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Dipeptide transport in brush-border membrane vesicles isolated from normal term human placenta
CURRENT INVESTIGATION
Dipeptide transport in brush-border membrane vesicles isolated from normal term human placenta Malliga E. Ganapathy, M.B., B.S., Virendra B. Mahesh, Ph.D., D.Phil., Lawrence D. Devoe, M.D., Frederick H. Leibach, Ph.D., and Vadivel Ganapathy, Ph.D. Augusta, Georgia We studied the transport of glycylsarcosine by the brush-border membrane vesicles isolated from normal term human placentas. This dipeptide resisted hydrolysis by placental membrane vesicles and was transported intact into an osmotically responsive intravesicular space. The transport process was Na• independent and probably occurred down a concentration gradient. Many peptides inhibited the transport of glycylsarcosine whereas amino acids had no effect. These results demonstrate, for the first time, the presence of a dipeptide transport system in the human placenta. (AM J OBSTET GYNECOL 1985;153:83-6.)
Key words: Dipeptide transport, Na - independence, human placenta, brush-border vesicle Transfer of nutrients from mother to fetus across the placenta is very important for adequate fetal growth. Placental transfer of amino acids is crucial for both fetal functional development and growth, as disturbed protein biosynthesis could result in abnormalities in either of these processes. Transfer of amino acids across the placenta can occur in the form of free amino acids as well as small peptides. While there are ample data to support the role of transport of small peptides in the adult intestine in the overall maintenance of human protein nutrition,' there are no analogous studies on the importance of placental transfer of small peptides in the maintenance of intrauterine nutrition. Our preliminary data strongly indicate, for the first time, that the brush-border membrane of the syncytiotrophoblast of human placenta possesses a dipeptide transport system which is distinct from the wellestablished amino acid transport systems.
Methods Brush-border membrane vesicles from normal term human placenta were prepared according to method of Smith et al. 2 One important difference, however, is
From the Departments of Endocrinology, Obstetrics and Gynecology, and Cell and Molecular Biology, Medical College of Georgia. This study was supported by National Institutes of Health Grant AM 28389 to F. H. L. This is contribution No. 0887 from the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia. Reprint requests: V. Ganapathy, Ph.D., Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912.
the inclusion of a Ca+· -precipitation step in our procedure, which resulted in better reproducibility of the method. We adapted this step from the methods applied to brush-border membrane vesicles isolated from the intestine and kidney. Placentas from normal pregnancies were obtained within 30 minutes of delivery. The maternal decidua was removed and the central portion between the maternal and fetal surfaces of the placenta was used for the preparation of the membrane. The placental tissue was cut into small pieces and collected in a I L beaker. All subsequent steps were carried out at 4° C. The tissue was washed three times in 200 ml of Hepes/Tris buffer, 10 mmol/L, pH 7.0, containing mannitol, 300 mmol/L. After washing, the tissue was placed in 300 ml of the same buffer and agitated with a magnetic stirring bar for 30 minutes. The tissue was removed with forceps, and the liquid was filtered through six layers of cotton gauze. The filtrate was centrifuged at 1000 x g for 10 minutes in order to remove blood and cellular debris. The supernatant was centrifuged at 10,000 x g for 15 minutes. The supernatant was collected and centrifuged again at 86,000 x g for 30 minutes. The pellets were collected in 50 ml of the Hepes/Tris-mannitol buffer and homogenized in a Dounce glass homogenizer with I 0 strokes. Then 0.5 ml of calcium chloride, 1 mol/L, was added to this homogenate and the mixture was stirred for 10 minutes and allowed to stand for an additional I 0 minutes. This was centrifuged at 3000 x g for 15 minutes. The supernatant contained brush-border membranes that were collected by centrifugation
83
84
Ganapathy et al.
September I. 1985 Am .J Obstet Gynecol
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Fig. I. Effect of medium osmolality on the equilibrium uptake of glycylsarcosine.
at 86,000 x g for 30 minutes. The membrane pellets were suspended in a small volume of preloading buffer by five passages through a 25-gauge needle. The preloading buffer varied with each experiment and the composition of buffer is given in the respective legend to the figure or table. Uptake of [ 1-"C]glycylsarcosine was measured by a rapid filtration technique" with the use of Metricel membrane filters (pore size, 0.45 µm) and hydrolysis of the dipeptide by the membrane preparations was determined as described earlier. 1 Results Alkaline phosphatase and 5' -nucleotidase were used as marker enzymes to assess the purity of the brushborder membranes. 2 Alkaline phosphatase was enriched 19.1-fold ± 2.2-fold and 5'-nucleotidase was enriched 18.5-fold ± 2.8-fold over the homogenate of the washed placental tissue. Freshly prepared membrane vesicles were used throughout the study. Glycylsarcosine is highly resistant to hydrolysis by tissue peptidases and, therefore, has been widely used in peptide transport studies. Hydrolysis of glycylsarcosine by placental brush-border membranes was assayed by a highly sensitive radiometric method. No detectable hydrolysis occurred during incubation periods
•
•
6 4 10 8 Incubation time (min)
Fig. 2. Time course of glycylsarcosine uptake. Membrane vesicles were suspended in Hepes/Tris buffer, 10 mmol/L, pH 7.5, containing mannitol, 300 mmol/L. Cptakc was measured in Hepes/Tris buffer, 10 mmol/L, pH 7.5, containing either sodium chloride, 150 mmol/L, or potassium chloride, 150 mmol/L. The final concentration of labeled glycylsarcosine was 5 mmol/L. •-•, K • gradient. o-o, Na - gradient.
as long as 3 hours. Therefore, any uptake of glycylsarcosine, observed under these conditions, represented uptake of the intact dipeptide. To test whether the dipeptide was transported from the incubation medium into the intravesicular space as opposed to bound to the vesicles, we measured the uptake at 90 minutes (equilibrium uptake) while varying the medium osmolality by the addition of mannitol. The uptake decreased as a linear function of the reciprocal of increasing medium osmolality and indicated that there was negligible binding of the dipeptide to the vesicles (Fig. 1). This relationship, however, did not hold when the medium osmolality was less than 200 mosm, probably because of rupture of the vesicles under these conditions. The results show that intact vesicles contained an osmotically responsive intravesicular space that disappeared with membrane disruption. The time course of glycylsarcosine uptake into these vesicles is shown in Fig. 2. The initial rates of uptake in the presence of either an Na• gradient or a K • gradient were identical. With longer periods of incubation, the uptake under K • gradient conditions was greater than that measured under Na• gradient conditions. However, the equilibrium uptake measured at 90 min-
Volume 153 Number I
utes was the same whether an Na· or a K • gradient was present. These results contrast markedly with those obtained for amino acid uptake, which is many times greater in the presence of an Na• gradient than in the presence of a K • gradient. Moreover, uptake of amino acids in the presence of an Na• gradient exhibits a transient overshoot, which is indicative of active transport that was absent when we studied glycylsarcosine uptake. These data show that glycylsarcosine uptake by placental brush-border membrane vesicles is Na' independent and probably occurs down a concentration gradient. We also studied the ability of unlabeled peptides and amino acids to inhibit the uptake of labeled glycylsarcosine. Uptake was measured at pH 5.5 because it was maximal for glycylsarcosine at this pH (data not shown). The free amino acids glycine and sarcosine minimally inhibited the uptake of glycylsarcosine while all peptides, except for carnosine, caused significant inhibition (Table I). These inhibition studies clearly show that glycylsarcosine uptake by placental brush-border membrane vesicles is a carrier-mediated process rather than a simple diffusion. If it were a simple diffusion process, it could not have been inhibited by other peptides. Since free amino acids did not inhibit glycylsarcosine uptake, it appears that the transport systems involved in the placental transfer of amino acids and dipeptides are different. We used a I-minute incubation in these inhibition experiments. However, it should be noted that shorter incubation periods (as low as I or 2 seconds) would be required for more rigorous kinetic analysis.
Comment We have demonstrated, for the first time, that the brush-border membrane of the human syncytiotrophoblast possesses a transport system for small peptides. This system is distinct from those used for amino acid transport and may, in vivo, serve as a significant conduit for amino acids. Normal human plasma contains appreciable amounts of small peptides. The peptide content of plasma significantly increases during assimilation of dietary proteins as many small peptides arising from protein digestion escape further hydrolysis and cross the intestine in intact form. 5 During pregnancy, maternal metabolism undergoes adaptive changes to ensure a proper nutrient supply to the fetus. Alterations in hormonal balance result in elevated levels of circulating insulin in the mother. 6 The maternal response to insulin changes during pregnancy. Early in gestation, the anabolic effects of insulin result in increased fat deposition and increased glycogen and protein synthesis while later there is increased muscle protein catabolism, pos-
Dipeptide transport in brush-border membrane vesicles
85
Table I. Effect of amino acids and peptides on glycylsarcosine uptake GlycJlsarcosine uptake nmollmg
Addition None Glycine Sarcosine Glycylsarcosine Glycylproline Carnosine Glycylleucine [3-Alanylglycylglycine Prolylglycylglycine
Protein 2.98 2.44 2.49 1.32 1.25 3.33 0.91 2.00 2.21
::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+:
0.28 0.09 0.19 0.02 0.09 0.11 0.03 0.09 0.18
o/c 100 82 44 42 112 31 67 74
Membrane vesicles were preloaded with Mes/Tris buffer, 10 mmol/L, pH 5.5, containing mannitol 125 mmol/L, and potassium chloride, 80 mmol/L. Uptake was initiated by adding 50 µI of membrane suspension to 200 µI of Mes/Tris buffer, 10 mmol/L, pH 5.5, containing sodium chloride, 80 mmol/L, labeled glycylsarcosine, and unlabeled peptides or amino acids. The final concentration of labeled glycylsarcosine was 5 mmol/L and the concentration of unlabeled peptides or amino acids was 100 mmol/L. Osmolality was maintained constant by varying the mannitol concentration. Incubation time was 1 minute. The data represent means of triplicates ::+:SD.
sibly due to the increased insulin resistance of maternal tissues." The increased breakdown of muscle proteins occurs at a time when the fetus gains most of its body mass and may represent an adaptive process important for assuring adequate fetal growth. While it is generally assumed that only free amino acids are released into the circulation during intracellular protein catabolism, no experimental evidence exists to support this assumption. Therefore, it is quite possible that small, hydrolysis-resistant peptides are generated as well and may gain access to the maternal circulation. If this, in fact, takes place, then the concentration of small peptides in maternal plasma may increase in the last trimester of pregnancy and may become an important source of amino acids for the fetus. Furthermore, placental tissue contains peptidases and may augment the supply of amino acids available to the fetus, once small peptides are transported across the placental brush-border membrane. The peptide transport system may also participate in the transfer of biologically active peptides of maternal origin, whether derived from dietary or endogenous proteins. These compounds, following maternal-fetal transfer, may exert a wide range of physiologic effects on the fetus, including circulatory homeostasis, neuroregulation, and liver function. Clinical conditions such as maternal hypertension, which alter placental anatomy, morphology, and function, may also influence the peptide transport system. Considering the important role this transport system may play in fetal metab-
86
Ganapathy et al.
olism and development, it merits further study in normal and pathologic gestations. REFERENCES I. Silk OBA, Hegarty J E, Fairclough PD. Clark \1L. Characterization and nutritional significance of peptide transport in man. Ann Nutr \letab 1982;26:'.1'.~7. 2. Smith CH. l\;elson OM, King BF, Donohue D\I, Ruzycki SM, Kellev LK. Characterization of a microvillous membrane prej)aration from human placental svncvtiotrophoblast: a morphologic, biochemical and physiologic study. A~tJ 011sn:rGY:\EC01. 1977;128:190.
September I, 1985 Am J Obstet Gynccol
3. Ganapathy V, Mendicino JF, Leibach FH. Transport ol glycyl-1.-proline into intestinal and renal brush border vesicles from rabbit. J Biol Chem 1981;256:118. 4. Ganapathv V, Burckhardt G, Leibach HI. Characteristics of glyrylsarcosine transport in rabbit intestinal brush-border membrane vesicles. J Biol Chem I 984;259:8954. 5. Gardner MLG. Intestinal assimilation of intact peptides and proteins from the diet-a neglected field? Biol Rev I 984;59:289. 6. Freinkel N. Banting Lecture, 1980, Of pregnancy and progeny. Diabetes 1980;29: I 023.
Bound volumes available to subscribers Bound volumes of the AMERICAr\ Jot;Rr\AL OF OBSTETRICS A;-.;D GY!'\ECOLOCY are available to subscribers (only) for the 1985 issues from the Publisher, at a cost of $74.50 ($95.05 international) for Vol. 151 (January-April), Vol. 152 (May-August), and Vol. 153 (September-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertising is removed. Copies are shipped within 30 days after publication of the last issue in the volume. The binding is durable buckram with the Jot;RNAI. name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact The C. V. Mosby Company, Circulation Department, 11830 Westline Industrial Drive, St. Louis, Missouri 63146, USA; phone (800) 325-4177, ext. 351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular JOURNAL subscription.
Dipeptide transport in brush-border membrane vesicles isolated from normal term human placenta Malliga E. Ganapathy, M.B., B.S., Virendra B. Mahesh, Ph.D., D.Phil., Lawrence D. Devoe, M.D., Frederick H. Leibach, Ph.D., and Vadivel Ganapathy, Ph.D. Augusta, Georgia We studied the transport of glycylsarcosine by the brush-border membrane vesicles isolated from normal term human placentas. This dipeptide resisted hydrolysis by placental membrane vesicles and was transported intact into an osmotically responsive intravesicular space. The transport process was Na• independent and probably occurred down a concentration gradient. Many peptides inhibited the transport of glycylsarcosine whereas amino acids had no effect. These results demonstrate, for the first time, the presence of a dipeptide transport system in the human placenta. (AM J OBSTET GYNECOL 1985;153:83-6.)
Key words: Dipeptide transport, Na - independence, human placenta, brush-border vesicle Transfer of nutrients from mother to fetus across the placenta is very important for adequate fetal growth. Placental transfer of amino acids is crucial for both fetal functional development and growth, as disturbed protein biosynthesis could result in abnormalities in either of these processes. Transfer of amino acids across the placenta can occur in the form of free amino acids as well as small peptides. While there are ample data to support the role of transport of small peptides in the adult intestine in the overall maintenance of human protein nutrition,' there are no analogous studies on the importance of placental transfer of small peptides in the maintenance of intrauterine nutrition. Our preliminary data strongly indicate, for the first time, that the brush-border membrane of the syncytiotrophoblast of human placenta possesses a dipeptide transport system which is distinct from the wellestablished amino acid transport systems.
Methods Brush-border membrane vesicles from normal term human placenta were prepared according to method of Smith et al. 2 One important difference, however, is
From the Departments of Endocrinology, Obstetrics and Gynecology, and Cell and Molecular Biology, Medical College of Georgia. This study was supported by National Institutes of Health Grant AM 28389 to F. H. L. This is contribution No. 0887 from the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia. Reprint requests: V. Ganapathy, Ph.D., Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912.
the inclusion of a Ca+· -precipitation step in our procedure, which resulted in better reproducibility of the method. We adapted this step from the methods applied to brush-border membrane vesicles isolated from the intestine and kidney. Placentas from normal pregnancies were obtained within 30 minutes of delivery. The maternal decidua was removed and the central portion between the maternal and fetal surfaces of the placenta was used for the preparation of the membrane. The placental tissue was cut into small pieces and collected in a I L beaker. All subsequent steps were carried out at 4° C. The tissue was washed three times in 200 ml of Hepes/Tris buffer, 10 mmol/L, pH 7.0, containing mannitol, 300 mmol/L. After washing, the tissue was placed in 300 ml of the same buffer and agitated with a magnetic stirring bar for 30 minutes. The tissue was removed with forceps, and the liquid was filtered through six layers of cotton gauze. The filtrate was centrifuged at 1000 x g for 10 minutes in order to remove blood and cellular debris. The supernatant was centrifuged at 10,000 x g for 15 minutes. The supernatant was collected and centrifuged again at 86,000 x g for 30 minutes. The pellets were collected in 50 ml of the Hepes/Tris-mannitol buffer and homogenized in a Dounce glass homogenizer with I 0 strokes. Then 0.5 ml of calcium chloride, 1 mol/L, was added to this homogenate and the mixture was stirred for 10 minutes and allowed to stand for an additional I 0 minutes. This was centrifuged at 3000 x g for 15 minutes. The supernatant contained brush-border membranes that were collected by centrifugation
83
84
Ganapathy et al.
September I. 1985 Am .J Obstet Gynecol
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Fig. I. Effect of medium osmolality on the equilibrium uptake of glycylsarcosine.
at 86,000 x g for 30 minutes. The membrane pellets were suspended in a small volume of preloading buffer by five passages through a 25-gauge needle. The preloading buffer varied with each experiment and the composition of buffer is given in the respective legend to the figure or table. Uptake of [ 1-"C]glycylsarcosine was measured by a rapid filtration technique" with the use of Metricel membrane filters (pore size, 0.45 µm) and hydrolysis of the dipeptide by the membrane preparations was determined as described earlier. 1 Results Alkaline phosphatase and 5' -nucleotidase were used as marker enzymes to assess the purity of the brushborder membranes. 2 Alkaline phosphatase was enriched 19.1-fold ± 2.2-fold and 5'-nucleotidase was enriched 18.5-fold ± 2.8-fold over the homogenate of the washed placental tissue. Freshly prepared membrane vesicles were used throughout the study. Glycylsarcosine is highly resistant to hydrolysis by tissue peptidases and, therefore, has been widely used in peptide transport studies. Hydrolysis of glycylsarcosine by placental brush-border membranes was assayed by a highly sensitive radiometric method. No detectable hydrolysis occurred during incubation periods
•
•
6 4 10 8 Incubation time (min)
Fig. 2. Time course of glycylsarcosine uptake. Membrane vesicles were suspended in Hepes/Tris buffer, 10 mmol/L, pH 7.5, containing mannitol, 300 mmol/L. Cptakc was measured in Hepes/Tris buffer, 10 mmol/L, pH 7.5, containing either sodium chloride, 150 mmol/L, or potassium chloride, 150 mmol/L. The final concentration of labeled glycylsarcosine was 5 mmol/L. •-•, K • gradient. o-o, Na - gradient.
as long as 3 hours. Therefore, any uptake of glycylsarcosine, observed under these conditions, represented uptake of the intact dipeptide. To test whether the dipeptide was transported from the incubation medium into the intravesicular space as opposed to bound to the vesicles, we measured the uptake at 90 minutes (equilibrium uptake) while varying the medium osmolality by the addition of mannitol. The uptake decreased as a linear function of the reciprocal of increasing medium osmolality and indicated that there was negligible binding of the dipeptide to the vesicles (Fig. 1). This relationship, however, did not hold when the medium osmolality was less than 200 mosm, probably because of rupture of the vesicles under these conditions. The results show that intact vesicles contained an osmotically responsive intravesicular space that disappeared with membrane disruption. The time course of glycylsarcosine uptake into these vesicles is shown in Fig. 2. The initial rates of uptake in the presence of either an Na• gradient or a K • gradient were identical. With longer periods of incubation, the uptake under K • gradient conditions was greater than that measured under Na• gradient conditions. However, the equilibrium uptake measured at 90 min-
Volume 153 Number I
utes was the same whether an Na· or a K • gradient was present. These results contrast markedly with those obtained for amino acid uptake, which is many times greater in the presence of an Na• gradient than in the presence of a K • gradient. Moreover, uptake of amino acids in the presence of an Na• gradient exhibits a transient overshoot, which is indicative of active transport that was absent when we studied glycylsarcosine uptake. These data show that glycylsarcosine uptake by placental brush-border membrane vesicles is Na' independent and probably occurs down a concentration gradient. We also studied the ability of unlabeled peptides and amino acids to inhibit the uptake of labeled glycylsarcosine. Uptake was measured at pH 5.5 because it was maximal for glycylsarcosine at this pH (data not shown). The free amino acids glycine and sarcosine minimally inhibited the uptake of glycylsarcosine while all peptides, except for carnosine, caused significant inhibition (Table I). These inhibition studies clearly show that glycylsarcosine uptake by placental brush-border membrane vesicles is a carrier-mediated process rather than a simple diffusion. If it were a simple diffusion process, it could not have been inhibited by other peptides. Since free amino acids did not inhibit glycylsarcosine uptake, it appears that the transport systems involved in the placental transfer of amino acids and dipeptides are different. We used a I-minute incubation in these inhibition experiments. However, it should be noted that shorter incubation periods (as low as I or 2 seconds) would be required for more rigorous kinetic analysis.
Comment We have demonstrated, for the first time, that the brush-border membrane of the human syncytiotrophoblast possesses a transport system for small peptides. This system is distinct from those used for amino acid transport and may, in vivo, serve as a significant conduit for amino acids. Normal human plasma contains appreciable amounts of small peptides. The peptide content of plasma significantly increases during assimilation of dietary proteins as many small peptides arising from protein digestion escape further hydrolysis and cross the intestine in intact form. 5 During pregnancy, maternal metabolism undergoes adaptive changes to ensure a proper nutrient supply to the fetus. Alterations in hormonal balance result in elevated levels of circulating insulin in the mother. 6 The maternal response to insulin changes during pregnancy. Early in gestation, the anabolic effects of insulin result in increased fat deposition and increased glycogen and protein synthesis while later there is increased muscle protein catabolism, pos-
Dipeptide transport in brush-border membrane vesicles
85
Table I. Effect of amino acids and peptides on glycylsarcosine uptake GlycJlsarcosine uptake nmollmg
Addition None Glycine Sarcosine Glycylsarcosine Glycylproline Carnosine Glycylleucine [3-Alanylglycylglycine Prolylglycylglycine
Protein 2.98 2.44 2.49 1.32 1.25 3.33 0.91 2.00 2.21
::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+:
0.28 0.09 0.19 0.02 0.09 0.11 0.03 0.09 0.18
o/c 100 82 44 42 112 31 67 74
Membrane vesicles were preloaded with Mes/Tris buffer, 10 mmol/L, pH 5.5, containing mannitol 125 mmol/L, and potassium chloride, 80 mmol/L. Uptake was initiated by adding 50 µI of membrane suspension to 200 µI of Mes/Tris buffer, 10 mmol/L, pH 5.5, containing sodium chloride, 80 mmol/L, labeled glycylsarcosine, and unlabeled peptides or amino acids. The final concentration of labeled glycylsarcosine was 5 mmol/L and the concentration of unlabeled peptides or amino acids was 100 mmol/L. Osmolality was maintained constant by varying the mannitol concentration. Incubation time was 1 minute. The data represent means of triplicates ::+:SD.
sibly due to the increased insulin resistance of maternal tissues." The increased breakdown of muscle proteins occurs at a time when the fetus gains most of its body mass and may represent an adaptive process important for assuring adequate fetal growth. While it is generally assumed that only free amino acids are released into the circulation during intracellular protein catabolism, no experimental evidence exists to support this assumption. Therefore, it is quite possible that small, hydrolysis-resistant peptides are generated as well and may gain access to the maternal circulation. If this, in fact, takes place, then the concentration of small peptides in maternal plasma may increase in the last trimester of pregnancy and may become an important source of amino acids for the fetus. Furthermore, placental tissue contains peptidases and may augment the supply of amino acids available to the fetus, once small peptides are transported across the placental brush-border membrane. The peptide transport system may also participate in the transfer of biologically active peptides of maternal origin, whether derived from dietary or endogenous proteins. These compounds, following maternal-fetal transfer, may exert a wide range of physiologic effects on the fetus, including circulatory homeostasis, neuroregulation, and liver function. Clinical conditions such as maternal hypertension, which alter placental anatomy, morphology, and function, may also influence the peptide transport system. Considering the important role this transport system may play in fetal metab-
86
Ganapathy et al.
olism and development, it merits further study in normal and pathologic gestations. REFERENCES I. Silk OBA, Hegarty J E, Fairclough PD. Clark \1L. Characterization and nutritional significance of peptide transport in man. Ann Nutr \letab 1982;26:'.1'.~7. 2. Smith CH. l\;elson OM, King BF, Donohue D\I, Ruzycki SM, Kellev LK. Characterization of a microvillous membrane prej)aration from human placental svncvtiotrophoblast: a morphologic, biochemical and physiologic study. A~tJ 011sn:rGY:\EC01. 1977;128:190.
September I, 1985 Am J Obstet Gynccol
3. Ganapathy V, Mendicino JF, Leibach FH. Transport ol glycyl-1.-proline into intestinal and renal brush border vesicles from rabbit. J Biol Chem 1981;256:118. 4. Ganapathv V, Burckhardt G, Leibach HI. Characteristics of glyrylsarcosine transport in rabbit intestinal brush-border membrane vesicles. J Biol Chem I 984;259:8954. 5. Gardner MLG. Intestinal assimilation of intact peptides and proteins from the diet-a neglected field? Biol Rev I 984;59:289. 6. Freinkel N. Banting Lecture, 1980, Of pregnancy and progeny. Diabetes 1980;29: I 023.
Bound volumes available to subscribers Bound volumes of the AMERICAr\ Jot;Rr\AL OF OBSTETRICS A;-.;D GY!'\ECOLOCY are available to subscribers (only) for the 1985 issues from the Publisher, at a cost of $74.50 ($95.05 international) for Vol. 151 (January-April), Vol. 152 (May-August), and Vol. 153 (September-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertising is removed. Copies are shipped within 30 days after publication of the last issue in the volume. The binding is durable buckram with the Jot;RNAI. name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact The C. V. Mosby Company, Circulation Department, 11830 Westline Industrial Drive, St. Louis, Missouri 63146, USA; phone (800) 325-4177, ext. 351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular JOURNAL subscription.