Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.

Metal binding properties of thiols; complexes with horse liver alcohol dehydrogenase.

The metal binding properties of thiols were investigated fluorimetrically and spectrophotometrically using horse liver alcohol dehydrogenase as a model metalloenzyme. The steady-state kinetics revealed that in the presence of the coenzyme the primary interaction of a thiol with the enzyme is by thiolate competing with alcohol for the active zinc site. The experiments with 2-mercaptoethanol and ethanethiol showed that at physiological pH it is enzyme-NAD-thiol complexes which are kinetically important with enzyme-thiol complexes only significant at higher pH. The dissociation constants for the binding of thiols in the ternary enzyme-NAD-thiol complexes showed tighter binding as the pH increases, with dithiols binding more tightly than monothiols. The primary binding to zinc was less dependent on the pKa value of each thiol than on mutual stabilization of zinc bound thiolate by the positive charge on the pyridinium ring of NAD, and by monodentate binding and with some dithiols perhaps bidentate binding. The tighter binding to the enzyme of the thiol when it is more hydrophobic or less polar indicates that the thiols interact not only with the active zinc site but also with a neighboring hydrophobic site. This is important for tight binding thiols which are rigidly held by multivalent binding through being anchored hydrophobically and to positively charged centers like zinc.

The effects of disulfiram and related compounds on equine hepatic alcohol dehydrogenase.

The reversible inhibition and the irreversible inactivation of equine hepatic alcohol dehydrogenase by disulfiram have been investigated. Disulfiram was found to be a potent competitive reversible inhibitor with KEO,I values at pH 7.0 and 10.0 of 50 microM and 30 microM, respectively. Reversible monodentate binding to the active site zinc is indicated by comparison with related compounds. Disulfiram was also found to chemically modify and inactivate the enzyme in an irreversible reaction, which proceeds via the formation of a reversible enzyme-disulfiram binary complex with a dissociation constant at pH 7.0 of 30 microM. The inactivation reaction has been studied over the pH 6.0 to 10.0 range. The dissociation constants for binding to the enzyme and the apparent first-order rate constants for inactivation have been determined as a function of pH. A pKa of 8.3 for the free enzyme has been assigned to the zinc-water ionization. Similar inhibition and affinity labelling kinetics are exhibited by diethyldithiocarbamate and by 2,2'- and 4,4'-dipyridyl disulphide, which have similar enzyme "on" velocity pKa values of 8.3 and 8.2, respectively. The enzyme is competitively protected from inactivation with disulfiram by 2,2'-dipyridyl, 1,7'-phenanthroline, acetone, and ethanol, all of which combine with the active site zinc to form binary complexes. Acetate gave mixed protection against inactivation due to an additional interaction with the anion binding site of the enzyme. In view of the effect of disulfiram on ethanol metabolism and the polyol pathway, its importance as an aversive drug are considered.

The effects of disulfiram on equine hepatic alcohol dehydrogenase and its efficiency against alcoholism: vinegar effect.

The effects of disulfiram, its metabolite diethyldithiocarbamate and dithiodipyridine on alcohol metabolism of equine hepatic alcohol dehydrogenase (EC.1.1.1.1.) have been investigated. They were found to form enzyme-NAD(+)-inhibitor complexes which were competitive inhibitors of alcohol metabolism with dissociation constants (KEO,I) at pH 7.0 of 50 microM, 1.3 mM, and 260 microM, respectively. Acetate and vinegar behaved similarly in forming an inhibitory enzyme-NAD(+)-acetate ternary complex competitive with ethanol, with at pH 7.0 essentially identical dissociation constants of 4.0 mM and 3.8 mM, respectively. Disulfiram, diethyldithiocarbamate and dithiodipyridine were also found to exhibit affinity-labelling kinetics with liver alcohol dehydrogenase. The liver enzyme is chemically modified and inactivated in a similar manner by all three reagents via binary enzyme complexes with dissociation constants of 30 microM, 200 microM and 50 microM, respectively. Used as a protector against enzyme inactivation by DL-alpha-bromo-beta-(5-imidazolyl)-propionic acid, disulfiram, diethyl-dithiocarbamate and dithiodipyridine were found to form competitive binary enzyme complexes by binding to the active zinc site with KE,I values of 30 microM, 170 microM and 50 microM, respectively. The disulfiram and acetate binding to zinc results in the formation of binary and ternary complexes which inhibit alcohol metabolism at the enzyme level. Due to many unwanted side-effect), and the easy removal of its anti-drinking effects by drinking vinegar (vinegar effect), disulfiram may still be questioned as an effective drug against alcoholism.

Inhibition and inactivation of horse liver alcohol dehydrogenase with the imidazobenzodiazepine Ro 15-4513.

The imidazobenzodiazepine ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine -3-carboxylate (Ro 15-4513) is important as a potential "drink and drive" drug due to effects on receptors in brain neurones, resulting in alcohol intoxication-antagonistic properties. Because of the molecule's importance its effect on alcohol metabolism in liver has been investigated. Ro 15-4513 was found to be, like its parent compound the 8-fluoro analogue flumazenil, a reversible alcohol competitive inhibitor of horse liver alcohol dehydrogenase (EC 1.1.1.1) with a dissociation constant of 345 microM at pH 7.0. Due to its azido group Ro 15-4513 was developed as a potential photoaffinity-labeling reagent for benzodiazepine receptors. Used with horse liver alcohol dehydrogenase, the enzyme is chemically modified and inactivated in a Michaelis-Menten type reaction via a reversible enzyme-Ro 15-4513 complex with a dissociation constant of 8.6 mM at pH 7.0. The inactivation reaction has been studied over the pH 6.0-10.0 range. The dissociation constants for the binding of Ro 15-4513 to the enzyme and the first-order rate constants for inactivation have been determined as a function of pH. These give pKa values of 7.2 and 8.8 for the free enzyme, the latter being assigned to the zinc-water ionization. The enzyme is protected from inactivation in a competitive manner by flumazenil and by many heterocyclic and thiol compounds which combine with the active-site zinc. Flumazenil has a similar binding affinity as Ro 15-4513 with an enzyme-flumazenil dissociation constant of 6.0 mM at pH 7.0. Ro 15-4513 may also have potential as a photoaffinity-labeling reagent for other metallo enzymes. Whether the effects of Ro 15-4513 on alcohol-metabolizing enzymes are also of clinical significance remains to be determined.

[Rheumatoid factor IgM analyses in Danish laboratories. An interlaboratory study]. / Rheumafaktor-IgM-analyse på danske laboratorier. Interlaboratorieundersøgelse.

The aim this study was to compare the correlation between the rheumatoid factor analyses in Danish laboratories. Concentrations of rheumafactor IgM were found in 10 different sera at 17 different laboratories. Thirteen laboratories used the ELISA technique and four the nephelometry technique. Correlation between the laboratories using the same technique was acceptable in the interval 10 to 200 IU/ml. Concentrations were higher when the nephelometry technique was used compared with the ELISA technique.

Comparative study of assays detecting complement activation. Complement-split product C3d (rocket immuno-electrophoresis) and C3d neodeterminants (ELISA).

The aim of the study was to investigate the correlation between two types of assay measuring specific products of complement C3 activation and their clinical application. Complement C3d split product was estimated using double-decker rocket immunoelectrophoresis (DD-RIE) and measurements of C3d neodeterminants exposed after C3 activation was carried out with an enzyme-linked immunosorbent assay (ELISA). A total of 595 blood samples were measured in parallel in the DD-RIE and the ELISA test systems. The samples originated from blood donors (44), uraemic patients undergoing dialysis (135), serial samples from rheumatoid arthritis (RA) patients during steroid treatment (88) and 328 randomly collected patient samples. The mean values for DD-RIE and ELISA (+/- 1 SD) for the 595 samples were 48 (+/- 20) mU/l and 48 (+/- 28) mU/l respectively. The interassay coefficient of variation (CV) in the ELISA was 18%. The Spearman rho-correlation coefficient between the two assays was 0.63 for all 595 samples. The mean values using ELISA and DD-RIE were practically identical for the 328 successively incoming samples, the samples from the 135 dialysis patients and the 44 donors. In RA patients a higher mean value was found for the 88 samples using DD-RIE than ELISA. In the majority of patient samples there was a good correlation between the two assays. However, the ELISA appears to be more sensitive in detecting acute complement activation and to give lower levels in RA patients with chronic complement activation.