Showing papers on "Enzyme assay published in 1968"
TL;DR: The spectrophotofluorometric determination of hydroxylated benzo[a]pyrene products is sufficiently sensitive to detect 10-12 mole per ml and has great utility in measuring the hydroxymatic activity of cells grown in culture.
1,664 citations
Journal Article•
TL;DR: Cardioactive steroids appear to inhibit (Na+ + K+)-ATPase by reducing the difference between the conformational energies ot the phosphorylated and nonphosphorylated forms of the enzyme.
Abstract: The combination of ouabain with (Na+ + K+)-ATPase is essentially irreversible at physiological temperature and pH. The rate of inhibition of the enzyme by different cardioactive steroids varies widely. The amount of steroid bound parallels the inhibition of enzyme activity. Mg2+, Na+, K+, nucleotides, and orthophosphate markedly influence the rate of interaction. In the presence of nucleotides, the rate is accelerated by Mg2+ and Na+ and retarded by K+. In the presence of orthophosphate, Mg2+ increases, K+ slows, and Na+ markedly decreases the rate of interaction. The ratio of ouabain binding to Na+-dependent phosphorylation is 0.50 for Electrophorus electric organ ATPase. In contrast to the native enzyme, the ouabain-treated enzyme rapidly incorporates orthophosphate. Cardioactive steroids appear to inhibit (Na+ + K+)-ATPase by reducing the difference between the conformational energies ot the phosphorylated and nonphosphorylated forms of the enzyme.
341 citations
TL;DR: The formation and activity of nitrogenase2 in Azotobacter vinelandii OP was examined using a cell-free assay system and the question of whether N2 is required for the formation of the enzyme could be answered as this gas could not be completely eliminated from the growth system.
Abstract: The formation and activity of nitrogenase2 in Azotobacter vinelandii OP was examined using a cell-free assay system. A lag period of about 30 min occurred between the exhaustion of the combined nitrogen source and growth on N2. Cells grown on ammonium acetate or potassium nitrate had no detectable nitrogenase activity. Nitrogenase activity appeared in cells, grown under a flowing gas phase of 20% O2 – 60% He, about 45 min after the exhaustion of ammonia. Nitrogenase formation was inhibited in a closed system with an atmosphere containing 40% O2 but not by one containing 20% O2. Hydrogen did not inhibit enzyme formation. The question of whether N2 is required for the formation of the enzyme could not be answered as this gas could not be completely eliminated from the growth system. Chloramphenicol prevented the formation of the enzyme and inhibited nitrogen fixation in whole cells, but had no effect on cell-free enzyme activity. A brief rise in turbidity which occurred during nitrogenase formation appeared...
291 citations
TL;DR: These studies suggest that the induction of aryl hydroxylase activity involves an activation of specific genes and changes in their transcription into messenger RNA.
255 citations
TL;DR: The initial stability of the enzyme in the absence of further synthesis and the inhibition of enzyme disappearance by high concentrations of cycloheximide suggest that early phases of induction involve synthesis of enzyme protein in the presence of turnover and that a system capable of degrading or inactivating the lyase subsequently forms in the tissue.
Abstract: The light induced synthesis of phenylalanine ammonia-lyase in disks cut from potato tubers is very sensitive to cycloheximide. Synthesis is inhibited 50% in disks cultured on 5 mum cycloheximide instead of water and almost completely in disks aged in the presence of 10 mum inhibitor. Inhibition is irreversible. Fresh disks exposed only 1 hour to 10 mum cycloheximide do not synthesize enzyme during the subsequent 24 hours.Normally a maximal enzyme activity develops in disks about 24 hours after being cut from the tuber. Thereafter enzyme activity declines. The disappearance of enzyme is not affected by concentrations of cycloheximide sufficient to inhibit the synthesis of enzyme initially. No disappearance of enzyme is noted during the initial phase of induction if enzyme synthesis is inhibited by cycloheximide. However, enzyme does disappear from the tissue if more than half the maximal enzyme content is allowed to form before synthesis is inhibited. If cycloheximide at a concentration 10-fold that needed to inhibit synthesis completely is added to disks after they have attained a maximal enzyme level, then subsequent loss of enzyme activity from the tissue is prevented. The initial stability of the enzyme in the absence of further synthesis and the inhibition of enzyme disappearance by high concentrations of cycloheximide suggest A) that early phases of induction involve synthesis of enzyme protein in the absence of turnover, B) that a system capable of degrading or inactivating the lyase subsequently forms in the tissue, and C) that the formation of the degrading or inactivating system requires protein synthesis.The effect of cycloheximide on uptake and incorporation of l-isoleucine-U-(14)C into soluble and insoluble proteins of tuber disks was also examined. During induction the rate of uptake increased 3 to 4-fold, and the rate of incorporation into protein, corrected for change in uptake, increased 25-fold. Cycloheximide inhibited incorporation of isoleucine-(14)C into proteins of fresh disks more than 80%. It did not prevent activation of general protein synthesis during induction and inhibited incorporation in induced disks only 20%. At all times incorporation of amino acid into the soluble, lyase-rich, protein fraction was more sensitive to cycloheximide than the insoluble fraction.
209 citations
TL;DR: The binding of non-occluded choline acetyltransferase to synaptosome membranes is a reversible process that is primarily dependent on the pH and ionic strength of the suspending medium and Varying the temperature or prolonged hypo-osmotic treatment does not release the membrane-bound enzyme.
Abstract: 1. The binding of non-occluded choline acetyltransferase to synaptosome membranes is a reversible process that is primarily dependent on the pH and ionic strength of the suspending medium. 2. The distribution of soluble enzyme bound to synaptosome membranes was studied by density-gradient centrifuging. 3. Choline acetyltransferase shows enzyme activity both in the free and in the membrane-bound form. 4. Varying the temperature or prolonged hypo-osmotic treatment does not release the membrane-bound enzyme. 5. The release of choline acetyltransferase from membranes by different anions, thiols, adenosine nucleotides and enzyme substrates was studied.
190 citations
TL;DR: Human skin collagenase also acts on collagen in the native fibrillar form at 37° with a pH optimum of 7 to 8.0, and is inhibited by low concentrations of EDTA, cysteine and human serum but not by soybean trypsin inhibitor.
188 citations
TL;DR: A sensitive radioassay has been developed and successfully applied to normal mammalian tissues of tryptophan hydroxylating enzyme, which shows complete dependence on the presence of oxygen, a reduced pteridine, and ferrous iron.
Abstract: Publisher Summary The development of a quantitative assay system and appropriate assay conditions for mammalian tryptophan hydroxylase has permitted a preliminary characterization of this enzyme. Tryptophan hydroxylase from brainstem and pineal tissue is either soluble or easily solubilized from mitochondrial fraction and exhibits the characteristics of a typical aromatic ring hydroxylase. This rate-limiting enzyme is extremely useful in understanding the hydroxyindole pathway of tryptophan metabolism and eventually the role of 5-HT in tissues. Hydroxylation of tryptophan at the 5-position is the first step in the hydroxyindole pathway of tryptophan metabolism. This pathway leads to the formation of physiologically active substances such as 5-HT and melatonin. The tryptophan hydroxylating enzyme activity shows complete dependence on the presence of oxygen, a reduced pteridine, and ferrous iron. Relatively high levels of 2-mercaptoethanol (0.05 M) are also required for optimal in vitro enzyme activity. With the availability of this reproducible source of tryptophan hydroxylating enzyme, it has been possible to develop a sensitive radioassay, which now has been successfully applied to normal mammalian tissues.
186 citations
TL;DR: In both conditions a steady state is maintained by a balance between enzyme degradation and synthesis, which shows that the accumulation of aminotransferase on induction is due to an increased rate of its synthesis from free amino acids and not due to a decreased rate of degradation.
144 citations
TL;DR: The marked similarity in the observed inhibition of diverse enzymes by long chain unsaturated fatty acids to that of reported inhibitions by fatty acyl-CoA esters suggests that inhibitory effects are due to detergent properties of these inhibitors.
138 citations
TL;DR: It is suggested that the acetyl-CoA carboxylase from the tumors is the same enzyme as that from host liver, and that the increased activity in the tumor is due to an increased amount of enzyme rather than a structurally altered enzyme.
TL;DR: The increases in the activities of hepatic citrate-cleavage enzyme and ;malic' enzyme occurred simultaneously with a marked increase in lipogenesis, suggesting a relationship of these enzymes to lipogenesis in chick liver.
Abstract: The activities of several enzymes possibly implicated in lipogenesis were measured in the soluble fraction of homogenates of liver and adipose tissue of embryonic and growing chicks. The activities of adipose-tissue enzymes showed little or no change. The activities of hepatic hexose monophosphate-shunt dehydrogenases, malate dehydrogenase, 3-phosphoglyceraldehyde dehydrogenase and NAD-linked α-glycerophosphate dehydrogenase also showed little or no change. Isocitrate dehydrogenase activity in liver rose to a peak on the day of hatching and fell to half the peak value during the next 12 days, where it remained to 26 days after hatching. The activities of ‘malic’ enzyme and citrate-cleavage enzyme showed very low stable values in embryonic liver and remarkable rises during the early part of the post-hatching period. An 85-fold increase in the activity of ‘malic’ enzyme activity was completed in 7 days and a 15-fold increase in that of citrate-cleavage enzyme in 5 days. The activities then attained were maintained up to 26 days after hatching. 2. The increases in the activities of hepatic citrate-cleavage enzyme and ‘malic’ enzyme occurred simultaneously with a marked increase in lipogenesis, suggesting a relationship of these enzymes to lipogenesis in chick liver. By contrast, activity of the hexose monophosphate-shunt dehydrogenases does not appear to be thus associated.
TL;DR: Three times crystallized metapyrocatechase has a specific activity of about 110 µmoles per min per mg of protein at 24° and contains 1 g atom of iron per mole of enzyme, based on molecular weight of 140,000.
TL;DR: The evidence from these experiments suggests that all mitochondria are in a dynamic state and that alteration of the enzyme complement is independent of mitochondrial genesis.
TL;DR: Tests against antibody showed that the tyrosine transaminases in fetal and adult liver are immunologically identical and proved that the increase in enzyme activity in explants exposed to hydrocortisone was due to an increase in enzymes protein.
TL;DR: This work has shown that the modifying effect on enzyme activity probably results from an induced conformational change in the enzyme structure.
Abstract: THE regulation of enzyme activity by cellular metabolites in micro-organisms is usually exerted by feedback control, an initial enzyme of a pathway being controlled by an end-product of that pathway. Such control of enzyme activity has, in many cases, been shown to be “allosteric”—that is, the end-product effector binds to the enzyme at a site distinct from the catalytic (active) site. The modifying effect on enzyme activity probably results from an induced conformational change in the enzyme structure.
TL;DR: The decline in enzyme activity during the neonatal period was correlated with a shift in the enzyme properties from the foetal to the adult type, and may reflect the decrease in the proportion of haemopoietic tissue in the liver.
Abstract: 1. The activity of thymidine kinase in rat liver supernatant decreased with development to a value in the adult that was 1% of that in the 17-day foetus. 2. The foetal enzyme was more stable than the adult to gel filtration on Sephadex G-25 at 0°. 3. The greater stability of the foetal enzyme to incubation at 45° was attributable to the presence of higher concentrations of nucleotides in foetal liver supernatant. 4. The Km values for foetal and adult enzymes were approx. 2·5μm- and 2·1μm-thymidine respectively. 5. The foetal enzyme was more sensitive to inhibition by thymidine triphosphate. 6. The decline in enzyme activity during the neonatal period was correlated with a shift in the enzyme properties from the foetal to the adult type, and may reflect the decrease in the proportion of haemopoietic tissue in the liver.
TL;DR: The results suggest that increased substrate delivery to the liver is the principal stimulus to the increased rate of glucose metabolism observed in newly hatched chicks and that changes in the activities of ;malic' enzyme and citrate-cleavage enzyme are secondary to an increased flow of metabolites through the glucose-to-fatty acid pathway.
Abstract: 1. The conversion of [U-14C]glucose into carbon dioxide, cholesterol and fatty acids in liver slices and the activities of `malic' enzyme, citrate-cleavage enzyme, NADP-linked isocitrate dehydrogenase and hexose monophosphate-shunt dehydrogenases in the soluble fraction of homogenates of liver were measured in chicks that were starved or starved then fed. 2. In newly hatched chicks the incorporation of [U-14C]glucose and the activity of `malic' enzyme did not increase unless the birds were fed. The response to feeding of [U-14C]glucose incorporation into fatty acids increased as the starved chicks grew older. 3. Citrate-cleavage enzyme activity increased slowly even when the newly hatched chicks were unfed. On feeding, citrate-cleavage enzyme activity increased at a much faster rate. 4. In normally fed 20-day-old chicks starvation decreased the incorporation of [U-14C]glucose into all three end products and depressed the activities of malic'enzymeandcitrate−c≤ava≥enzyme.Re−feed∈g∈creasedalloftheseprocesses→∥a∥lorhigher−than−∥a∥l≤vels.5.In⊥h≠wlyˆchedand20−day−oldχcks⋆vation∈creasedtheactivityofisocitratedehydro≥naseandfeed∈gorre−feed∈gdecreasedit.6.Veryli≤chan≥∈hexosemonophospˆe−shuntdehydro≥naseactivitywasobserveddur∈gthediηrymanipa––tions.7.Therest–s∈dicatetˆ∈creased⊂strate∂ivery→theliveristhepr∈cipalstiμlus→the∈creasedrateofglucosemηbolismobserved∈≠wlyˆchedχcks.Therest–salsosug≥sttˆchan≥s∈theactivitiesofmalic′enzymeandcitrate-c≤ava≥enzyme.Re-feed∈g∈creasedalloftheseprocesses→∥a∥lorhigher-than-∥a∥l≤vels.5.In⊥h≠wlyc^hedand20-day-oldχcks⋆vation∈creasedtheactivityofisocitratedehydro≥naseandfeed∈gorre-feed∈gdecreasedit.6.Veryli≤chan≥∈hexosemonophospe^-shuntdehydro≥naseactivitywasobserveddur∈gthediηrymanipations.7.Therests∈dicatet∈^creased⊂strate∂ivery→theliveristhepr∈cipalstiμlus→the∈creasedrateofglucosemηbolismobserved∈≠wlyc^hedχcks.Therestsalsosug≥sttc^han≥s∈theactivitiesofmalic' enzyme and citrate-cleavage enzyme are secondary to an increased flow of metabolites through the glucose-to-fatty acid pathway and that the dehydrogenases of the hexose monophosphate shunt play a minor role in NADPH production for fatty acid synthesis.
TL;DR: The kinetic data suggest that the mechanism of nucleotide activation results in a two-stage allosteric transition of the enzyme, viz., a change in both catalytic efficiency and enzyme affinity toward glucose-1-P.
TL;DR: The glutamic acid decarboxylase has been purified from the lobster central nervous system and potassium ion and β‐mercaptoethanol were essential for enzyme activity.
Abstract: — The glutamic acid decarboxylase has been purified from the lobster central nervous system. Potassium ion (0-075 m) and β-mercaptoethanol (0-025 m) were essential for enzyme activity. Enzyme had about 60 per cent of its optimal activity in the absence of added pyridoxal phosphate. Carbonyl reagents (10−4m-hydroxylamine or amino oxyacetic acid) would abolish this residual activity. The pH optimum of the enzyme was about 8-0.
Standard Michaelis-Menten kinetics were applied to the decarboxylation of glutamate and a Km of 0.02 m was calculated. GABA inhibited the reaction (Ki= 1.25 × 10−3m), but the inhibition showed anomalous behaviour when graphed by the method of Lineweaver and Burk (1934). The GABA inhibition resembled competitive inhibition, but curves rather than straight lines intersecting at a common point on the velocity axis were obtained. This effect remains unexplained.
Preliminary studies failed to reveal any subunit structure of the enzyme. The sedimentation coefficient (.S20.w) was 6-55 in a sucrose density gradient in an ultracentrifuge. This was unchanged by the addition of any of the agents that influence enzyme activity.
The subcellular localization of the decarboxylase was explored in crude homogenates of lobster central nervous system prepared in various ways. The major proportion (about 90 per cent) of the enzyme activity was in the soluble fraction.‘Particulate’enzyme could be prepared, but gentle suspension of this material in buffer liberated most of the activity.
A contaminant in the radioactive substrates led to the production of radioactive GABA without the simultaneous evolution of CO2. In this case, GABA production required active enzyme but was not an exclusive property of the glutamic decarboxylase activity.
TL;DR: Kinetic studies with the partially purified enzyme suggest that decreased levels of NAD, such as occur after ethanol ingestion in vivo, would have a marked effect on enzyme activity, which may be important in oxidizing biologically active aldehydes derived from endogenous sources.
TL;DR: Though phosphoenolpyruvate and fructose 1,6-diphosphate readily protect the enzyme against Cu(2+) inhibition, little evidence of significant reversal of the inhibition by these compounds could be found.
Abstract: 1. Preincubation of partially purified rat liver L-type pyruvate kinase at 25° for 10min. causes a marked increase in co-operativity with respect to both the substrate, phosphoenolpyruvate, and the allosteric activator, fructose 1,6-diphosphate. 2. The results are consistent with the existence of two forms of liver L-type pyruvate kinase, designated forms LA and LB. It is postulated that form LA has a low Km for phosphoenolpyruvate (about 0·1mm) and is not allosterically activated, whereas form LB is allosterically activated by fructose 1,6-diphosphate, exhibiting in the absence of the activator sigmoidal kinetics with half-maximal activity at about 1mm-phosphoenolpyruvate. In the presence of fructose 1,6-diphosphate, form LB gives Michaelis–Menten kinetics with Km less than 0·1mm. It is further postulated that preincubation converts form LA into form LB. 3. The influence of pH on the preincubation effect was studied. 4. The inhibition of pyruvate kinase by Cu2+ was studied in detail. Though phosphoenolpyruvate and fructose 1,6-diphosphate readily protect the enzyme against Cu2+ inhibition, little evidence of significant reversal of the inhibition by these compounds could be found. 5. The effects of starvation, fructose feeding and preincubation on the pyruvate kinase activity of crude homogenates of various tissues of the rat were also studied.
TL;DR: Biosynthetic l-threonine deaminase has been purified approximately 250-fold from crude extracts of nutritionally derepressed Salmonella typhimurium, capable of removing the radioactivity from a crude extract containing label in all the proteins except native threonineDeaminase.
TL;DR: The rat liver lysosome seems to to be heterogeneous, pharmacologically, and the dose-response relationship for enzyme release from the heavy lysOSomes, was studied.
TL;DR: A study of threonine deaminase from Saccharomyces cerevisiae, which has been purified 50-fold, with a low resolution of pyridoxal phosphate coenzyme, finds that Affinity of the enzyme for its substrate is even more strongly modified by pH.
Abstract: A study of threonine deaminase from Saccharomyces cerevisiae has been undertaken The enzyme has been purified 50-fold, with a low resolution of pyridoxal phosphate coenzyme The purified enzyme is very unstable under all conditions tried so far Properties have been studied with crude extracts Optimum pH has been found to be 82 Isoleucine has complex effects on the enzyme activity At a concentration of 01 mM, it is able to stimulate the enzyme At higher concentrations, isoleucine inhibits The Ki values increase with increasing pH, but 100% inhibition can be reached at all ranges of pH studied Affinity of the enzyme for its substrate is even more strongly modified by pH
a) Substrate cooperativity, nil at pH 7, intermediate at pH 8, seems to be maximum at pH 9 At any pH, the substrate cooperativity can be suppressed by addition of L-valine In addition, rather low concentrations of isoleucine also act partly as valine does
b) Apparent Km values, measured in the presence of valine, vary with pH in the opposite direction to Ki values Hill's coefficients for substrate and inhibitor reflect these findings For threonine alone, they vary from 12 at pH 7 to 3 at pH 9 In the presence of valine, they remain close to 1, at all pH Isoleucine, 01 mM, produces an incomplete effect compared to valine For isoleucine inhibition, the cooperativity coefficients remain close to 3 at all pH The molecular weight of the enzyme has been determined to be roughly 12 × 105, at pH 68 and at pH 87 Thermal inactivation is unimodal and fails to show desensitization of the enzyme in any of the conditions tried
The implications of these results on the structure of the enzyme have been discussed
TL;DR: Using acid deoxyribonuclease digest as the substrate, the pH optimum of the enzyme is close to 5.5 in succinate buffer, whereas a higher value is found in acetate buffer; pH-activity curves are barely affected by the presence of Mg2+.
TL;DR: A form of phosphofructokinase isolated in the absence of stabilizers was found to be more sensitive to ATP inhibition, as evidenced by a lower apparent Ki, and gave curves for fructose-6-P which were more sigmoid than those of the native enzyme.
TL;DR: A procedure for release of more than 80% of the total polymerase activity from rat liver nuclei is reported, which would allow meaningful conclusions to be drawn about the quantities of each type of activity present in intact nuclei.