N-Cyclo-N'-(4-Dimethylamino-alpha-Naphthyl)Carbodiimide Inhibits Membrane-Bound and Partially Purified Tonoplast ATPase from Maize Roots

Certain carboxylic acid groups within the primary structure of proton translocating proteins are thought to be involved in the proton pathway. In this report, the effects of a lipophilic carboxylic acid reactive reagent, N-cyclo-N′(4-dimethylamino-α-naphthyl)carbodiimide (NCD-4), on the two types of proton pumps in maize (Zea mays L.) root microsomes were investigated. NCD-4 was found to inhibit the vacuolar-type H⁺-ATPase in microsomal preparations; however, the plasma membrane-type H⁺-ATPase was unaffected. The H⁺-ATPase in highly purified tonoplast vesicles was also inhibited by NCD-4. Inhibition was dependent on the concentration and length of exposure to the reagent. However, there was little, if any, increase in the fluorescence of treated vesicles, indicating few carboxylic acid residues were reacting. Inhibition of the tonoplast H⁺-ATPase by NCD-4 was examined further with a partially purified preparation. The partially purified H⁺-ATPase also showed sensitivity to the NCD-4, supporting the hypothesis that this carboxylic acid reagent is an inhibitor of the tonoplast ATPase from maize roots.     

Ligninolytic properties of different white-rot fungi

Summary Seven white-rot fungi were examined for the production of ligninase, manganese peroxidase and laccase. All these enzymes were found inTrametes gibbosa andTrametes hirsuta. Only manganese peroxidase and laccase were produced byPycnoporus cinnabarinus,Coriolopsis polyzona,Stereum hirsutum,Dichomitus squalens andGanoderma valesiacum. All fungi decolorized Poly B-411 and Indulin AT plates with low-N medium. The differences in enzyme pattern indicate that different species of fungi may employ different modes of lignin metabolism.     

abetd-Xylose metabolism in Aureobasidium pullulans: effects of aeration and vitamins

Summary The yeast-like organism Aureobasidium pullulans efficiently converted abetd-xylose to cell mass (Y _X/S=0.45 g·g^–1) with negligible production of polyols (Y _P/S=0.003 g·g^–1) under aerobic conditions. A. pullulans grown semiaerobically exhibited different fermentation capacities in seven basal (vitaminless) medium and medium containing a mixture of seven vitamins. It was found that under semiaerobic conditions a mixture of vitamins significantly enhanced production of ethanol from abetd-xylose, resulting in a 15-fold higher yield coefficient of ethanol (Y _E/S=0.22 g·g^–1) as compared to that achieved in vitaminless medium. This increase in ethanol production was accomplished at the expense of cell mass. A. pullulans produced extremely low amounts of polyols throughout all aerobic and semiaerobic experiments. A. pullulans displayed strictly NADPH-linked xylose reductase and NAD⁺-linked xylitol dehydrogenase activities.     

Transformation of a new Gram-positive methylotroph,Brevibacterium methylicum,by plasmid DNA

Summary Brevibacterium methylicum is a newly isolated Gram-positive facultatively methylotrophic bacterium that uses the NAD⁺-dependent methanol dehydrogenase for methanol oxidation and assimilates its carbon via the ribulose monophosphate cycle. Protoplasts prepared by lysozyme treatment of B. methylicum cells grown in the presence of glycine were transformed by plasmid shuttle vectors pCEM500 (16.5 kb; Sm^r/Sp^r, Km^r/Gm^r) and pEC71 (7.1 kb; Km^r/Nm^r) constructed on the basis of B. lactofermentum plasmid pAM330 and replicating in Escherichia coli and in amino-acid-producing coryneform bacteria. The resistance markers were found to be expressed in B. methylicum and autonomous plasmid DNAs of various size were isolated from the transformants. The presence of the pAM330 replicon in these plasmids was demonstrated by DNA-DNA hybridization experiments. Offprint requests to: J. Nevera     

Assessment of membrane potential changes using the carbocyanine dye,diS-C3-(5): synchronous excitation spectroscopy studies

The fluorescence of the voltage sensitive dye, diS-C₃-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C₃-(5) fluorescence from cells and from the supernatant. It has been found that diS-C₃-(5) fluorescence in the supernatant can be selectively monitored at _exc = 630 nm and _em= 650 nm, while the cell associated fluorescence can be observed at _exc= 690 nm and _em = 710 nm. A modified theory for the diSC₃-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, In I/I°, and the underlying change in the plasma membrane potential, _p=_p-°_p. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K⁺ gradients. It has been demonstrated that the membrane potential change, _p,can be measured on an absolute scale. Offprint requests to: J. Plasek     

Further investigation on nuclear transplantation in different orders of teleost: the combination of the nucleus of Tilapia (Oreochromis nilotica) and the cytoplasm of Loach (Paramisgurnus dabryanus).

The nucleus of a blastula cell from Tilapia (Oreochromis nilotica, family Cichlidae, order Perciformes) was transplanted into an enucleated egg of Loach (Paramisgurnus dabryanus, family Cobitidae, order Cypriniformes). From among 3747 nucleo-cytoplasmic hybrid (NCH) eggs two NCH larval fish (0.05%) were obtained; one died on the 6th day and the other died on the 12th day after the operation. Morphological examinations showed that both NCH larval fish had developed normally with an opened mouth except they could not take food after complete utilization of their egg yolk on the 5th day of development. The possible mechanisms for obtaining such inter-order NCH larval fish are discussed. This is the first report indicating that inter-order NCH larval fish can be obtained in spite of their evolutionary divergence.     

Microflora participating in the decomposition of carboxymethyl cellulose continuously added to the soil

The microbial community in the soil was analyzed during four weeks of a continuous enrichment of structural chernozem soil samples with a 0.1% solution of carboxymethyl cellulose (CMC) under aerobic and semianaerobic conditions. During the first 14 d, the total amount of the aerobic and anaerobic, cellulose-degrading microorganisms increased significantly. Various metabolic pathways were u‘ed te decompose the substrate: diverse metabolic systems were activated and different groups of microorganisms preferred in dependence on the presence of oxygen or the source of mineral nitrogen. In the later phases of cultivation, a decrease in the concentration of zymogenous microflora and in the level of substrate mineralization was observed ovon though CM-cellulase activity remained high. During the fourth week of cultivation, a conspicuous increase in the numbers of oligothropic bacteria occurring in the colcnies of the microorganisms degrading cellulose was found. The representatives of prosthecobacteria (Caulobacter, Hyphomicrobium, Prosthecomicrobium spp.) andSeliberia sp. were thus identified. This “microflora of dispersion” attends the zymogenous microbes degrading CMC and indicates later phases of the process of decomposition.     

Effect of detergents on aspartate ammonia-lyase activity inEscherichia alcalescens

The effect of twelve detergents on aspartate ammonia-lyase activity of Escherichia alcalescens used for the production of L-aspartic acid was tested. Best permeabilization was found with Triton X-100, Slovafol 910 and Corona, a mixed commercial preparation. In contrast to Triton X-100 and Slovafol 910, a much narrower range of suitable concentrations was observed with Corona.     

Studies of electron-transfer properties of salicylate hydroxylase from Pseudomonas cepacia and effects of salicylate and benzoate binding

The pH dependence of the redox behavior of salicylate hydroxylase from Pseudomonas cepacia as well as the effects of salicylate, benzoate, and chloride binding is described. At pH 7.6 in 0.02 M potassium phosphate buffer E1(0')(EFl ox/EFl.-) is -0.150 V and E2(0')(EFl.-/EFl red H-) is -0.040 V versus the standard hydrogen electrode (SHE). A maximum of 5% of FAD anion semiquinone is thermodynamically stabilized under these conditions. However, in coulometric and dithionite titrations more semiquinone is kinetically formed, indicating slow transfer of the second electron. The potential/pH dependence is consistent with a two-electron, one-proton transfer. Upon salicylate binding the midpoint potential is shifted 0.020 V negative from -0.094 to -0.114 V vs SHE at pH 7.6. A maximum of 7% of the neutral semiquinone is stabilized both in potentiometric and coulometric titrations. This small potential shift indicates that the substrate is bound nearly to the same extent to all three oxidation states of the enzyme. It is clear that the substrate binding does not make the reduction of the flavin thermodynamically more favorable. In contrast to salicylate, the potential shift caused by the effector, benzoate, is much more significant. (A maximum potential shift of -0.07 V is calculated.) Benzoate binds most tightly to the oxidized form and is least tightly bound to the two-electron-reduced form of the enzyme. For the reduction of the free enzyme the transfer of the second electron or the transfer of the proton is rate limiting, as is shown by the kinetic formation of the anionic semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)