Enzymes involved in adenine nucleotide metabolism of developing chick embryo liver.

1. This paper describes the changes in the activity of adenylate deaminase, adenylate and inosinate phosphatase, and adenosine deaminase in the developing chick embryo liver. 2. The adenylate and inosinate phosphatase and adenosine deaminase activity appears considerably higher in chick embryo liver with respect to other chick embryo tissues previously examined. 3. During development the control exerted by ATP on AMP breakdown undergoes variations. Consequently, in the first period of incubation AMP is degraded by the direct pathway (AMP-IMP) and in the last period of incubation by the indirect pathway (AMP-adenosine). In the intermediate period (from the 12th to the 15th day of incubation) both pathways may be followed. 4. The ability to synthesize purine nucleotides through "salvage pathway" seems to be acquired by embryonic liver at least at the 15th day.     

CHICK BRAIN CREATINE KINASE ONTOGENY BEFORE THE ADVENT OF CREATINE AND INSULIN

The ontogeny of brain creatine kinase (CK) was studied during chick embryo development. The cytosolic activity increased 270% in 10 h from the 2nd to the 3rd days of incubation; this was followed by a plateau phase throughout development and at the end of incubation there appeared to be another increase of cytosolic and mitochondrial CK activities. Therefore, early embryonic chick brain CK is another‘constitutive’enzyme like the early embryonic chick heart CK since creatine has not been enzymatically detected in the embryo until day 4 of incubation. Insulin does not appear to stimulate the early increase of brain CK activity since the hormone is not present in the embryo until day 5 of incubation. It is likely that CK increase is associated with neuronal multiplication at early stages and possibly to neuronal maturation before hatching.     

Gene activation of alcohol dehydrogenase in Japanese quail and chicken-quail hybrid embryos

No preferential activation of the maternally derived alcohol dehydrogenase (ADH) allele was found in any of the chicken male x Japanese quail female hybrids examined. ADH activity in the liver was, in fact, found to exist in two different cathodal zonal regions on starch gel electropherograms; the zone II bands appeared at day 5 of incubation in the quail embryo (day 6 in the hybrid embryo) and the zone I bands appeared in 9-day quail embryos (10-day hybrid embryos). By day 13 of incubation, only the faster-migrating zone I bands could be detected in both quail and hybrid embryos.     

Developmental changes in the activity of membrane-bound gamma-glutamyl transpeptidase and in the sialylation of synaptosomal membranes from the chick embryonic brain.

gamma-Glutamyl transpeptidase (GGT) is a membrane-bound sialoglycoprotein. The developmental changes in GGT activity and in sialic acid content were determined in a crude synaptosomal membrane fraction from the cerebral hemispheres of the chick embryo between days 11 and 19 of incubation. The GGT activity increased almost eightfold during the examined developmental period, while sialic acid content rose significantly only between days 11 and 15. Cortical administered on day 13 significantly increased GGT activity. On the other hand, the content of membrane bound sialic acid was not substantially affected. The value of the GGT apparent Michaelis constant (Kmapp) for gamma-glutamyl-p-nitroanilide in the presence of 20 mmol.l-1 glycylglycine was 1.5 mmol.l-1 and cortisol did not influence it. However, Vmax was increased by this hormone. The affinity of GGT to concanavalin A (ConA) did not change during development. Neither the administration of cortisol nor neuroaminidase treatment had any effect on the interaction of GGT with ConA. Desialylation of crude synaptosomal fraction did not change GGT activity. The results presented here suggest no developmental nor functional relationship between the activity of GGT and the level of sialylation in synaptosomal membranes from the cerebral hemispheres of the chick embryo.     

Toxic effects of phencyclidine on developing chick embryo brain

We studied the effects of Phencyclidine (PCP, Angel Dust) on the developing chick embryo brain. In Group-1, the eggs were injected with PCP on the 7th day of incubation and the embryo brains were studied on the 10th day. In Group-2, eggs were injected twice; first on the 7th day and then on the 10th day of incubation. Group-2 brains were then studied on the 16th day of incubation. PCP significantly depressed the development of embryo brains. Cerebral hemisphere weight, total protein and total DNA were significantly lower on day 10 of incubation in Group-1. Similar results were observed in Group-2. Concomitantly, the concentration of brain serotonin at day 10 was also significantly reduced when PCP was injected into the eggs on the 7th day of incubation. Since serotonin has been reported to influence development of the chick embryo brain, the present finding of the effect of PCP on brain development might be a secondary phenomenon. The possible implications of the effects of PCP on human brain development are also discussed.     

Creatine regulation in the embryo and growing chick

1. The absence of creatine was demonstrated enzymically in the hen's-egg yolk and in the albumin contrary to former reports. 2. A comparison of the results obtained by enzymic and colorimetric methods to measure creatine is presented. 3. Creatine phosphate was not detected in the yolk extracts. 4. The content of free arginine enzymically assayed was 15.7mumol in the yolk and 3.38mumol in the albumin. Arginine amounts to practically all of the guanidine compounds in the yolk and one-half of those in the albumin. 5. No glycine amidinotransferase activity was found in the egg-yolk homogenates. 6. The heart of the chick embryo does not receive creatine from the egg and the creatine kinase activity present in this organ starting from the 27th hour of incubation suggests that the enzyme is a constitutive one working probably as an adenosine triphosphatase in a way similar to the kinase isolated from rabbit skeletal muscle. 7. Liver glycine amidinotransferase activity appeared clearly after day 5 of incubation. The specific activity reached a maximum at day 12 and then declined; however, the activity per total mass of liver increased steadily during all the prenatal period. Concomitantly with this steady increase a rise in the creatine content of the whole embryo was observed. An analogous increasing relationship between total liver amidinotransferase activity and liver creatine content was also detected during the postnatal period. 8. Repression of amidinotransferase by creatine cannot be accepted as occurring under physiological conditions since an inverse relationship between the two parameters was not observed. 9. Repression of liver amidinotransferase is observed only when pharmacological concentrations of the exogenous creatine are present in the chick liver.     

Vitamin A economy of the developing chick embryo and of the freshly hatched chick

1. The changes in the net amounts of retinol, retinyl esters and retinal in both the developing chick embryo and the newly hatched chick were investigated. The embryo requires about 68nmol of the vitamin for its growth, whereas the baby chick requires about 108nmol during the first 7 days after hatching. 2. Retinal was present in the egg in fairly high concentrations at the beginning of the incubation but it virtually disappeared from the extra-embryonic tissue after day 17 of incubation. It was not found in the liver of the embryo or of the newly hatched chick up until day 7.     

Studies on enzymes of nitrogen metabolism, and nitrogen end products in the developing chick (Gallus domesticus) embryo.

1. A total of 450 fertilized eggs were used to study the concentrations of uric acid, urea and ammonia in allantoic and amniotic fluids, and some enzymes of nitrogen metabolism in the liver and kidney during the development of the chick embryo from the 5th to 21st day of incubation. 2. Concentrations of the compounds studied were higher in allantoic fluid. The molar concentration of allantoic uric acid increased steadily with time. The pattern of urea and ammonia in both allantoic and amniotic fluids were the same. 3. Arginase (E.C.3.5.3.1) activity in both embryonic kidney and definitive kidney was higher than that in the liver. The specific activity of arginase (mumole urea formed/hr per g wet wt kidney) dropped during development. 4. Little arginine synthetase activity (argininosuccinate synthetase, E.C.6.3.4.5; and argininosuccinate lyase, E.C.4.3.2.1) was found in kidney, but none in the liver. 5. The complete urea cycle function was absent in both the liver and the kidney of the chick embryo.     

Glycogen metabolic enzymes in the skeletal muscles of the developing chick embryo

In the skeletal muscles of the chick embryo from the 10th till the 15th day of embryogenesis, phosphorylase (EC. 2.4.1.1) is represented by two isozymes one of which corresponds, by electrophoretic mobility, to the liver phosphorylase and another to phosphorylase of the skeletal muscles of the adult rat. From the 17th day of embryogenesis on only one isozyme of phosphorylase is found in the skeletal muscles which is identical with that of the skeletal muscles of the adult bird. The isozyme spectrum of phosphorylase of the whole 4 days old embryo contains, besides phosphorylase L, a special "embryonic" isozyme which differs from that of the skeletal muscles by immunochemical characteristics and electrophoretic mobility. From the 10th day of embryogenesis till hatching, the activity of phosphorylase of the skeletal muscles increases more than 50 times and that of glycogen synthetase (EC. 2.4.1.11) only 4 times.     

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: ontogeny in chick embryo liver

Aryl hydrocarbon hydroxylase (AHH, cytochrome P1-450) is induced in chick liver very early during embryonic development if embryos are treated with 3-methylcholanthrene-type compounds such as 3,4,3'4'-tetrachlorobiphenyl. In mammals, AHH induction is known to be mediated by the Ah receptor. Liver from embryonic and newly hatched chicks was found to contain a cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which has properties that are very similar to properties of the Ah receptor previously characterized in mammalian tissues. In chick embryo liver, cytosolic binding sites for TCDD were of high affinity (Kd for [3-H]-TCDD = 0.2 nM) and were specific for 3-methylcholanthrene-type inducers. The specific binding component sedimented at about 9S on sucrose density gradients prepared at low ionic strength. A high level of Ah receptor was detected in chick embryo liver by the fifth day of incubation (5 DI); this is at least 24 hours prior to the onset of AHH inducibility. The Ah receptor concentration increased from 5 DI to 8 DI, the period when chick liver is undergoing early morphological differentiation. After 8 DI, Ah receptor levels dropped substantially and remained low into the posthatching period. In contrast, AHH inducibility was high by 7 DI and remained high throughout embryonic development and into the posthatching period. The discrepancy between Ah receptor levels and the degree of AHH inducibility suggests that only a small fraction of the Ah receptor population is required for maximal AHH induction.     

Choline acetyl transferase activity in chick embryo neuroretinas during development in ovo and in monolayer cultures

The specific activity of the enzyme choline acetyl transferase (CAT) in chick neuroretinas was investigated during in ovo development and in monolayer cultures. The enzyme activity was barely detectable on the 6th day of incubation but increased markedly between the 7th and 11th days. The activity increased sharply between the 15th and 17th days and then slowly until hatching. When cell suspensions from 6- to 7-day neuroretinas were cultured as monolayers, CAT specific activity increased rapidly. After 4–5 days in culture, the activity of the enzyme was identical to that found in the neuroretina on the 11th day of incubation. Cells from 9-day neuroretinas also differentiate in monolayer cultures, but with a more irregular pattern. These data show that cholinergic neurons from chick embryo neuroretina differentiate in monolayer cultures without a lag and at the same rate as in vivo.     

Induction of UDPglucose dehydrogenase during development, organ culture, and exposure to phenobarbital. Its relation to levels of UDPglucuronic acid and overall glucuronidation in chicken and mouse.

Liver UDPglucose in early chick-enbryo has, by the 19th day of incubation, reached levels existing in young hatched (White Leghorn) chicks. In developing ASH/TO mouse liver, the dehydrogenase is low, but increases sharply at late foetal and weaning stages; adult activity is greater in females than males. The UDPglucuronic acid content of embryo liver from at least 12 days resembles that of adult chicken; in mouse liver it rises over birth and infancy. These differences in relative rates of development of enzyme and nucleotide in the 2 species can explain why overall glucuronidation by liver appears in chick rapidly after hatching, but in mouse only gradually during infancy. UDPglucose dehydrogenase increases in embryo liver, probably by induction, 2-3-fold during culture with phenobarbital and some 5-fold when exposed to the drug in ovo. Phenobarbital treatment also increases the enzyme in late foetal and adult mice, abolishing the sex difference. Differences between induction of UDPglucose dehydrogenase and UDPglucuronyl transferase during development, culture and phenobarbital treatment indicate that control mechanism for these two enzymes are not directly linked.     

Lipid and carbohydrate metabolism in euthyroid and hypothyroid chick embryo (Gallus domesticus).

1. The effect of propylthiouracil (PTU)-induced hypothyroidism on carbohydrate and lipid metabolism was studied in the chick embryo. 2. A single dose of PTU (250 micrograms/embryo) was administered on day 11 and embryos sacrificed on day 20 of incubation. 3. Thyroid glands were significantly enlarged (6 fold) by PTU administration. 4. Increased thyroid weight was associated with growth retardation and decreased plasma thyroxine levels. 5. Plasma glucose level was lower and phospholipids were significantly higher in the hypothyroid embryo. 6. Liver lipid concentrations in the control and hypothyroid embryos were not different but were significantly higher in both groups when compared to previously reported values in the young chick. 7. In contrast to PTU treatment after hatching, liver glycogen levels were not increased in the hypothyroid chick embryo. This was attributed to the high lipid nutrient condition of the chick embryo since a high lipid diet in the young chick decreased hepatic glycogen accumulation significantly.     

Developmental Changes in the Activity of Xanthine Dehydrogenase and closely Related Enzyme in Chick Tissues

Xanthine-oxidizing activities in the chick tissues were measured by a couple of assay procedures during development of chick embryo. With the usual assay using pterine and NAD⁺, no detectable level of XDH activity was observed in the liver and little in the duodenum before hatching, whereas an appreciable activity was detected in the kidney of chick embryo. When assayed with xanthine and dichlorophenol indophenol, an XDH-like enzyme activity was significantly detected in the embryonic liver, while no further enhancement of the activity was detected in the kidney and duodenum. Electrophoregrams obtained with samples from various developmental stages, followed by activity staining with tetrazolium dye, supported the above results and revealed that the embryonic XDH-like enzyme is not distinguishable from XDH of adult tissue in molecular size. This XDH-like enzyme, pre-existing in the liver before hatching, however, exhibited no cross reaction with antibody against intact XDH. The nature of this material was discussed in comparison with deflavinated XDH.     

Hepatic alcohol dehydrogenase activity in chick hepatocytes towards the major alcohols present in commercial alcoholic beverages: comparison with activities in rat and human liver

We have compared hepatic alcohol dehydrogenase activities in chick, rat and human liver with the major alcohols in commercial alcoholic beverages. 1. Chick and rat hepatic alcohol dehydrogenase was greater when assayed at a physiological pH in buffer containing chloride ions, as compared with the activity in pyrophosphate buffer at alkaline pH. 2. In contrast to reports of instability of ADH to freezing, we found the enzyme from all three species stable to freezing in 0.25 M sucrose. 3. Rat liver enzymatic activity was unstable in the presence of substrate, where as that of chick and human was not. 4. For all three species, the Km of hepatic ADH for substrate decreased with increasing chain length of alcohols. In both chick and human samples, the Vmax values for the higher chain alcohols were similar to that with ethanol, while in rat samples, ADH activity was dramatically lower with the higher chain alcohols compared to ethanol.     

Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.     

Developmental Changes in β-Citryl-L-G1utamate Concentration and Its Synthetic and Hydrolytic Activities in Neuronal Cells Cultured from Chick Embryo Optic Lobes

Developmental changes in the concentration of beta-citryl-L-glutamate(beta-CG) have been examined in the cerebrum and optic lobe of the developing chick brain and in primary cultured neuronal cells from the chick embryo optic lobes with gas chromatographic and HPLC methods originated in our studies. A sharp peak was shown by beta-CG, with a maximal concentration at 13 days of incubation in the optic lobe of the developing chick brain but decreasing markedly to adult levels. The developmental change in primary cultured neurons was similar to that in the optic lobe of the developing chick brain. Changes in synthetic and hydrolytic activities of beta-CG were studied during growth of primary cultured neurons. Incorporation of radioactivities from radiolabeled pyruvate and alanine into beta-CG increased significantly on day 3 of culture, reaching a plateau on day 6, whereas that from radioactive glutamine and glutamate increased gradually from day 3 to day 12 of culture. The hydrolyzing enzyme activity of beta-CG during neuron growth was low until day 3 of culture, when it increased significantly until day 12. Similar developmental changes were observed in the developing chick embryo optic lobes.     

The presence of peroxisomal carnitine palmitoyltransferase in chick embryo liver

Hepatic peroxisomes and mitochondria from 20-day-old chick embryo were separated by sucrose density gradient centrifugation and the characteristics of carnitine acyltransferases in these organelles were studied. The carnitine acyltransferase activities in peroxisomes were increased markedly by the treatment of chick embryo with clofibrate, while those in mitochondria did not change. In the liver of clofibrate-treated chick embryo, approximately 50% of total liver carnitine palmitoyltransferase (CPT) activity was present in the peroxisomal fraction. Peroxisomal CPT activity was easily solubilized, in contrast with mitochondrial CPT. The solubilized protein solutions from isolated peroxisomes and mitochondria were separately chromatographed on a column of Blue Sepharose CL-6B after the gel filtration on Sephadex G-25. Peroxisomal CPT was completely bound to a Blue Sepharose CL-6B column and was eluted below 0.25 M KCl, whereas mitochondrial CPT was not retained on the column. The substrate specificity profile of peroxisomal CPT with long-chain acyl-CoAs (C8 to C18) was similar to that of mitochondrial CPT, and the apparent Km value of peroxisomal CPT for palmitoyl-CoA was 5.2 microM, being similar to that of mitochondrial CPT. It is concluded that carnitine long-chain acyltransferase, which is different from mitochondrial CPT and is induced by clofibrate treatment, is present in peroxisomes of chick embryo liver.     

Physical and enzymatic properties of a class III isozyme of human liver alcohol dehydrogenase: chi-ADH

chi-Alcohol dehydrogenase (chi-ADH), a class III isozyme characterized by its anodic electrophoretic mobility and lack of inhibition by 4-methylpyrazole, has been isolated from human liver and purified to homogeneity in a reducing medium. chi-ADH resembles other human liver ADH isozymes of classes I and II with respect to its molecular weight, dimeric structure, stoichiometry of zinc and NADH binding, and pH optima for the oxidation of alcohols. This homodimer exhibits subtle differences in its absorption spectrum and amino acid composition relative to those of other human isozymes but differs markedly from their specificity toward alcohols and aldehydes. chi-ADH oxidizes ethanol very poorly. The reaction is bimolecular, and an apparent Km cannot be discerned up to 2.3 M ethanol. The enzyme is inactive toward methanol, ethylene glycol, digitoxigenin, digoxigenin, and gitoxigenin , but alcohols with carbon chain lengths greater than four are oxidized rapidly with Km values decreasing with increasing carbon chain length. Taken jointly, the composition, structure, and enzymatic properties of the ADH isozymes purified and studied so far strongly imply that their metabolic roles, yet to be discovered, will give a new perspective to ethanol metabolism and pathology.