Significant Change In Specific Activity Research Articles

Induction of Neurolathyrism in Guinea Pig Fed with Lathyrus sativus Diet

Abstract Objectives To induce Neurolathyrism in guinea pig fed with Lathyrus sativus diet. Methods Twenty four guinea pigs were randomly distributed into 3 groups, 8 animals each. Groups I control fed with control diet, Group II low toxin group fed with 90% Lathyrus diet containing 100 mg β-ODAP/100 g Lathyrus and Group III fed with 90% Lathyrus diet with 1060 mg β-ODAP/100 gLathyrus. The experiment was for 2 months. Diet intake, body weight, protein carbonyl, catalase, MDA, specific activity of electron transport complexes I, II, III and IV in cortex and cerebellum were analyzed. Histopathological and morphological changes were studied by electron microscopy Results The diet intake and bodyweight was significantly reduced in both the experimental groups (II & III) as compared group I. Protein carbonyls levels were significantly increased in motor cortex region of group II & III as compared to group I. The catalase activity in cortex region was significantly decreased in group II & III compared to group I. The MDA levels in motar cortex region were significantly higher in the group II & III as compared to group I. Whereas the MDA levels in the cerebellum was significantly higher in group III as compared to group I. The activity of complex I and II of cortex was decreased significantly in group II and III as compared to group I. However, there was no significant change in specific activity of electron transport complex II and complex III. The nitrate levels in the group II & III were significantly higher than group I in the cortex region. However, there were no significant differences in the nitrate levels in the cerebellum region. Electron microscopic study of motor cortex showed that the extensive degeneration of motor neuron was observed in group II and III compared to group I. Conclusions Consumption of ninety % Lathyrus sativus dietfor 2 months induces neurolathyrism in guinea pigs. Funding Sources Indian Council of Medical Research, National Institute of Nutrition, Hyderabad, India.

Seasonal Responses of Extracellular Enzyme Activity and Microbial Biomass to Warming and Nitrogen Addition

Soil microbial responses to climate warming in temperate regions may interact with the effects of increased atmospheric N deposition. In addition, the combined effects of these factors on microbial activity during the plant growing season may differ from the effects over winter, when reduced plant soil C inputs and soil freezing can alter microbial nutrient availability and demand. We examined seasonal changes in soil extracellular enzyme activity (EEA), microbial biomass C and N, and soil fungal and bacterial content in a warming and N addition experiment in a temperate old field. For EEA, we examined both hydrolases (organic C degrading enzymes, a chitinase and phosphatase) and ligninases (phenol oxidase and peroxidase). While both hydrolase and ligninase activities exhibited significant seasonal variation, EEA was unresponsive to the experimental treatments. Microbial biomass C increased with warming year round, however, and microbial biomass N increased with N addition but only over summer. Despite increased microbial biomass in response to warming, phosphatase was the only enzyme that exhibited a significant change in specific activity (enzyme activity per unit of microbial biomass) in response to warming. Likewise, soil fungal and bacterial biomass varied seasonally, but treatment effects on these variables were minimal. Overall, while the effects of N addition on microbial N varied seasonally, microbial responses were relatively insensitive to the warming and N addition treatments in our experiment. This insensitivity was unexpected given the large treatment effects on plant productivity and soil N dynamics documented during the same time frame in the field experiment.

Long-term nutritional effects on the primary liver and kidney metabolism in rainbow trout, Oncorhynchus mykiss (Walbaum): adaptive response to a high-protein/non-carbohydrate diet and starvation of glucose 6-phosphate dehydrogenase activity

The adaptive response of the kinetics of glucose 6-phosphate dehydrogenase (G6PDH) was studied in trout liver and kidney after feeding (control or high-protein/non-carbohydrate diet) or starving for 30 days, as well as influences on growth and other nutritive parameters. Typical Michaelis-Menten kinetics were found for the hepatic catalysis of G6PDH under all conditions studied, without evidence of sigmoidicity. The administration of a high-protein/non-carbohydrate diet (61.0% protein, 7.9% lipids) produced no significant changes in specific activity or other kinetic parameters of the liver enzyme. This nutritional situation caused a significant daily increase in relative growth (20%) and feed efficiency (13.5%), as well as a 13% decrease in the protein-conversion efficiency, with respect to the control diet (46% protein, 8% lipids, 22% carbohydrates). On the other hand, long-term starvation (30 days), as a lipolytic condition, significantly decreased the activity and catalytic efficiency of hepatic G6PDH, by almost 45%, without significant changes in the Km and activity-ratio values. These changes agree with a fall in the intracellular concentration of the enzyme as a consequence of a protein-repression process. The activity of the renal G6PDH was less than in the liver of control fish, and no variation in kinetics was found under the high-protein diet or starvation. This behaviour clearly demonstrates that the kidney pentose-phosphate pathway showed no adaptive response in relation to synthesis of fatty acids and other lipids.

Characterization of free and immobilized amine oxidases.

Bovine plasma amine oxidase was covalently bound to CH-Sepharose 4B by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The immobilized enzyme showed no significant change in specific activity when spermidine was the substrate, while the enzyme affinity toward benzylamine and propylamine increased significantly. Similarly, the pig kidney diamine oxidase physically adsorbed to Con A-Sepharose showed large changes in affinity toward substrates such as p-dimethylaminoethylbenzylamine with respect to the native enzyme. These changes are discussed in terms of active site modification as a consequence of the enzyme immobilization.

Inhibitors of sterol synthesis. Studies of the distribution and metabolism of 5α-[2,4− 3H] cholest-8(14)-en-3β-ol-15-one after intragastric administration to rats

5α-[2,4− 3H]Cholest-8(14)-en-3β-ol-15-one was administered to a series of male Sprague-Dawley rats by intragastric intubation in the form of an emulsion in a mixture of triolein, sodium taurocholate, bovine serum albumin, and glucose. [4− 14C]Cholesterol was similarly administered to a second series of rats. The distribution of 3H and 14C was studied at 12 and 48 h after the administration of the sterols. The results demonstrated that the 15-ketosterol is absorbed and metabolized to material with the Chromatographic properties of fatty acid esters of the 15-ketosterol, to cholesterol, and to fatty acid esters of cholesterol. The [ 3H]cholesterol formed from the 15-ketosterol was characterized by its behavior on silicic acid-Super Cel column chromatography, by the chromatographic behavior of its acetate derivative on alumina-AgNO 3 column chromatography, and by purification by way of its dibromide derivative without significant change in specific activity. The general distribution of 3H was similar to that of 14C. No unusual concentration of 3H in any of the organs studied was observed.

Quantitative tritium exchange of [ 3H] aflatoxin B 1 during penetration through isolated human skin

Tritium labelled aflatoxin B1 ([G-3H]AFB1) underwent an almost total tritium exchange with water during penetration through isolated human skin. The process was not enzymatic and the site of exchange appeared to be within the epidermis. The mechanism which mediated this extensive exchange was not determined. However, the tritium in [G-3H]AFB1 was found to be very susceptible to chemical conditions which favored carbanion formation at the alpha-carbon of the cyclopentenone ring. The relative effectiveness of the various solvents in mediating the loss of the tritium label was 1 N3NaOH much greater than methanol greater than 1 N HCl greater than water. This work serves as a warning that [G-3H]AFB1 can easily undergo significant changes in specific activity in biological tissues and under relatively mild experimental conditions. It is possible that conditions within the skin favor carbanion formation.

The Extent of Starch Turnover in Mature Pepper Leaves in the Light

The extent of starch turnover in pepper leaves in the light has been estimated. After leaves were labelled with 14CO2 at constant specific activity for 4–7 h, the irradiance was reduced to a level which caused no net change in the starch content of the leaf, and the supply of 14CO2 was removed. There was no significant change in specific activity of starch over the following 6–10 h, thus there was no exchange of 14C-starch with 12C-assimilates entering the chloroplasts.

Ethanol and hypobarometric simulated high altitude: a gonadal-hepatic toxicity study in the male rat.

Male rats were maintained on water or on 10% ethanol drinking fluid. They were pairfed for 30 d prior to exposure to simulated high altitude (approximately 6000 m) and for 78 d, during which they were exposed to simulated high altitude on alternate days. Corresponding controls were maintained at ambient pressure. The high-altitude animals showed loss in liver and epididymis weights compared to respective water controls as contrasted with increased spleen weight in ethanol-drinking rats exposed to high altitude. Hepatic mitochondrial aldehyde dehydrogenase (ALDH) was decreased, compared to controls, by hypobarometric pressure in water-drinking animals. Ethanol intake negated this effect. The kinetics of this inhibition show changes in Vmax without concomitant changes in the apparent Km. Hepatic alcohol dehydrogenase did not change by either treatment. Testicular and epididymal ALDH showed, statistically, no significant changes in specific activity as a function of exposure to high altitude. However, combined ethanol drinking and altitude exposure increased epididymal ALDH compared to water-drinking rats subjected to the same experimental conditions. The changes in liver and testicular weight and in the enzyme involved in the biotransformation of ethanol-derived acetaldehyde suggest the contribution of endocrinological and biochemical factors to hypoxia and to ethanol-evoked adverse responses studied.

Effect of methionine, glycine and serine on serine hydroxymethyltransferase activity in rat glioma and human neuroblastoma cells.

Human neuroblastoma SK-N-SH-SY5Y (5Y) and rat glioma (C6) cells were cultured with supplemental methionine, glycine, or serine for three to six days. Serine hydroxmethyltransferase (SHMT: L-serine: tetrahydrofolate 5, 10-hydroxymethyltransferase, EC 2.12.1) was assayed radiometrically in whole cell homogenates, crude supernatant fractions and crude particulate fractions. No significant changes in specific activity or cellular morphology were noted at methionine, glycine, or serine concentrations up to 16 mM. Serine concentrations of 20 and 40 mM led to significantly lower gliomal enzyme specific activities. This activity was unevenly distributed between soluble and particulate fractions, with 190 and 398 nmoles of HCHO formed per mg of protein per hour, respectively. Growth stage and time of incubation were major determinants of enzyme specific activity. C6 cells' specific activity rose slowly with increasing time in culture until cellular confluence. At this time there was a pronounced elevation in specific activity, occurring more rapidly in cells grown in 1.2 mM methionine. Intracellular amino acid analysis of C6 cells demonstrated a significant rise in methionine after four days in media containing 0.2 mM methionine. No appreciable diminution in the intracellular levels of glycine or serine occurred following incubation in excess methionine. It is concluded that SHMT-specific activity in C6 and 5Y cells is not regulated by glycine, serine, or methionine levels and that high concentrations of these amino acids (> 30 mM) are not detrimental to these cells derived from the CNS.

Colorimetric determination of succinic acid using yeast succinate dehydrogenase

An enzymatic method for the rapid determination of succinic acid in biological fluids was developed utilizing yeast mitochondria as a source of succinate dehydrogenase. The yeast enzyme catalyzes a complete stoichiometric reduction of 2-( p-iodophenyl)-3-( p-nitrophenyl)-5-tetrazolium chloride to a red formazan. The formazan is extracted into ethylacetate and its absorbance measured at 490 nm. The method is simple, specific, reproducible, and very sensitive (0.01 to 0.14 μmol). The yeast enzyme can be stored in liquid nitrogen for periods of at least 30 days with no significant change in specific activity. In this respect it is superior to a variety of succinate dehydrogenase preparations from animal tissues. The method was applied to measurement of succinic acid excreted by nonproliferating yeast cells metabolizing glucose. Derepressed yeast cells secreted several-fold as much succinic acid as repressed cells submitted to identical test conditions.

Thyrotropin stimulation of a thyroid contractile protein phosphorylation.

Contractile proteins were isolated from preincubated dog thyroid slices by a procedure identical to the one used for the preparation of muscle actomyosin. At least two of these proteins (molecular weight = 26000 and 15000) were phosphorylated in the intact cell system. After one hour of incubation of the slices with thrytropin (10 mU/ml) and [32P]phosphate, the specific activity of the 26000-Mr protein was increased by a factor of three while no significant change in specific activity was observed in the 1500-Mr contractile protein. This effect of thyrotropin was already observed after 30 min of action, was elicited at hormone concentrations of the same order as those required to induce secretion and has been reproduced by dibutyryl adenosine 3':5'-monophosphate. Cycloheximide (0.35 mM) which almost totally inhibited protein synthesis in thyroid slices, did not affect the thyrotropin stimulation of the phosphorylation of the 26000-Mr protein. The phosphorylation of serine residue(s) has been demonstrated in this protein but the presence of radioactive phosphothreonine could not be detected. On the basis of its molecular weight, the 26000-Mr protein could be similar to the inhibitory component of muscle troponin complex (TN-I).

Metabolism of biogenic amines in neuroblastoma and glioma cells in culture.

The kinetic parameters of monoamine oxidase (MAO; E.C 1.4.3.4) and catechol-O-methyltransferase (COMT; EC 2.1.1.6) were evaluated in extracts of adrenergic and non-adrenergic mouse neuroblastoma cells and in rat glioma cells. Using the naturally-occurring substrates tyramine, tryptamine, serotonin and norepinephrine, the affinity of MAO for a given substrate was independent of the presence of the catecholaminergic pathway or cell type used, with apparent Km values ranging from 8-14 microM for tryptamine to 510-580 microM for norepinephrine. The MAO activity in glioma cells was substantially greater than in either neuroblastoma clone, but Vmax values varied little with substrate among cell lines. Both the neuronal and glial COMT had a similar Km for 1-norepinephrine (200 microM); the corresponding Vmax values were also similar among the different cell lines, but represented only 2-10% of the maximal MAO activity. Neuroblastoma and glioma cells, when grown from early logarithmic to stationary phase, showed no significant changes in specific activity of either MAO or COMT. Growth of cells for 3 days with 1 mM-N6,O2'-dibutyryl adenosine-3',5'-cyclic monophosphate resulted in no marked change in either MAO or COMT activity. These results suggest that in neurons neither MAO nor COMT plays a major role in the type of transmitter inactivation that is analogous to that of acetylcholinesterase in cholinergic synapses. The occurrence of considerable MAO and acetylcholinesterase activities in glioma cells may indicate a role for these cells in neurotransmitter inactivation.

Thyrocalcitonin injection to rats increases the liver inorganic phosphate.

The mechanism of the hypophosphatemic effect of thyrocalcitonin (TCT) is uncertain and may involve removal of inorganic phosphate (P1) from the plasma by heretofore unidentified organs. Rats, 192 g. thyroparathyroidectomized 16-24 h earlier, received 100 MRC mU TCT or vehicle (V) SC and 100 muCi 32PO4 iv. One h later plasma and tissue samples were collected. Compared to rats which received V, TCT-treated rats had lower plasma P1 (-0.36 plus or minus 0.08 mumol/ml, P smaller than 0.001) and Ca, higher liver P1 (+0.66 plus or minus 0.19 mumol/g, P smaller than 0.01) and unchanged red blood cell and rectus abdominus P1. TCT had no significant effect on the amount of perchloric acid soluble phosphate in any of the tissues. TCT-treated rats had lower plasma 32P1 (cpm/ml) and higher liver 32P1 (cpm/g) with no significant changes in specific activity (SA). TCT caused a small decrease in muscle 32P1 SA. There were no significant changes in the 32P (cpm/g) or 32P SA of the acid soluble phosphate fraction of any of the tissues. The results suggest that a portion of the hypophosphatemic effect of TCT can be accounted for by the increased P1 level in the liver.

Esterases of Drosophila. II. Biochemical studies of esterase-5 in D. pseudoobscura.

In vitro enzyme hybridization was carried out with combinations of six allozymic variants of Esterase-5 from Drosophila pseudoobscura. Studies on heat stability and specific activity changes accompanying hybridization were done to examine the possible expression of overdominance at the biochemical level. In 11 of 15 combinations no significant change in specific activity was found following hybridization. In two cases hybridization resulted in a decrease in activity in the mixture, while in two cases esterase activity was elevated. Heat stability studies, in several cases, revealed reduced rates of inactivation in in vitro and in vivo heterozygotes compared with homozygotes. From these and other data a model for the molecular mechanism of heterosis is presented.

Lysosomes of the arterial wall: II. Subcellular fractionation of aortic cells from rabbits with Experimental Atheroma

Cells were isolated from the aortas of control rabbits and of rabbits with experimental atheroma induced by cholesterol feeding. They were homogenized and fractionated by three different techniques relying mainly on differences in equilibrium density of cell particles in sucrose gradients. After cholesterol feeding, the aortic smooth muscle cells transformed progressively into foamy cells. Their protein to DNA ratio was unchanged. The mitochondrial marker cytochrome oxidase and the presumed plasma membrane markers 5′-nucleotidase and leucyl-β-naphthylamidase showed no significant change in specific activity, nor in density distribution, even in severely atheromatous lesions. Only minimal changes were seen for α-glucosidase, an enzyme probably associated largely with microsomes. Catalase was increased up to 7-fold; one-third of this activity was in particles of about 1.18 density, the rest was soluble. The specific activity of all lysosomal acid hydrolases increased with increasing severity of atheroma, up to a little more than 2-fold for N-acetyl-β-galactosaminidase and α-mannosidase, 3- to 4-fold for N-acetyl-β-glucosaminidase, acid phosphatase, and cathepsin C, more than 10-fold for β-galactosidase and β-glucuronidase. At the same time the lysosome became less fragile and their density distribution underwent a progressive broadening in the direction of decreasing density, extending to a value as low as 1.03. Incipient changes in lysosome density could be detected already in the early stages of the cholesterol-induced atheromatous transformation and in animals with moderate atheroma induced by a diet rich in saturated fatty acids. The cholesterol content of the cells increased up to 16-fold and the distribution of this substance was altered from a plasma membrane type in normal smooth muscle cells to one resembling that of lysosomal enzymes in foamy cells. Most likely, cholesterol storage is responsible for the decrease in lysosome density, and cholesterol-containing vacuoles in foamy cells are of lysosomal nature. Much of this cholesterol was in esterified form. In other experiments, no age- or sex-related changes in the aortic cell organelles could be demonstrated. After repeated intravenous injection of Triton WR-1339 there was a significant decrease in equilibrium density of the lysosomes indicating uptake of the detergent by this organelle. No change was observed after repeated injection of dextrans of various molecular weight, injection of neutral red, or starvation.

Amino-acid metabolism during osmotic stress in isolated axons of [formula omitted

During hypo-osmotic stress, the amino-acid concentration of isolated axons of C. sapidus decreases while the specific antivity of these compounds labelled from glucose increases. On the contrary, the amino-acid content increases and there is no significant change in specific activity during hyperosmotic shock. These results are discussed in terms of the mechanisms involved in the control of the amino-acid pool in the process of isosmotic intracellular regulation.

Adenosine triphosphatase activity in glial cells and in neuronal perikarya of edematous rat brain

Summary The activity of Na,K-ATPase and the ouabain insensitive ATP-hydrolyzing activity was studied in fractions of glial cells and of neuronal perikarya isolated in bulk from cortex and subcortical white matter of rats, previously treated with intraperitoneal administration of water. With increasing water load, the cortical Na,K-ATPase activity markedly decreased in glial cells and was slightly elevated in neuronal perikarya of edematous brain. The Na,K-ATPase of the glial cells and of the neuronal perikarya of white matter, on the other hand, exhibited no significant changes in specific activity. The ouabain-insensitibe ATPase activity behaved similarly. It was markedly decreased in cortical glial cells isolated from rats receiving water equaling 20% or 30% of their body weight, respectively, whereas no change of the cortical ouabain-insensitive ATPase was observed in the neuronal perikarya.

A study of some enzymes of glycerolipid biosynthesis in rat liver after subtotal hepatectomy.

1. The accumulation of triglyceride in the liver remnant after subtotal hepatectomy (removal of 82% of the liver) exceeded that described for partial hepatectomy (removal of 70% of the liver). 2. Palmitoyl-CoA synthetase, glycerol phosphate acyltransferase and diglyceride acyltransferase activities were measured in the microsomal fraction, and phosphatidate phosphohydrolase activity was measured in the particle-free supernatant fraction, prepared from the liver remnant at various times after subtotal hepatectomy. 3. The only enzyme showing a significant change in specific activity was phosphatidate phosphohydrolase. The specific activity was approximately fivefold that of the control value at 6h after operation and threefold that of the control at 10, 16 and 24h after operation. A smaller increase in the specific activity of the enzyme in sham-operated animals occurred only at 6h after operation. 4. However, at this time the total phosphohydrolase activity of the remaining liver in the sham-operated rats was approximately threefold that in hepatectomized rats. 5. Injection of actinomycin D prevented the increase in activity of phosphatidate phosphohydrolase but did not prevent the accumulation of triglyceride.

Studies on the mechanism of active intestinal transport of glucose

d-[2- 3H]Glucose is actively accumulated by hamster intestinal rings and everted sacs. The isolated glucose from the tissue and the serosal fluid showed no significant change in specific activity. These data rule out an active transport mechanism involving a covalent bond with the oxygen of the hydroxyl on C-2 of glucose. At the same time an exchange reaction of the 3H with water catalyzed by brush borders has been observed. This reaction is probably independent of glucose transport.

Ribonucleic acid biosynthesis in methylcholanthrene-treated rats

The incorporation of [ 14C]orotic acid into whole liver RNA and nuclear RNA was examined at various times after a single administration of 3-methylcholanthrene. No significant change in specific activity was observed. Zonal ultracentrifugation patterns of nuclear RNA were also unaffected by pretreatment of the rats with 3-methylcholanthrene.