Reduction In Specific Activity Research Articles

Analysis of nonspecific reactions in enzyme-linked immunosorbent assay testing for human rotavirus

Solid-phase enzyme immunoassays can be utilized to detect antigens directly in clinical specimens. However, a small number of stools which we tested for human rotavirus by enzyme-linked immunosorbent assay (ELISA) were found to have nonspecific activity in the absence of rotaviral antigen. Similar nonspecific activity was found in eight of eight sera which contained rheumatoid factor. This nonspecific activity was markedly reduced by pretreatment of the specimens with reducing agents, normal goat serum, and anti-human immunoglobulin M (IgM). Thus, it is likely that these specimens contain an IgM antibody capable of reacting nonspecifically with the other components of the assay. Although pretreatment with the mild reducing agent N-acetylcysteine markedly reduced this nonspecific activity, such treatment did not reduce the specific ELISA activity due to rotavirus. Other treatments did produce a reduction in specific activity. Thus pretreatment with N-acetylcysteine offers a practical means to increase the specificity of ELISA systems without reducing their sensitivity.

Expression of KB Cell Alkaline Phosphatase Isoenzymes During Growth in Immunosuppressed LEW Rats<xref ref-type="fn" rid="FN2">2</xref>

Alkaline phosphatase isoenzyme expression of human tumor xenografts was studied by the growing of KB cells in immunosuppressed neonatal LEW rats. In culture these cells produced the oncoamniotic (FL) isoenzyme as the major form and the Regan isoenzyme as a minor fraction as well as a "hybrid" that shared properties of both of the other isoenzymes. Despite a reduction in specific activity, this isoenzyme pattern was essentially unchanged during in vivo growth. KB cells "pretreated" in culture with the glucocorticoid prednisolone in hyperosmolal medium exhibited a decrease in the levels of the oncoamniotic (FL) isoenzyme and an increase in the Regan isoenzyme. During growth of pretreated cells in vivo, a time-dependent resumption in the expression of the oncoamniotic (FL) isoenzyme was associated with the disappearance of the Regan isoenzyme. This shows that the expression of the oncoamniotic (FL) isoenzyme is not restricted to human tumor cells monophenotypic with respect to alkaline phosphatase.

Effect of Sciatic Nerve Crush on Enzyme Activities of Skeletal Muscles of the Rat

Weight loss and reduction in specific activity of cytochrome oxidase of both red (soleus) and white muscles (gastrocnemius and plantaris) of the rat was greatest 5 days after sciatic nerve crush (p less than 0.001) and then became minimal. In neither was there a significant, concomitant loss of protein. By 19 days, the specific activity of cytochrome oxidase was the same in both muscle types. The specific activity of lactate dehydrogenase was reduced significantly (p less than 0.001) in the white muscles, to a value approaching that of the red soleus by 19 days postoperatively, but remained unaltered in the soleus. Nerve crush is proposed as a model experimental system for studying neural regulation of skeletal muscle metabolism.

Intracellular uptake and degradation of extracellular tracers in mouse skeletal muscle in vitro: The effect of denervation

Innervated and chronically denervated mouse skeletal muscles have been incubated under various conditions in a Ringer solution containing one of the three macromolecules: [ 3H]α-neurotoxin, [ 3H]inulin and horseradish peroxidase. Following extensive wash-out for 4 h of the extracellular compartment, the amount of each macromolecule retained intracellularly was obtained. Intracellular uptake of a [ 3H]monoacetylated α-neurotoxin in vitro at 37°C was found to be increased in denervated mouse extensor digitorum longus muscles compared to innervated control muscles. Similarly, the uptake in vitro at 37°C of [ 3H]inulin and horseradish peroxidase was also increased in denervated muscles. At 4°C the uptake of [ 3H]inulin and horseradish peroxidase was markedly reduced. Protamine was found to stimulate the uptake of [ 3H]inulin at 37°C but not at 4°C. Reduction in specific activity by addition of 50-fold excess of unlabelled inulin failed to affect the uptake of [ 3H]inulin suggesting that this uptake process obeyed bulk kinetics. Furthermore, the endocytized [ 3H]inulin was found to be strongly retained in the muscles since prolonged washing or addition of unlabelled inulin to the washing solution did not reduce the uptake. Characterization of [ 3H]inulin taken up by the muscles was performed by gel chromatography on Sephadex G-25. Using a purified [ 3H]inulin solution it was observed that about 45% of the total radioactivity remaining in the muscles was eluted as [ 3H]inulin. Additional radioactivity consisted of lower molecular weight compounds. These degradation products of [ 3H]inulin were only present in the muscle homogenate and were not detected in the incubation solution. The results suggest that intracellular uptake of different macromolecules by endocytosis in skeletal muscles increases following denervation, and that following uptake, degradation of the endocytized material may occur.

In vitro studies of skeletal muscle membranes. Adenylate cyclase of fast and slow twitch muscle and the effects of denervation.

Sarcolemmal membranes were prepared from slow-twitch (red) and fast-twitch (white) skeletal muscle of the rat. A sensitive adenylate cyclase assay was used and basal, fluoride- and catecholamine-stimulated activities measured. The greater in vivo sensitivity of red muscle to the effects of catecholamines correlates, in the present study, with approximately a twofold stimulation of its sarcolemmal adenylate cyclase with isoproterenol (10 micronm). The white muscle enzyme, on the other hand, is only minimally stimulated (20%) at the same concentration of beta-adrenergic agonist. Fast-twitch muscle is known to be physiologically insensitive to catecholamine in vivo. A course of sciatic nerve denervation was followed to further distinguish these two metabolic types of skeletal muscle and their respective adenylate cyclases. The slow-twitch muscle enzyme activities were completely and permanently lost on denervation. The white muscle enzyme, however, recovered almost completely after an initial reduction in specific activity the first week. Interestingly, the NaF-stimulated activity lagged behind both the basal and hormone-stimulated activities of the white muscle enzyme, in returning to control levels. The activities of cyclic nucleotide phosphodiesterase were evaluated in homogenates of the two muscle types in innervated rats and following denervation, in order to further define the neural influence on skeletal muscle cyclic nucleotide metabolism. The results suggest that the motor nerve may regulate some of the metabolic properties of slow-twitch muscle (which may involve cyclic AMP) by controlling the responsiveness of its sarcolemmal-bound adenylate cyclase system.

Comparison of cytoplasmic superoxide dismutase in liver, heart and brain of aging rats and mice

Comparisons of the specific activity of cytoplasmic superoxide dismutase, in homogenates of liver, brain and heart demonstrate considerably reduced activity in livers of aging rats and mice, a very small reduction in specific activity in the heart and no reduction in the brain of aging animals. Antisera elicited in rabbits against purified superoxide dismutase from liver of either young or old rats revealed complete cross-reactivity with the heart and brain enzymes. They also exhibited complete cross-reactivity with the mouse enzymes from all three organs. This finding has, for the first time, enabled a comparison of possible age-dependent alterations in the same enzyme antigen in different cell types. Despite the differences in age-related changes in specific activity in homogenates of liver, heart and brain the enzyme shows a considerable decline in catalytic activity per antigenic unit in all three organs in both aging rats and mice.

Phosphoglycerate kinase from young and old Turbatrix aceti

Phosphoglycerate kinase (ATP:3-phospho- d-glycerate-1-phosphotransferase, EC 2.7.2.3) from young and old Turbatrix aceti has been purified to homogeneity. The “old” enzyme exhibits a marked reduction in specific activity both in crude homogenates and in pure form when compared to preparations from young nematodes. The specific activities for pure “young” and “old” enzymes are 650–750 and 300–400 units/mg, respectively. All other properties of “young” and “old” enzymes were nearly identical, including molecular weight (43 000), K m, behavior on columns, thermal stability and mobility during gel electrophoresis at three pH values. The results are discussed in terms of the possible mechanism of formation of “altered” enzymes. In addition, certain properties of the nematode phosphoglycerate kinase are compared with those of the enzyme from yeast and rabbit muscle.

Regulation of palmitic acid synthesis in cultured glial cells: effects of glucocorticoid on fatty acid synthetase, acetyl-CoA carboxylase, fatty acid and sterol synthesis.

Abstract— C‐6 glial cells in culture were utilized to define the role of glucocorticoid in the regulation of palmitic acid synthesis and the important lipogenic enzymes, fatty acid synthetase and acetyl‐CoA carboxylase. Particular emphasis was given to fatty acid synthetase which exhibited more than a 50% reduction in specific activity when cells were exposed to hydrocortisone (10 μg/ml) for 1 week. Coordinate changes in acetyl‐CoA carboxylase activity and in palmitic acid (and sterol) synthesis from acetate accompanied the alterations in fatty acid synthetase. Immunochemical techniques were utilized to show that the decrease in synthetase activity involved an alteration in enzyme content, not in catalytic efficiency. The changes in content of fatty acid synthetase were caused by alterations in enzyme synthesis. Glucocorticoids may regulate fatty acid synthetase in C‐6 glial cells by a mechanism similar to that suggested for adipose tissue. The inhibition of palmitic acid synthesis may be relevant to other effects of glucocorticoids on developing brain.

The effect of piromen on the flial-mesenchymal scar in the cortex of hamsters

In the present work the effect of Piromen is studied on the formation of the glial-mesenchymal scar in the experimentally injuried cortex of hamsters. The uniform effect of the bacterial polysaccharide is suggested in a strong and lasting stimulation of proliferating reactive cells in the traumatic area. The excessive proliferation is combined with a decrease of differentiation in the reactive cells. A reduction of specific activities (phagocytosis, production of collagen and glial filaments) of these cells was also observed. Finally a porous mesenchymal scar develops with a great many of dedifferentiated cells. On the other hand a spongy glial scar is formed, which is due to a desintegration of astrocytic processes in the peritraumatic area. A so far not precisely understood transformation of reactive astrocytes plays an important role herein. The changes of the glial-mesenchymal scar after administration of Piromen, reported in the literature, were on the whole confirmed.

Variation in the activity levels of selected enzymes of Erwinia amylovora 595 in response to changes in dissolved oxygen tension and growth rate of D-glucose-limited chemostat cultures.

Chemostat cultures of Erwinia amylovora 595, grown in mineral salts-nicotinic acid medium at 30 degrees C, and limited by D-glucose concentrations in the presence of dissolved oxygen tensions (D.O.T.) greater than about 6mm Hg, became limited by oxygen availability below about 4 mm Hg. This latter limitation was accompanied by a marked increase in acid production as the D.O.T. was depressed. The transition between D-glucose- and oxygen-limitation was also characterized by a maximum in succinate oxidase activity, and a minimum in the in situ respiration. D-Glyceraldehyde-3-phosphate dehydrogenase and D-fructose-1, 6-diphosphate aldolase showed small reductions in specific activity in the region 4-6 mm Hg D.O.T., but further reduction to 2 mm Hg resulted in a marked increase in the specific activity of aldolase. Malate dehydrogenase followed the converse trend, and attained very low activity levels when the D.O.T. decreased beyond the lower limits of detection. The in situ respiration was maximal at 2 mm Hg D.O.T., while potential respiration values were minimal at 2 mm Hg, and maximal at about 8 mm Hg D.O.T. The insitu respiration rate was proportional to dilution rate (D), in presence of excess oxygen, up to 0.18 h-1, after which a marked diminution occurred and continued until the wash-out rate was attained. Succinate oxidase activity decreased with increase in dilution rate, but remained constant above D equals 0.18 h-1. Malate dehydrogenase showed a persistent decline with increase in dilution rate, while D-glyceraldehyde-3-phosphate activity increased somehwat at higher dilution rates. The data are interpreted in terms of two transition points, at 6 and 2 mm Hg D.O.T., and of a change from respiratory to fermentative metabolism at low D.O.T., and at high dilution rates.

Modulation of leucine transaminase activity by dietary means.

The specific activity of leucine transaminase was measured in supernatants of liver, skeletal muscle (gastrocnemius), and kidney homogenates obtained from fed, starved, and protein-deprived rats. After 12 h of starvation, there were slight reductions in leucine transaminase activity of both muscle and kidney tissues. When starvation was prolonged to 1 full day, the activity of this enzyme increased by approximately twofold in both muscle and kidney. Prolongation of fasting to 5 days resulted in an additional increase in specific activity of leucine transaminase in muscle. During the entire 5 days of starvation, leucine transaminase activity remained unaltered in liver of starved rats. Protein deprivation for 1 or 5 days resulted in significant reductions in specific activity of leucine transaminase in skeletal muscle. Protein deprivation did not produce a remarkable effect on the activity of this enzyme in kidney or liver tissue. The results of this study, together with those previously obtained, indicate that within our experimental conditions increased oxidation of leucine in skeletal muscle of starved rats is not initially related to an alteration in activity of leucine transaminase. When caloric deficiency is prolonged, the potential for transamination is also increased. These adaptive changes increase the ability of skeletal muscle to use leucine as a metabolic fuel.

Metabolic DNA in the hepatocyte nuclei in newborn rats.

The behaviour in time of labelled nuclear DNA in the hepatocytes of newborn rats was studied using autoradiographic and biochemical techniques in two groups of experiments. In the first group H-3-thymidine was injected to the mothers at the 16th day of pregnancy and the amount of labelled DNA was evaluated in the newborns after delivery. In the second group H-3-thymidine was injected to the newborns two hours after birth and the labelled DNA was studied at the same time intervals as the first group. The amount of labelled thymidine incorporated into the first group of animals remains constant for the first three days of life, thereafter a reduction in specific activity of DNA is observed concomitant with an increase of the percentage of labelled nuclei and a decrease of the number of grains per nucleus. These results show that mitotic divisions, which are absent during the first three days of life, take place between the third and sixth days of life. The decrease of the specific activity is therefore due to dilution and not to loss of labelled DNA. In the second group of experiments the DNA labelled with H-3-thymidine shows a decrease by about 30--40% per day during the first three days of life accompanied by a decrease in the number of grains per nucleus without changes in the percentage of labelled nuclei. These data show that DNA synthesized during the first day after birth is metabolically unstable, unlike that synthesized during foetal life.

Light-induced reduction in specific activity of malate enzyme in Euglena gracilis Z

When dark grown Euglena are exposed to more than about 400 foot candles of white light, there is an exponential reduction in the specific activity of malate enzyme. The original activity is reduced by more than 90%. This reduction in malate enzyme is not inversely co-ordinate, in an Ames-Garry plot, which the production of chlorophyll.

Change in the structure of Shope papilloma virus-induced arginase associated with mutation of the virus.

The change in the state of the virus-induced enzyme associated with a mutation in the virus provides additional evidence that the enzyme is synthesized from virus rather than rabbit genetic information. This change in structure results in differences in stability of polymerization, degree of optical rotary dispersion (ORD) specific rotation, change in elution characteristics from carboxymethyl cellulose, and a reduction in specific activity of the arginase. Liver arginase differs markedly in ORD characteristics from the virus-induced enzyme. In contrast to the virus-induced enzyme, it showed no negative Cotton effect at 233 nm until it was activated with manganese. Manganese had no influence on the ORD spectrum of virus-induced arginase. In addition, liver arginase is denatured by 4 M urea, while the virus-induced enzyme requires 10 M urea for denaturation.

Changes in the amount and properties of adenyl cyclase in brown adipose tissue during acclimation of rats to cold.

Adenyl cyclase of interscapular brown adipose tissue of rats undergoing acclimation to cold was measured in order to find out whether the amount of the nor adrenaline-stimulated enzyme might be increased in cold-acclimated rats, in which a characteristic large increase in the overall calorigenic response to noradrenaline (nonshivering thermogenesis) is induced by the prolonged exposure to cold (4–8 weeks). It was found that the growth of the brown adipose tissue in the acclimating rats is accompanied by an increase in the total amount of noradrenaline-stimulated adenyl cyclase activity. Since this increase does not keep pace with the growth of the tissue a reduction in specific activity of the noradrenaline-stimulated adenyl cyclase was observed. Thus no evidence was obtained for an increased concentration of noradrenaline-responsive structures in the cell membrane of the brown adipose tissue of cold-acclimated rats.In contrast, fluoride-stimulated adenyl cyclase activity increased almost twofold after only 2 days of exposure to cold and before the growth of the tissue had started. Thereafter the activity of this enzyme increased in parallel to the growth of the tissue. The extra activity that developed during the first 2 days persisted throughout the period of acclimation to cold (8 weeks); it disappeared within 1 day when the cold-acclimated rats were returned to room temperature.These changes in adenyl cyclase activity are more rapid than any hitherto reported. They presumably reflected a change in the properties of the cell membrane in response to an environmental stress. It is not known whether the increase in measurable fluoride-stimulated adenyl cyclase activity reflects an increase in the synthesis of a specific protein or an unmasking of the activity of an enzyme already present.No change in the specific activity of cyclic AMP phosphodiesterase was observed; during acclimation to cold the total amount of this enzyme increased in parallel with the growth of the tissue.

Bacillus subtilis Glutamine Synthetase: SPECIFIC CATALYTIC CHANGES ASSOCIATED WITH LIMITED SULFHYDRYL MODIFICATION

Abstract Bacillus subtilis glutamine synthetase readily reacts with iodoacetamide, producing very specific changes in the catalytic parameters characteristic of the native enzyme. The alkylation reaction is influenced both by substrates and feedback inhibitors of the enzyme; whereas ammonia and glutamate are without effect, Mg2+-ATP enhances the alkylation reaction, and Mn2+-ATP effectively prevents the catalytic changes that accompany incubation of the enzyme with the alkylating agent. Histidine, tryptophan, and glutamine also potentiate the response of the enzyme to iodoacetamide. With limited alkylation, a marked increase in activity is seen using Mn2+ as divalent cation, a sharp reduction in specific activity is seen in the assay using Mg2+ as divalent cation, and the response of the enzyme to feedback inhibitors is altered. Whereas these changes in B. subtilis catalytic activity are qualitatively similar to changes seen with enzymatic adenylylation of Escherichia coli glutamine synthetase, no role in vivo for sulfhydryl modification of B. subtilis enzyme has been found. When assayed with Mn2+ as divalent cation, alkylated glutamine synthetase has a higher apparent Km for glutamate than does native enzyme; the substrate saturation function with the alkylated enzyme is hyperbolic and has lost the inhibition by high concentrations of glutamate, which is characteristic of the native enzyme. When assayed with Mg2+ as divalent cation, the alkylated enzyme shows loss of apparent affinity for glutamate at high glutamate concentrations. Sulfhydryl titration indicates that 1 cysteine residue is readily alkylated per subunit, with two to three additional groups titrated after dissociation of the enzyme. The alkylation reaction is specific for cysteine residues. These results thus provide by chemical modification of the B. subtilis glutamine synthetase a system that is qualitatively analogous to the enzymatic adenylylation of the E. coli glutamine synthetase; the results also exhibit a clear structural distinction between the two enzymes. Under similar conditions of study, the sulfhydryl groups of the B. subtilis enzyme are readily titrated by iodoacetamide, whereas those of E. coli glutamine synthetase are not accessible to the alkylating agent.

Enzymatic removal of diacetyl from beer. 3. Enzyme protection and regeneration of cofactor.

Use of diacetyl reductase, a reduced nicotinamide adenine dinucleotide (NADH)-requiring enzyme, to eliminate diacetyl off-flavor in beer was studied. The crude enzyme was extracted from Aerobacter aerogenes and partially purified by ammonium sulfate precipitation or Sephadex chromatography. In the semipure state, the enzyme was inactivated by lyophilization; in a crude state, the lyophilized extract remained stable for at least 4 months at - 20 C. A 50% reduction in specific activity within 5 min was observed when crude diacetyl reductase was suspended (5 mg of protein/ml) in phosphate buffer at pH 5.5 or below; a similar inactivation rate was observed when the crude enzyme was dissolved in a 5% aqueous ethyl alcohol solution. Effective crude enzyme activity in beer at a natural pH of 4.1 required protection of the enzyme in 10% gelatin. Incorporation of yeast cells with the gel-protected enzyme provided regeneration of NADH. Combinations of yeast, enzyme, and gelatin were tested to obtain data analyzed by regression analysis to determine the optimal concentration of each component of the system required to reduce the level of diacetyl in spiked (0.5 ppm) beer to less than 0.12 ppm within 48 hr at 5 C. The protected enzyme system was also effective in removing diacetyl from orange juice (pH 3.8) and some distilled liquors.

Effect of Thiamine Deficiency in Rats on Adipose Tissue Lactate Dehydrogenase Isozyme Distribution

Lactate dehydrogenase (LDH) (EC. 1.1.1.27) was present in five isozymic forms in rat epididymal adipose tissue from rats fed a complete diet; LDH-I (most anodal electrophoretically) was present in low concentration. Thiamine deficiency resulted in a reduction of specific activity (units per milligram soluble protein) of total LDH, but a marked increase in LDH-I and LDH-II. Feeding thiamine-deficient rats a complete diet restored the LDH-isozyme distribution pattern to control characteristics. The shift in LDH-isozyme distribution pattern with thiamine deficiency was suggestive of an increase in the activity of the gene responsible for the synthesis of the H subunit.

Effective Specific Activity Dilution in Labeled DNA of Cultured Mammalian Cells (L5178Y)

Upon labeling the DNA of cultured mammalian cells with radio-active thymidine it was found that there was a large reduction in the specific activity of radioactive thymidine in DNA when this specific activity was compared to the specific activity of the exogenous supply. This reduction in specific activity may be determined once the effective dilution factor for thymidine in mammalian cells is known. The average effective dilution factor was found to be 2 x 10(-4) M for L5178Y cells in log growth.

Tryptophan-niacin relationship in Xanthomonas pruni.

Wilson, R. G. (Oklahoma State University, Stillwater) and L. M. Henderson. Tryptophan-niacin relationship in Xanthomonas pruni. J. Bacteriol. 85:221-229. 1963.-The observation that Xanthomonas pruni, a bacterial pathogen for the peach, requires niacin for growth and can use tryptophan or 3-hydroxyanthranilic acid as a substitute was confirmed. To determine whether niacin is synthesized via the tryptophan-3-hydroxyanthranilic acid pathway, experiments using labeled metabolites were undertaken. Labeled tryptophan, 3-hydroxyanthranilic acid, quinolinic acid, and nicotinic acid were supplied in the basal medium in amounts sufficient to insure maximal growth. Nicotinic and quinolinic acids were isolated from the cells after the growth period. The isotope was incorporated from the first three labeled compounds into niacin with dilutions approximately the same in all cases, ranging from 7.6 to 17.1. The dilution of isotopic niacin was 3.1- to 5.9-fold. Only labeled quinolinic acid gave rise to labeled quinolinic acid in the cell, but this acid gave rise to niacin with 10- to 12-fold reduction in specific activity. The results indicate that if quinolinate participates as an obligatory intermediate in the synthesis of niacin from tryptophan, its concentration within the cell is very small and it does not equilibrate readily with exogenous quinolinate. The results confirm the conclusion, drawn from growth studies, that niacin is needed to permit tryptophan synthesis at a sufficient rate to promote growth. In the absence of an external source of niacin, tryptophan or some of its metabolites can promote growth by acting as precursors of niacin.