Amino
Acids
(1970)
Uptake and W.
Department
of Biochemistry
Medical (Submitted
of Basic and
lmino
Acids
Aromatic
by Larvae
D. G. Haynes
and Chemistry, St. Bartholomew’s College, London, England for publication,
10 January
Hospital
1969)
Haynes, W. D. G. 1970. Taenia crassiceps: Uptake of Basic and Aromatic Amino Acids and Imino Acids by Larvae. Experimental Parasitology 27, 25&264. A study was made of the uptake of lysine, tyrosine, phenylalanine, and proline by metacestode larvae of Taeniu crassiceps (cestoda, cyclophyllidea) by observing in each case the rate of uptake, the kinetics of uptake, and the effects upon uptake of the presence of a variety of other compounds. Lysine was absorbed by a mechanism of active transport specific for molecules with a charged basic group. Tyrosine and phenylalanine were absorbed by an active mechanism specific for neutral aromatic amino acids, Proline was absorbed by a mechanism showing affinity for both imino and amino acids, but the involvement of active processes was not conclusively demonstrated for this mechanism. INDEX DESCRIPTORS: amino acids; proline; imino acids; Cestoda; lysine; tyrosine; phenylalanine; physiology; metabolism.
In a number of recent studies, tapeworms have been shown to absorb amino and imino acids by a variety of active transport mechanisms. The most detailed work has been carried out on adult Hymenolepis diminuta, and separate mechanisms have been demonstrated in this species for the active uptake of neutral aliphatic amino acids, aromatic amino acids, basic amino acids, acidic amino acids, and imino acids (Read, Rothman, and Simmons 1963, Kilejian 1966). Larvae of Taenia crassiceps have been shown to absorb L-proline, L-glutamic acid, and a number of neutral aliphatic amino acids (Haynes and Taylor 1968). In the lyresent work a study has been made of the uptake of basic amino acids, aromatic amino acids, and imino acids by the larvae, and transport mechanisms specific for each groups are described. 256
MATERIALS
Biological
Taeniu crassiceps;
AND METHODS
Materials
Larval stages of the TO1 strain of Taenia crassiceps were maintained in the peritoneal cavities of white mice (TO strain), where they multiplied rapidly by ascolar budding. Transfer from host to host was effected by intraperitoneal injection, an initial injection of about 10 larvae resulting, about 6 months later, in an infection of 1000-3000. Two main types of larvae were obtained, a solid, infective, metacestode stage about l-2- by M-mm in size, and a cysticercus stage. Because of the greater fragility of the cysticerci, only metacestodes were used in the present study. Chemicals Radiochemicals were obtained from the Radiochemical Centre, Amersham; all other
UPTAKE
OF AMINO
ACIDS BY
chemicals were of analytical grade and obtained from British Drug Houses Ltd., Poole, Dorset. Only the L-isomers of amino and imino acids were used. All solutions were made up in Hanks’s saline (in gm: NaCl 8.0; KC1 0.4; CaClz 0.14; MgSO**7 HZ0 0.10; MgC1,*6 Ha0 0.10; NaZHPO*. 12 HZ0 0.12; KH,POI 0.06. The solution was made up to 1 liter with distilled water). The pH of this saline was adjusted to 7.4 by the addition of small amounts of sodium bicarbonate, and the maintenance of this pH was checked by observation of the color of phenol red included as an indicator. Taylor (1963) has shown that the larvae can survive for several days in this saline. Uptake was studied of the basic amino acid lysine, the aromatic amino acids tyrosine and phenylalanine, and the imino acid proline. Procedure for Experiments For each experiment larvae were removed from a single host mouse and washed at least three times in saline at room temperature. The metacestodes were then separated and preincubated in a large volume of saline (pH 7.4) at 37°C for at least 15 minutes. Larvae were incubated in groups of lO15, using stainless-steel gauze baskets and 5-ml aliquots of medium. All incubations were at 37°C and pH 7.4 and at least five replicate incubations were carried out in each medium used. The compound under study was labeled either with l”Carbon at 0.1 v C./ml or tritium at 0.5-2 u C./ml. After incubation in the labeled compound, the larvae were washed three times in saline at room temperature, dried on hard filter paper, then extracted for at least 48 hours in at least 100 times their own volume (normally 2.0 ml) Of 70% ethanol or methanol. Read, Rothman, and Simmons (1963) have shown that treatment of tapeworms in this manner results
257
Taenia CrUSSiCepS
in the complete extraction of all their free amino acids. Because of the large numbers of larvae that could be obtained from a single host, it was possible to study the uptake of a compound from a number of media in a single experiment. Thus, when studying the inhibition of uptake of a compound in the presence of a wide variety of inhibitors, it was possible to test all of the inhibitors in only one or two experiments. Calculation
of Uptake
The radioactivity in the alcoholic larval extracts was measured as desoribed by Haynes and Taylor ( 1968), using a Beckman LS 100 scintillation counter with external standard. Uptake was normally related to larval dry weight, obtained by heating the extracted larvae for at least 5 hours at 105110°C. However, uptake could also be related to larval volume by means of the formula Uptake/ml
larvae = uptake/gm specific gravity
dry weight
X
wet weight/dry
weight ratio.
Since the specific gravity of the larvae is known to be 1.06 (Haynes and Taylor 1968 ) , and since the wet weight/dry weight ratio was found to be 5.25, the formula can be modified as follows: Uptake/ml
larvae = uptake/gm dry weight
Uptake/ml
larvae = uptake/gm dry weight
1.06 X 5.25 X 0.202.
Since it has been shown that metabolism of amino acids by tapeworms is minimal during short incubations (Read et al. 1963, Haynes and Taylor 1968), this value for uptake will be a fair approximation of the (absorbed) concentration of the compound achieved within the larvae. However, it
258
W.
D. G. HAYNES
will tend to be an underestimate of total internal concentration, as no account is taken of the concentration within the larvae prior to incubation.
TABLE II The Apparent Internal Concentrations of Lysine, Tyrosine, Phenylalanine, and Proline Achieved by Taenia crassiceps Larvae after 4-64-Minute Incubations in 0.1 mM Solution@ Length
REXJLTS
4
Observed Rates of Uptake Uptake was studied initially after 4-minute incubations, using 0.1 mM solutions, and the results are shown in Table I. Comparison is made in the table with the uptake of a group of nine neutral aliphatic amino acids, all of which are known to be absorbed by the larvae by active processes (Haynes and Taylor 1968, Haynes, unpublished experiments). Lysine, tyrosine, and phenylalanine appear to be absorbed at about the same rate as the neutral aliphatic amino acids, but proline is taken up much more slowly. In Table II the results of a number of experiments are shown in terms of p moles of compound absorbed/ml larvae, using the formula given in Materials and Methods. As discussed previously, these figures will be approximately values for the internal (mM) concentrations of the compounds achieved by the larvae. Results are shown for the 4-minute experiments alTABLE I The Absorption of Lysine, Tyrosine, Phenylanine, and Proline by Taenia crassiceps Larvae, from 0.1 mM Solutions in 4-Minute Incubations: a Comparison with the Uptake of Some Neutral Aliphatic Amino Acids Commund absorbed I
Neutral aliphatic amino acidsb Lysine Tyrosine Phenylalanine Proline
Uptake
( 2 SD)a
0.17 to 0.65 0.26 + 0.093 0.41 & 0.088 0.13 SI 0.024 0.027 f. 0.0045
5 Uptake is expressed as pmoles/gm dry weight larvae. t Haynes and Taylor, 1968; Haynes, unpublished.
of incubation 8
Lysine
0.053
0.051
Tyrosine Phenylalanine
0.082 0.026
0.14
Proline
0.0055
16 0.084
a All the figures refer to apparent centration within the larvae (umoles larval volume).
(minutes) 32
64
0.12
0.21
0.048
0.077
(mM) conabsorbed/ml
ready described and for a number of experiments of longer duration. The larvae appear to achieve internal concentration of lysine and tyrosine greater than the concentration in the medium: this occurs within 16 to 32 minutes of incubation for lysine and within 8 minutes for tyrosine. Proline, by contrast, is never at a greater concentration in the larvae than in the medium, even after 64 minutes of incubation. Kinetics of Uptake The kinetics of uptake were studied by the method of Lineweaver and Burk (Dixon and Webb 1965), by measuring uptake of a compound from different external concentrations in a fixed time period (4 minutes) and plotting the reciprocal of uptake against reciprocal concentration. The concentrations used were 0.1-0.4 or 0.5 mM for lysine, tyrosine, and phenylalanine, and l-5 mM for proline. For each compound a straight line graph was obtained (a Lineweaver-Burk plot), with a finite maximum rate of uptake (uptake at infinite external concentration). The lines of the graphs were calculated by the method of least mean squares and values were obtained for both the maximum uptake rate (V,,,) and transport
UPTAKE
TABLE
OF AMINO
ACIDS BY
III
The Kinetics of Uptake of Lysine, Tyrosine, Phenylalanine, and Proline by Taenia crassiceps Lafvae in 4-Minute Incubations: a Comparison with the Uptake of Some Neutral Aliphatic Amino Acids
259
Taenia CrasSiCepS
used if no inhibition was observed at the initial concentration. Four minute incubations were used throughout, and the results are shown in Tables IV-VI; only those inhibitions that are statistically significant the method of standard deviations) shown.
of up(K,) was calculated for the inhibitor, as deK, value Compound scribed by Read et al. (1963). Ki for a comabsorbed (mM) pound is a reciprocal measure of its ability Neutral to inhibit the uptake of other compounds: aliphatic” in the case of simple competition between 0.52-1.2 0.29-2.7 amino acids 0.1-0.5 two compounds for a single uptake mech0.082 0.14 0.1-0.4 Lysine anism, K, and Ki for one of the compounds 0.25 0.20 0.1-0.5 Tyrosine 0.055 0.1-0.5 0.067 Phenylalanine should be synonymous. KS values are of 0.40 5.3 Proline l-5 importance in membrane transport studies, a Maximum uptake rates are expressed as first, because they allow a direct comparison kmoles absorbed/gm dry weight of larvae/minute of a compounds properties during absorpof incubation. tion and when acting as an inhibitor, and b Haynes and Taylor, 1968, Haynes, unpubsecond, because a small difference in a lished. percentage inhibition of uptake often corresponds to a comparatively large differconstant (K,) of each compound: the Kt of ence in Ki of the inhibitor. Thus the difa compound is the concentration at which ferences in inhibitory ability within a group uptake is half maximal and is analogous to of compounds are often much more clearly the Michaelis constant of an enzymic shown by a study of Ki values than by a process. The results are shown in Table table of percentage inhibitions under any III, and comparison is again made with one set of conditions. The Ki values obresults obtained using the nine neutral tained in the present work are shown in aliphatic amino acids. Tables IV-VI. Lysine, tyrosine, and phenylalanine have Inhibition of lysine uptake (Table IV) very low values for both Kt and V,,,. only occurred in the presence of other basic Proline, by contrast, has a high Kt value, amino acids. Of these inhibitors, only and a V,,, of the same order as the lysine and arginine have Ki values of the neutral aliphatic amino acids. same order as the Kt for lysine, the other Ki values being much higher. Inhibition of Uptake Inhibition of tyrosine uptake (Table V) occurred both in the presence of neutral A number of amino acids and other compounds were tested as inhibitors of the aliphatic amino acids and in the presence uptake of lysine, tyrosine, and proline. In of the aromatic amino acids whether neutral or slightly charged. There was no these experiments the compound absorbed was at a concentration in the same range inhibition in the presence of basic amino as its Kt value, while the inhibitors were acids, imino acids or nonamino organic acids. Of those compounds which did ininitially at 10 times this concentration: higher concentrations of inhibitor were hibit tyrosine uptake, the aromatic amino Concentration range used (mM)
Whenever a significant
Maximum uptake rate (V max)a
take
occurred,
an inhibition
inhibition
(by are
constant
260
W. D. G. HAYNES TABLE
IV
TABLE
Inhibition of the Uptake of 0.1 mM Lysine by Taenia crassiceps Larvae in the Presence of 1 mM Concentrations of Some Amino Acids and Imino
Acids in
4-Minute Incubatio@ 70 Inhibitor
Inhibition observed 61 74 32 8.9b 17b
Lysine Arginine Hydroxylysine Histidine Ornithine
Ki Value of inhibitor (mM) 0.36 0.20 1.2 6.1 2.8
a No inhibition was observed in the presence of taurine, threonine, valine, serine, aspartic acid, proline, or hydroxyproline. b Calculated from the results of experiments in which 5 mM inhibitor concentrations were used.
acids, and possibly valine, have Ki values of about the same order as the K, for tyrosine, while threonine and glycine have much higher Ki values. Inhibition of proline uptake (Table VI) occurred in the presence of the neutral aliphatic and aromatic amino acids, the imino acids, and several nonamino organic TABLE
V
Inhibition of the Uptake of 0.1 mM Tyrosine by Taenia crassiceps Larvae in the Presence of 1 mM Concentrations of Amino Acids and Some Other Compounds in I-Minute Incubations
% Inhibitor
Inhibition observed
Ki Value of inhibitor (mM)
Tyrosine Phenylalanine Tryptophan Histidine
50 62 87 50
0.66 0.41 0.098 0.66
Threonine Valine Glycine Lysine
24 46 21 0
2.1 0.78 2.5 -
0
-
0
-
0
-
Proline p-Aminobenzoic acid Phenylacetic acid
VI
Inhibition of the Uptake of 1 mM Proline by Taenia crassiceps Larvae in the Presence of 10 mM Concentrations of Amino Acids and Some Other Compounds in 4-Minute Incubations % Inhibitor
Inhibition observed
& Value of inhibitor (mM)
Proline Hydroxyproline
46 26
7.1 24
Methionine Valine Glycine Tyrosine
38 32 19 11
14 18 37 67
Omithine Histidine Lysine Citrulline Hypoxanthine Thymine Cytosine Adenine
31 0 0 N.S.a 0 0 0 0
19 -
38
14
39 38
13 14
p-Aminobenzoic acid 4-Amino-n-butyric acid Succinic acid a N.S. = Inhibition
not statistically
significant.
acids. All the ZG values obtained, with the exception of that for proline, were considerably above the proline &. DISCUSSION
Lysine Uptake Taenia crassiceps larvae absorb lysine by means of a mechanism in which active processes are probably involved. The rate of uptake is similar to that of other actively transported compounds, for example the neutral aliphatic amino acids, and the kinetics of uptake resemble those of mediated processes rather than those of simple diffusion. The ability to concentrate lysine against a gradient is indicated by the apparent achievement of lysine concentrations within the larvae greater than the concentration in the medium (Table II),
UPTAKE
OF AMINO
ACIDS BY
but is not shown with complete certaintv because of the length of the incubations involved ( 32 or 64 minutes ) and the absence of detailed knowledge of amino acid metabolism in cestodes (for exallrple the presence of lysine carboxylase activity). The mechanism appears to be specific for amino acids with a side chain that contains a charged basic group at physiological pH: affinity for the mechanism, as indicated by the ability to inhibit lysine uptake (Table IV), can be related both to the nature of this basic group and to the length of the side chain. Histidine appears to have the weakest affinity among the basic amino acids for the lysine uptake mechanism; it is also the only basic amino acid that has any affinity for the mechanisms of neutral amino acid uptake ( Haynes and Taylor 1968, and present work). The basic charge on the histidine molecule is caused by the presence of an imine group in the side chain. This group has a pK value of between 6 and 7, which means that at the experimental pH (7.4) only a small fraction of the imine groups will be charged and only a small fraction of the histidine molecules will be acting as true basic amino acids. Inhibition of the uptake of neutral amino acids in the presence of histidine could be due to the fact that those histidine molecules in which the side chain is uncharged will be acting as neutral amino acids. All of the other basic amino acids possess a side chain with either an imidine group (arginine) or an amine group (lysine, hydroxylysine, omithine ) . Roth groups have a very high pK value of 10-12, which means that at the experimental pH virtually all of the molecules will have a basic charge; these amino acids can therefore bc expected to inhibit lysine uptake to a greater degree than histidine. The fact that none of these strongly basic amino acids show any aflinity for the mechanisms of neutral amino acid uptake, despite the
Taenia cmassicep
261
fact that they all possess a normal amino zwitterion, indicates that the affinity between a charged basic group and the uptake sites specific for it must be considerably stronger than the affinity between the amino zwitterion and the uptake sites specific for the neutral amino acids. This is perhaps confirmed by the fact that the K, for lysine uptake (Table III) is considerably below that of any of the neutral aliphatic amino acids, The affinity for uptake of a basic amino acid appears to be considerably decreased by the presence on the side chain of a (weakly acidic) hydroxyl group. Thus hydroxylysine inhibits lysine uptake considerably less than does lysine itself. The affinity of a basic amino acid for the lysine uptake mechanism can also be directly related to the length of its side chain. Thus the greatest inhibition of lysine uptake occurs in the presence of arginine, which has a side chain of one nitrogen and five carbon atoms; rather less inhibition occurs in the presence of lysine (a side chain of five carbon atoms only), while ornithine, with a side chain of only three carbon atoms, is a still weaker inhibitor. Since the basic group is always attached to the last carbon atom of the side chain, this effect could be simply a matter of the distance between the basic group and the amino zwitterion: however, no firm conclusions can be drawn in the absence of detailed studies of the pattern of absorption for all the basic amino acids. The very low V,,,,, for lysine uptake (Table III) could indicate that there are fewer sites available for the uptake of basic amino acids than for compounds, such as the neutral aliphatic amino acids, which have comparatively high V,,,,, values. Read et al. (1963) have described a mechanism for the uptake of basic amino acids in adults of H. climinuta which very closely resembles the mechanism described above for T. crassiceps larvae. The K, for
262
W.
D. G. HAYNES
mechanisms with overlapping affinities, lysine uptake in H. diminuta is 0.11 mu, absorbing aromatic as compared to 0.14 mM in T. crassiceps, one preferentially amino acids, the other the neutral aliphatic while lysine, arginine, ornithine, and histidine inhibit lysine uptake in the same amino acids. Uptake of a compound by means of the relative order in both species. Histidine is mechanism for the neutral aliphatic amino the only basic amino acid to show any acids has been shown to be initially specific affinity for the mechanisms for neutral amino acid uptake in H. diminuta, just as for molecules with an amino zwitterion. The inability of proline to inhibit tyrosine in T. crassiceps. The conditions of incubation used in the uptake could indicate a similar specificity in the mechanism for aromatic amino acid work on H. diminuta differed considerably uptake. The effect of the side chain on affrom those employed in the present study, finity for the latter mechanism is clearly especially as regards length of incubation much less specific since affinities for this and external concentration of the absorbed mechanism are shown by amino acids havcompounds, so that a detailed comparison of uptake rates in the two species is not ing a wide variety of aromatic side chain possible. However, lysine is certainly more groups, including compounds, such as tyrosine, in which the side chain is slightly rapidly absorbed in H. diminuta, probably about five times as rapidly as in T. was- charged. Both tyrosine and phenylalanine have siceps; a difference of this order could be very low values for Kt and V,,, (Table due simply to the greater surface area/ III). This could indicate that these comvolume ratio of the adult tapeworm form, pounds have very high afhnities for a comas discussed previously by Haynes and paratively small number of uptake sites, Taylor ( 1968). as was suggested in the case of Iysine. It Tyrosine and Phenylulanine Uptake is, however, difficult to see why the aromatic amino acids should have a much The uptake of tyrosine and phenylalanine greater affinity for uptake than the neutral by T. crassiceps larvae appears to involve aliphatic amino acids, when uptake in each active transport processes. This is indicated, as in the case of lysine uptake, by the rates case is specific for the amino zwitterion. It of uptake, the kinetics of uptake, and the can, therefore, be suggested that the low apparent ability of the mechanism to cause Kt values of tyrosine and phenylalanine concentration against a gradient. Con- merely reflect the fact that if fewer uptake sites are available for the preferential abcentration against a gradient of tyrosine appears to occur within 8 minutes of in- sorption of these molecules, then all of the cubation, as compared with 32 minutes for sites will be saturated at a lower external concentration. This, in turn, would suggest lysine (Table II ) . that the common practice of using the K, Affinity for the mechanism, as indicated by the ability to inhibit tyrosine uptake, is value as a measure of a compounds affinity for uptake has no great validity, unless shown most strongly by the aromatic amino other factors, for example the V max of the acids (Table V). The neutral aliphatic amino acids have lower affinities. The compound, are taken into consideration. The mechanism present in T. crassiceps mechanism by which the larvae preferlarvae for the uptake of aromatic amino entially absorb the neutral aliphatic amino acids is very similar to a mechanism deacids is similar in having a definite but weak affinity for the aromatic amino acids scribed in adults of H. diminuta by Read ( Haynes and Taylor 1968) ; thus, it seems et al. (1963). In H. diminutu, as in T. c~a.ssiceps, tyrosine and phenylalanine were ablikely that the larvae possess two uptake
UPTAKE
OF AMINO
ACIDS BY
sorbed at much the same rate as the other amino acids, and had very low values for Kt, (0.17 mM for tyrosine, 0.14 mA4 for phenylalanine ) : there was again a clear preference for the aromatic amino acids, although tryptophan and histidine were not tested as inhibitors of tyrosine uptake. The rate of tyrosine and phenylalanine uptake in H. diminuta appeared to be about five times that in T. crassiceps, but a direct comparison is not possible, as was discussed in the section on lysine uptake.
Proline Uptake The rate of proline uptake in T. crassiceps larvae appears to be about 10% of that of most of the amino acids (Table I). Since, in addition, the concentration of proline against a gradient was never demonstrated (Table II), it is possible that proline uptake does not involve active transport. However, the kinetics of uptake do indicate the presence of a mediated process, while the effect of other compounds on proline uptake (Table VI) indicates the presence of considerable specificity in this process. Affinity for the mechanism involved, as indicated by the ability to inhibit proline uptake, is shown by imino acids and by neutral amino acids, whether aromatic or aliphatic. The greatest affinity is shown by proline itself, while hydroxyproline has a much greater affinity for the proline uptake mechanism than for the mechanism for uptake of neutral aliphatic amino acids (Haynes and Taylor 1968); thus it seems possible that the larvae possess a mechanism preferentially absorbing the imino acids, but that this mechanism has affinities that overlap with those of the mechanisms specific for the uptake of neutral amino ac1‘d s. The imino acids are aromatic compounds that possess both an imine group and a carboxvl group attached to the same carbon atom. Since both of these groups are fully
Taenia crassiceps
263
ionized at the experimental pH, there is a close similarity in this arrangement to that of the amino zwitterion. However, the structural differences of the two types of molecule are such that the imine group of an imino acid is considerably further from the carboxyl group than is the amine group of an amino acid. It is therefore of interest that nonamino organic acids are able to inhibit the uptake of imino acids (Table VI), but not apparently that of neutral amino acids (Haynes and Taylor 1968). This suggests the possibility that the imine and carboxyl groups of an imino acid may be sufficiently far apart for the carboxyl group to have some affinity for sites specific only for this group; an investigation is at present being carried out on the effect of imino acids on the uptake of several nonamino organic acids. If proline does not have affinity both for the mechanisms for neutral amino acid uptake and for a mechanism specific for molecules with a carboxyl group, it is possible that the larvae may not in fact possess a mechanism specific for imino acids as such; the slow rate of proline uptake and the high K, value observed could be the result of the fact that proline possesses neither a true amino zwitterion nor a completely free carboxyl group. The lack of interference during uptake between proline and the basic amino acids (Table VI, and Haynes and Taylor 1968), indicates that the imine group present in proline has little or no affinity for the mechanism of basic amino acid uptake. The uptake of proline by T. crassiceps larvae, as described above, differs considerably from the pattern of proline uptake in H. diminuta adults, as described by Kilejian ( 1966). In the latter species the Kt for proline uptake was comparatively low, at about 0.3 mM, while the rate of proline uptake was greater that that of most of the amino acids. There appeared to be a similar overlap in the affinities of the mechanisms for proline and neutral amino acid uptake, but the effect of non-
264
W. D. G. HAYNES
amino organic acids on proline uptake was not tested. ACKNOWLEDGMENT I would like to thank the Science Research Council for a grant for the purchase of a Beckman LS 100 scintillation counter. REFERENCES DIXON, M., AND Longmans, HAYNES, W. D. Sstudies on
WEBB, E. C. 1965. “Enzymes.” Green, London. Pp. 63-75. G., AND TAYLOR, A. E. R. 1968. the absorption of amino acids by
larval tapeworms ( Cyclophyllidea: Taenia crassiceps) . Parasitology 58, 47-59. KILEJIAN, A. 1966. Permeation of L-proline in the cestode Hymenolepis diminuta. Journal of Parasitology 52, 1108-1115. READ, C. P., ROTHMAN, A. H., AND J. E. SIMMONS, JR. 1963. Studies on membrane transport, with special reference to hostparasite integration. Annals of the New York Academy of Sciences 113, 154-205. TAYLOR, A. E. R. 1963. Maintenance of larval Taenia crassiceps ( Cestoda: Cyclophyllidea) in a chemically defined medium. Experimental Parasitology 14, 304-310.