Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation)

Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due to the introduction of a sense transgene(s) of Aox1, the nuclear gene encoding AOX, were used to investigate mechanisms regulating AOX activity. After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer. High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain [alpha]-keto acids, particularly pyruvate. Reduction of AOX to its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate. Our evidence suggests that NADPH may be specifically required for AOX reduction. All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well. Transgenic leaves lacking AOX due to the introduction of an Aox1 antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOX-regulatory mechanisms. Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act to prevent redirection of carbon metabolism, such as to fermentative pathways.     

Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria

Import of the synthetic precursor of the alternative oxidase from soybean was shown to be dependent on a membrane potential and ATP. The membrane potential in soybean mitochondria may be formed either by respiration through the cytochrome pathway, or through the alternative oxidase pathway with NAD⁺-linked substrates. Import of the alternative oxidase precursor in the presence of succinate as respiratory substrate was inhibited by KCN. Import in the presence of malate was insensitive to KCN and SHAM added separately, but was inhibited by KCN and SHAM added together (inhibitors of the cytochrome and alternative oxidases respectively). Import of the alternative oxidase was accompanied by processing of the precursor to a single 32 kDa product in both cotyledon and root mitochondria. This product had a different mobility than the two alternative oxidase bands detected by immunological means (34 and 36 kDa), suggesting that the enzyme had been modified in situ. When the cDNA clone of the alternative oxidase was modified by a single mutation (–2 Arg changed to –2 Gly), the processing of the precursor was inhibited.     

Serotonergic Regulation of Acetylcholine Release in Rat Frontal Cortex

Abstract: The extent to which serotonin regulates the activity of cortically projecting cholinergic neurons was studied using in vivo microdialysis to monitor interstitial concentrations of acetylcholine in the frontal cortex of freely moving rats. Systemic administration of the serotonin release-inducing agent fenfluramine (3 or 10 mg/kg, i.p.) increased acetylcholine release by 110–130%. The fenfluramine-induced increase in acetylcholine release was significantly attenuated by pretreatment with the selective serotonin uptake inhibitor fluoxetine (10 mg/kg, i.p.). Pretreatment with the selective dopamine D₁ receptor antagonist SCH-23390 (0.3 mg/kg, s.c.) failed to prevent the fenfluramine-induced increase in acetylcholine release. In contrast, the serotonin 5-HT_2A receptor antagonist ketanserin (5 mg/kg, i.p.) blocked fenfluramine-induced increases in acetylcholine release. In contrast to previous studies that have concluded that serotonin has inhibitory actions on cortical acetylcholine release, the present results indicate that fenfluramine increases cortical acetylcholine release in vivo by its ability to enhance serotonin transmission and that serotonin produces these effects at least in part via actions at serotonin 5-HT_2A receptors.     

Induction of alternative oxidase synthesis by herbicides inhibiting branched-chain amino acid synthesis

Sycamore suspension cells ( Acer pseudoplatanus L.) were incubated in the presence of sulfonylurea and imidazolinone herbicides. These inhibitors of acetolactate synthase (ALS), a key enzyme of branched-chain amino acid synthesis, triggered a dramatic induction of the alternative oxidase (AOX). AOX activity increased in treated cells, eventually exceeding cytochrome (cyt) pathway activity. This induction of AOX activity was correlated with the accumulation of a 35 kDa AOX protein in isolated mitochondria, detected by Western blotting with a monoclonal antibody against Sauromatum guttatum AOX. It was preceded by the accumulation of putative 1.6 kb AOX mRNA, detected using an Aox cDNA probe from soybean. The metabolic perturbations induced by the herbicides rather than the herbicide molecules themselves were responsible for this induction of AOX. However, α-oxobutyrate (one of the substrates of ALS) and its transamination product, α-aminobutyrate, which accumulated after herbicide treatment, were not involved. The inhibition of branched-chain amino acid synthesis was probably somehow responsible for the AOX induction since: (i) a mixture of those amino acids (leucine, isoleucine, valine) prevented AOX induction by ALS inhibitors; (ii) the herbicide Hoe 704, a potent inhibitor of acetolactate reducto-isomerase (the enzyme following ALS in the branched-chain amino acid pathway), also triggered AOX induction.     

Immobilization of tyrosinase for use in nonaqueous media: Enzyme deactivation phenomena

Summary We have investigated features for minimizing the inactivation of tyrosinase (E.C. 1.14.18.1) that is caused by immobilization on glass beads and Celite^®. The degree of inactivation is dependent on the enzyme loading and the carrier's surface area. Addition of a sacrificial protein during the immobilization procedure offers a protective effect with increased residual activity on the basis of comparable enzyme loading.     

Format determinants of synthetic myelin basic protein peptide S82 mimicked by a mixture of synthetic peptides S8 and S79

Antibodies to synthetic myelin basic protein peptide S82 (TTHYG-SLPQKAQGHRPQDEG) did not react with synthetic peptide S8 (GSLPQKAQGHRPQDENG) and only partially so with synthetic peptide S79 (AQGHRPQDEG); however, the antibodies did react to a considerable extent with an equimolar mixture of S8 and S79. Since the anti-S82 antibodies had previously been shown to be directed to a non-sequential format determinant dependent on the conformation of secondary structure, it seems probable that the mixture of S8 and S79 assumed a format that neither one individually possessed to any great degree.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.Supported by Research Grants NS-10237 (Duke) and NS-15322 (St. Luke's) from the National Institutes of Health and by RG1197-B7 from the National Multiple Sclerosis Society.     

A thermophilic glucoamylase fromCephalosporium eichhorniae

A novel exocellular glucoamylase produced by a thermophilic fungus,Cephalosporium eichhorniae, was purified by a combination of membrane filtration and Sephadex chromatography. The enzyme was a glycoprotein, 28% carbohydrate by weight. It was composed of a single polypeptide chain with a molecular weight of 26,850. The enzyme was thermostable with optimum activity between 45 and 62°C. It had a substrate preference of amylose>amylopectin. Analysis by thin-layer and gas-liquid chromatography showed the major hydrolytic product of starch was glucose, classifying this enzyme as a thermophilic glucoamylase.     

Slide staining device for use during space flight.

A slide staining device is described that performs Gram and Wright stains during space flight. Reagents and liquid wastes are contained within a closed system.     

Prostaglandin F2α — A fertility drug in dairy cattle?

Lactating dairy cows were injected once or twice with the Tham salt or an analogue of prostaglandin F(2alpha) (PGF) in a series of nine trials. In specific comparisons involving only those cows observed in estrus during the 24 h before they were inseminated, the average pregnancy rate to first insemination for over 2000 PGF treated cows was 69%, compared to 60% in a comparable number of untreated herd mates. The one trial in which this fertility effect did not occur was one in which semen of low fertility was used only with PGF treated cows. Exploitation of this fertility effect associated with the use of PGF requires accurate diagnosis of estrus. This is necessary because of variation in the interval from injection to estrus and can be achieved by the use of tail paint. This simple technique also allows single PGF injection regimens to be successfully used. The fertility advantages associated with the use of PGF have also been reported by others but may not have received due recognition because of the quest for a procedure which dispensed with the need for estrus detection.