Development and Control of Intestinal and Hepatic Fructokinase

Home , Fructokinase, Hepatic fructokinase

INTESTINAL AND HEPATIC FRUCTOKINASE 765

expansion on intrarenal distribution of plasma flow in the dog. 42. The authors acknowledge the expert assistance of Mrs. Ann- Amer. J. Physiol., 223: 125 (1972). Christine Eklof and Mr. Gothe Carlebjork. 37. West, G. R., Smith, H. W., and Chasis, H.: Glomerular filtration 43. This research was supported by Grant no. B74-19X-3644-03Bfrom rate, effective renal blood flow and maximal tubular excretory the Swedish Medical Research Council and by a grant from the capacity in infancy. J. Pediat., 32: 10 (1948). Prenatal Research Funds of Expressen. 38. Laevosan Gesellschaft, Linz, Austria. 44. Requests for reprints should be addressed to: P. Herin, M.D., 39. 3M Company, St. Paul, Minn. Karolinski Institutet, Pediatriska Kliniken, St: Goran's Barn- 40. Sage Instruments, Inc., White Plains, N. Y. liniker, Box 12500, 112 81 Stockholm, Sweden. 41. Gilford Instrument Labs., Inc., Oberlin, Ohio. 45. Accepted for publication April 4, 1974.

Pediat. Res. 8: 765-770 (1974) Developmental biochemistry intestine fetus liver fruct okinase Development and Control of Intestinal and Hepatic Fructokinase

RICHARD J. GRAND,(^') MARIA I. SCHAY, AND STEPHANIE JAKSINA Department of Pediatrics, Harvard Medical School, and the Department of Medicine (Division of Clinical Nutrition), the Children's Hospital Medical Center, Boston, Massachusetts, USA

Extract experimental animals (1, 2, 20, 26) and in man (I 5), although the mechanism of this regulation has not yet been elucidated. The patterns of development of intestinal and hepatic In the rat, adrenalectomy prevents the rise in the activity of fructokinase have been studied in the rat, in the rabbit, and in the hepatic enzyme expected after fructose feeding; hydro- man, and information regarding the mechanism of control of cortisone administered to the intact animal greatly enhances this enzyme in mature organs has been obtained. Fructokinase activity. The intestinal enzyme has not been so studied. activity was detectable 4 days before term in fetal rat jejunum (1.0 nmol/min/mg protein) and rose progressively to a plateau During gestation and postnatal development in many species, fetal hepatic fructokinase activity is low (3, 21, 23), 20-fold greater by the 20th day of life (20 nmol/min/mg protein). The enzyme in rat ileum and liver and in rabbit liver even when fructose levels in fetal blood are appreciable (23), and the developmental pattern of fructokinase activity in the behaved similarly (rising from 0.5 to 4.0 nmol/min/mg protein liver has been shown to correlate with the utilization of (ileum) and from 0.5 to 8.5 nmol/min/mg protein (liver)). In exogenously administered (' 4C)fructose (3). human fetal liver, there was a threefold rise in fructokinase Because no data are available regarding the fetal levels or activity from approximately the 10th to 24th week of gestation (increasing from 2.1 to 7.8 nmol/min/mg protein); postnatal development of fructokinase in the intestine, the jejunal activity rose slightly, but not significantly, during this present studies were undertaken to compare such patterns in period. As expected, feeding of mature rats with sucrose after the rat, in the rabbit, and in the human fetus. In addition, the availability of a highly specific assay technique for the enzyme prolonged carbohydrate starvation led to a twofold increase in jejunal (from 6.05 to 13.0 nmol/min/mg protein) and hepatic (25) has made it worthwhile to re-examine similar patterns in the liver and to study further possible mechanisms of control (from 14.1 to 24.2 nmol/min/mg protein) fructokinase of the activity of this enzyme. The data show a progressive rise activity. The administration of actinomycin before the in the specific activity of fmctokinase during development and introduction of sucrose inhibited the carbohydrate-stimulated rise in fructokinase activity in both organs. Similarly, injection suggest that its activity in the mature animal is regulated at the of actinomycin during sucrose feeding produced a significant level of transcription. fall in enzyme activity which greatly exceeded that produced by fasting alone. The data suggest that the substrate-dependent METHODS activity of this enzyme may be regulated at the level of transcription. ANIMALS AND TISSUE PREPARATION Fetuses were obtained from timed pregnancies in New Speculation Zealand white rabbits and in albino Charles Riverr (Sprague- These studies support the hypothesis that the genetic Dawley derived) rats. For rabbits, only the second through control of certain enzymes involved in carbohydrate metabo- fifth pregnancies in a given doe were utilized; in rats, any but lism can be influenced by exogenous factors, among them the first pregnancy was acceptable. Mature tissues were dietary carbohydrate. obtained from adult animals of the same species. Human fetal liver and intestine were obtained from nonviable fetuses during therapeutic abortion, according to practices in operation at the Substrate-dependent control of the activity of hepatic and time of the study and approved by the Committee on Human intestinal fructokinase has been demonstrated in mature Investigation, Children's Hospital Medical Center. 766 GRAND, SCHAY, AND JAKSINA

Animals were anesthetized either by rapid cooling or with Intestinal disaccharidases were assayed by a modification (5) ether, and exsanguinated bx severing the aorta. Livers were of the method of Messer and Dahlqvist (13). Results were rapidly excised and kept at 4 until the intestine was removed, expressed as nanomoles of glucose liberated per minute per stripped of its mesentery, flushed with iced saline solution (pH milligram of protein in the EDTA homogenate. 7.4), and the mucosa scraped with glass slides on an iced glass plate. Fetal intestine was not scraped, but was used as FEEDING STUDIES AND ACTINOMYCIN D stripped. The first third of the intestine measured from the ligament of Treitz was termed jejunum and the last third, In all experiments, adult rats (120-150 g) were fed a ileum. Histologic study of the fetal intestine showed that the carbohydrate-free diet (86% protein, 10% fat, 4% salt mixture stripped preparation represented intact mucosa and approxi- (30)) for 5-7 days before each study. They were then fasted mately one-half the thickness of the muscularis. for 48 hr, and subsequently re-fed a solid sucrose-rich ,test diet Tissues (liver or intestine) were then weighed, minced and, (68% sucrose, 18% protein, 8% fat, 4% salt mixture, 2% yeast when fructokinase was to be assayed, homogenized in 10 (30)) with additional sucrose (30% w/v) in the drinking water volumes of 25 mM potassium phosphate buffer, pH 7.4, for various periods of time. Whenever the effect of actino- containing 5 mM EDTA and 0.5 mM dithiothreitol, using a mycin D was studied, the drug was dissolved in distilled water glass homogenizer with 10 passes of a Teflon pestle (25). A (250 pg/ml) and administered intraperitoneally according to high speed supernatant was prepared by centrifugation at the schedules shown in the legends to the figures and tables; 20,000 X g for 30 min. (Sorvall model RC-2B). Preliminary control animals received sterile 0.9% NaCl solution. Previous studies revealed that the fructokinase preparation from this studies (5) have demonstrated that the additives in the material had activity identical with that in the 105,000 X g actinomycin, as prepared by the manufacturer, have no effect supernatant in the original method (25). When intestinal on intestinal enzyme levels. disaccharidases were to be measured, an additional homogenate was prepared in 100 volumes of 5 mM EDTA, pH 7.4 (5). ESTIMATION OF RNA SYNTHESIS Previous studies from this (5, 6) and other laboratories (9) ENZYME ASSAYS have characterized the effects of actinomycin on total jejunal Fructokinase activity was measured as described by Weiser RNA synthesis, and many data are also available regarding the and Quill (25) with modifications; in this assay, fructose effects of the drug on hepatic RNA synthesis (14). In the 1-phosphate is the only reaction product. The high speed present study, hepatic RNA synthesis was measured only as an supernatants were acidified to pH 6.1 with 0.05 N HC1, indication of the activity of the actinomycin preparation. centrifuged at 20,000 X g for 20 min, and then returned to pH Eight and 20 hr after the injection of actinomycin D, 7.4 with 0.05 N KOH. This acidified supernatant usually (14C)orotic acid (0.1 pCi/g, body wt) was administered contained 2.0-3.0 mg protein/ml (intestine) or 3.5-4.5 intravenously. After 45 min, rats were killed, the livers excised mg/ml (liver) as determined by the method of, Lowry et al. and homogenized, and incorporation of radioactivity into cold (12). The incubation mixture contained 50 mM Tris-HC1 trichloroacetic acid (TCA)-precipitable material was measured buffer, pH 7.4, 10 mM MgCl,, 10 mM ATP, 50 mM as described previously (5). Results were expressed as counts N-acetylglucosamine, 10 mM D-fructose. The fructose for the per minute per milligram of protein in the alkaline digest. incubation mixture was prepared by diluting 100 mM D-(' C-Wfructose with enough unlabeled 100 mM D-fructose MATERIALS to give a solution which contained 750-900 cpm/nmol. The Disaccharides and actinomycin D were obtained as described final volume of the incubation mixture was 0.2 ml. The usual previously (5): (I4 C)Orotic acid (57.7 mCi/mmol) and concentration of enzyme was 0.2-0.3 mg/assay. Incubation D-(' C)fructose (5.1 -9.8 mCi/mmol) were products of New (performed in duplicate) was begun by adding 0.1 ml enzyme England Nuclear Corporation (31). ATP and N-acetylglucosa- suspension and was continued for 30 min at 37'. The reaction mine were purchased from Worthington Biochemical Corpora- was stopped by boiling for 2 min. Precipitated protein was tion (32) and Calbiochem (33), respectively. sedimented at 1,000 X g for 5 min. Of this supernatant, 20 pl were then spotted on squares (2 by 2 cm) of Whatman RESULTS DEAE-cellulose paper (29). The papers were washed by swirling twice in 1.0 mM ammonium formate, once in distilled FRUCTOKINASE DEVELOPMENT water, and once in absolute ethanol. A paper blank was washed with each set of assay papers and served as background The patterns of development of intestinal and hepatic control. Reaction controls containing (14C)fructose, but fructokinase in the rat are shown in Figure 1. In the jejunum lacking either ATP or enzyme, were also prepared with each (Fig. la), activity was undetectable until the 18th-19th day set of assays and showed a low level and reproducible quantity of gestation; it then rose progressively, increasing 20-fold, with of (14C)fructose bound to the papers. This was used for the a plateau occurring at approximately 17 days postnatally. The final calculation of activity as shown below. After drying, the levels of enzyme in the ileum (Fig. lb) were considerably papers were placed in scintillation vials containing 15 ml lower, but showed a similar pattern with maturation, toluene-2,s-diphenyloxazole (PP0)-1,4-bis[(5-phenyloxazolyl- increasing 8-fold by day 20. In the liver (Fig. Ic), fructokinase 2)lbenzene (POPOP) and counted in a Packard liquid activity increased nearly 20-fold from the 18th day of scintillation spectrometer. Correction for background was gestation to 20 days postnatally, achieving a plateau which was made and the amount of fructose 1-phosphate formed during maintained throughout adult life. Rabbit hepatic fructokinase the assay was calculated from the specific activity of the activity showed similar changes during development (Fig. 2), fructose in the incubation mixture by the formula. increasing nearly 9 times from the 19th fetal day to 25 days postnatally (the adult level). cpm assay - cpm reaction control The changes in fructokinase activity in the human fetus cpm/nmol fructose substrate = nmoles fructose 1-phosphate/assay from approximately the 10th to the 24th week of gestation This value was corrected for incubation time, sample size, and (4.4-17.5 cm crown-rump length) are shown in Figure 3. concentration of protein in the original enzyme suspension, There was a three-fold rise in activity during this time in the and fructokinase activity was expressed as nanomoles of liver and the calculated linear regression line was statistically fructose 1-phosphate formed per minute per milligram of significant (P < 0.05). In the jejunum, the rise was less protein. dramatic, and the linear regression analysis was not significant. INTESTINAL AND HEPATIC FRUCTOKINASE

DAYS DAYS

DAYS Fig. 1. Developmental pattern of rat jejunal (a), ileal (b), and hepatic (c)fructokinase activity. The arrow indicates the day of birth.

0 2 4 6 8 10 12 14 16 18 C-R LENGTH IcmI Fig. 3. Development of fructokinase activity in human fetal jejunum (0- - -0) and liver (0-0). The calculated linear regression lines DAYS are shown: jejunum, r = +0.4212, P not significant; liver, r = +0.5782,p Fig. 2. Developmental pattern of fructokinase activity in rabbit liver. < 0.05. C-R: crown-rump.. The arrow indicates the day of birth. incorporation of (14C)orotic acid into cold TCA-insoluble REGULATION OF FRUCTOKINASE BY CARBOHYDRATE material (control, 590 It: 83 cpmlmg protein; actinomycin- Previous studies in this laboratory suggested that the rise in treated, 200 + 70 cpm/mg protein, n = 7, P < 0.01). By 20 hr fructokinase activity brought about by large quantities of after treatment, incorporation was reduced only 24% (450 f ingested sucrose could be inhibited by pretreating animals with 88 cpm/mg protein, P not significant). actinomycin D (6). Further experiments were carried out to Treatment of the animals with this dose of actinomycin (1.0 extend these observations. pg/g body wt) before beginning sucrose feeding (Table 1) Table 1 demonstrates the effect of actinomycin on inhibited the sucrose-dependent fructokinase response ob- carbohydrate-stimulated fructokinase activity. Neither glucose tained in the absence of the drug. By contrast, sucrase activity feeding nor administration of actinomycin to starved animals increased in these animals, although to a lesser degree than affected fructokinase levels significantly. As expected, sucrose that described previously (6). feeding for 20 hr produced significant increases in enzyme In order to determine whether actinomycin would affect the activity, which were further augmented after 44 hr of feeding. maximally stimulated activity of fructokinase, rats were fed Prolonged and sustained reductions in the incorporation of the sucrose-rich diet for 7 days and were then killed at the (3H)uridine into cold TCA-insoluble material in the jejunum start of the experiment or 8 hr after receiving actinomycin are produced by treatment of rats with actinomycin (5, 6). In (1.0 pglg body wt) (Table 2). In response to the drug, the the present studies in the liver, 8 hr after the administration of specific activity of fructokinase in the jejunum fell markedly, the drug (1.0 pg/g body wt), there was a 65% reduction in the although sucrase activity was unaffected. By contrast, levels of 768 GRAND, SCHAY, AND JAKSINA

Table 1. Effect of diet and pretreatment with actinomycin on fructokinase

Specific activity

Condition of Experiment Jejunum Liver

Dose of Time on Statistical Statistical Jejunum, actinomycin, diet, Fructokinase ~ignificance,~ Fructokinase ~ignificance,~ sucrase Diet' /Ig/g hr activity2 P activity3 P activityZ

Starved (36)4 Starved (3) 1.O Glucose (3) 20 Sucrose (15) 20 Sucrose (3) 44 Sucrose (7) 1 .O 20

' Animals were starved for 48 hr before killing or before being fed sugar. Actinomycin was injected 4 hr before beginning sugar feeding. Nanomoles per minute per milligram of protein. 3Two-tailed Students t test. Numbers in parentheses refer to the number of animals studied. Compared with starved control animals. Compared with sucrose-fed, uninjected animals. hepatic fructokinase changed only modestly, but significantly, Table 2. Effect on actinornycin on fully stimulated activity after the injection of actinomycin (Table 2 P < 0.05). That the of fructokinase' responses in the jejunum and liver were not caused by starvation alone was verified by a separate experiment in Specific activity, nmol/min/mg protein which a 24-hr fast after 7 days on the sucrose-rich diet produced no significant changes in jejunal or hepatic fructo- Jejunum kinase activities (7-day sucrose-fed controls: jejunum 7.1, liver 19.9 nmol/min/mg protein; 7-day sucrose-fed, followed by 24 Conditions of Liver, hr starvation: jejunum 7.6, liver 19.3 nmol/min/mg protein). experiment Fructokinase Sucrase fructokinase

DISCUSSION Control 11.1 t 0.4 31.1 + 5.2 23.4 t 1.1 Actinomycin-treated 8.6 t 0.7' 29.3 + 3.1 20.6 + 0.53 Fructose is ...phosphorylated in both the intestine and liver by two ~athways:conversion to fructose 6-phosphate by 1 ~~t~ were fed the suaose-rich diet for 7 days and were killed at the kinase and to fructose ]-phosphate by fructokinase- In tissue start of the experiment or 8 hr after injection of actinomycin (1.0 /I~/~ extracts from mature animals, with fructose as the substrate, a body wt). Homogenates were and enzymes assayed as relatively large proportion of the total ~hos~hor~latingdescribed under Metho&. Results are mean c SE (n = 6). activity is due to hexokinase (1, 2, 26). However, its high zp< O.Ol. K, for fructose (6 mM) compared with that for glucose sp < 0.05. (<0.1 mM) suggests that hexokinase is unlikely to account for appreciable fructose utilization under normal conditions (1, 2). By contrast, fructokinase has a low K, for fructose (intestine, induction. Whether or not induction by hormones controls the 0.125 mM (27); liver, 0.2-0.5 mM (2)) and, during fructose development of hepatic fructokinase is not known. Although feeding, accounts for nearly 70% of the total fructose the precocious development of many hepatic enzymes can be phosphorylating activity in the intestine (26). The specific stimulated by hormones (by mechanisms which appear to be activity of the enzyme in the mature intestine is 5-6 times dependent upon RNA synthesis (7)), some of the key greater in the midjejunum than in the ileum. Previous workers enzymes of gluconeogenesis are resistant to such stimulation have failed to detect activity in fetal liver (21, 23). (28). The present study confirms the postnatal rise in hepatic On the basis of the small number of assays of fructokinase fructokinase activity in the rat and rabbit. The experiments activity in human fetal liver in this study, it is not possible to also demonstrate that activity is present in fetal liver. compare the pattern of development to that in animals. Inasmuch as the experimental methods in this and earlier However, it is clear that, in the human, fructokinase activity is studies are similar, it can be assumed that the sensitivity and easily detectable earlier in gestation (the first trimester) than specificity of the assay used here account for the observations in any other mammal studied, except the rhesus monkey (24). in the fetuses. Fetal liver contains an appreciable and gradually This contrasts with the finding that hepatic glucokinase is decreasing quantity of hematopoietic elements (1 I), and the undetectable in human fetal liver for as long as 24 weeks from possibility exists that the increasing specific activity of conception (17). fructokinase during development represents diminishing con- The development pattern of intestinal fructokinase has not tamination by such cells. However, Ballard and Oliver (3) have been reported previously. The present studies demonstrate a shown that the incorporation of (I4 C)fructose into glycogen marked, age-related increase in the specific activity of in developing rat liver increases concomitantly with the fructokinase in the jejunum, and an obvious but smaller increase in fructokinase activity. This pattern is similar in increase in the ileum, patterns similar to those found in the many species and is not changed by the presence of fructose in liver (see Figs. 1 and 2). As in the liver, activity in the intestine fetal blood (24). Thus, the timing of the appearance of the was detectable in the fetus presumably because of the enzyme during development is probably not due to substrate sensitivity of the assay technique. Other carbohydrate-depen- INTESTINAL AND HEPATIC FRUCTOKINASE 769 dent jejunal enzymes show increasing activities during the first day of birth. Similarly, in the rabbit liver, activity is present in 3 postnatal weeks in the rat (4, 10) and hormonal control of the second trimester, starting 11 days before birth. In human such changes has been postulated (8, 10). Precocious fetal liver, there is a threefold rise in fructokinase activity development of sucrase has been stimulated both by cortisone from the 10th to 24th week of gestation, while the jejunal and early sucrose feeding (lo), but jejunal fructokinase has not enzyme rises only slightly during this period. been so studied. Carbohydrate feeding after prolonged carbohydrate starva- Although fructokinase activity in mature liver and intestine tion is associated with a 2-fold increase in hepatic and jejunal is dependent upon dietary carbohydrate, the mechanism of fructokinase activity, a rise which is inhibited by pretreatment control has not been elucidated previously. In both organs, of animals with actinomycin. These data suggest that the fructose or sucrose feeding after carbohydrate or total substrate-dependent activity of this enzyme is regulated at the starvation produces marked increases in enzyme activity (2, level of transcription. 20, 26), and it has been shown that the responses of hepatic fructokinase to dietary fructose are abolished by adrenalec- REFERENCES AND NOTES tomy (2). The present studies also demonstrate increases in the specific activity of fructokinase in mature jejunum and liver in 1. Adelman, R. C., Ballard, F. J., and Weinhouse, S: Purification and response to dietary sucrose. These changes are of approxi- properties of rat liver fructokinase. J. Biol. Chem., 242: 3360 mately the same magnitude as those reported by others (see (1 967). 2. Adelman, R. C., Spolter, P. D., and Weinhouse, S: Dietary and Table 1 and References 2 and 26). In addition, the hormonal regulation of enzymes of fructose metabolism in rat experiments with actinomycin suggest possible mechanisms for liver. J. Biol. Chem., 241: 5467 (1966). the control of this enzyme. Actinomycin produces a prolonged 3. Ballard, F. J., and Oliver, I. T.: Ketohexokinase, isoenzymes of and sustained reduction of RNA synthesis in the jejunum, glucokinase and glycogen synthesis from hexoses in neonatal rat liver. Biochem J., 90: 261, (1964). although it is not complete (5, 6). In the liver, the effect is 4. Doell, R. G., and Kretchmer, N.: Intestinal invertase: Precocious more transient, with a return of synthetic activity by 20 hr development of activity after injection of hydrocortisone. after administration of the drug. Nevertheless, the effect of the Science, 143: 42 (1 964). drug in both organs is to interrupt the rise in fructokinase 5. Grand, R. J., Chong, D. A,, and Isselbacher, K. J.: Intracellular processing of disaccharidases: The effect of actinomycin D. activity after sucrose feeding; the rise in intestinal sucrase Biochim. Biophys. Acta, 261: 34 1 (1 972). activity is not abolished. Comparable effects of actinomycin 6. Grand, R. J., and Jaksina, S.: Additional studies on the regulation on the adaptive responses of two additional jejunal glycolytic of carbohydrate-dependent enzymes in the jejunum: Changes in enzymes (pyruvate kinase and fructose diphosphate aldolase) amino acid pools, protein synthesis and the effect of actinomycin D. Gastroenterology, 64: 429 (1973). have been described previously (19). 7. Greengard, 0.: Enzymic differentiation in mammalian liver. Under the conditions of the present experiments, evidence is Science, 163: 891 (1969). avail?ble which suggests that actinomycin has a direct action 8. Herbst, J. J., and Koldovsky, 0.: Cell migration and cortisone on the regulation of fructokinase levels and that the changes in induction of sucrase activity in jejunum and ileum. Biochem. J., 126: 471 (1972). specific activity are not toxic side effects of the drug. Thus, 9. Imondi, A. R., Lipkin, M., and Balis, M. E.: Enzyme and template although jejunal and hepatic fructokinase activities fail to stability as regulatory mechanisms in differentiating intestinal increase in response to carbohydrate in the presence of epithelial cells. J. Biol. Chem., 245: 2194 (1970). actinomycin, jejunal sucrase activity rises. The increase in the 10. Lebenthal, E., Sunshine, P., and Kretchmer, N.: Effect of carbohydrate and corticosteroids on activity of alpha-gluco- level of the latter enzyme is not as great as in control animals, sidases in intestine of the infant rat. J. Clin. Invest., 51: 1244 but is comparable with changes reported previously (6). A (1 972). similar dissociation between the effects of actinomycin on 11. Leeson, C. R., and Cutts, J. H.: The post-natal development of the fructokinase and sucrase activities can be found in the studies rabbit liver. Biol. Neonate, 20: 404 (1972). 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: obtained during the high sucrose intake (Table 2), where Protein measurement with the Folin phenol reagent. J. Biol. actinomycin produced a marked and rapid fall in jejunal Chem., 193: 265 (195 1). fructokinase levels without affecting sucrase activity. Since a 13. Messer, M., and Dahlqvist, A.: A one-step ultramicro method for reduction in enzyme activity as a result of actinomycin the assay of intestinal disaccharidases. Anal. Biochem., 14: 376 (1 966). administration (9) or the failure to increase enzyme activity in 14. Prosky, L., Roberts, B., Jr., O'Dell, R. G., and Imblum, R. L.: the presence of the drug (16) have been interpreted as Differential effects of actinomycin-D on nucleic acid and protein reflecting changes in the function of mRNA for that enzyme synthesis in rat liver. Arch. Biochem. Biophys., 126: 393 (1968). (22), it is tempting to postulate that fructokinase is regulated 15. Rosensweig, N. S., Stifel, F. B., Herman, R. H., and Zakim, D.: The dietary regulation of the glycolytic enzymes. 11. Adaptive at the level of transcription. However, actinomycin exhibits a changes in human jejunum. Biochim. Biophys. Acta, 170: 228 dose-related effect on the synthesis of ribosomal RNA (14), (1968). and recent evidence suggests that the drug also interferes with 16. Salas, M., Viiiuela, E., and Sols, A.: Insulin-dependent synthesis of the initiation of translation in mammalian cells without liver glucokinase in the rat. J. Biol. Chem., 238: 3535 (1963). 17. Sigman, B., Hawkins, R. L., and Tildon, J. T.: Fetal development affecting the stability of mRNA (18). Thus, changes in protein of the multiple forms of rat hexokinase. Biochem. Med., 6: 29 synthesis (or enzyme levels) in the presence of the drug (1 972). cannot be necessarily ascribed to transcriptional events. 18. Singer, R. H., and Penman, S.: Stability of HeLa cell mRNA in Nevertheless, if in the present studies, actinomycin produced actinomycin. Nature, 240: 100 (1972). 19. Stifel, F. B., Herman, R. H., and Rosensweig, N. S.: Dietary its effect by interfering with initiation or ribosomal RNA regulation of glycolytic enzymes. XI. Effect of inhibitors of synthesis, a change in the levels of sucrase, as well as protein synthesis on the adaption of certain jejunal glycolytic fructokinase, could have been expected. The failure of sucrase and folate-metabolizing enzymes to diet and sex steroids. activity to change coordinately with that .of fructokinase Biochim. Biophys. Acta, 273: 484 (1971). 20. Stifel, F. B., Rosensweig, N. S., Zakim, D., and Herman, R. H.: makes an actinomycin-induced lesion in initiation or ribosomal Dietary regulation of glycolytic enzymes. I. Adaptive changes in RNA synthesis unlikely. The data thus support the proposal rat jejunum. Biochim. Biophys. Acta, 170: 22 1 (1 968). that the control of fructokinase is probably at the level of 21. Swiatek, K. R.: Development of gluconeogenesis in pig liver slices. transcriptian. Biochim. Biophys. Acta, 252: 274 (1971). 22. Tomkins, G. M., Gelehrter, T. D., Granner, D., Martin, D., Jr., Samuels, H. H., and Thompson, E. B.: Control of specific gene expression in higher organisms. Science, 166: 1474 (1 969). SUMMARY 23. Walker, D. G.: The post-natal development of hepatic fructokinase. Biochem. J., 87: 576 (1963). Fructokinase activity is detectable in the fetal rat jejunum 24. Walker, D. G.: Development of enzymes for carbohydrate and liver 4 days before delivery, and in the rat ileum on the metabolism. In the Biochemistry of Development, p. 77 (J. B. 770 BOIME, CORASH, AND GROSS

Lippincott Co., Philadelphia, 197 1). 32. Freehold, N. J. 25. Weiser, M. M., and Quill, H.: Estimation of fructokinase in crude 33. Los Angeles, Calif. tissue preparations. Anal. Biochem., 43: 275 (1 97 1). 34. The authors are indebted to Dr. Yvonne Bishop for aid with 26. Weiser, M. M., Quill, H., and Isselbacher, K. J.: Effects of diet on statistical analyses, and to Dr. Shirley G. Driscoll, Boston rat intestinal soluble hexokinase and fructokinase activities. Hospital for Women, for providing human fetal tissues. Amer. J. Physiol., 221: 844 (1971). 35. Dr. R. J. Grand is recipient of Academic Career Development 27. Weiser, M. M., Quill, H., and Isselbacher, K. J.: Isolation and Award no. 5-K07-AM-44590 from the National Institute of properties of intestinal hexokinase, fructokinase and N-acetylglu- Arthritis, Metabolism and Digestive Diseases. cosamine kinase. J. Biol. Chem., 246: 2331 (1971). 36. This research was supported by a Research Grant no. AM-14523 28. Yeung, D., Stanley, R. S., and Oliver, I. T.: Development of from the National Institute of Arthritis, Metabolism and gluconeogenesis in neonatal rat liver. Biochem. J., 105: 1219 Digestive Diseases. (1967). 37. Requests for reprints should be addressed to: R. J. Grand, M.D., 29. H. Reeve Angel, Clifton, N. J. 300 Longwood Ave., The Children's Hospital Medical Center, 30. Nutritional Biochemicals Co., Cleveland, Ohio. Boston, Mass. 021 15 (USA). 31. Boston, Mass. 38. Accepted for Publication April 11, 1974.

Pediat. Res. 8: 770-774 (1974) Developmental biochemistry protein synthesis placenta ribosomes Protein Synthesis in Cell-free Extracts from First and Third Trimester Human Placenta

IRVING BOIME(~) Departments of Obstetrics and Gynecology and Pharmacology, Washington University School of Medicine, Saint Louis, Missouri, USA LAURENCE CORASH AND ERHARD GROSS Section on Molecular Structure, Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Marj~land,USA

Extract known about the biosynthesis of these hormones. It is not clear as to whether or not human chorionic gonadotropin and Ribosomal and cell sap fractions were prepared from first human placental lactogen are synthesized in the form of and third trimester human placentas. High endogenous activity precursor molecules as are some other hormones (15). was observed with ribosomes prepared from first trimester or Furthermore, although peak levels of human chorionic third trimester placenta when incubated either in the presence gonadotropin are observed during the first trimester of of cell sap derived from first trimester placenta tissue or from pregnancy, it has not been established whether there is an Krebs I1 ascites tumor cells. The homologous placental system increase in (I) the de novo synthesis of the hormone (2) the is capable of translating poly(U) and globin mRNA, although rate of mobilization of a precursor, or (3) the cellular secretion the latter is translated at about 10% of the efficiency as of the hormone. In order to answer these and other questions observed in the homologous ascites tumor system. The regarding the regulation of placental protein synthesis, it is placental cell sap may be deficient in a factor necessary for the necessary to have an active, cell-free, protein-synthesizing translation of globin mRNA. The preparation of active system from placental tissue taken at various stages of ribosomes from first and third trimester polysomes shows that gestation. it will be possible to study the endogenous synthesis of We wish to report the preparation of such a system from the placental proteins throughout pregnancy. first and third trimester human placenta. These consist of ribosomes from placenta and ribosome-free supernatant Speculation fractions prepared from the same tissue or from ascites tumor These results indicate the feasibility of studying the cell-free cells. synthesis of placental peptide hormones. Human chorionic gonadotropin and human placental lactogen reach peak levels of synthesis at different gestational periods and thus the MATERIALS AND METHODS relation between the syn;hesis of the hormones and their secretion can be elucidated. Rat liver tRNA was a gift from Dr. Dolph Hatfield and crude rabbit nlobin mRNA was a gift from Dr. Philip Leder. (3 ~)~eucinebas purchased from the New ~n~land-~uclear Although the secretion of various placental peptide hor- Company (18) and sucrose was obtained from Schwarz-Mann mones during gestation is well documented (16), little is (19).