Cellular localization of aldolase C subunits in human brain.

An antiserum specific for the brain-type aldolase C subunit has been used to investigate the cellular localization of this protein in human brain. Immunoperoxidase labeling at the light microscope level showed heavy staining of Purkinje cells in the cerebellum and of astrocytes in the cerebrum. Faint staining of occasional large neurons in the gray matter of the cerebral cortex was also seen.

A two-site immunoradiometric assay for the MB isoenzyme of creatine kinase.

A two-site immunoradiometric assay for myocardial creatine kinase MB isoenzyme is described. The method utilizes immobilized anti-human creatine kinase BB antibodies and 125I-labelled anti-human creatine kinase MM antibodies and can specifically detect creatine kinase MB in the presence of approximately 1000-fold excess of creatine kinase MM or BB. Native creatine kinase MB prepared from human heart and creatine kinase MB prepared by hybridisation of purified human creatine kinase MM and creatine kinase BB appeared to react identically in the assay. Serum estimations on patients with suspected myocardial infarction correlated with the presence of an MB band on electrophoresis but preliminary results suggest that the two-site immunoradiometric assay may be more sensitive.

Human brain aldolase C4 isoenzyme: purification, radioimmunoassay, and distribution in human tissues.

The nervous system specific isoenzyme of frustose-1, 6-diphosphate aldolase (E.C.4.1.2.13), aldolase C4, has been purified from human brain, and a sensitive radioimmunoassay has been developed for its detection. This assay is also capable of detecting other hybrid isoenzymes containing the C subunit but not the A4 isoenzyme. A systematic survey of human organs has shown that immunoreactive aldolase C is present in all human organs but at levels less than 2% of those found in human brain; especially low levels occur in kidney, skeletal muscle, lung, and thyroid tissue. The presence of aldolase C in other organs apart from nervous tissue is unlikely to be explicable by innervation alone since significant quantities are found in erythrocytes. The high degrees of localisation of aldolase C4 in nervous tissue makes it a suitable marker for cell damage within the central nervous system.

Immunoreactive aldolase C in cerebrospinal fluid of patients with neurological disorders.

Nervous-system specific aldolase C has been detected in human cerebrospinal fluid (CSF) by radioimmunoassay. Measurement of 138 samples of CSF showed a mean level of 92 +/- 28 ng/ml. There was no correlation between the level of CSF aldolase C and the CSF total protein, albumin, IgG, or IgA levels. Aldolase C immunoreactivity present in concentrated CSF diluted out in parallel with the standard curve in the assay and showed an elution profile on ion-exchange and gel filtration chromatography similar to that of aldolase present in whole human brain extracts. Addition of known quantities of purified aldolase C4 to CSF gave quantitative recovery on subsequent radioimmunoassay. Measurement of aldolase C in the CSF of 66 patients with neurological disorders showed several patients with levels considerably in excess of 120 ng/ml, but there was no statistically significant difference in the mean levels between groups of patients with different diseases.

A RIA combined with SPE for the determination of a dual D2-receptor and beta2-adrenoceptor agonist, AR-C68397XX, in human plasma.

A radioimmunoassay has been developed for the determination of AR-C68397XX, a dual D2-receptor and beta2-adrenoceptor agonist, in human plasma. The method incorporates solid phase sample extraction and is suitable for the determination of the analyte at pg ml(-1) concentrations. The antiserum was raised in Suffolk cross sheep following primary and booster immunisations with an immunogen prepared by conjugating a carboxyphenylmethyl derivative of AR-C68397XX, to bovine serum albumin. The radioligand was prepared by the 125I-labelled iodination of a derivative of AR-C68397XX. The solid phase extraction procedure, using octadecyl sorbent, was introduced to remove matrix interferences in the plasma and to enhance method sensitivity. The calibration range is 20-500 pg ml(-1), using 0.5 ml of undiluted human plasma sample.

A radioimmunoassay combined with solid-phase extraction for the determination (pg ml-1) of AR-C15849XX) in human plasma.

A radioimmunoassay has been developed for the determination of AR-C15849KF, a CCK-8 analogue, in human plasma. The method incorporates solid-phase sample extraction, is suitable for the determination of the analyte at pg ml-1 concentrations and is based on a method developed and validated for dog plasma. The solid-phase extraction, using ion-exchange aminopropyl and octadecyl sorbents sequentially, was retained for this procedure to remove matrix interferences in the plasma and to enhance method sensitivity. The calibration range is 10-500 pg ml-1, using a 1 ml sample of undiluted human plasma. The method has been successfully used to generate early human pharmacokinetic data during a programme of exploratory development.

A radioimmunoassay combined with solid-phase extraction for the determination of a novel anti-obesity agent, ARL 15849XX, in dog plasma.

A radioimmunoassay has been developed for the determination of ARL 15849XX, a cholecystokinin-8 (CCK-8) analogue, in dog plasma. The method incorporates solid-phase sample extraction and is suitable for the determination of the analyte at picogram per millilitre concentrations. The antiserum was raised in Suffolk-cross sheep following primary and booster immunisations with an immunogen prepared by conjugating ARL 16935XX, an analogue of ARL 15849KF, to bovine serum albumin. The radioligand was prepared by the no-carrier-added 125I iodination of a non-sulphated derivative, ARL 15745XX. The solid-phase extraction procedure, carried out using ion-exchange aminopropyl and octadecyl sorbents sequentially, was introduced to remove matrix interferences in the plasma and to enhance the method sensitivity. The calibration range is 20-1000 pg ml-1, using a 1 ml sample of undiluted dog plasma.

A time-resolved fluorescence immunoassay for the determination of a novel respiratory therapeutic agent, AR-C68397XX (Viozan) in human plasma.

A dissociation-enhanced lathanide fluorescence immunoassay (DELFIA) method has been developed for the determination of AR-C68397XX, a novel respiratory therapeutic agent, in human plasma. The method is a 'direct' immunoassay and provides an alternative to the solid phase extraction RIA described in a previous publication, which employs the same specific antiserum. The DELFIA method is suitable for the determination of the analyte at pg ml(-1) concentrations. The non-isotopic label was prepared by complexation of a DTPA derivative of AR-C68397XX with free europium cation (Eu3+). Plasma samples were diluted at least 5-fold prior to analysis to eliminate matrix interference. The calibration range is 10-2000 pg ml(-1), and the LOQ of the method is 50 pg ml(-1) using 50 microl of diluted human plasma sample.

The activation of ox-brain NAD+-dependent isocitrate dehydrogenase by magnesium ions.

Two independent methods were used to assess the dependence of the activity of ox brain NAD+-dependent isocitrate dehydrogenase on the concentration of magnesium ions. The results indicated the complex between magnesium and isocitrate to be the true substrate for the enzyme. Free isocitrate is neither a substrate nor an inhibitor of the enzyme but free magnesium ions inhibit competitively with respect to the magnesium-isocitrate complex. The inhibition of the enzyme by ATP and citrate appears to be largely explicable in terms of their effects on the concentration of the complex between Mg2+ and isocitrate. The dependence of the activation of the enzyme by ADP on the concentration of magnesium ions suggests that free ADP, rather than its complex with Mg2+, is the activator.

The effect of pH on the allosteric behaviour of ox-brain NAD+-dependent isocitrate dehydrogenase.

The sigmoid dependence of the initial rate of the reaction catalysed by NAD+-dependent isocitrate dehydrogenase on the concentration of isocitrate is greatly reduced as the pH is decreased. The apparent pK for this process is 7.3. At pH values of 6.0 and 6.5 the enzyme exhibits hyperbolic kinetics with respect to isocitrate at relatively high concentrations of NAD+, but the dependence becomes sigmoid at lower NAD+ concentrations. The allosteric activators ADP and citrate have only small effects on the activity of the enzyme at pH 6.5 but in the latter case the effects are increased by decreasing the concentration of NAD+. The dependence of initial velocity on the NAD+ concentration is hyperbolic at all pH values in the pH range 6--8.5. NADH is a competitive inhibitor of enzyme activity with respect to NAD+, and its presence induces a sigmoid dependence of initial velocity on isocitrate concentration at pH 6.5 and in the presence of high concentrations of NAD+. Kinetic studies at pH 6.5 indicate that the apparent maximum velocity of the reaction with respect to NAD+ is insensitive to changes in the isocitrate concentration and that with isocitrate as the substrate is relatively insensitive to changes in the NAD+ concentration under conditions where the behaviour appears to be hyperbolic. threo-Ds-Isocitrate is the true substrate for the enzyme and the corresponding Ls isomer is neither a substrate nor an inhibitor.

The effect of ligands on the irreversible inhibition of the NAD+-dependent isocitrate dehydrogenase from ox brain.

The effects of ligands on the irreversible inhibition of the NAD+-dependent isocitrate dehydrogenase from ox brain were studied. Isocitrate in the presence of Mg2+ ions was found to protect against denaturation at 40 degrees C and this protection was enhanced by ADP which also protected on its own. None of the substrates or the activator ADP afforded protection against inhibition by diethylpyrocarbonate. Inactivation by this compound and by elevated temperatures did not obey first-order kinetics with respect to time. Inhibition by iodoacetate appeared to obey first-order kinetics both with respect to time and to inhibitor concentration. Protection against inhibition was afforded by isocitrate, NAD+ and ADP. The dependence of the extent of protection on the concentration of the magnesium-isocitrate complex was sigmoid at both pH 6.5 and pH 7.5 indicating the apparent homotropic cooperativity seen in initial-rate kinetic experiments to be a reflection of cooperative binding of this substrate. ADP reduced the affinity of the enzyme for this substrate without affecting the degree of this cooperativity. The dependence of the extent of protection by ADP upon its concentration obeyed Michaelis-Menten kinetics. The significance of these observations in terms of the kinetic and allosteric mechanism followed by this enzyme is discussed in the light of previous kinetic studies.