Galactosyltransferase activities during embryonic development of chich neural tissue

Galactosyltransferase specific activities in embryonic chick retina, optic tectum, and telecephalon were found to decline during embryonic development. Incorporation of galactose from nucleotide sugar into exogenously added glycoprotein acceptor was measured in the presence of excess of glycoprotein acceptor. This ensured that the specific activity measurements were reflections of true enzyme specific activity rather than availability of acceptor. Moreover, we have shown that the decline in specific activity is not due to degradation of the nucleotide sugar, UDP-galactose, under our in vitro assay conditions.Enzymatic specific activity declined sharply with embryonic age for all tissues tested. This decline was not affected by the presence of 5-bromodeoxyuridine during in vitro culture of embryonic chick neutral retina above that caused by the culturing alone. Galactosyltransferase activity was not found to be associated with the plasma membrane fraction from homogenized tissue but rather with the microsomal fraction. Thus, the changes in galactosyltransferase specific activity detected here do not reflect changes at the cell surface.     

Cellular energy metabolism during fetal development. IV. Fatty acid activation, acyl transfer and fatty acid oxidation during development of the chick and rat

We have investigated developmental profiles of ATP-dependent palmityl-CoA synthetase, acetyl-CoA synthetase, palmitylcarnitine transferase, and fatty acid oxidation in heart and liver of developing chicks and rats. Palmityl-CoA synthetase activity of rat liver and heart homogenates increased 6- to 10-fold during the first postnatal week. Chick embryo heart activity peaked between 13 and 16 days of development. The activity of embryonic chick livers was bimodal with highest activity seen at 7 and 16 days of development. Posthatching values were approximately 50–75% of the peak embryonic levels. Acetyl-CoA synthetase activity of rat liver and heart homogenates was low but also showed developmental increases following birth. Acetyl-CoA synthetase activity of chick embryonic hearts was greatest at 16 days of development. Palmitylcarnitine transferase activity of rat liver and heart homogenates showed a striking increase during the first week of life. Chick heart activity was similar to that observed for palmityl-CoA synthetase with a peak between 13 and 16 days of embryonic development. Coincident with the postnatal rise in fatty acid activation and palmitylcarnitine transferase activity in developing rats, the oxidation of palmityl-CoA plus carnitine and of palmitylcarnitine increased from barely measurable levels at birth to adult levels by 30 days of age. The increases that we observe probably relate to changes in the specific activity of the enzymes as well as to an increase in the absolute number of mitochondria during development.     

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat.

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.     

The subcellular location, maturation and response to increased plasma glucagon of Ruthenium Red-insensitive calcium-ion transport in rat liver

1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca(2+) transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5'-nucleotidase and Ruthenium Red-sensitive Ca(2+) transport. Initial rates of Ruthenium Red-insensitive Ca(2+) transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca(2+) transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5'-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca(2+) transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca(2+) transport activity, glucose 6-phosphatase and 5'-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca(2+) transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3-4 days, but then rapidly decreases to approach adult values. Maximal activity of 5'-nucleotidase in the microsomal and nuclear fractions is seen about 4-6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca(2+) transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2-3-fold the initial rate of Ruthenium Red-insensitive Ca(2+) transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1mug of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca(2+) transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca(2+) transport are an integral part of the physiological network in the liver cell.     

大白鼠DNA拓扑异构酶Ⅰ生物学性质的研究

对大白鼠组织作DNA拓扑弄构酶Ⅰ(拓扑酶Ⅰ)活力测定,见酶活力出现在胚胎早期,在胚胎发育过程及出生后不同年龄期,酶活力基本稳定;几种成年大鼠组织的酶活力彼此无显著差异;肝细胞再生及癌变,酶活力亦无显著变化。     

The effect of TEPC-183 plasmacytoma growth on beta-glucuronidase activity in serum and tissues of tumor-bearing mice

beta-Glucuronidase activity increased in the serum of BALB/c mice during the growth of the IgM-secreting plasmacytoma, TEPC-183. The increase appeared to correlate with tumor burden. The beta-glucuronidase activity in tissue homogenates of spleen, liver, and kidney from tumor-bearing mice also increased significantly compared to the levels found in corresponding tissues from normal control mice. Assays of lysosomal and microsomal fractions from livers of TEPC-bearing mice indicated that approximately 70% of the enzyme activity was associated with the lysosomal fraction and the remainder with the microsomal fraction. A similar distribution was found in homogenates prepared from the plasmacytoma itself. In contrast to this the beta-glucuronidase activity in livers from normal BALB/c mice is nearly equally distributed between lysosomal and microsomal fractions.     

Developmental aspects of uridine diphosphate galactose 4-epimerase in rat intestine

Kinetic and developmental characteristics of rat intestinal UDP-galactose 4-epimerase activity have been examined. The enzyme in the adult rat had a V_max. value 2–3 times higher than that of the newborn animal, but the K_m values for the enzyme in the newborn and adult rat were the same (0.17mm). No differences in epimerase activity were found along the length of the jejuno-ileum of adult animals, but higher activity was detected in the lower portion of the villi and crypts. The specific activity of the enzyme in the newborn rat began to rise at about 17 days of age, reaching a peak at 29 days of age, and then became constant at adult values. Total epimerase activity in the newborn rat liver was 2–5 times higher than the total activity in the intestine, and total epimerase activity in the adult intestine was 3–4 times higher than the total activity in the liver. Cortisone injection did not enhance the increase of epimerase normally seen during development, but caused a decrease in activity of this enzyme in the jejunum in rats up to 17 days of age. After 17 days, cortisone treatment had no effect on epimerase activity.     

Developmental Changes in Rat Liver Xanthine Oxidase(s) and Its Intracellular Distribution

Developmental change and subcellular distribution of xanthine oxidase in the rat liver were examined.

The specific activity of the fetal liver xanthine oxidase increased sharply to the levels of the adult liver on the day of the birth. After birth, the activity dropped rapidly and on the 14th day after birth it was about 1/4 of adult level. Then the activity was regained and around 28th day after birth it was about the same as in adult level.

In the livers from 80 days old rats, about 60% of total xanthine oxidase activity was found in soluble fraction and the rest was distributed among particulate fractions including microsomal, lysosomal, mitochondrial and nuclear fractions.

In contrast to the adult livers 80% of total xanthine oxidase activity in fetal liver was found to be in particulate fractions.

From kinetic studies of xanthine oxidases in particulate and soluble fractions it was suggested that xanthine oxidase in soluble fraction and xanthine oxidase in particulate fraction might be different in their natures of protein molecule.     

Developmental changes in alcohol-dehydrogenase activity in rat and guinea-pig liver

1. Alcohol-dehydrogenase activity is first detectable in the rat foetus on about the eighteenth day of gestation, after which time it increases to about 25% of the adult activity at birth. Adult activity is reached at about 18 days after birth. The ethanol-oxidizing capacity of liver slices from rats correlates well with the increase of the enzyme activity in vitro. 2. In the guinea pig there is a steady linear increase from about 17 days before term to 5 days after birth. Adult activity is reached between the sixth and eighth postnatal day. 3. Some kinetic properties of liver alcohol dehydrogenase are very similar in newborn and adult rats. 4. Administration of ethanol to pregnant rats during the latter half of gestation had no effect on alcohol-dehydrogenase activity in the liver of the newborn offspring. Intraperitoneal injections of ethanol to newborn and young rats had no effect on the alcohol-dehydrogenase activity of the livers. 5. Intraperitoneal injections of hydrocortisone and triamcinolone to newborn and adult non-adrenalectomized rats had no significant effect on the increase of the alcohol-dehydrogenase activity as studied up to 4 days after the injection.     

Isoprenaline-induced changes in rat parotid and submandibular glands are age- and dosage-dependent.

Neonatal rats treated with chronic injections of isoprenaline (isoproterenol) for 10 days revealed differential induction of proline-rich proteins and glycoprotein synthesis between the parotid and submandibular glands. Biosynthesis of proline-rich proteins (Mr 17000-35000) and a Mr-220000 glycoprotein were detectable by solubilization in 10%-trichloroacetic acid extracts from parotid glands 14 days after birth. The enzyme lactose synthase (UDP-galactose: 2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase) (EC 2.4.1.22) is also induced 4-7-fold in specific activity compared with control neonatal rats, but again only after 14 days post partum, with isoprenaline treatment. This is in accord with the ability of the parotid gland to respond to beta-receptor stimulation and subsequent increases in intracellular cyclic AMP necessary for induction of protein synthesis [Grand, Chong & Ryan (1975) Am. J. Physiol. 228, 608-612]. Induction of the proline-rich proteins and a Mr-190000 glycoprotein in the soluble fraction from the submandibular gland were not detected until 49 days after birth under identical conditions in the same animal. Cyclic AMP in the submandibular gland undergoes increases on beta-receptor stimulation similar to those achieved in the adult animal, 1 day after birth (Grand et al., 1975). This same differential induction between parotid and submandibular gland was obtained with a range of isoprenaline dosages in adult animals. Trichloroacetic acid-soluble proline-rich proteins were isolated from parotid glands at a dosage of 4.0 mg of isoprenaline/kg body wt., but 7.0 mg/kg was required to induce also biosynthesis of these proteins in the submandibular gland. Gland hypertrophy showed the same differential dosage kinetics, based on gland weight, between the two glands; however, hypertrophy could be accomplished at a lower dosage of isoprenaline than that used to induce proline-rich-protein biosynthesis.     

Mutagenicity of benzo(a)pyrene in uninduced tissues from BALB/c mice and Sprague-Dawley rats as an index of possible health risks using the Salmonella mutagenicity assay

The mutagenicity of benzo(a)pyrene [B(a)P] in uninduced tissues from Sprague-Dawley rats and BALB/c mice resulted in no age, sex or tissue-related differences when S9 preparations from lung, kidney and spleen were used in the Salmonella mutagenicity assay. Liver S9 fractions from both species resulted in a significantly greater number of His+ revertants (TA98) per plate than observed on the control plates (spontaneous reversion rate). Liver homogenates from adult Sprague-Dawley rats showed a significantly lower potential to activate B(a)P than homogenates from adult BALB/c mice. In both species, male liver microsomal enzymes had a greater potential to activate B(a)P than female microsomal enzymes. These data indicated that in uninduced tissues B(a)P may not be a very powerful mutagenic agent. More detailed in vitro and in vivo studies are needed to assess the precise health risks associated with this environmental pollutant.     

Factors affecting hepatic glycolysis and some changes that occur during development

1. Glycolysis by the supernatant fraction of homogenates of liver from guinea pigs and rats at various stages of development (foetal, newborn and adult) has been examined in a suitably fortified medium by measurement of inorganic phosphate uptake and production of lactate and glycerol 1-phosphate. 2. Starting with glucose as substrate, two rate-determining steps in glycolysis occur at the stages of glucose phosphorylation and the phosphofructokinase reaction in liver tissue from animals of all ages. Effects of the post-natal development of glucokinase are recorded. 3. The appearance of microsomal glucose 6-phosphatase activity around birth has an effect on glycolysis owing to competition for glucose 6-phosphate. 4. A stimulating effect of the nuclear fraction, especially from foetal liver, on glycolysis by the supernatant fraction is interpreted as being due to stimulation by adenosine-triphosphatase activity at the 3-phosphoglycerate-kinase stage.     

The effect of phenobarbital on the submicrosomal distribution of uridine diphosphate glucuronyltransferase from rat liver

1. The detergent Triton X-100 activates UDP glucuronyltransferase from rat liver in vitro six- to seven-fold with p-nitrophenol as substrate. The enzyme activity when measured in the presence of Triton X-100 is increased significantly by pretreatment of male rats with phenobarbital for 4 days (90mg/kg each day intraperitoneally). If no Triton X-100 is applied in vitro such an increase could not be shown. In all further experiments the enzyme activity was measured after activation by Triton X-100. 2. The K(m) of the enzyme for the substrate p-nitrophenol does not change on phenobarbital pretreatment. 3. When the microsomal fraction from the liver of untreated rats is subfractionated on a sucrose density gradient, 47% of the enzyme activity is recovered in the rough-surfaced microsomal fraction, which also has a higher specific activity than the smooth-surfaced fraction. 4. Of the increase in activity after the phenobarbital pretreatment 50% occurs in the smooth-surfaced fraction, 19% in the rough-surfaced fraction and 31% in the fraction located between the smooth- and rough-surfaced microsomal fractions on the sucrose density gradient. 5. The latency of the enzyme in vitro, as shown by the effect of the detergent Triton X-100, is discussed in relation to the proposed heterogeneity of UDP glucuronyltransferase.     

Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells.

Administration of dexamethasone to pregnant rats at 19 days gestation increased phosphatidylcholine synthesis (45%) from radioactive choline in type II cells. This enhanced synthesis of phosphatidylcholine was accompanied by an increased conversion of choline phosphate into CDP-choline. Similar results were obtained by incubating organotypic cultures of 19-day-fetal rat lung with cortisol. The increased conversion of choline phosphate into CDP-choline correlated with an enhanced choline-phosphate cytidylyltransferase activity (31% after dexamethasone treatment; 47% after cortisol exposure) in the cell homogenates. A similar increase (26% after dexamethasone treatment; 39% after cortisol exposure) was found in the microsomal-associated enzyme. No differences in cytosolic enzyme activity were observed. The specific activity of the microsomal enzyme was 3-4 times that of the cytosolic enzyme. Most of the enzyme activity was located in the microsomal fraction (58-65%). The treatments had no effect on the total amount of enzyme recovered from the cell homogenates. These results, taken collectively, are interpreted to indicate that the active form of cytidylyltransferase in type II cells is the membrane-bound enzyme and that cytidylyltransferase activation in type II cells from fetal rat lung after maternal glucocorticoid administration occurs by binding of inactive cytosolic enzyme to endoplasmic reticulum.     

Glucagon stimulation of ruthenium red-insensitive calcium ion transport in developing rat liver.

The maturation of glucagon-stimulated Ruthenium Red-insensitive Ca2+-transport activity was determined in livers of rats ranging in age from 5 days preterm to 10 weeks of adult life. Previous indications are that this activity is confined to vesicles derived mainly from the endoplasmic reticulum. Perinatal-rat liver contains near-adult values of Ruthenium Red-insensitive Ca2+-transport activity, and exhibits large transient increases in the rate of this activity at two stages of development, immediately after birth, and at 2-5 days after birth. The administration of glucagon to foetal rats, at developmental stages after 19.5 days of gestation (2.5 days before birth), results in a large stable increase (greater than 100%) of Ca2+-transport activity in a subsequently isolated 'heavy' microsomal fraction. That this fraction was enriched in vesicles derived from the rough endoplasmic reticulum was indicated by both an electron-microscopic examination and a marker-enzyme analysis of the subcellular fractions. The administration of glucagon into newborn animals only hours old does not enhance further the initial rate of Ca2+-transport activity, and from day 1 to 10 weeks after birth the administration of the hormone results in the moderate enhancement of Ca2+ transport. Experiments with cyclic AMP and inhibitors of phosphodiesterase activity suggest that cyclic AMP plays a key role in the enhancement by glucagon of Ruthenium Red-insensitive Ca2+ transport, and arguments are presented that this transport system has an important metabolic role in the redistribution of intracellular Ca2+ in liver tissue.     

Different developmental changes in latency for two functions of a single membrane bound enzyme: glucose-6-phosphatase activities as a function of age.

(1). The capacity for the synthesis of glucose 6-phosphate from PPi and glucose as well as for glucose-6-P hydrolysis, catalyzed by rat liver microsomal glucose-6-phosphatase, increases rapidly from low prenatal levels to a maximum between the second and fifth day, then slowly decreases to reach adult levels. When measured in enzyme preparations optimally activated by hydroxyl ions, the maximum neonatal activities were 4--5-fold higher than in adult animals and several-fold higher than had previously been observed for the unactivated enzyme. (2) The latencies of two catalytic activities associated with the same membrane-bound enzyme show strikingly different age-related changes. The latency of PPi-glucose phosphotransferase activity reaches high levels (60--80% latent) soon after birth and remains high throughout life, while the latency of glucose-6-P phosphohydrolase decreases with age. The phosphohydrolase is 2--3 times more latent in the liver of the neonatal animal than in the adult. (3). The well established neonatal overshoot of liver glucose-6-phosphatase is almost entirely due to changes in the enzyme in the rough microsomal membranes. The enzyme activity in the rough membrane reaches a maximum and then decreases after day 2, while that in the smooth membrane is still slowly increasing. Despite the great differences in absolute specific activities and in the pattern of early enzyme development between the rough and smooth microsomes, enzyme latency in the two subfractions remains parallel, glucose-6-P phosphohydrolase being only slightly more latent, while PPi-glucose phospho-transferase is much more latent in smooth than in rough membranes throughout life. (4). Kidney glucose-6-P phosphohydrolase and PPi-glucose phosphotransferase activities were found to change in a parallel fashion with age, showing a small neonatal peak between days 2 and 7 before rising to adult levels. Kidney phosphotransferase activity, like that of liver, remained highly latent throughout life. In contrast to liver, the glucose-6-P phosphohydrolase of kidney did not show a characteristic decrease in latency with age and in the adult remained appreciably more latent than in liver. (5). An improved method was devised for the separation of smooth microsomes from liver homogenates.     

Studies on the stearic acid dehydrogenase in the liver and brain of rats of various ages (author's transl)]

Tissue slices from the liver and brain of 7-day-old rats incubated with [1-14C]stearic acid desaturate the stearate to oleate. The activities of the two tissues are different but of the same order of magnitude. With increasing age, the activity in the liver increases markedly, while the brain activity decreases. The postmitochondrial supernatant from adult (3-month-old) liver contains 2 to 3 orders of magnitude more stearoyl-CoA dehydrogenase activity than the brain postmitochondrial fraction. The washed microsomal fraction from liver had about the same activity as the postmitochondrial supernatant, but no dehydrogenase activity could be detected in the washed microsomal fraction from the brain. The acyl-CoA synthetase and the palmitoyl-CoA hydrolase activities measured in the washed microsomes from adult brain were both lower than in liver microsomes. The concentration of stearoyl-CoA (the substrate for the stearoyl-CoA dehydrogenase) resulting from the ratio of these activities was too high, however, for the lack of desaturase activity to have been simulated by lack of substrate.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase. A comparison of the modulation in vitro by phosphorylation and dephosphorylation to modulation of enzyme activity by feeding cholesterol- or cholestyramine-supplemented diets

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase) was considerably inhibited during incubation with ATP+Mg²⁺. The inactivated enzyme was reactivated on further incubation with partially purified cytosolic phosphoprotein phosphatase. The inactivation was associated with a decrease in the apparent K_m of the reductase for hydroxymethylglutaryl-CoA, and this was reversed on reactivation. The slight increase in activity observed during incubation of microsomal fraction without ATP was not associated with a change in apparent K_m and, unlike the effect of the phosphatase, was not inhibited by NaF. Liver microsomal fraction from rats given cholesterol exhibited a low activity of hydroxymethylglutaryl-CoA reductase with a low apparent K_m for hydroxymethylglutaryl-CoA. Mícrosomal fraction from rats fed cholestyramine exhibited a high activity with a high K_m. To discover whether these changes had resulted from phosphorylation and dephosphorylation of the reductase, microsomal fraction from rats fed the supplemented diets and the standard diet were inactivated with ATP and reactivated with phosphoprotein phosphatase. Inactivation reduced the maximal activity of the reductase in each microsomal preparation and also reduced the apparent K_m for hydroxymethylglutaryl-CoA. There was no difference between the preparations in the degree of inactivation produced by ATP. Treatment with phosphatase restored both the maximal activity and the apparent K_m of each preparation, but never significantly increased the activity above that observed with untreated microsomal fraction. It is concluded that hydroxymethylglutaryl-CoA reductase in microsomal fraction prepared by standard procedures is almost entirely in the dephosphorylated form, and that the difference in kinetic properties in untreated microsomal fraction from rats fed the three diets cannot be explained by differences in the degree of phosphorylation of the enzyme.     

Hepatic lipid metabolism. Age-related changes in triglyceride metabolism.

Age-related changes in hepatic triglyceride formation have been described in developing rats. Triglyceride formation was measured in vitro in the presence of [14C]glycerol-3-phosphate, palmitate, ATP, CoA, and Mg2+ by using liver homogenates and microsomal fractions derived from various age groups of animals. Triglyceride formation was most active in one-day-old rats and then decrease with age. The increase in triglyceride formation following birth was prevented by the administration of puromycin or by denying suckling. In addition, changes in plasma and hepatic triglyceride concentrations, were also determined as functions of age. These studies suggest that the age of the animal significantly influences triglyceride metabolism.