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.     

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.     

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     

Differences in the Biophysical Properties of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex in the Long-Sleep and Short-Sleep Mouse Lines

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.     

Constitutive appearance of peroxisomes in a regulatory mutant of the methylotrophic yeast Hansenula polymorpha

Summary A selection by glucosamine for mutants of Hansenula polymorpha insensitive to glucose repression of methanol assimilation is described. Constitutive synthesis of enzymes is established in standard batch cultures of glucosegrown cells. Upon prolonged glucose metabolism the phenotype is masked by catabolite inactivation and degradation of enzymes. Addition of the substrate methanol remarkably improves constitutive synthesis by preventing catabolite inactivation and delaying degradation. Regular peroxisomes of reduced number are formed in mutant cells under repressed conditions. No constitutive synthesis is detectable using ethanol as a carbon source. In addition, this alcohol is detrimental to growth of the mutants, indicating that H. polymorpha is constrained to repress synthesis of enzymes involved in the C₁-metabolism when ethanol is present as a substrate.