Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously. The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R[( 2KB]/[pyruvate]), was found to be 40 +/- 3 under a wide variety of conditions. Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme. At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration. On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions. In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids. 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate. However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism. The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relations between anxiolytic effects of diazepam and changes in the EEG spectral power

The influence of diazepam (1; 5; 10 mg/kg, i. p.), chlorpromazine (1 mg/kg) and amphetamine (1; 5 mg/kg) on the Fourier's spectral EEG power of sensomotor cortex and dorsal hippocampus and conflict behavior freely moving albino and cotton (Sigmondon hispidus) rats was studied. Effects of diazepam (5 mg/kg) in cotton rats were similar, but influence on the theta-activity was more expressed. Correlation between slowing of theta-activity and extent of anxiolytic effect in conflict situation was showed. On the basis of the results obtained the authors discuss possible mutual relations between the influence of diazepam on EEG and anxiolytic effect of benzodiazepines.     

Purification and partial characterization of goose ceruloplasmin

The preparation and properties of ceruloplasmin from goose blood plasma are described. Ammonium sulfate was used to precipitate the crude protein followed by adsorption and elution from DEAE-Sephadex A-50. Further treatment with an ethanol-chloroform mixture and Sephadex G-200 yielded an intensely blue protein possessing a high degree of chemical purity and biological activity. Goose ceruloplasmin, existing in two forms, appears to be a single polypeptide, apparent Mr121,300, with an A610/A280 ratio of 0.07. Copper represented 0.32%, which corresponded to six atoms of copper per protein molecule. Although the amount of EPR-detectable copper was the same as in mammalian ceruloplasmins there were some differences in EPR parameters, mainly in A parallel. Goose ceruloplasmin's amino acid composition, although similar in many residues to human ceruloplasmin, was lower in tyrosine, cystine/cysteine, and acidic amino acids. Valine was found as the N-terminal amino acid. Hexose, hexosamine, sialic acid, and fucose accounted for 6.65% of the weight. Goose protein contained only half the sialic acid of human ceruloplasmin. Two values for Km using either p-phenylenediamine (0.64 and 0.053 mM) or o-dianisidine (0.76 and 0.15 mM) were evaluated from Lineweaver-Burk plots. EPR studies on reactions with water radiolysis products at cryogenic temperatures allowed us to discover that goose ceruloplasmin, like human and bovine ceruloplasmins, possesses superoxide dismutase activity.     

Down-regulation of murine collagen-induced arthritis by a T cell hybridoma

T cell hybridoma cell lines were generated by somatic cell fusion of BW 5147 myeloma cells and splenic cells from mice suppressed for collagen-induced arthritis (CIA). Two cell lines were characterized for their cell surface phenotype, antigen recognition and capacity to down-regulate the erythema and edema associated with CIA. Cell line T101N was determined to portray the cell surface phenotype Ly1+2- L3T4- Thy1+ by a direct binding assay. Cell line T104B1 was determined to express only the Thy1+ alloantigen. Panning studies, measurement of IL-2 production in vitro and the suppression of antibodies to type I and type II collagen in vivo indicate that the hybridoma cells are not isotype specific in their recognition of the polymorphic interstitial collagens. Down-regulation of the erythema and edema of CIA occurred on injection of 1 X 10(5) T101N cells but not T104B1 cells. Histology of the tarsus region of the hind paw of CIA mice 33 days after the administration of T101N cells showed contrasting histopathology compared to that of CIA mice. The joints of CIA mice given T101N cells showed aligned articular surfaces resembling normal joint structure and only residual pannus. The data indicate that collagen-specific cloned T cell lines can modulate the gross pathology and joint architecture of joints exhibiting CIA.     

Reduction in postprandial energy expenditure during pregnancy.

Energy expenditure was measured during pregnancy in seven primigravid women at 12-15, 25-28, and 34-36 weeks and after the cessation of lactation. On each occasion the resting metabolic rate and the increase in metabolic rate after ingestion of a liquid test meal were measured by indirect calorimetry. In absolute terms the resting metabolic rate increased steadily during pregnancy but when expressed per unit of body weight no change was found. The energetic response to a mixed constituent meal was significantly reduced by 28% in the middle trimester of pregnancy. These findings suggest a possible maternal adaptation to increase energetic efficiency at a time when the energy demands of the fetus are high.     

Effects of Suanzaorentang on behavior changes and central monoamines

Previously, it was found that the ancient Chinese remedy of Suanzaorentang could be a promising anxiolytic drug (Chen and Hsieh, 1985a, Chen and Hsieh, 1985b). To understand the mechanism of the action of Suanzaorentang, the effects of Suanzaorentang on behavior changes and central monoamines and their metabolites were studied in rats. It was found that Suanzaorentang significantly (1) prolonged the period from the onset of clonic to tonic convulsions induced by pentylenetetrazol or picrotoxin, (2) prolonged the sleep duration induced by hexobarbital, (3) reduced locomotor activity, (4) enhanced the hypomotility induced by alpha-MT, (5) reduced the locomotor stimulation produced by levodopa plus benserazide, and (6) reduced central HVA, VMA, and 5-HIAA, but had no significant effects on central DA, NA, and 5-HT. These facts implied that Suanzaorentang decreased the turnover rate of central monoamines and central catecholaminergic activity.