Showing papers on "Enzyme assay published in 1972"

The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme.

Abstract: 1. The ;xanthine oxidase' activity of rat liver supernatant, most of which behaves as an NAD(+)-dependent dehydrogenase (type D) can be rapidly converted into an oxidase (type O) by thiol reagents such as tetraethylthiuram disulphide, copper sulphate, 5,5'-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercuribenzoate. Treatment with copper sulphate, if prolonged, leads to almost complete inactivation of the enzyme. The effect of these reagents is prevented by dithioerythritol, and in all cases but that of N-ethylmaleimide is reversed by the same thiol. 2. Dithioerythritol prevents and reverses the conversion of xanthine oxidase from type D into type O brought about by storage of rat liver supernatant at -20 degrees C, preincubation under anaerobic conditions, treatment with carbon or with diethyl ether, and reverses, but does not prevent, the conversion obtained by preincubation of the whole liver homogenate. 3. Conversion of the enzyme from type D into type O is effected by preincubation of rat liver supernatant with the sedimentable fraction from rat liver but not from chick or pigeon liver. The xanthine dehydrogenase activity of chick liver supernatant is not changed into an oxidase by preincubation with the sedimentable fraction from rat liver. 4. The enzyme activity of rat liver supernatant is converted from type D into type O during purification of the enzyme: the purified enzyme can be reconverted into type D by dithioerythritol. 5. The enzyme appears as an oxidase in the supernatant of rat heart, intestine, spleen, pancreas, lung and kidney. The enzyme of all organs but intestine can be converted into a dehydrogenase by dithioerythritol.

The activities of phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase and the glycerol 3-phosphate dehydrogenase in muscles from vertebrates and invertebrates

Abstract: 1. The maximum activities of hexokinase, phosphorylase and phosphofructokinase have been measured in extracts from a variety of muscles and they have been used to estimate the maximum rates of operation of glycolysis in muscle. These estimated rates of glycolysis are compared with those calculated for the intact muscle from such information as oxygen uptake, glycogen degradation and lactate formation. Reasonable agreement between these determinations is observed, and this suggests that such enzyme activity measurements may provide a useful method for comparative investigations into quantitative aspects of maximum glycolytic flux in muscle. 2. The enzyme activities from insect flight muscle confirm and extend much of the earlier work and indicate the type of fuel that can support insect flight. The maximum activity of hexokinase in some insect flight muscles is about tenfold higher than that in vertebrate muscles. The activity of phosphorylase is greater, in general, in vertebrate muscle (particularly white muscle) than in insect flight muscle. This is probably related to the role of glycogen breakdown in vertebrate muscle (particularly white muscle) for the provision of ATP from anaerobic glycolysis and not from complete oxidation of the glucose residues. The activity of hexokinase was found to be higher in red than in white vertebrate muscle, thus confirming and extending earlier reports. 3. The maximum activity of the mitochondrial glycerophosphate dehydrogenase was always much lower than that of the cytoplasmic enzyme, indicating that the former enzyme is rate-limiting for the glycerol 3-phosphate cycle. From the maximum activity of the mitochondrial enzyme it can be calculated that the operation of this cycle would account for the reoxidation of all the glycolytically produced NADH in insect flight muscle but it could account for only a small amount in vertebrate muscle. Other mechanisms for this NADH reoxidation in vertebrate muscle are discussed briefly.

Control of Circadian Change of Serotonin N-Acetyltransferase Activity in the Pineal Organ by the β-Adrenergic Receptor

Abstract: Serotonin N-acetyltransferase (EC 2.3.1.5) activity in the rat pineal organ is enhanced 50-fold at night. Rats exposed to light at night or kept in darkness during the daytime do not show any elevation of enzyme activity. Treatment with reserpine, a compound that depletes norepinephrine from nerves, 1-propranolol, a β-adrenergic blocking agent, or cycloheximide, an inhibitor of protein synthesis, abolishes the nocturnal increase in serotonin N-acetyltransferase activity, indicating that the enzyme activity is modulated by neural release of norepinephrine from sympathetic nerves via β-adrenergic receptors, and that the increase in enzyme activity is due to synthesis of new enzyme molecules. When rats are exposed to light at night or injected with 1-propranolol, there is a precipitous fall in serotonin N-acetyltransferase activity (half-life 5 min). Cycloheximide administered at night results in a slow fall in enzyme activity (half-life 60 min). When rats are kept in darkness and then exposed to light for 10 min, L-isoproterenol rapidly initiates the elevation of serotonin N-acetyltransferase activity to the initial level in 60 min. On the other hand, when the rats are kept in continuous light, L-isoproterenol initiates an increase in serotonin N-acetyltransferase activity after a lag phase of 60 min. The results indicate that there are two types of changes in serotonin N-acetyltransferase activity; a rapid increase and decrease mediated by the β-adrenergic receptor, and a slow increase and decrease in enzyme activity that appears to represent the turnover of the enzyme.

Study of optimum buffer conditions for measuring alkaline phosphatase activity in human serum

Abstract: We have compared 23 compounds, with and without transphosphorylating properties, as buffer systems for human serum alkaline phosphatase activity, with p -nitrophenylphosphate as substrate. Relative enzyme activity in four representative buffers at near-optimal conditions was ethylaminoethanol > diethanolamine > 2-amino-2-methyl-1-propanol > carbonate. Transphosphorylation was demonstrated in the two buffers in which the enzyme was most active, ethylaminoethanol and diethanolamine. The optima for pH, buffer concentration, and substrate for these four systems were studied in detail. 2-Ethylaminoethanol supports the highest enzyme activity of any of the compounds tested. Diethanolamine was shown to have many of the favorable characteristics required for a reference enzyme procedure.

Assay and properties of digitonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver

Abstract: 1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0°C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of Pi) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis–Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis–Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn2+ was slightly more, and Ca2+ somewhat less, stimulatory than Mg2+. The Mg2+-dependent fraction showed Michaelis–Menten kinetics with respect to the added Mg2+. 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.

Genetic expression of aryl hydrocarbon hydroxylase induction. IV. Interaction of various compounds with different forms of cytochrome P-450 and the effect on benzo(a)pyrene metabolism in vitro.

Abstract: The phenomena of increased type II binding, measured by pyridine interaction with oxidized cytochrome P-450 in vitro , and of decreased type I binding, determined by hexobarbital combination with oxidized P-450 in vitro , are related to aryl hydrocarbon hydroxylase induction by aromatic hydrocarbons in genetically responsive mice and do not occur in 3-methylcholanthrene-treated genetically nonresponsive mice. A method for determining specific binding between P-450 and compounds absorbing in the 350-450 nm region is described. Various lipophilic compounds preferentially inhibit the hydroxylase activity from control or 3-methylcholanthrene-treated genetically nonresponsive mice, whereas other compounds selectively block the 3-methylcholanthrene-induced enzyme activity. By observing the preferential inhibition of one or the other form of hydroxylase activity, one may be able to determine the form of cytochrome P-450 with which a given compound binds. Hence we suggest that α-naphthoflavone, β-naphthoflavone, 2,5-diphenyloxazole, and lindane (γ-hexachlorocyclohexane) interact with a spectrally distinct type a species of P-450, the formation of which is associated with hydroxylase induction by aromatic hydrocarbons. Phenylimidazoles, 2-diethylaminoethyl-2,2-diphenyl valerate HCl (SKF 525-A), metyrapone, 2,2-bis( p -chlorophenyl)-1,1,1-trichloroethane, 17β-estradiol , δ9-tetrahydrocannabinol, cholecalciferol, pyridine, n -octylamine, and aniline inhibit aryl hydrocarbon hydroxylase activity by competing with benzo[ a ]pyrene at a type b P-450 active site. A third class of compounds inhibits both type a and b hydroxylase activities equally, and a fourth class of compounds does not affect either form of the enzyme system. Hexachlorobenzene, α- and β-naphthoflavone, 2,5-diphenyloxazole, lindane, and derivatives of 2-phenylbenzothiazole interact differently with the hepatic enzyme in phenobarbital-treated mice and in control mice. In microsomes from mouse kidney and from rat liver or kidney, the control and 3-methylcholanthrene- or phenobarbital-inducible hydroxylase activities are preferentially inhibited by many of these same compounds in the same manner, indicating that the two forms of the enzyme active site are probably the same in the liver and kidney of the mouse and rat.

Occurrence and Distribution of Aromatic L-amino Acid (L-DOPA) Decarboxylase in the Human Brain

Abstract: —The enzymatic decarboxylation of l-DOPA was measured in isotonic dextrose homogenates of different regions of the human brain by estimating 14CO2 evolved from tracer amounts of d l-DOPA[carboxy1-14C]. Enzyme activity was linear with respect to tissue concentration and time of incubation. The reaction exhibited a pH maximum at 7·0, was completely dependent upon the presence of high concentrations of pyridoxal phosphate, proceeded at the same rate in an atmosphere of air and nitrogen, and produced dopamine in addition to CO2 as a reaction product. The enzyme preparation behaved like an aromatic l-amino acid decarboxylase: it also decarboxylated o-tyrosine and when incubated with 5-hydroxytryptophan, serotonin was isolated as the reaction product; but it was devoid of activity towards d-DOPA[carboxy1-14C]. Within the human brain, l-DOPA decarboxylase was most active in the putamen and caudate nucleus; the pineal gland, hypothalamus, and the reticular formation and dorsal raphe areas of the mesencephalon exhibited considerable activity. Areas of cerebral cortex exhibited very low enzymatic activity and in regions composed predominantly of white matter, l-DOPA decarboxylase activity was not significantly above blank values. The activity of l-DOPA decarboxylase in the human putamen and caudate nucleus tended to decrease with the age of the patients; in comparatively young subjects (46 yr old) the enzyme activity compared favourably with that found, by means of the same assay technique, in the caudate nucleus of the cat.

Fructose-1,6-Diphosphate-Dependent Lactate Dehydrogenase from a Cariogenic Streptococcus: Purification and Regulatory Properties

Abstract: The lactate dehydrogenase (LDH) from Streptococcus mutans NCTC 10449 is under stringent metabolic control. The partially purified enzyme was specifically activated by high concentrations of fructose-1,6-diphosphate (FDP) and was inhibited by adenosine triphosphate. There appeared to be at least two binding sites for the activator which interacted in a cooperative manner. The interaction between the FDP sites was independent of the pH of the assay system, although the relative affinity of the enzyme for the activator was influenced by pH. There also appeared to be at least two pyruvate binding sites on the S. mutans LDH with some cooperative interaction between them, and the interaction between these sites was also independent of the hydrogen ion concentration. Two pyruvate analogues had different effects on the interaction of pyruvate with the LDH. One of the analogues, α-ketobutyrate, stimulated enzyme activity at limiting pyruvate concentrations, but had no significant effect at saturating concentrations of the substrate. The net effect of α-ketobutyrate was to shift the pyruvate saturation curve from sigmoidal to hyperbolic and to decrease the Hill coefficient from about 2.0 to 1.0. The other pyruvate analogue, oxamate, inhibited enzyme activity at all pyruvate concentrations but had no effect on the sigmoidal nature of the pyruvate saturation curve or on the apparent kinetic order of the reaction with respect to substrate. These results suggested that there may be two types of pyruvate binding sites on the LDH from S. mutans. Other kinetic properties of the S. mutans NCTC 10449 enzyme were studied and compared with those exhibited by the LDH from several other strains of the organism.

Synthesis and turnover of nitrate reductase in corn roots.

Abstract: The induction and reinduction of nitrate reductase in root tip or mature root sections show essentially a similar pattern: a lag, a period of rapid increase in enzyme activity and finally a period of relatively minor change. Both inductions are sensitive to 6-methylpurine and cycloheximide. Kinetic studies with 6-methylpurine suggest that the half-life of the messenger RNA for nitrate reductase in both sections is about 20 minutes. The rate of decay of nitrate reductase activity induced by transfer to a nitrate-free medium is slower in root tips (t½ = 3 hours) than in mature root sections (t½ = 2 hours). The enzyme from mature root sections is also less stable to mild heat treatments (27 C; 40 C) than the enzyme from root tip sections. The results indicate that factors regulating enzyme turnover show important changes as root cells mature and may be significant in determining steady state levels of the enzyme.

Narcotic Drugs: Effects on the Serotonin Biosynthetic Systems of the Brain

Abstract: The effects of short- and long-term administration of morphine on the activity of two measurable forms of rat brain tryptophan hydroxylase were studied. Morphine administration produced an immediate decrease and a longterm increase in the nerve ending (particulate) enzyme activity but did not change the cell body (soluble) enzyme activity. Cocaine administration demnonstrated a short-term decrcease in measurable nerve eniding enzyme activity that was due to the inhibition of the high affinity uptake (the Michaelis constant, K(m) is 10-(5) molar) of trytophan, the serotonin precursor. Cocaine did not aflect the low affinity uptake K(m) = 10-(5) molar) of tryptophan. Both the uptake of the precursor and the enizymiie activity appeared to be drug-sensitive regullatory processes in the biosynthlesis of serotonin.

Partial Purification and Properties of Glycerophosphate Acyltransferase from Rat Liver

Abstract: 1 Glycerophosphate acyltransferase was partially purified from rat-liver microsomes which were resolved with a nonionic detergent, Triton X-100, in glycine buffer pH 8.6. The purification procedure involves molecular-sieve chromatography and sucrose density gradient centrifugation. 2 The partially purified enzyme requires Ca2+ for its activity. Divalent cations, such as Mg2+, Mn2+ and Co2+, are able to substitute for Ca2+ with varying degrees of effectiveness. Ca2+ in higher concentrations is inhibitory, but this inhibition is abolished by the addition of small amounts of phospholipids. Moreover, phospholipids stimulate the enzyme activity at all concentrations of Ca2+ tested. The pH optimum of the enzyme is broad, extending over a pH range from 6.6 to 9.0. 3 The apparent Michaelis constant of the partially purified enzyme for sn-glycerol 3-phosphate is 0.2 mM, which approximates the value found with unresolved microsomes (0.5 mM). 4 Among the acyl donors examined, palmityl-CoA is utilized most efficiently by the partially purified enzyme. Unsaturated fatty acyl-CoA thioesters, such as oleyl-CoA, linoleyl-CoA and arachidonyl-CoA, are poor substrates. 5 The partially purified enzyme catalyzes the formation of monoacylglycerol 3-phosphate from sn-glycerol 3-phosphate and palmityl-CoA, esterifying preferentially position 1 of the glycerol moiety. Little phosphatidic acid is produced even in the presence of both palmityl-CoA and linoleyl-CoA. Thus the acyltransferase preparation is free of the enzyme responsible for acylation of 1-acylglycerol 3-phosphate. 6 The results presented indicate that phosphatidic acid synthesis in mammalian liver proceeds through a sequential acylation of sn-glycerol 3-phosphate mediated by two distinct acyltransferases. The data also show that acylation of sn-glycerol 3-phosphate to monoacylglycerol 3-phosphate occurs in a non-random manner, thus contributing to the asymmetric distribution of fatty acids in glycerolipids.

Glycosyltransferases in human blood.I. Galactosyltransferase in human serum and erythrocyte membranes.

Abstract: Human serum and hemoglobin-free erythrocyte membranes were found to contain a galactosyltransferase which catalyzes the transfer of galactose from UDP-galactose to specific large and small molecular weight acceptors. The requirements for enzyme activity were found to be similar for the enzymes from both sources. However, the membrane-bound enzyme depended on a detergent for maximal activity. Mn++ was an absolute requirement for transfer and uridine nucleoside phosphates were inhibitors. The most effective acceptor for galactose was a glycoprotein containing N-acetylglucosamine residues in the terminal position of its oligosaccharide side chains, N-acetylglucosamine was also an acceptor. While the presence of α-lactalbumin in the incubation medium resulted in a significant decrease in the transfer of galactose to N-acetylglucosamine, glucose, which was not an acceptor for galactose in the absence of α-lactalbumin, became an excellent acceptor. The serum enzyme catalyzed the transfer of 54 nmoles of galactose per milliliter of serum per hour and its apparent Km for UDP-galactose was 7.5 × 10-6M. The membrane enzyme had a similar apparent Km. Using a quantitative assay system the enzyme was found to be present in all individuals studied, regardless of their blood type, secretor status, or sex.

Inducible Formation of Glutamate Dehydrogenase in Rice Plant Roots by the Addition of Ammonia to the Media

Abstract: The activity of glutamate dehydrogenase (l-glutamate: NAD oxidoreductase, EC 1.4.1.2.; GDH) of rice plants changes in response to the nitrogen source supplied to the culture solution. The activity of NADH-GDH(aminating) in roots is rapidly increased by the addition of ammonia, whereas the activity in shoots is much less affected by nitrogen supply. The activity increased with increasing concentration of ammonia at least up to 14.3 mM. In roots GDH activity was found in both the mitochondrial and soluble fractions. The increase of NADH-GDH activity caused by the ammonia treatment occurs mainly in the latter fraction. The new band with GDH activity was detected on the zymogram of polyacrylamide gel electrophoresis and this inducible enzyme is active with both NAD and NADP. On the other hand, the constitutive enzyme activity active with NAD is also increased by the ammonia treatment. The increase of enzyme activity is prevented by the addition of cycloheximide or chloramphenicol to culture medium. The incorporation of 14C-leucine(U) into GDH proteins was also studied using polyacrylamide gel electrophoresis. Higher radioactivity was found in induced samples than in non-induced ones. These results show that the increase of GDH activity in roots by ammonia treatment seems to depend on de novo protein synthesis.

Effect of collagenolytic activity in basal cell epithelioma of the skin on reconstituted collagen and physical properties and kinetics of the crude enzyme.

Abstract: Human basal cell epithelioma was shown to contain collagenolytic enzymes. With the use of reconstituted radioactive collagen substrate, the crude enzyme (tumor homogenate) was shown to release radioactivity 4 to 70 times greater than the release from normal control skin. Disc electrophoresis of the same incubation mixture demonstrated degradation products of collagen such as αA and βA. Kinetic studies with the pooled specimens revealed a linear increase of collagenolytic activity with respect to crude enzyme concentration or the length of incubation time. The optimal pH ranged between 7 and 8.5. Normal human serum, cysteine, and ethylenediaminetetraacetate significantly inhibited the enzyme activity, whereas a trypsin inhibitor (soybean) did not. Viscometric studies demonstrated that basal cell epithelioma crude enzyme decreased the specific viscosity of acid-soluble calf skin tropocollagen. Polarimetric studies indicated that, at temperatures below the denaturation temperature of tropocollagen, the ordered structure of the triple helical macromolecule was not affected during this treatment. The denaturation temperature of tropocollagen was decreased by 5° as a result of incubation with basal cell epithelioma homogenates.

Microphotometric determination of enzyme activity in single cells in cryostat sections i. application of the gel film technique to microphotometry and studies on the intralobular distribution of succinate dehydrogenase and lactate dehydrogenase activities in rat liver

Abstract: A gel film technique was adopted for kinetic enzyme activity determination in single cells using cryostat sections by means of a microscope photometer. The main principle of the method is a comparative activity determination, based on bipositional recording of initial reaction kinetics in two preselected measuring fields in the same tissue section. Systematic model experiments were performed to prove the fulfillment of the following conditions: (a) validity of Beer’s law; (b) optimal con centrations of substrates, cosubstrates and dye within the reaction film, including evidence that the reaction rate is not limited by insufficient diffusion of these compounds; (c) proportionality between local enzyme concentration or thickness of tissue sections and the recorded reaction rate; and (d) specificity of the method as demonstrated by studying reaction rates in the absence of the substrate as well as in the presence of substrate and a specific inhibitor. The validity of the method was also examined by comparing the levels of succinate dehydrogenase activity in various rat tissues, as measured microphotometrically, with the enzyme levels as determined in homogenates. Microphotometric assays for nicotinamide adenine dintscleotide phosphate isocitrate dehydrogenase, lactate dehydrogenase and succinate dehydrogenase are described. Using these techniques it was found that: the penportal and central areas of the hepatic lobule in rat contain the same level of lactate dehydrogenase activity; succinate dehydrogenase activity is 1.6 times higher in the periportal area as compared to the centrilobular area. In experimental thyrotoxicosis, the ratio of enzyme activities in the periportal and central areas was 1.1.

Biochemical changes in intestinal mucosa after experimental small bowel by-pass in the rat.

Abstract: 1. After experimental small bowel by-pass the excluded segment of intestine, whether jejunum or ileum, shows a decrease (expressed in units/cm of intestine) in mucosal weight, protein, RNA and activity of all eight enzymes studied (leucyl β-naphthylamidase, alkaline phosphatase, β-galactosidase, α-glucosidase, aryl sulphatase, β-glucuronidase, cytochrome oxidase and catalase). There is a fall in DNA content and there are no consistent changes in enzyme activity when expressed as units/mg of DNA. 2. The intestine remaining in continuity after small bowel by-pass shows an increase (expressed in units/cm of intestine) in mucosal weight, protein, RNA and enzyme activity. There is an accompanying rise in DNA content in the intestine in continuity. 3. The increased values of all variables measured in the intestine in continuity are much more marked in ileum than in jejunum. 4. The adaptive changes seen in the by-passed intestine and the intestine in continuity are a result of ‘hypoplasia’ and ‘hyperplasia’ respectively.

Physical properties and subunit structure of L-asparaginase isolated from Erwinia carotovora.

Abstract: 1. l-Asparaginases from Erwinia carotovora and Escherichia coli (EC2 enzyme) are both capable of inhibiting and eliminating certain types of tumour cells. The Er. carotovora enzyme is a more basic protein, however, and in contrast with the EC2 enzyme it contains neither tryptophan nor cystine, and disulphide bonds are therefore absent. The molecule is very stable in solution from pH3.0 to about pH12.0, and is somewhat more stable at alkaline pH than is the Esch. coli enzyme. Calculations based on a s(0) (20,w) 7.43S and a sedimentation-equilibrium molecular weight of 135000+/-10000 give a frictional ratio (f/f(0)) of 1.08. The molecular conformation is therefore very compact in solution, and the electron microscope shows the negatively stained molecules as almost spherical particles with a diameter of 7.2+/-0.7nm. 2. Sedimentation-velocity and equilibrium ultracentrifugation, in 5-8m solutions of urea and guanidinium chloride, and also electrophoresis in sodium dodecyl sulphate-polyacrylamide gel, reveal a dissociation of the native protein molecule into four subunits of similar molecular weight in the range 32500-38000. The enzymically inactive subunits can be physically reassembled into an active tetramer when urea is removed by dialysis. Although the subunit structures of the Er. carotovora enzyme and the Esch. coli enzyme molecules are similar, the secondary bonding forces holding the subunits together in the tetramer are somewhat stronger in the Er. carotovora enzyme. 3. The optical-rotatory-dispersion (o.r.d.) parameters that characterize the Cotton effects arising from ordered structure in the molecule are [m'](233)=-3522+/-74 degrees and [m'](200)=9096+/-1700 degrees . These show very marked changes as the secondary structure is disrupted and the molecule dissociates into subunits. A correlation pathway was traced on the basis of o.r.d. parameters and enzyme activity as the polypeptide chains were denatured and renatured (and reconstituted) into active molecules after the dilution of solutions in urea. Subunits resulting from treatment with sodium dodecyl sulphate do not show the typically disordered o.r.d. profile, but nevertheless they are inactive.