A comparative study of nuclear and chloroplast DNA dependent RNA polymerases from wheat leaves

• 1.1. Three DNA dependent RNA polymerases have been purified from chromatin and chloroplast fractions of wheat leaves.
• 2.2. The purified enzymes were completely dependent on exogenous DNA after purification by glycerol gradient, DEAE-Sephadex and phosphocellulose chromatography.
• 3.3. The nuclear enzymes, I and II, showed a strong preference for denatured nuclear DNA, whereas the chloroplast enzyme preferred denatured chloroplast DNA.
• 4.4. The three enzymes require either Mg²⁺ or Mn²⁺ for activity.
• 5.5. α-amanitin specifically inhibited RNA polymerase II but has no effect on polymerase I and chloroplast polymerase.
• 6.6. Enzyme I is most active at very low ionic strength (0.10 mM KC1), whereas enzyme II and chloroplast enzyme show maximum activity at 150mM and 50 mM KC1 respectively.

     

Phosphatidylinositol-cleaving activity in smooth muscle from rat vas deferens

• 1.1. Phosphatidylinositol-cleaving activity was studied in subcellular fractions from smooth muscle of rat vas deferens.
• 2.2. In the presence of calcium ions and deoxycholate most of the endogenous phosphatidylinositol was broken-down in 60 min, whilst the other phospholipids were stable.
• 3.3. The enzymatic activity responsible for this breakdown catalyses a phospholipase C-type cleavage of the glycerol-phosphate bond, the water soluble products from exogenous [³²P]-labelled phosphatidylinositol being d-myoinositol 1:2-cyclic phosphate (702-80%) and d-myoinositol 1-phosphate (202-30%).
• 4.4. Activity was abolished by 1 mM ethanedioxybis(ethylamine)tetra-acetate (EGTA) and in the presence of deoxycholate both the soluble and total particulate fractions showed maximum activity at pH 6.52-6.8. The soluble fraction showed a second peak of activity at pH 5.52-5.8 that was independent of deoxycholate; this was not observed in the particulate fraction.
• 5.5. About two-thirds of the activity was soluble. The remaining activity was particulate, with a preferential concentration in the microsomal fraction.

     

On the mechanism of decreasing the lithogenicity of bile in Camelus dromedarius

• 1.1. Analysis of camel bile revealed that it is highly concentrated.
• 2.2. The molar fraction of phospholipids in camel bile was low and cholesterol was very high relative to rat bile.
• 3.3. Cholate is the main primary bile acid secreted. Moderate quantities of the secondary bile acid deoxycholate were found but no lithocholate was detectable possibly secondary to rapid recycling of the bile acid pool in the enterohepatic circulation.
• 4.4. Glycoconjugated bile acids predominated over tauroconjugates.
• 5.5. The hepatic bile from the camel may be concentrated as part of the general mechanism of water conservation exhibited by that species. The increased concentration of bile acids helps maintain cholesterol in solution, thus reducing lithogenicity.

     

Isolation and characterization of two forms of malate dehydrogenase from human placenta

• 1.1. The purification and characterization of the cytoplasmic and mitochondrial forms of malate dehydrogenase from human placenta are described.
• 2.2. Both enzymes are composed of two subunits and have similar molecular weights and similar pH optima.
• 3.3. However, they differ with respect to thermal stability, excess substrate inhibition and electrophoretic mobility.

     

Poly(d(A—T)) dependent RNA polymerase activity after treatment of nuclei with micrococcus nuclease

• 1.1. Rat liver nuclei were incubated with or without 20 units micrococcus nuclease (EC3.1.4.7)/mg nuclear DNA.
• 2.2. The soluble poly(d(A—T)) dependent RNA polymerases were reduced in activity to 15–20% that of the controls after treatment with micrococcus nuclease.
• 3.3. RNA polymerases I plus III activities were completely, RNA polymerase II activity partially reversible on removal of the DNA released into the soluble fraction by treatment of nuclei with micrococcus nuclease.
• 4.4. Inhibitory constants obtained with the solubilized DNA were 17.1 μM and 20.7 μM nucleotide-DNA for RNA polymerases I plus III and RNA polymerase II, respectively. The corresponding inhibitory constants obtained with native salmon DNA were 23.0 μM and 34.4 μM nucleotide-DNA.

     

Activity of some enzymes involved in the metabolism of polyamines in the liver of streptozotocin-diabetic rats

• 1.1. The effect of diabetes on some enzymes of polyamine metabolism was studied in male rats 1–12 days after administration of streptozotocin.
• 2.2. Hepatic ornithine decarboxylase activity decreased in the first days after the administration, but increased thereafter. The decrease was not due to an alteration of the ODC-antizyme concentration, nor to a posttranslational modification catalyzed by transglutaminase.
• 3.3. S-adenosylmethionine decarboxylase and ornithine transaminase were both increased.
• 4.4. Spermicline acetyltransferase activity was practically unchanged, while its inactivating factor was markedly decreased.

     

Comparison of nuclear DNA-dependent rna polymerases from mouse tumors and tissues of normal swiss mice

• 1.1. Five major forms of nuclear DNA-dependent RNA polymerases from seven transplantable murine tumors and from five tissues from normal, adult, male Swiss mice (Mus musculus) were separated chromatographically on DEAE-Sephadex A-25. Forms Ia and Ib were insensitive to α-amanitin, whereas forms IIa and IIb were highly sensitive and form III was slightly sensitive.
• 2.2. The polymerases from all tumors or ascites tumors only were compared statistically with those from the normal tissues in regard to elution patterns, specific activities, activities per mg of nuclear DNA. degrees of purification and yields. Similarly, individual tumors were compared with homologous normal tissues. All parameters were characterized by relatively large variance.
• 3.3. Activities of all RNA polymerases per mg DNA were higher in all tumors compared with normal tissues. The order of statistical significance of these differences was lib > III > IIa > Ib > Ia.
• 4.4. Activities per mg DNA of all RNA polymerases from 6C3HED and L1210 tumors, with the exception of L1210 tumor enzyme III, were significantly greater than those from spleen, but only the activities of Lewis lung tumor enzyme IIb and of Taper hepatoma enzymes Ia and III were higher than those from the homologous tissues, lung and liver, respectively.

     

δ-Aminolevulinic acid dehydratase inactivation by uroporphyrin I in light and darkness

• 1.1. The action of uroporphyrin I on erythrocytic ALA-D activity under dark and light conditions was examined.
• 2.2. Photo and non-photoinactivation of ALA-D induced by uroporphyrin I were observed.
• 3.3. Both effects were dependent on uroporphyrin concentration, temperature and time of exposure of the protein to the porphyrin.
• 4.4. Light-dependent effect of uroporphyrin I is related with the phototoxicity of porphyrins and could be produced by primary amino acid photooxidation followed by secondary cross-linking of the protein.
• 5.5. Light-dependent effect of uroporphyrin I could be ascribed to a direct enzyme inhibition due to binding of the porphyrin to the protein inducing structural changes at or near its active site.

     

The characterization of the linoleic acid desaturation and elongation system in ovine placental tissue

• 1.1. The desaturation and elongation of linoleic acid has been studied in homogenates and in subfractions of ovine placental tissue.
• 2.2. The reaction was characterized in terms of pH and temperature optima, time course and protein concentration.
• 3.3. Activity was found to be confined to the 11,000g supernatant fraction of the tissue and the results suggest that the enzymes are membrane bound.
• 4.4. The cytosolic fraction and ATP were required for full activity and the reaction was inhibited by cyanide.
• 5.5. The properties of the reaction are compared with those of other desaturation systems and their implications with regard to possible reaction mechanisms are discussed.

     

The haemocytes and the activities of leucine aminopeptidase and alkaline phosphatase during development of Ceratitis capitata: Specific secretion of a leucine aminopeptidase isozyme

• 1.1. The types of haemocytes during larval development were studied.
• 2.2. The developmental profile of leucine aminopeptidase and alkaline phosphatase was studied. The maximum LAP activity was found to be in early larval development, while the maximum alkaline phosphatase during the white pupal stage.
• 3.3. These activities were compared with those determined in cell-free haemolymph.
• 4.4. Both hydrolytic enzymes have been found histochemically in the prohaemocytes and in the plasmatocytes.
• 5.5. In cultured haemocytes experiments it was found that 64% of the total LAP activity was secreted into the incubation medium, while electrophoretic analysis of released LAP activity demonstrated that only LAP A isozyme was secreted.
• 6.6. Based on the above results we suggest that both hydrolytic enzymes are functionally important throughout larval development.

     

Kinetic analysis of reversible closed bicyclic enzyme cascades covering the whole course of the reaction

A kinetic analysis of the closed bicyclic enzyme cascades is presented.

• 1.1. It includes the dependence on time from the onset of the reaction, of the concentration of the modified and unmodified enzyme species involved and the time course equations of the modificational fractions of the interconvertible enzymes.
• 2.2. The transient phase equations obtained allow the definition of new regulatory modification properties.
• 3.3. The expressions for concentrations of the unmodified and modified forms of the interconvertible enzymes, as well as those of the fractional modifications in the steady state are derived as particular cases of the general equations.
• 4.4. These steady state expressions coincide with those obtained by other authors.
• 5.5. The analytical results obtained are discussed in relation to the Escherichia coli glutamine syntethase cascade.

     

Purification of two lipases with high phospholipase A1 activity from guinea-pig pancreas

• 1.1. Two cationic lipases (Ia and Ib) were purified from homogenates of fresh guinea-pig pancreas by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose (twice for the latter) followed by gel filtration on Sephadex G-100.
• 2.2. Both enzymes were homogeneous upon polyacrylamide gel electrophoresis. Their molecular weights are 37000 and 42000 for lipases Ia and Ib, respectively, as determined by gel filtration on Sephadex G-100. Very close values for isoelectric points were found in the pH range 9.3–9.4.
• 3.3. The cationic lipases are characterized by a high phospholipase A activity (500 IU/mg protein using a potentiometric assay with egg yolk lecithin as substrate), resulting in an unusual phospholipase/lipase activity ratio of 1.
• 4.4. Using doubly labelled phosphatidylcholine, a specificity, A₁, was described for the two enzymes, which are unaffected by N-ethylmaleimide, diisopropylfluorophosphate and p-bromophenacylbromide. The enzymes are insensitive to EDTA and slightly inhibited by CaCl₂and MgCl₂, whereas sodium deoxycholate is required for maximal activity.

     

Properties of enzymes hydrating epoxides in human epidermis and liver

• 1.1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different.
• 2.2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity Chromatographic properties.
• 3.3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities.
• 4.4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes.
• 5.5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts.
• 6.6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.

     

The effects of osmotic stressors on the stenohaline carp (Cyprinus carpio)

• 1.1. Carp were acclimatized to different concentration of urea and mannitol.
• 2.2. The fish survived in 300 mOsm urea and 262 mOsm mannitol for a longer period. Higher concentrations were only tolerated for a short time.
• 3.3. Urea penetrated into the animals. The internal concentration of urea in plasma was nearly equal to the outside concentration after 7 days. Therefore a very high internal osmolality was adjusted (sum of normal and urea osmolality).
• 4.4. Urea treatment only resulted in changes of Ca level, while the concentration of other electrolytes was not clearly varied.
• 5.5. Extracellular space of muscle was reduced while the intracellular space remained unchanged after urea treatment.
• 6.6. Mannitol treatment resulted in changes of electrolyte concentrations due to dehydration.
• 7.7. After 1 day of treatment the concentration of Na in plasma decreased which might indicate the limitation of tolerance.
• 8.8. Immediate shrinkage of ICS and, later, reduction of ECS were clear reactions to mannitol influence.

     

Application of electrical analogs to metabolic control analysis: Linear pathways with multiple feedback

• 1.1. Using electrical analogs, we have presented a systematic procedure for calculating the flux control coefficients of linear metabolic pathways with multiple feedback loops.
• 2.2. In this method, an electrical analog circuit is constructed first for the unregulated pathway.
• 3.3. This circuit is subsequently modified in a step-by-step fashion to take into account the effect of each feedback loop in the pathway.
• 4.4. An analog circuit consists of resistances which are connected in series (or parallel) with a voltage (or current) source.
• 5.5. The flux control coefficients of the enzymes are represented by voltages across (or currents through) the resistances and are determined by an application of Ohm's law.
• 6.6. We have investigated the possible patterns in linear pathways with two feedback loops.
• 7.7. This is followed by an analysis of a linear pathway with an arbitrary pattern of feedback inhibition.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Inhibition of dihydrofolate reductase by palmitoyl coenzyme A

• 1.1. Palmitoyl-CoA was found to inhibit bovine liver dihydrofolate reductase.
• 2.2. 50% inhibition of the enzyme was obtained with 1.5 μM palmitoyl-CoA.
• 3.3. The inhibition was reversed by addition of bovine serum albumin, β-cyclodextrin or spermine.

     

Elasmobranch fish (Scyliorhinus canicula and Raja clavata) muscle AMP deaminase: Substrate specificity and effect of EDTA and adenylate energy charge

• 1.1. The AMP deaminases from skeletal muscles of dogfish and skate were shown to be specific to 5′-AMP. Among several adenine nucleotide analogs, only dAMP was deaminated to an extent lower than 5%.
• 2.2. Similar to vertebrates AMP deaminases, these enzymes were inhibited when incubated in the presence of EDTA solutions.
• 3.3. The activity of the enzymes was regulated by adenylic energy charge variations, depending on the size of the total adenine nucleotide pool.
• 4.4. The shape of the adenylate energy charge response curves of the dogfish and skate muscle AMP deaminases do not distinguish the two enzymes.

     

Enzymes of fructose metabolism in the intestinal mucosa of the rat (Rattus norvegicus). Localization along the small intestine; consequences of dietary fructose

• 1.1. The activities of all the eight enzymes of conversion of fructose to glucose, of all the three key enzymes of glycolysis and of the two dehydrogenases of pentose shunt were determined in proximal and distal mucosa of small intestine.
• 2.2. With the exception of hexokinase, all of these enzymes have an activity significantly higher in the proximal than distal mucosa.
• 3.3. The gradient along the intestine is particularly important for the three enzymes which are typical for fructose metabolism (ketohexokinase, triokinase and fructose-1-phosphate aldolase), for glucose-6-phosphatase and for phosphofructokinase.
• 4.4. The effects of fructose diet on the enzyme activities are compatible with the results, described in other papers, concerning the final products of metabolism.
• 5.5. The increase of fructose metabolism appears to result mainly from the stimulation of the activities of ketohexokinase and fructose-1-phosphate aldolase which control all the pathways of ketohexose utilization.
• 6.6. The activation of glucose-6-phosphatase, in comparison with the other enzymes which are involved in glucose-6-phosphate metabolism, explains the appearance of the ability to synthesize glucose with fructose as substrate. This enzyme is the only key enzyme of fructose to glucose conversion which responds to fructose feeding in distal mucosa.
• 7.7. The activities of hexokinase and phosphofructokinase are not increased by fructose feeding.
• 8.8. The activity of pyruvate kinase. the only key glycolytic enzyme which is necessarily implicated when fructose is the substrate, is stimulated but less than the typical enzymes of fructose metabolism.
• 9.9. But, because of its quantitative importance, the glycolytic pathway is responsible for the most part of the observed increase of fructose utilization.
• 10.10. The responses of pyruvate kinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities to fructose feeding are similar in the two parts of small intestine.
• 11.11. The activities of ketohexokinase, triokinase and glucose-6-phosphate isomerase are stimulated only in the proximal small intestine mucosa.
• 12.12. The other enzyme activities which are stimulated in proximal segment are also increased in distal segment.
• 13.13. All segments of small bowel show adaptive changes to dietary manipulation but not necessarily for all their functions.
• 14.14. The gradient of enzyme activities from the proximal to the distal small intestine persists despite dietary modification, but the data do not determine that this gradient is intrinsic or that it is not intrinsic.

     

α,α-trehalase from the brine shrimp Artemia salina. purification and properties

• 1.1. Trehalase (α,α-trehalose 1-d-glycohydrolase, E.C. 3.2.1.28) from cryptobiotic Artemia salina embryos was purified and characterized.
• 2.2. Most of the enzyme activity was present in an insoluble form and could be solubilized by deoxycholate treatment at high ionic strength and sonication.
• 3.3. The enzyme had a molecular weight of 75,000 and its isoelectric point was 6.2. It was optimally active at pH 5.6 with a K_m = 4.3 × 10⁻³ M for its natural substrate.
• 4.4. The enzyme was highly specific for trehalose, only lactose and cellobiose being hydrolyzed to a limited extent.