Photoinactivation of δ-aminolevulinic acid dehydratase from maize by flavin mononuclotide

The -aminolevulinic acid dehydratase activity was irreversibly inactivated by irradiation of the enzyme in presence of flavin mononucleotide. The loss of enzyme activity was dependent on time of irradiation, concentration of FMN and intensity of irradiance. It required oxygen and was markedly enhanced in heavy water. The presence of levulinic acid (a competitive inhibitor of -ALAD) during irradiation prevented the inactivation considerably indicating photooxidative damage at or near the active site. Superoxide dismutase, sodium benzoate and sodium formate offered no protection, but singlet oxygen quenchers like azide and tryptophan were effective. NADH, electron donor to excited flavins, also prevented the loss of enzyme activity. These results indicate that singlet oxygen produced by light absorption of FMN was responsible for the photooxidative inhibition of the enzyme.Abbreviations ALAD -aminolevulinic acid dehydratase - FMN flavin mononucleotide - O₂ ^- superoxide - H₂O₂ hydrogen peroxide - 10₂ singlet oxygen - LA levulinic acid - PBG porphobilinogen - BSA bovine serum albumin - BME 2-mercaptoethanol - SOD superoxide dismutase - pHMB para-hydroxymercuribenzoate - DTT dithiothreitol - FAD flavin adenine dinucleotide - NADH nicotinamide adenine dinucleotide     

Susceptibility of protein kinase C to oxidative inactivation: loss of both phosphotransferase activity and phorbol diester binding

Exposure of protein kinase C to low concentrations of either N-chlorosuccinimide or H2O2 resulted in rapid and parallel loss of phosphotransferase activity and phorbol ester binding. This oxidative inactivation of protein kinase C also occurred in intact cells exposed to a low concentration of H2O2. With H2O2 treatment the rate of inactivation of protein kinase C in the cytosol of MCF-7 cells was rather slower than that which occurred in the cytosol of PYS cells. However, in both cell types, the oxidative inactivation of membrane-associated protein kinase C occurred rapidly in comparison to the enzyme in the cytosol. Prior treatment of cells with phorbol ester to induce membrane association (stabilization) of protein kinase C, followed by exposure to H2O2, resulted in increased inactivation of protein kinase C, suggesting that membrane association of protein kinase C increases its susceptibility to oxidative inactivation.     

Isolation of S-100 binding proteins from brain by affinity chromatography

S-100-binding proteins, and calmodulin-binding proteins were isolated from S-100- and calmodulin-depleted bovine brain extract by Ca2+-dependent affinity chromatography using S-100- and calmodulin-coupled Sepharose columns respectively. The majority of the protein (80 to 90%) including calcineurin that bound to S-100 also bound to calmodulin and vice versa, suggesting both proteins may regulate common targets. However these two regulatory proteins also bind few other proteins specific for each. These include cyclic nucleotide phosphodiesterase, 55k, and 220k proteins for calmodulin and 24k, 42k, and 90k proteins for S-100. Certain proteins also specifically bound to S-100 both in Ca2+-dependent and independent ways. In glial cells S-100 protein may replace calmodulin in regulating Ca2+-influenced functions.     

Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.     

Irreversible oxidative inactivation of protein kinase C by photosensitive inhibitor calphostin C.

Isolated protein kinase C (PKC) was irreversibly inactivated by a brief (min) incubation with calphostin C in the presence of light. This inactivation required Ca2+ either in a millimolar range in the absence of lipid activators or in a submicromolar range in the presence of lipid activators. In addition, an oxygen atmosphere was required suggesting the involvement of oxidation(s) in this inactivation process. Furthermore, PKC inactivation might involve a site-specific oxidative modification of the enzyme at the Ca(2+)-induced hydrophobic region. Physical quenchers of singlet oxygen such as lycopene, beta-carotene, and alpha-tocopherol all reduced the calphostin C-induced inactivation of PKC. In intact cells treated with calphostin C, the inactivation of PKC was rapid in the membrane fraction compared to cytosol. This intracellular PKC inactivation was also found to be irreversible. Therefore, calphostin C can bring prolonged effects for several hours in cells treated for a short time. Taken together these results suggest that the calphostin C-mediated inactivation of PKC involves a site-specific and a 'cage' type oxidative modification of PKC.     

Selective resistance of bacterial polyadenylate-containing RNA to hydrolysis by guanosine 3'-5'-monophosphate-sensitive nuclease of Bacillus brevis

Earlier experiments had shown that the degradation of newly synthesized RNA in permeable cells of Bacillus brevis is mediated primarily by a guanosine 3′,5′-monophosphate-sensitive 3′-exonuclease [N. Sarkar and H. Paulus (1975) J. Biol. Chem. 250, 684–690]. More recently, we found that a substantial fraction of pulse-labeled RNA in B. brevis is polyadenylylated [N. Sarkar, D. Langley, and H. Paulus (1978) Biochemistry 17, 3468–3474], and it was thus of interest to examine the effect of polyadenylylation on the susceptibility of RNA to degradation by the 3′-exonuclease. Purified 3′-exonuclease from B. brevis hydrolyzed the unadenylylated fraction of pulse-labeled RNA from B. brevis much more rapidly than poly(A)-containing RNA. Similar results were obtained with the pulse-labeled unadenylylated and polyadenylylated RNA fractions from Bacillus subtilis. Control experiments showed that the differential hydrolysis of the labeled RNA preparations by 3′-exonuclease was not due to the presence of inhibitors or activators. These results suggest that the stability of mRNA in Bacillus species may be regulated by polyadenylylation.     

Weighted average regression and environmental calibration as a tool for quantifying climate-driven changes in vegetation

Aims Studies of the climatic responses of plant assemblages via vegetation-based environmental reconstructions by weighted averaging (WA) regression and calibration are a recent development in modern vegetation ecology. However, the performance of this technique for plot-based vegetation datasets has not been rigorously tested. We assess the estimation accuracy of the WA approach by comparing results, mainly the root mean square error of prediction (RMSEP) of WA regressions for six different vegetation datasets (total species, high-frequency species and low-frequency species as both abundance and incidence) each from two sites.