Cytosolic and mitochondrial isoenzymes of branched-chain amino acid aminotransferase during development of the rat.

The isoenzymic forms of branched-chain amino acid aminotransferase in mitochondria of rat tissues were compared with the better-known cytosolic forms in order to find any regular pattern of expression of these isoenzymes during development. Mitochondria of all tissues examined except brain contained only a type-I isoenzyme differing from the cytosolic type-I isoenzyme in heat stability and activation by mercaptoethanol. Foetal and adult brain mitochondria contained isoenzymes type III as well as type I. The large excess of type-I isoenzyme in foetal liver was localized in mitochondria, apparently of haematopoietic cells. The activity of this isoenzyme declined precipitously (by 80%) from day 19 of gestation at the same period and rate as does the volume fraction of haematopoietic cells that are then leaving the liver. Cortisol treatment accelerated the loss of these cells, and proportionally accelerated loss of the mitochondrial isoenzyme I. A development succession of type-I isoenzyme by the unique type II of liver parenchymal cell cytosols could not be demonstrated, since small, about equal, amounts of types I and II were always present in cytosols of foetal and adult liver. Developmental succession of isoenzymes within tissues was limited to cytosols and was demonstrated by the presence of cytosolic isoenzyme III in foetal and newborn skeletal muscle and kidney, organs which contain only isoenzyme I in the adult.     

Adenylate kinase isoenzyme patterns in normal and neoplastic human lung, and in various adult as compared to fetal rat tissues

The isozyme pattern and total activity of adenylate kinase were studied in normal adult and fetal human and rat tissues using starch gel electrophoresis. Three adenylate kinase isoenzymes were identified in human tissues. Although normal adult lung exhibited higher adenylate kinase activity than did its fetal or neoplastic variant, isozyme patterns in the three types of tissues were indistinguishable from each other and from that in fetal human liver. The pattern of these three isozymes in rat lung (as in spleen) also did not change between fetal and adult life. However, adult kidney and heart of this species did appear to contain isozymes not present in fetal life. Brain (both adult and fetal) was striking different from all the other tissues in that it contained only one adenylate kinase isozyme. The total adenylate kinase activity per gram of adult rat liver, kidney and lung was significantly higher than in the cognate fetal organs, whereas that in brain or spleen did not change with age. The activity in adult heart (similar to the fetal one) was higher than in any other tissue examined.     

Isoenzyme patterns in parenchymal and non-parenchymal cells isolated from regenerating and regenerated rat liver

Parenchymal and non-parenchymal cells were isolated from adult rat liver that had been fully regenerated after a 70% partial hepatectomy. The characteristics of the parenchymal cell preparations from regenerated rat liver indicated that they were a homogeneous population and comparable with parenchymal cells isolated from intact liver. The parenchymal cells from regenerated adult rat liver contain glucokinase, hexokinase, pyruvate kinase type I and aldolase B. The non-parenchymal cells contain hexokinase, pyruvate kinase type III and aldolase B. When cells were isolated at different times of the day from rats on controlled feeding schedules, variation of tyrosine aminotransferase activity and liver glycogen content were observed in the parenchymal cells in keeping with the reported diurnal oscillations found in whole liver extracts. When parenchymal cells were isolated from rats 48 and 72h after partial hepatectomy, different isoenzyme patterns were observed. These cells appeared to synthesize pyruvate kinase type III, a function that was assigned previously to non-parenchymal cells or to foetal rat liver hepatocytes.     

Hexokinase isoenzymes in tissues of the adult and developing guinea pig

Changes in the activities and isoenzyme distribution of hexokinase were determined in a number of tissues during the development of the guinea pig. The total activity in the fetal liver showed a large fall during the second half of gestation to reach adult values by term. With normal diet the fetal, neonatal, and adult livers had isoenzymes I and III but little or no detectable IV (glucokinase). The fetal liver had predominantly type I, but the proportion of type III increased during development. The kinetics of the guinea pig isoenzymes were similar to those reported for the rat. Two additional isoenzymes with mobility between I and II were detected in the fetal liver and blood. They appear to have kinetic properties similar to type I. Detectable liver glucokinase activity was induced by glucose administration to adult guinea pigs. The total activity in kidney, brain and skeletal muscle showed a postnatal rise while in the fetal heart it was high and declined after birth. These tissues contained predominantly type I with varying proportions of type III hexokinase. The ratio of particulate-bound to soluble hexokinase varied from tissue to tissue. All except the liver showed a significant increase in binding after birth. The changes are discussed in relation to the control of glucose utilization in the fetal and neonatal periods.     

The distribution of enzyme and isoenzyme activities between parenchymal and haematopoietic cells in the liver of the foetal guinea pig

The hepatocyte and haematopoietic cell contents of the liver of the foetal guinea pig were measured over the latter half of gestation. Hepatocytes represented about 30% of liver volume at mid-gestation and this increased to 70-80% by term; cell volume remained fairly constant until 5-7 days before term, then more than doubled. Haematopoietic cells represented about 5% of liver volume at mid-gestation and this progressively fell to <1% by term. At 75% of gestation hepatocytes and haematopoietic cells were prepared from perfused foetal livers by collagenase digestion. Enzyme activity of the hepatocyte was, without exception, similar to that of the whole liver. In general, enzyme activity in the haematopoietic cells was similar to that in erythrocytes, with relatively low values for aldolase, glycerol 3-phosphate dehydrogenase, phosphoglycerate mutase, enolase, lactate dehydrogenase, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, isocitrate dehydrogenase, ;malic' enzyme, glutamate dehydrogenase and aspartate aminotransferase. The haematopoietic cell contribution to total enzyme activity in the foetal liver was usually much less than 10% and could thus not account for the major changes in hepatic enzyme activity over the latter half of gestation. Hepatocytes contained hexokinase isoenzymes I and III, aldolase isoenzymes A and B and pyruvate kinase isoenzymes 1, 2 and 4. The haematopoietic cells contained hexokinase isoenzyme I and two additional bands of activity with slightly greater mobility, aldolase isoenzyme A and pyruvate kinase isoenzymes 2 and 4.     

Uridine kinase activities in developing, adult and neoplastic rat tissues.

Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3--5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.     

Role of adenylate kinase, AMP deaminase and 5'-nucleotidase in the metabolism of adenylic nucleotides

The activities of adenylate kinase, AMP-deaminase and 5'-nucleotidase in various tissues of the rat were studied. The activity of the forward adenylate kinase reaction (ATP + AMP----2 ADP) against the back one (2 ADP----ATP + AMP) was predominant. The liver was shown to contain two, while the blood serum--three adenylate kinase isoenzymes. In the skeletal muscles, the catabolism of adenylic acid involving AMP-deaminase and 5'-nucleotidase predominantly occurred via deamination, in the liver--via dephosphorylation, while in the leucocytes, erythrocytes and blood serum the activity of these processes was essentially the same. In vitro, ATP enhanced the activity of AMP-deaminase in the liver, leucocytes and erythrocytes and decreased it in the blood serum. Under effects of ATP, the activity of 5'-nucleotidase in the leucocytes and blood serum was markedly elevated, that in the liver and erythrocytes was unaffected.     

Glycolytic isoenzymes and glycogen metabolism in regenerating liver from rats on controlled feeding schedules

Intact rats trained on a controlled feeding and lighting schedule designated ;8+16' exhibited diurnal oscillations in liver weight, glucokinase activity and liver glycogen content. Glucokinase activity expressed as units/g of liver decreased to 30% of that from unoperated controls during the first 48h after partial hepatectomy and returned to near normal values in 2 weeks. When the glucokinase activity was expressed as units/liver per 100g body wt., a decrease to 50% of control activity was observed between 24 and 48h after the operation. A similar pattern was found for pyruvate kinase type I. In contrast, pyruvate kinase type III activity increased after partial hepatectomy. It is suggested that the newly divided cells after partial hepatectomy do not synthesize glucokinase and pyruvate kinase I but do synthesize pyruvate kinase III. Glycogen was found to accumulate as early as 24h after partial hepatectomy, and normal concentrations were reached after 48h if the operation was performed at times other than during the feeding periods.     

Multiple forms of uridine kinase in normal and neoplastic rat liver

Two species of uridine kinase with molecular weights of approximately 120000 (I) and 30000 (II) have been identified in the rat liver system. Species I predominates in the 7-day postnatal and adult rat liver and increases in the regenerating remnant of the latter after partial hepatectomy; the concentration of species II is low in these tissues. Species I also predominates in the slow-growing hepatomas 5123D and 7800. In contrast, II is the predominant form in the foetal rat liver and accounts for 40% of the total activity in the rapidly growing Novikoff ascites hepatoma. In contrast to species II, which was stable, species I was inactivated by preincubation for 30min at 37 degrees C, before assay at 23 degrees C.     

Calcium . calmodulin-dependent protein kinase II and calcium . phospholipid-dependent protein kinase activities in rat tissues assayed with a synthetic peptide

Rat tissue levels of Ca2+ . calmodulin-dependent protein kinase II (protein kinase II) and Ca2+ . phospholipid-dependent protein kinase (protein kinase C) were selectively assayed using the synthetic peptide syntide-2 as substrate. The sequence of syntide-2 (pro-leu-ala-arg-thr-leu-ser-val-ala-gly-leu-pro-gly-lys-lys) is homologous to phosphorylation site 2 in glycogen synthase. The relative Vmax/Km ratios of the known Ca2+-dependent protein kinases for syntide-2 were determined to be as follows: protein kinase II, 100; protein kinase C, 22; phosphorylase kinase, 2; myosin light chain kinase, 0.005. Levels of protein kinase II were highest in cerebrum (3.36 units/g tissue) and spleen (0.85 units/g) and lowest in testis (0.05 units/g) and kidney (0.04 units/g). Protein kinase II activity was localized predominantly in the 100,000g particulate fraction of cerebrum and testis, in the supernatant fraction of heart, liver, adrenal, and kidney, and about equally distributed between particulate and supernatant in spleen and lung. Likewise, protein kinase C activity was highest in cerebrum (0.56 units/g) and spleen (0.47 units/g), and the majority of activity was present in the cytosolic fraction for all tissues measured except for cerebrum and testis in which the kinase activity was equal in both fractions. Finally, the ratios of protein kinase II to protein kinase C were different in various rat tissues and between particulate and supernatant fractions. These results suggest somewhat different functions for these two Ca2+-regulated, multifunctional protein kinases.     

Increase in liver and kidney deoxycytidine kinase activity linked to neoplastic transformation

Deoxycytidine kinase activity in normal rat liver cytosol was low (0.8 nmol/hr/mg protein); it increased 2–26-fold in 12 lines of chemically-induced, transplantable rat hepatomas of different growth rates. The increased kinase activity correlated positively with the hepatoma growth rate. The kinase activity did not change in the regenerating liver and the activity in the differentiating, neonatal rat liver was similar to values in adult liver. Deoxycytidine kinase activity in 2 chemically-induced, transplantable rat kidney tumors was increased to twice the value found in normal renal cortex. Among 15 normal rat tissues examined the highest kinase activities were observed in thymus, bone marrow and spleen. Of the normal and malignant rat tissues tested, only testis had detectable cytidine deaminase activity.     

Thymidine kinase in rat liver during development

1. The activity of thymidine kinase in rat liver supernatant decreased with development to a value in the adult that was 1% of that in the 17-day foetus. 2. The foetal enzyme was more stable than the adult to gel filtration on Sephadex G-25 at 0 degrees . 3. The greater stability of the foetal enzyme to incubation at 45 degrees was attributable to the presence of higher concentrations of nucleotides in foetal liver supernatant. 4. The K(m) values for foetal and adult enzymes were approx. 2.5mum- and 2.1mum-thymidine respectively. 5. The foetal enzyme was more sensitive to inhibition by thymidine triphosphate. 6. The decline in enzyme activity during the neonatal period was correlated with a shift in the enzyme properties from the foetal to the adult type, and may reflect the decrease in the proportion of haemopoietic tissue in the liver.     

Differential activation of type I and type II 3',5'-cyclic adenosine monophosphate-dependent protein kinases by growth hormone-releasing factor

Rat GH-releasing factor (rGRF) stimulated GH release and intracellular cAMP accumulation in cultured rat anterior pituitary cells with EC50 values of approximately 10 and 150 pm, respectively. Consistent with an effect on cellular cAMP levels, rGRF stimulated the adenylate cyclase activity of rat anterior pituitary membranes with an EC50 value of approximately 60 pm. Using antisera directed against the regulatory subunits of type I and II cAMP-dependent protein kinases, these enzymes were immunoprecipitated from the cytosolic fraction of cultured cells in order to monitor the degree of their activation by rGRF. Both isoenzymes were rapidly activated in cells incubated with rGRF but with different kinetics; full activation of protein kinase I was evident within 3-5 min and activation of protein kinase II occurred between 5 and 15 min. The magnitude of activation was differentially regulated by rGRF in a concentration-dependent manner. Somatostatin only partially attenuated rGRF-stimulated GH release, cAMP accumulation, and adenylate cyclase activation. Somatostatin was effective in partially antagonizing activation of protein kinase II at all concentrations of rGRF and of protein kinase I only at intermediate concentrations of rGRF. The significance of this rGRF-induced differential activation of the two isoenzymes of cAMP-dependent protein kinase is discussed in terms of the multiple effects of rGRF on somatotropic cells of the rat anterior pituitary.     

Antithrombin III mRNA in adult rat liver and kidney and in rat liver during development

Using Northern blots the size of antithrombin III (AT III) mRNA in rat liver was found to be 1650 nucleotides. Adult rat kidney also contained a slightly smaller mRNA at about 20% the level in liver. The ontogeny of AT III mRNA in the liver was assessed by dot blot hybridization. The mRNA was detectable at the earliest age examined (14th day of gestation) at about 15% of the adult levels. After the 17th day of gestation the levels of antithrombin III mRNA rise reaching 50% of adult levels at birth. After birth the mRNA levels rise to 75% of adult levels by the 5th day and reach adult levels by 40 days after birth. We suggest that foetal AT III is produced by both the foetal liver and by placental transfer of the maternal inhibitor.     

Distribution of glutamine hexosephosphate aminotransferase in rat tissues; changes with state of differentiation

The activity of glutamine hexosephosphate aminotransferase (L-glutamine: D-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was determined with an improved assay method in some three dozen rat tissues: adult, developing and neoplastic. The highest activities (20–200 units/g) were seen in colon, mammary (during late lactation), submaxillary, sublingual and parotid glands, placenta and liver. The activity increased strikingly along the length of the intestine; glucose feeding inhibited it in ileum and colon. In liver and intestine the activity increased with age but in brain, muscle, heart and kidney the activity was considerably higher during fetal (7.1–12.8 units/g) than in adult life (0.8–3.5 units/g). Renal, mammary and muscle tumors (but not hepatomas) had much higher activities (4–20.5 units/g) than the cognate normal adult tissue.The distribution pattern among tissues indicates that glutamine hexosephosphate aminotransferase is of general importance to all growing, undifferentiated tissues and of special importance to the differentiated function of particular adult organs. The latter are organs which engage in glycoprotein secretion. The results support the assumption that glutamine hexosephosphate aminotransferase activity is essential for glycoprotein synthesis.     

Comparison of the properties of two forms of pyruvate kinase in rat liver and determination of their separate activities during development

1. Two forms of hepatic pyruvate kinase, designated type L and type M, were distinguished on the basis of kinetic, chromatographic, electrophoretic and immunological criteria. They were partially purified and their properties compared with each other and with the purified enzyme from skeletal muscle. 2. In contrast with type L, the type M enzyme showed no marked evidence of co-operative interactions with phosphoenolpyruvate and was not stimulated by fructose diphosphate. 3. The activity profiles of type L and type M enzymes were determined in developing rat liver by utilizing differences in the kinetic properties of the two forms. The high activity of type M enzyme in the early foetal rat decreased in late gestation and immediately after birth to reach a low value, which remained essentially constant for the remainder of the developmental period. The activity of type L enzyme, in contrast, was low in the early foetal and neonatal liver but increased markedly at the onset of weaning. 4. Possible roles of the two forms of hepatic pyruvate kinase in the control of glycolysis and gluconeogenesis are discussed.     

Developmental regulation of type II calcium/calmodulin-dependent kinase isoforms in rat cerebellum

Two distinct isoforms of a Type II calcium/calmodulin-dependent protein kinase were separated from high-speed supernates (cytosol) of rat neonatal [postnatal day 10 (P10)] and adult [postnatal day 40 (P40)] cerebellum using cation-exchange chromatography. The isoenzymes contained variable amounts of three subunits of apparent Mr's of 50 kDa (alpha), 58 kDa (beta'), and 60 kDa (beta). The specific activity of calmodulin-dependent kinase (CaM kinase II) in crude homogenates increased sixfold between P10 and P40 using exogenous MAP 2 as substrate. Cytosol from cerebellum at P40 contained a predominant isoform (approximately 40% of total cytosolic activity) with a 1:5 molar ratio of alpha:beta',beta subunits that eluted with 150 mM NaCl (designated 150) and a less abundant isoform (approximately 20% of total cytosolic activity) containing a 1:8 molar ratio of alpha:beta',beta subunits that eluted with 350 mM NaCl (designated 350). In neonatal cerebellum at P10, the relative abundance of the two isoforms was reversed such that approximately 50% of the cytosolic calmodulin-dependent kinase activity was recovered in the 350 isoform, whereas only 20% of the total cytosolic kinase activity was recovered in the 150 isoform. Previous studies indicate that cerebellar granule cells may contain an all beta',beta isoform of CaM kinase II that lacks alpha subunit. Thus, to assess the cell-specific localization of kinase isoforms within cerebellum, cytosol prepared from primary cultures of rat cerebellar granule cells was applied to cation-exchange chromatography and analyzed for calmodulin-dependent kinase activity. The cells contained both isoforms of the kinase that were present in fresh tissue suggesting that granule cell-enriched cultures express all three kinase subunits. The data demonstrate that rat cerebellum contains unique mixtures of CaM kinase II isoenzymes and that their expression is developmentally regulated.     

Hepatotropin mRNA expression in human foetal liver development and in liver regeneration

A 569 bp probe against the β-chain of hepatotropin was used to examine expression of RNA for this growth factor in human adult and foetal liver, foetal kidney and pancreas, and rat liver after partial hepatectomy. Low level expression of a 6kb RNA occurred in human adult and normal rat liver. 70% hepatectomy increased expression, peaking at 10 h and returning to near normal levels 24 h after resection. The 6 kb band was strongly expressed in human foetal liver, as compared with adult, but not in foetal kidney or pancreas, suggesting a major role for hepatotropin in both foetal development and regeneration of the liver.     

Development of gluconeogenesis in neonatal rat liver. Effect of triamcinolone

1. The normal development of the key enzymes of gluconeogenesis in rat liver, glucose 6-phosphatase, hexose diphosphatase, phosphopyruvate carboxylase and pyruvate carboxylase, was measured during the neonatal period. 2. Glucose 6-phosphatase, hexose diphosphatase and pyruvate carboxylase are all present in the late foetal liver, but all the enzymes show an increase in activity after birth. 3. Phosphopyruvate carboxylase is not present in liver extracts from foetal rats, but activity appears immediately after birth and increases rapidly over the first day and then more slowly to reach its maximum at the fourth postnatal day. 4. The fluorinated synthetic glucocorticoid, triamcinolone, was administered to foetal rats at various gestation times by intraperitoneal injection in utero and the animals were killed at intervals between 4 and 48hr. later. 5. The administration of triamcinolone results in slight depression of glucose 6-phosphatase, and a more significant depression of hexose diphosphatase to about one-half its normal activity in foetal rat liver. 6. Triamcinolone injection is without effect on pyruvate carboxylase activity and does not result in premature appearance of phosphopyruvate carboxylase in foetal rat liver. 7. Pyruvate kinase and aspartate amino-transferase activities in foetal rat liver are both depressed by triamcinolone treatment, whereas phosphofructokinase activity is elevated. 8. Tyrosine amino-transferase activity in foetal rat liver is markedly elevated in animals exposed to triamcinolone for 10hr. or more, but the effect is only observed in animals close to term. 9. The results are discussed in relation to mechanisms involved in the initial synthesis of tissue-specific enzymes in developing tissues, and it is concluded that glucocorticoids do not initiate the synthesis of the gluconeogenic enzymes.     

Aldehyde dehydrogenase in 2-acetaminofluorene-induced rat hepatomas. Ontogeny and evidence that the new isoenzymes are not due to normal gene de-repression

The pre- and post-natal ontogeny of Sprague-Dawley rat liver aldehyde dehydrogenase [aldehyde-NAD(P)(+) oxidoreductase, EC 1.2.1.5] is described. At no time in its ontogenetic development does normal liver aldehyde dehydrogenase exhibit any of the characteristics of a series of unique aldehyde dehydrogenases that can be isolated from 2-acetamidofluorene-induced rat hepatomas. Enzyme activity is first detectable in 15-day foetal liver and gradually increases throughout pre- and post-natal development until adult activities are attained by day 49 after birth. Electrophoretically, normal aldehyde dehydrogenase, throughout its ontogeny, exists as the same single isoenzyme found in normal adult liver. Isoelectric points for two normal liver isoenzymes demonstrable by isoelectric focusing are pH5.9 and 6.0. The immunochemical properties of aldehyde dehydrogenase during its ontogeny are identical with those of normal adult liver aldehyde dehydrogenase when tested against anti-(hepatoma aldehyde dehydrogenase) serum in Ouchterlony double-diffusion tests. The results indicate that the hepatoma-specific aldehyde dehydrogenases are not the result of the de-repression of genes normally repressed in adult rat liver or in some other adult tissue.