Changes In Enzyme Content Research Articles

Protopanaxadiol and Protopanaxatriol Ginsenosides Can Protect Against Aconitine-induced Injury in H9c2 Cells by Maintaining Calcium Homeostasis and Activating the AKT Pathway.

This study aimed to investigate the effects of protopanaxadiol and protopanaxatriol ginsenosides on aconitine-induced cardiomyocyte injury and their regulatory mechanisms. The effects of ginsenosides on aconitine-induced cardiomyocyte damage were initially evaluated using H9c2 cells, and the molecular mechanisms were elucidated using molecular docking and western blotting. The changes in enzyme content, reactive oxygen species (ROS), calcium (Ca2+) concentration, and apoptosis were determined. Furthermore, an aconitine-induced cardiac injury rat model was established, the cardiac injury and serum physiological and biochemical indexes were measured, and the effects of ginsenoside were observed. The results showed that ginsenoside Rb1 significantly increased aconitine-induced cell viability, and its binding conformation with protein kinase B (AKT) protein was the most significant. In vitro and in vivo, Rb1 protects cardiomyocytes from aconitine-induced injury by regulating oxidative stress levels and maintaining Ca2+ concentration homeostasis. Moreover, Rb1 activated the PI3K/AKT pathway, downregulated Cleaved caspase-3 and Bax, and upregulated Bcl-2 expression. In conclusion, Rb1 protected H9c2 cells from aconitine-induced injury by maintaining Ca2+ homeostasis and activating the PI3K/AKT pathway to induce a cascade response of downstream proteins, thereby protecting cardiomyocytes from damage. These results suggested that ginsenoside Rb1 may be a potential cardiac protective drug.

Dietary supplementation with N-acetylcysteine, α-tocopherol and α-lipoic acid prevents age related decline in Na+,K+-ATPase activity and associated peroxidative damage in rat brain synaptosomes

This study has shown that in aged rat brain (22-24 months) crude synaptosomes in comparison to that in young animals (4-6 months), a striking decrease in the activity of Na(+),K(+)-ATPase occurs along with decreased K (m) and V (max) but without any change in enzyme content as seen by immunoblotting. This is associated with an accumulation of peroxidative damage products in aged brain. When rats are given antioxidant supplementation in the diet with a combination of N-acetylcysteine, alpha-tocopherol and alpha-lipoic acid daily from 18 months onwards and sacrificed at 22-24 months for experimentation, the age associated decrease of Na(+),K(+)-ATPase activity, alterations of its kinetic parameters and accumulation of peroxidative damage products in brain synaptosomes are prevented nearly completely. Because of the critical importance of Na(+),K(+)-ATPase in neuronal functions, the results of this study may be of potential implications in controlling age-related functional deficits of the brain.

The peroxisomal protein import machinery

Peroxisomes are unique organelles whose physiological functions vary depending on the cellular environment or metabolic and developmental state of the organism. These changes in enzyme content are accomplished by the dynamically operating membrane and matrix protein import machineries of peroxisomes that rely on the concerted function of at least 20 peroxins. The import of folded matrix proteins is mediated by cycling receptors that shuttle between the cytosol and peroxisomal lumen. Receptor release back to the cytosol represents the ATP-dependent step of peroxisomal matrix protein import, which consists of two energy-consuming reactions: receptor ubiquitination and dislocation.

Eosinophil-Mediated Cholinergic Nerve Remodeling

Eosinophils are observed to localize to cholinergic nerves in a variety of inflammatory conditions such as asthma, rhinitis, eosinophilic gastroenteritis, and inflammatory bowel disease, where they are also responsible for the induction of cell signaling. We hypothesized that a consequence of eosinophil localization to cholinergic nerves would involve a neural remodeling process. Eosinophil co-culture with cholinergic IMR32 cells led to increased expression of the M2 muscarinic receptor, with this induction being mediated via an adhesion-dependent release of eosinophil proteins, including major basic protein and nerve growth factor. Studies on the promoter sequence of the M2 receptor indicated that this induction was initiated at a transcription start site 145 kb upstream of the gene-coding region. This promoter site contains binding sites for a variety of transcription factors including SP1, AP1, and AP2. Eosinophils also induced the expression of several cholinergic genes involved in the synthesis, storage, and metabolism of acetylcholine, including the enzymes choline acetyltransferase, vesicular acetylcholine transferase, and acetylcholinesterase. The observed eosinophil-induced changes in enzyme content were associated with a reduction in intracellular neural acetylcholine but an increase in choline content, suggesting increased acetylcholine turnover and a reduction in acetylcholinesterase activity, in turn suggesting reduced catabolism of acetylcholine. Together these data suggest that eosinophil localization to cholinergic nerves induces neural remodeling, promoting a cholinergic phenotype.

Evaluating the Relationship Between Variance in Enzyme Expression and Toxicant Concentration in Health Risk Assessment

ABSTRACT The replacement of default uncertainty factors with those based on chemical-specific data is a topic of interest to a growing number of government-based organizations and those in affiliated professional societies. The division of the uncertainty factors for animal-to-human extrapolation and human interindividual variance (UFA and UFH, respectively) into their pharmacodynamic (PD) and pharmacokinetic (PK) components invites additional and specific considerations. Where data are available, or substantiated PK models have been developed, the animal-to-human chemical-specific differences have been quantified and utilized to replace the PK component of the uncertainty factor. The increasing degree to which the genome is being characterized has stimulated additional interest in describing the impact of genetic polymorphisms on susceptibility. Frequently, proteins for which the genes are being evaluated are the group of xenobiotic metabolizing enzymes. In-depth understanding of the genetic polymorphisms of genes coding for Aldehyde dehydrogenase, glucuronyl transferase and cytochrome P450 enzyme forms has been combined with information on the bioactivation or detoxication of environmental contaminants. The preliminary conclusion of some of these considerations is that alterations in enzyme content or enzyme activity result in a de facto alteration of risk. While this may be true of the “all-or-none” genetic alterations, the impact of more subtle changes in enzyme content and/or activity are more difficult to predict. The hepatotoxicity of trichloroethylene (TCE) is dependent upon an initial, cytochrome P450 2E1 (CYP2E1)-mediated oxidative step. Variance of CYP2E1 content of human liver has been characterized from a bank of tissues from human organ donors and combined with data describing the in vitro Michaelis-Menten kinetic parameters in order to extrapolate the metabolic capacity (and variance thereof) from in vitro to in vivo and assess its impact on PK through incorporation in a physiologically based pharmacokinetic (PBPK) model. This presentation summarizes that work, and demonstrates and discusses why extremes of CYP2E1-mediated metabolic capacity in adult humans has virtually no impact on the PK metric most closely related to hepatotoxic injury from TCE exposure.

Myogenin induces a shift of enzyme activity from glycolytic to oxidative metabolism in muscles of transgenic mice.

Physical training regulates muscle metabolic and contractile properties by altering gene expression. Electrical activity evoked in muscle fiber membrane during physical activity is crucial for such regulation, but the subsequent intracellular pathway is virtually unmapped. Here we investigate the ability of myogenin, a muscle-specific transcription factor strongly regulated by electrical activity, to alter muscle phenotype. Myogenin was overexpressed in transgenic mice using regulatory elements that confer strong expression confined to differentiated post-mitotic fast muscle fibers. In fast muscles from such mice, the activity levels of oxidative mitochondrial enzymes were elevated two- to threefold, whereas levels of glycolytic enzymes were reduced to levels 0.3-0.6 times those found in wild-type mice. Histochemical analysis shows widespread increases in mitochondrial components and glycogen accumulation. The changes in enzyme content were accompanied by a reduction in fiber size, such that many fibers acquired a size typical of oxidative fibers. No change in fiber type-specific myosin heavy chain isoform expression was observed. Changes in metabolic properties without changes in myosins are observed after moderate endurance training in mammals, including humans. Our data suggest that myogenin regulated by electrical activity may mediate effects of physical training on metabolic capacity in muscle.

Brain Acetyl-CoA Carboxylase: Isozymic Identification and Studies of Its Regulation during Development and Altered Nutrition

Acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, exists as an oligodendrocyte-associated enzyme in brain and plays an important role in supplying fatty acid for myelination. Rat brain acetyl-CoA carboxylase (ACC) has been identified as a single isozyme of M r = 265,000 daltons, indistinguishable immunologically from the isozyme in rat adipose tissue and liver. Total activity of brain ACC declines from birth to 4 weeks of age in the newborn rat. This change in activity can entirely be accounted for by changes in enzyme content, not enzyme specific activity, and is paralleled by decreases in ACC mRNA. In contrast, cardiac, skeletal muscle and liver ACC does not change in content over this developmental period. Unlike ACC in liver and adipose tissue, the enzyme content and specific activity of brain ACC is invariant during various states of nutrition. These data indicate that that brain ACC is subject to unique regulation, as compared to non-neural enzyme. The mechanisms underlying the control of neural ACC activity may be important to understanding the process of myelination during development and to a more general understanding of the factors regulating ACC expression/activity in other tissues.

Spatial and temporal fluctuation in biomass, nitrogen mineralizing reactions and mineral nitrogen in a soil cropped to barley

We compared changes in components of the N-mineralization cascade ranging from the very specific, such as a deaminase, to the highly integrated, such as biomass in a Black Chernozemic seeded to barley (Hordeum vulgare L.) under field conditions at Edmonton. Changes in enzyme content were related to soil [Formula: see text] to determine if the microbial environment changed sufficiently to exert feedback control on N mineralizing reactions and thereby to be detected. Histidase and protease were chosen as model systems for depolymerization and deamination respectively because information exists on their control in pure culture studies, on histidine content and control of histidase in soil, and assay procedures are available for soils. We observed an inverse relationship of labile histidase activity with [Formula: see text] in soils with high [Formula: see text] content and low [Formula: see text] ratio. This relationship provides indirect evidence for [Formula: see text] control of histidase content, but emphasizes that it is only one element of a complex control mechanism. Conversely, enzyme content was not rate limiting to net N mineralization, or sensitive to common control mechanisms. Biomass-C, an integrative measure of substrate supply, potential biological activity and enzymatic activity, describes net mineral-N production better than do indices of any single step. Regular spatial variability is exhibited by [Formula: see text] (and [Formula: see text]). [Formula: see text] is a product of mineralization; a substrate for immobilization, nitrification, plant uptake, and other reactions; and may also be a regulator of activity, or synthesis, of some enzymes. It is intriguing that none of the variables that influence mineral N, such as enzyme activity, biomass, or respiration, varied spatially in a statistically identifiable manner, yet [Formula: see text] did. Key words: Nitrogen mineralization, enzyme content, biomass, protease, histidase, ammonium regulation

Characterization of secretory responses in exocrine pancreas of genetically obese Zucker rats

Secretory responses of the exocrine pancreas in the obese Zucker rat with an inherited genetic disorder were compared with those in lean control rats. Amylase concentration in pancreatic acinar cells of obese rats was reduced to 62% of the control value, whereas trypsinogen concentration was increased to 269%. The activity ratio of amylase to trypsinogen in the pancreatic acini of obese rats was about one-fourth of that in controls. The activity ratio of amylase to trypsinogen in pancreatic juice released by stimulation with varying concentrations of CCK8 or carbachol in the obese rats was also reduced to one-fourth of that in control rats. The concentration of the secretagogs inducing half-maximal secretory responses (EC50) in the obese rats was almost identical to that in the controls. The downward shift of the dose-response relation for these secretagogs in the acini of obese rats was analogous to that in streptozotocin-induced diabetic rats, cold-exposed rats, or lactating rats, as demonstrated previously. The present results may be explained by the changes in enzyme content and secretory responses found in pancreatic acini of obese Zucker rats, which would be attributable mainly to congenital disturbance in "insulo-acinar axis."

Thyroid hormone stimulates expression of 6-phosphofructo-2-kinase in rat liver

The activity of liver 6-phosphofructo-2-kinase (PFK-2), the enzyme that catalyses the synthesis of fructose 2,6-bisphosphate, was markedly decreased in hypothyroid rats and partially restored after 3 days of treatment with triiodothyronine. The changes in PFK-2 activity were accompanied by parallel changes in enzyme content measured by immunotitration and in PFK-2 mRNA determined by dot blot and Northern blot hybridization with cDNA probes. It is concluded that thyroid hormone stimulates liver PFK-2 gene expression by a pre-translational mechanism.

Pancreatic Acinar Cell Amylase Gene Expression: Selective Effects of Adrenalectomy and Corticosterone Replacement*

To examine the role of glucocorticoids in the regulation of the acinar pancreas, adult male rats were adrenalectomized (Adx) and replaced with no corticosterone (B), normal B, or high B. Plasma B concentration, body weight gain, and thymus weight were used as independent measures of treatment efficacy. Compared to controls, Adx animals had a 75 +/- 0.5% (n = 30) reduction in pancreatic amylase content; a 50% decrease occurred within 1 day and the maximal 75% decrease was observed after 5 days. In Adx animals, amylase content was normalized by normal B replacement and was increased to 235 +/- 39% (n = 30) of control by high B replacement. Furthermore, in all Adx rats, pancreatic content of amylase and plasma B concentration was significantly correlated (r = 0.81, n = 30). The effect of adrenalectomy was selective for amylase; contents of ribonuclease, chymotrypsin, and elastase were not altered. However, the effects of high B replacement were not selective, and increased the content of all digestive enzymes. To determine whether the changes in enzyme content were associated with changes in messenger RNA (mRNA), pancreatic RNA was probed with 32P-labeled complementary DNAs for amylase, ribonuclease, and chymotrypsin. After adrenalectomy and B replacement there was a significant correlation only between amylase mRNA (r = 0.87, n = 13) and plasma B concentration. These data indicate that physiological levels of B have a selective effect on pancreatic amylase gene expression. In contrast, high levels of B have the separate, nonselective effect of increasing the content of all digestive enzymes without increasing corresponding mRNA levels.

The induction of mitochondrial l-3-glycerophosphate dehydrogenase by thyroid hormone

l-3-Glycerophosphate dehydrogenase was purified from porcine brain mitochondria by a shorter and simpler procedure than previously reported. Immunoblotting with antiserum to the porcine enzyme established that rat liver l-3-glycerophosphate dehydrogenase has the same Mr (76000) by SDS-polyacrylamide gel electrophoresis. In liver mitochondria from normal and hyperthyroid rats, changes in l-3-glycerophosphate dehydrogenase activity were parallelled by changes in enzyme content assayed by immunoblotting. Similar changes were found in the amount of enzyme synthesised in vitro by reticulocyte lysate programmed with rat liver mRNA, suggesting that thyroid hormone causes specific induction of l-3-glycerophosphate dehydrogenase mRNA.

Effects of ethionine on digestive enzyme synthesis and discharge by mouse pancreas.

The earliest changes noted during the evolution of pancreatitis induced by feeding mice a choline-deficient ethionine-supplemented (CDE) diet are an increase in the number of zymogen granules in pancreatic acinar cells and an increase in digestive enzyme content of the pancreas. We have studied the processes of protein and digestive enzyme synthesis and discharge at varying times after institution of the CDE diet, a choline-deficient diet (CD), and a diet containing ethionine but not choline-deficient (E). Both the CDE and E diets increased digestive enzyme content within 12 h of their institution. Both the CDE and E diets reduced the rate of protein and amylase synthesis and caused a marked reduction in the rate of protein and amylase discharge from the pancreas. These changes were greatest and were noted earliest in the CDE diet group. A marked reduction in secretagogue-induced in vivo and in vitro amylase discharge followed ingestion of either the CDE or E diet. These studies indicate that the increased pancreatic content of digestive enzymes noted after ingestion of the CDE and E diets results from an ethionine-induced decrease in the rat of digestive enzyme discharge. This phenomenon is enhanced by simultaneous choline deficiency. Subsequent intrapancreatic activation of zymogens may couple these changes in enzyme content to the development of hemorrhagic pancreatitis.

Hormonal regulation of fatty acid synthetase in chick embryo liver

Fatty acid synthetase activity in chick embryonic liver is negligible compared to that in newly hatched, fed chicks. The enzyme activity is prematurely induced 5–50-fold in 20-day-old embryos and in newly hatched chicks by the administration of insulin, hydrocortisone, growth hormone, glucagon or dibutyryl cyclic AMP. The induction of the enzyme activity is blocked by the administration of cycloheximide, indicating that new protein synthesis is required. Immunochemical titrations of different enzyme preparations from 5-day-old chicks, adult chicken and various inducer-treated embryos gave an identical equivalence point, indicating that the changes in synthetase activity after hormonal induction in embryos are related entirely to changes in content of enzyme. The increase in liver synthetase content after administration of insulin, glucagon or dibutyryl cyclic AMP is directly related to an increase in the rate of synthetase synthesis. The induction of the synthetase activity by suboptimal doses of glucagon or cyclic AMP is potentiated by the phosphodiesterase inhibitory theophylline. There is a very rapid decay of synthetase activity, with a half-life of about 4 h after elevation to higher levels following administration of insulin, glucagon or dibutyryl cyclic AMP. Glucagon and dibutyryl cyclic AMP induction of the synthetase activity is observed early in the embryonic development, whereas insulin induction is noted 2 days before hatching. Insulin, glucagon and cyclic AMP are potentially capable of altering the levels of glycolytic intermediates which may be involved in the induction of synthetase.

Fatty acid synthetase of mammalian brain, liver and adipose tissue regulation by prosthetic group turnover

The role of prosthetic group turnover in the regulation of the fatty acid synthetase of mammalian brain, adipose tissue and liver has been studied. The fatty acid synthetase was labeled in the 4'-phosphopantetheine prosthetic group of the acyl carrier protein by administration to rats of calcium [ 3H]pantothenate. By determining the time course of incorporation of radioactivity into both CoA, the precursor for the 4'-phosphopantetheine moiety of acyl carrier protein, and the fatty acid synthetase, it was possible to estimate the rate of prosthetic group turnover. The turnover rate was found to be extremely rapid in all three tissues. In brain, the prosthetic group is replaced completely in 10–11 h and this is an order of magnitude faster than the turnover of the enzyme complex, previously shown to occur with a t 1 4 of 6.4 days. In liver and adipose tissue, the prosthetic group turnover is slightly faster than in brain. Thus, the rapid turnover of the prosthetic group, first shown in Escherichia coli, also occurs in three different mammalian tissues and may represent a general biological phenomenon. Since turnover of the protein of fatty acid synthetase of brain does not change at all in various nutritional states when turnover of liver synthetase changes dramatically, we considered the question of whether the striking turnover of the prosthetic group in these tissues also responded differently to those states. Distinct changes in the efficiency of the exchange of the 4'-phosphopantetheine between CoA and fatty acid synthetase were observed only in the liver but not in the brain. Increased exchange in liver during fat-free feeding was discovered and the earlier observation of diminished exchange during starvation was confirmed. We also evaluated by immunochemical methods the possibility that rapid regulation of fatty acid synthetase is mediated by changes in catalytic efficiency. Quantitative precipitin analyses of liver extracts from animals during the first hours after onset of starvation or upon refeeding after starvation demonstrated that changes in enzyme activity were related entirely to changes in enzyme content. Thus, the data did not support the possibility that fatty acid synthetase activity is decreased by changes in catalytic efficiency due to removal of the 4'-phosphopantetheine prosthetic group or to inhibition by metabolites.

Fatty Acid Synthetase of Developing Brain and Liver: CONTENT, SYNTHESIS, AND DEGRADATION DURING DEVELOPMENT

Abstract Immunochemical techniques have been utilized to study the content, synthesis, and degradation of the fatty acid synthetase in liver and brain during development and in various nutritional states. The distinctive changes in synthetase activity during development of both tissues are related entirely to changes in content of enzyme. During fasting and fat-free feeding, in contrast to the lack of change in synthetase activity of brain, there are dramatic alterations in hepatic activity. These changes are related entirely to changes in content of enzyme in liver. During development of brain the rate of synthesis of the enzyme decreases. In developing liver, when synthetase activity rises at the time of weaning, there is a marked acceleration of synthesis of enzyme. During fasting, when hepatic synthetase concentration falls drastically, there is a considerable reduction in synthesis of enzyme, and when animals are refed a fat-free diet, a remarkable increase in enzyme synthesis occurs, confirming earlier reports. The rate of fatty acid synthetase degradation in brain of adult animals differs considerably from that in liver; the half-lives are 6.4 and 2.8 days, respectively. In young suckling animals the rate of degradation of the enzyme in brain is markedly accelerated (t1/2 = 1.9 days), whereas in liver there is little change (t1/2 = 2.3 days). The most profound change in degradation of synthetase in liver occurs during fasting when the t1/2 decreases to 18 hours. No changes were observed in degradation in brain during fasting or fat-free feeding. Rate constants for synthesis and degradation of fatty acid synthetase in liver and brain during development and in the various nutritional states were derived; these quantitatively accounted for the differences in content of the enzyme in these tissues. In developing liver the dramatic elevation in synthetase content apparent after weaning is associated with an acceleration of enzyme synthesis but no change in rate of enzyme degradation. The changes in enzyme content during development of brain are accompanied by alterations in the rates of synthesis and degradation of fatty acid synthetase. Both processes are more rapid in the brain of the young suckling animal than in the mature animal. These observations represent the first direct measurements of rates of synthesis and degradation of an enzyme in mammalian brain.

Anaerobic Accumulation of γ-Aminobutyric Acid and Alanine in Radish Leaves (Raphanus sativus, L.)

In leaves, the anaerobic accumulation of alanine was accompanied by a loss of aspartate, and these changes preceded gamma-aminobutyrate accumulation and glutamate loss. Changes in keto acid content did not appear to be the cause of amino acid changes. Accumulation of gamma-aminobutyrate was due to acceleration of glutamate decarboxylation and arrest of gamma-aminobutyrate transamination. Changes in enzyme content did not explain the changes in reaction rates in vivo. Most of the aspartate may be converted anaerobically to alanine via oxalacetate and pyruvate.

Synthesis and Degradation of Malic Enzyme in Chick Liver

Abstract The role of synthesis and of degradation in regulation of malic enzyme concentration in the liver of growing chicks has been studied. Malic enzyme was assayed by enzymic activity and by immunological analysis with immunoglobulin containing specific anti-malic enzyme. Quantitative immunoprecipitin curves and equivalence point determinations indicated that there was a constant relationship between immunologically precipitable protein and units of enzyme activity in livers of neonatal and growing chicks during a time period in which total enzyme activity increased 60-fold. Hence, changes in malic enzyme activity were due to changes in enzyme content of the liver rather than to activation or inhibition of preformed enzyme. The relative rate of malic enzyme synthesis was determined by measuring incorporation of counts into malic enzyme protein after pulse labeling total protein with l-[4,5-3H2]leucine. Malic enzyme protein was purified by quantitative precipitation of the enzyme by antibody followed by disc gel electrophoresis in the presence of sodium dodecyl sulfate. Radioactivity in the band corresponding to malic enzyme was estimated after electrophoresis of the immunoprecipitate. Relative to synthesis of total protein, the rate of synthesis of malic enzyme increased more than 50-fold when neonatal chicks were fed. Starvation of week-old chicks for 2 days caused a 60 to 70% decrease in synthesis of malic enzyme. Degradation of malic enzyme was very slow in unfed, neonatal chicks (tfrac12; = 350 hours). Feeding, which increased the synthesis and concentration of malic enzyme also caused an increase in the rate of degradation. In 8- and 11-day-old chicks degradation of malic enzyme was found to be first order with a tfrac12; of 55 hours. In fasting chicks a t½ of 28 hours was observed, but there was also an increase in the rate of degradation of total liver protein suggesting that part of the increased rate of degradation of malic enzyme was due to a general increase in protein breakdown. The temporal relationship between synthesis of malic enzyme and synthesis of fatty acids was studied in unfed neonatal chicks given a single glucose meal. Fatty acid synthesis from [1-14C]acetate was increased 7-fold at 1 ½ hours and 20-fold at 3 hours after the glucose meal. Neither the activity nor the synthesis of malic enzyme were increased at 3 hours but both were significantly increased at 6 hours (100% and 160%, respectively). These results suggest that the increased synthesis of malic enzyme may have been initiated by an increased flux through the pathway for fatty acid synthesis.

Studies of muscle development. IV. Some characteristics of RNA polymerase activity in isolated nuclei from developing chick muscle

1. 1. Nuclei isolated from developing chick leg muscle in a solution of 2.2 M sucrose, 0.003 M MgCl 2 retain morphological characteristics found in fixed tissue. The RNA/DNA ratio in the nuclei decreases from 0.39 in the 11-day embryo to 0.19 at 18 days of development. 2. 2. RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase, EC 2.7.7.6) activity was measured in the intact nuclei and Mg 2+-activated enzyme activity was found to change little during muscle development. In contrast, enzyme activity assayed in the presence of Mn 2+ decreased by one half from 11 to 18 days of embryonic development. These differences in enzyme activity persisted after (NH 4) 2SO 4 stimulation. 3. 3. The RNA product of this system was determined in vitro by comparing the relative incorporation of each of the four ribonucleotide triphosphates. The RNA synthesized in the presence of Mg 2+ was rich in guanine resulting in U G ratios below 1.0, while the product of the Mn 2+-activated system in the presence and absence of (NH 4) 2SO 4 was high in uridine, giving U G ratios over 1.0. The U G ratio was also above 1.0 in the Mg 2+ + (NH 4) 2SO 4 system after long periods of incubation. 4. 4. Preliminary experiments suggest that the observed differences in RNA synthesis during development are the result of changes in enzyme content or activity rather than DNA template availability.

DNA Polymerase and Carbohydrate Metabolizing Enzyme Content of Normal and Leukemic Glass Column Separated Leukocytes

Abstract1. Enzymes of normal and leukemic glass column separated leukocytes were assayed with fluorometric (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and isocitric dehydrogenase) and radioisotope (DNA polymerase) micromethods.2. Most results were obtained by direct assay of the specific cell type. Some required simple calculation. It was demonstrated that the enzyme activity of mixed cell suspensions was the sum of the enzyme content of the component cells, at least for the enzymes studied.3. Assays of mixed cell suspensions may give an erroneous picture. Thus changes in differential cell counts, as well as alterations in enzyme content of individual cells, must be taken into account for correct interpretation.4. Changes in enzyme content of mixed cell suspensions after therapy were due chiefly to alterations in the differential cell count rather than to changes in the enzyme content of individual cell types.5. The patterns of assays of the carbohydrate metabolizing enzymes studied, except isocitric dehydrogenase, paralleled each other. Highest values were found in mature PMN leukocytes and lowest in blasts. With isocitric dehydrogenase absolute values were much lower, while the highest assays were found in myelocytes.6. DNA polymerase content correlated well with the ability of a cell to divide. It was highest in blasts and lowest in mature PMN leukocytes.