Recombinant human interleukin-1 (IL-1) alpha and beta were found to activate a latent cytosolic form of the transcription factor NF kappa B in rat C6 glioma. IL-1 beta was 10 times more potent than IL-1 alpha for this activity and both were inhibited by the IL-1 receptor antagonist. The activation was detectable from 20 min and remained sustained for up to 24 h. The electrophoretic mobility of the activated complex was shown to be different from that of the corresponding complexes in another IL-1-responsive cell line, the murine thymoma line EL4.NOB-1. C6 cells, when transiently transfected with five NF kappa B consensus sequence repeats linked to the reporter gene chloramphenicol acetyltransferase (CAT), demonstrated increased CAT activity in response to IL-1 beta treatment. The activation of NF kappa B in glial cells may thus represent an early step in IL-1 signalling in brain and is likely to have consequences for IL-1-induced gene expression in these cells.

Photoaffinity labelling of rat adrenal mitochondrial preparations with [3H]PK 14105 resulted in a single 3H-labelled band on SDS/PAGE gels with an apparent-molecular-mass peak of 18 kDa. This represents a polypeptide associated with the peripheral-type benzodiazepine-binding site. Solubilization of photoaffinity-labelled membranes with 6 M-guanidine hydrochloride, followed by gel filtration and reversed-phase h.p.l.c. of the solubilized material, resulted in the purification to homogeneity of the [3H]PK 14105-labelled polypeptide. This purified polypeptide was used to raise a rabbit polyclonal antiserum which recognized the immunogen in pure form and exclusively recognized it in a crude preparation of rat adrenal mitochondria as judged by immunoblotting. By the same analysis the antiserum identified the corresponding polypeptide from rat kidney and salivary gland, demonstrating its cross-reactivity. Subsequent immunocytochemical studies localized the polypeptide to the cortex of the adrenal gland, the distal tubules of kidney, the interstitial cells of testis, the biliary epithelium of liver and the choroid plexus and ependyma cells within the brain. This selective localization within organs may provide an insight into the physiological role of the peripheral-type benzodiazepine acceptor.

Uroporphyrinogen III synthase purified from rat liver is a monomer of Mr 36,000 by gel filtration and 28,000 by SDS/polyacrylamide-gel electrophoresis. The enzyme exists in two interconvertible forms separable on h.p.l.c. Both forms of the enzyme could be renatured with full activity after SDS/polyacrylamide-gel electrophoresis, demonstrating the absence of a reversibly bound cofactor. The enzyme activity could be inhibited by pyridoxal 5′-phosphate in the absence and in the presence of NaBH4, consistent with (an) essential lysine residue(s). The enzyme thus shows great similarity to that from Euglena gracilis.

Hydroxymethylbilane synthase from human erythrocytes was purified 47,000-fold to greater than 95% homogeneity and 7.5% yield by a simple and rapid procedure using heat treatment (80 degrees C, in the presence of proteinase inhibitors, to convert one of two chromatographically separable forms into the other), DEAE-cellulose and Cibacron Blue F3G-A-Sepharose chromatographies and Sephadex G-75 gel filtration. The purified enzyme was similar to the enzyme purified from other species in showing hyperbolic dependence of velocity on substrate concentration, a non-linear progress curve for uroporphyrinogen appearance, and was monomeric, having an Mr of 44,000 by gel filtration on Sephadex G-100 and h.p.l.c. and an Mr of 45,000 on SDS/polyacrylamide-gel electrophoresis. The enzyme showed a sharp pH profile for Vmax, and various folates were shown to accelerate neither the enzymic formation of hydroxymethylbilane nor ring-closure of hydroxymethylbilane.

Phenylhydrazine treatment induced hydroxymethylbilane synthase activity (EC 4.3.1.8) in rat spleen, erythrocytes and liver by 40-fold, 7.5-fold and 6-fold respectively. Five multiple forms of the enzyme were resolved by DEAE-cellulose chromatography. In the presence of phenylmethanesulphonyl fluoride only three forms, two major and one minor, were resolved by the fractionation, suggesting that two of the original forms arose by proteolytic modification. Heat treatment (70 degrees C) in the presence of proteinase inhibitor converted one of the major forms into the other major form. Product isomer analysis suggested that this heat-labile form represented an enzyme-substrate covalent intermediate and not a hydroxymethylbilane synthase-uroporphyrinogen III synthase complex. Identical elution profiles and kinetic properties of the enzymes from rat spleen and erythrocytes suggested that the enzyme isolated from spleen was possibly from stored erythrocytes. Sephadex G-75 chromatography of the heat-stable DEAE-cellulose-purified form yielded pure enzyme as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr was found to be 43000 +/- 1500. Initial-velocity studies on all enzyme forms showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of a displacement-type mechanism. For the heat-stable form Vmax, varied with pH as a typical bell-shaped curve, indicating that two ionizable groups with pK values of 7.4 and 8.8 are important for catalysis. Km decreased with decreasing pH on the acid side of the pH optimum, suggesting the absence of ionization of a group with pK 7.4 in free enzyme or substrate.

1. Porphobilinogen deaminase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] from Euglena gracilis was purified more than 200-fold. 2. The enzyme has a molecular weight of 41 000 +/- 2000, does not contain a chromophoric prosthetic group, and appears not to require metal ions for activity. 3. The stoicheiometry of the overall reaction at pH 7.4 was shown to be: 4 Porphobilinogen leads to uroporphyrinogen-I + 4 NH4+. This stoicheiometry for porphobilinogen and uroporphyrinogen was also observed over a wide range of pH values. 4. Initial-velocity studies showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of a displacement-type mechanism. 5. Vmax. varied with pH as a typical bell-shaped curve, indicating that two ionizable groups with pK values of 6.1 and 8.9 are important for catalysis. A plot of Vmax./Km against pH showed a single ionization (pK 8.2) to influence binding of substrate.

1. The specific activity of yeast hexokinase A depends on the concentration of the protein in the solution being assayed. When a solution containing 13.5 mg of hexokinase A/ml is diluted 10-100-fold at various values of pH and temperature, there is a gradual decline in the specific activity of the enzyme until an equilibrium value is reached, which varies with the chosen experimental conditions. 2. The catalytic activity lost when hexokinase A (1 mg/ml) is incubated at 30degreesC is recovered by lowering the temperature to 25degreesC. 3. These concentration- and temperature-dependent phenomena are consistent with the existence of a monomer-dimer equilibrium in which the dimer alone is the catalytic form of the enzyme. 4. Glucose alone prevents the decline in specific activity of hexokinase A after dilution, but it does not re-activate dilute solutions solutions of the enzyme. It is concluded that glucose binds to both the dimer and the monomer and prevents both association and dissociation. 5. The progress curve describing the phosphorylation of glucose catalysed by hexokinase A does not attain a steady state. It is possible that dissociation of catalytically active dimers in a ternary complex with glucose and ATP (or glucose 6-phosphate and ADP) could explain the non-linearity of this progress curve.

Cytoplasmic aspartate aminotransferase and malate dehydrogenase were purified from pig heart. Kinetic parameters were determined for the separate reaction catalysed by each enzyme and used to predict the course of the coupled reaction: (see article). Although a lag phase should have been easily seen, none was detected. The same coupled reaction was also carried out by using radioactive aspartate in the presence of unlabelled oxaloacetate. The reaction was quenched with HClO4 after 70 ms and the specific radioactivity of the malate produced in this system was found to be essentially the same as that of the original aspartate. These results show that oxaloacetate produced by the aspartate aminotransferase is converted into malate by malate dehydrogenase before it equilibrates with the pool of unlabelled oxaloacetate and are consistent with a proposal that the enzymes are associated in a complex. However, no physical evidence of the existence of a complex could be found. An alternative means of compartmentation of the intermediate as an unstable isomer is considered.