Additives and Enzyme Stability

The polypeptide chain of an enzyme is folded so that the necessary functional groups are brought together in the active site. Conformational changes may disrupt this arrangement and cause loss of enzymic activity. The effect of soluble additives on the unfolding process is discussed. Additives may be classified as substrates and similar ligands, small uncharged organic molecules, specific and non-specific ionic species, and polymers.     

1H, 13C and 15N resonance assignment of phage T4 endoribonuclease RegB

RegB is involved in the control of the phage T4 life cycle. It inactivates the phage early mRNAs when their translation is no more required. We determined its structure and identified residues involved in substrate binding. For this, all backbone and 90% of side-chain resonance frequencies were assigned.     

Low rates of iridoid glycoside hydrolysis in two Longitarsus leaf beetles with different feeding specialization confer tolerance to iridoid glycoside containing host plants

Iridoid glycosides are plant defence compounds that are deterrent and/or toxic for unadapted herbivores but are readily sequestered by dietary specialists of different insect orders. Hydrolysis of iridoid glycosides by β‐glucosidase leads to protein denaturation. Insect digestive β‐glucosidases thus have the potential to mediate plant–insect interactions. In the present study, mechanisms associated with iridoid glycoside tolerance are investigated in two closely‐related leaf beetle species (Coleoptera: Chrysomelidae) that feed on iridoid glycoside containing host plants. The polyphagous Longitarsus luridus Scopoli does not sequester iridoid glycosides, whereas the specialist Longitarsus tabidus Fabricius sequesters these compounds from its host plants. To study whether the biochemical properties of their β‐glucosidases correspond to the differences in feeding specialization, the number of β‐glucosidase isoforms and their kinetic properties are compared between the two beetle species. To examine the impact of iridoid glycosides on the β‐glucosidase activity of the generalist, L. luridus beetles are kept on host plants with or without iridoid glycosides. Furthermore, β‐glucosidase activities of both species are examined using an artificial β‐glucosidase substrate and the iridoid glycoside aucubin present in their host plants. Both species have one or two β‐glucosidases with different substrate affinities. Interestingly, host plant use does not influence the specific β‐glucosidase activities of the generalist. Both species hydrolyse aucubin with a much lower affinity than the standard substrate. The neutral pH reduces the β‐glucosidase activity of the specialist beetles by approximately 60% relative to its pH optimum. These low rates of aucubin hydrolysis suggest that the ability to sequester iridoid glycosides has evolved as a key to potentially preventing iridoid glycoside hydrolysis by plant‐derived β‐glucosidases.     

Antimitotics induced cardiolipin cluster formation possible role in mitochondrial enzyme inactivation

We demonstrate here that drugs which inactivate cytochrome c oxidase are able to segregate cardiolipin essential for the enzyme activity, in a separate phase inaccessible for the enzyme. A molecular explanation of the drug-induced aggregation process is proposed.     

Interaction between nitrogen and photon flux density in birch seedlings at steady-state nutrition

Birch ( Betula pendula Roth.) was investigated under steady-state nutrition and growth at different incident photon flux densities (PFD) and different relative addition rates of nitrogen. PFD had a strong influence on the relative growth rate at optimum nutrition and on the nitrogen productivity (growth rate per unit of nitrogen) but little effect on the formal relationships between nitrogen and growth, i.e. PFD and nitrogen nutrition are orthogonal growth factors. At a given suboptimum nitrogen (the same distance from optimum), increased PFD increased the relative growth rate and, therefore, the relative uptake rate and the required relative addition rate in accordance with the theoretical equality between these three parameters at steady-state nutrition. Correspondingly, at a given suboptimum relative addition rate, increased PFD decreased nitrogen status (larger distance from optimum) at an unchanged relative growth rate. Nutrient uptake rate, dry matter content, and partitioning of biomass and nutrients are strongly influenced by nitrogen status. PFD influences these characteristics, but only to an extent corresponding to its effect on the nitrogen status. The influence of PDF on the relative growth rate at optimum and on nitrogen productivity is well described by hyperbolic relationships, similar to reported PFD/photosynthesis relationships. These expressions for plant growth as well as the productivities of leaf area and quantum appear to be valuable characteristics of plant responses to light and nutrition. Although the calculated PFD/growth relationships indicate saturation at high values of PFD, a more realistic estimate of PFD at which saturation occurs is about 30 mol m⁻² day⁻¹, where the highest relative growth rate and nitrogen productivity were experimentally determined. No significant effect was observed because of day length differences between the present and previous experiments.     

Sodium channel opening as a precursor to inactivation

The time constant of the process producing the delay in Na inactivation development as determined by the two pulse method (_delay) was extracted and compared to that of the slowest Na activation process ₃ for the I _Na during the conditioning pulse of that same determination. _delay and two pulse inactivation _c values were computer generated using a nonlinear least squares algorithm. _h and single pulse inactivation _h values were independently generated for each determination also with the aid of the computer using the same non-linear least squares algorithm. In one determination at 2 mV, _c was 4.68 and _delay 0.494 ms while _h was 4.70 and ₃ 0.491 ms for a _c/_h of 0.996 and a _delay/₃ of 1.006. Mean _delay/₃ from five determinations in four axons, both Cs and K perfused, and spanning a potential range of-27 to 2mV was 1.068. The precursor process to inactivation is channel opening. Some fraction of channels presumably inactivate via another route where prior channel opening is not required.     

Cyclic AMP,fructose-2,6-bisphosphate and catabolite inactivation of enzymes in the hydrocarbon-assimilating yeast Candida maltosa

The inactivation of fructose-1,6-bisphosphatase, isocitrate lyase and cytoplasmic malate dehydrogenase in Candida maltosa was found to occur after the addition of glucose to starved cells. The concentration of cyclic AMP and fructose-2,6-bisphosphate increased drastically within 30 s when glucose was added to the intact cells of this yeast. From these results it was concluded that catabolite inactivation, with participation of cyclic AMP and fructose-2,6-bisphosphate, is an important control mechanism of the gluconeogenetic sequence in the n-alkane-assimilating yeast Candida maltosa, as described for Saccharomyces cerevisiae.     

Characterization of a mutant of the yeast Candida maltosa defective in catabolite inactivation of gluconeogenetic enzymes

A spontaneous mutant of the yeast Candida maltosa SBUG 700 was isolated showing pseudohyphal marphology under all growth conditions tested. The C. maltosa PHM mutant takes up glucose with the kinetics of C. maltosa SBUG 700 and starved cells contain the same cyclic AMP concentration. Addition of glucose to the PHM mutant does not result in an increase of the intracellular cyclic AMP level and in catabolite inactivation of fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. However, addition of 2,4-dinitrophenol is followed by a rapid, transient increase of the cyclic AMP level in the mutant cells, but not by catabolite inactivation. These results show that a common mechanism might be responsible for catabolite inactivation and glucose-induced cAMP signaling or that glucose-induced cAMP signaling is required for catabolite inactivation in C. maltosa.     

Analysing partitioning of recently fixed and of reserve carbon in reproductive Phaseolus vulgaris L. plants

Abstract. Partitioning of recently-fixed carbon among plant organs and subsequent distribution of reserve carbon were studied by supplying whole shoots of bean plants ( Phaseolus vulgaris L.) with ¹⁴C-labelled CO₂ of constant specific radioactivity throughout a photoperiod. The gain of tracer carbon in each part revealed net accumulation of recently-fixed carbon from direct fixation, import or both. Growth rate coefficients describing the present pattern of plant growth were calculated from ratios of tracer carbon to total carbon present in plant organs and were used to project future plant form. The period 10–20 d after the start of flowering was marked by a major increase in partitioning of recently-fixed carbon to reproductive growth. Growth rates for the plant and its parts during this period were projected on the basis of growth rate coefficients and were found to be similar to rates measured by gravimetric growth analysis. Changes in tracer carbon recovered in individual organs after chase periods of various lengths revealed net decreases for leaves and stems. About 9% of the carbon distributed to fruits came from reserves even in the absence of obvious stress. Respiratory loss during the chase period was determined from the progressive drop in recovery of the original tracer carbon. The methods are being applied to measure current net accumulation rates in studies of sink organ physiology, and to compare partitioning of recently-fixed and of stored carbon in several plant species under defined growth conditions.     

Protection of photosynthetic O2 evolution against heat inactivation: the role of chloride,pH and coupling status

Heat inactivation of photosynthetic O₂ evolution was studied in isolated thylakoids from spinach (Spinacia oleracea) and mangrove (Avicennia marina) leaves. Different temperatures, salt, pH and uncoupler effects were investigated. From these results and others in the literature it was concluced that chloride loss from the membrane and, more specifically, the oxygen-evolving complex of photosystem II, may be the cause of inhibition of oxygen evolution during heat inactivation.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - Tricine N-2-hydroxy-1, 1-bis (hydroxymethyl) ethyl glycine - EDTA ethylenediaminetetraacetic acid - FeCN K-ferricyanide