Product yield in oxygenation of linoleate by soybean lipoxygenase: the value of the molar extinction coefficient in the spectrophotometric assay

Two iodimetric methods and a gravimetric method were used to determine the spectrophotometric molar absorptivity of the purified product of lipoxygenase-catalyzed dioxygenation of linoleate (13-LS-hydroperoxy-cis,trans-9,11-octadecadienoate). Earlier determinations had led to the use of values varying from 24,000 to 28,000 M-1 cm-1 for epsilon at 235 nm. In the current work, the two iodimetric values (spectrophotometric and titrimetric) average 22,500, while gravimetric analysis of scrupulously purified material gives 22,900. Final 235-nm absorbancies for lipoxygenase runs over a wide range of linoleic acid concentrations up to 200 microM give a constant final percentage completion. If one assumes a 100% reaction, epsilon is 23,600. Each method has less than 1.5% standard error; the average of the three independent methods is 23,000 +/- 580 (2.5%), all being lower than the previous values. In the enzyme-catalyzed reaction of linoleate at less than 200 microM substrate, only 235-nm-absorbing material is formed. Above 200 microM linoleate, yields at 235 nm decrease and yields of materials absorbing at 280 nm increase (the latter is known to arise from lipoxygenase-catalyzed reaction of linoleyl hydroperoxide). Below 200 microM substrate, linoleate purified by HPLC produces only one HPLC-observable product, 13-linoleyl hydroperoxide. At higher substrate concentrations other HPLC peaks arise, again with higher wave-length absorptions. Spectrophotometric data using the epsilon determined here agree with those from the O2 electrode. It is concluded that at S less than 200 microM, saturating air, and sufficient enzyme, soybean lipoxygenase-1 produces a sole product and the reaction proceeds to completion.     

Use of the green fluorescent protein for investigations of Paecilomyces fumosoroseus in insect hosts.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been expressed in a variety of prokaryotic and eukaryotic organisms and has been used extensively as a marker in the study of host-pathogen interactions. We have expressed GFP in the entomopathogenic fungus Paecilomyces fumosoroseus through co-transformation with a vector that confers resistance to glufosinate ammonium. All cell types express GFP and were readily detected by fluorescence microscopy. No correlation was observed between the amount of fluorescence and the pattern of vector integration as observed by Southern analysis. Fluorescent hyphae and conidia were easily distinguished on two insect hosts, the Russian wheat aphid, Diuraphis noxia, and the diamondback moth, Plutella xylostella, and blastospores were also detected in the hemolymph of the diamondback moth. GFP-tagged strains of P. fumosoroseus can be used to study the developmental fate of the fungus within its insect hosts.     

Inactivation viewed through single sodium channels

Recordings of the sodium current in tissue-cultured GH3 cells show that the rate of inactivation in whole cell and averaged single channel records is voltage dependent: tau h varied e-fold/approximately 26 mV. The source of this voltage dependence was investigated by examining the voltage dependence of individual rate constants, estimated by maximum likelihood analysis of single channel records, in a five-state kinetic model. The rate constant for inactivating from the open state, rather than closing, increased with depolarization, as did the probability that an open channel inactivates. The rate constant for closing from the open state had the opposite voltage dependence. Both rate constants contributed to the mean open time, which was not very voltage dependent. Both open time and burst duration were less than tau h for voltages up to -20 mV. The slowest time constant of activation, tau m, was measured from whole cell records, by fitting a single exponential either to tail currents or to activating currents in trypsin-treated cells, in which the inactivation was abolished. tau m was a bell-shaped function of voltage and had a voltage dependence similar to tau h at voltages more positive than -35 mV, but was smaller than tau h. At potentials more negative than about -10 mV, individual channels may open and close several times before inactivating. Therefore, averaged single channel records, which correspond with macroscopic current elicited by a depolarization, are best described by a convolution of the first latency density with the autocorrelation function rather than with 1 - (channel open time distribution). The voltage dependence of inactivation from the open state, in addition to that of the activation process, is a significant factor in determining the voltage dependence of macroscopic inactivation. Although the rates of activation and inactivation overlapped greatly, independent and coupled inactivation could not be statistically distinguished for two models examined. Although rates of activation affect the observed rate of inactivation at intermediate voltages, extrapolation of our estimates of rate constants suggests that at very depolarized voltages the activation process is so fast that it is an insignificant factor in the time course of inactivation. Prediction of gating currents shows that an inherently voltage-dependent inactivation process need not produce a conspicuous component in the gating current.     

Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.      

Incorporation of membrane proteins into large single bilayer vesicles. Application to rhodopsin

A general procedure to incorporate membrane proteins in a native state into large single bilayer vesicles is described. The results obtained with rhodopsin from vertebrate and invertebrate retinas are presented. The technique involves: (a) the direct transfer of rhodopsin-lipid complexes from native membranes into ether or pentane, and (b) the sonication of the complex in apolar solvent with aqueous buffer followed by solvent evaporation under reduced pressure. The spectral properties of rhodopsin in the large vesicles are similar to those of rhodopsin in photoreceptors; furthermore, bleached bovine rhodopsin is chemically regenerable with 9-cis retinal. These results establish the presence of photochemically functional rhodopsin in the large vesicles. Freeze-fracture replicas of the vesicles reveal that both internal and external leaflets contain numerous particles approximately 80 A in diameter, indicating that rhodopsin is symmetrically distributed within the bilayer. More than 75% of the membrane area is incorporated into vesicles larger than 0.5 micron and approximately 40% into vesicles larger than 1 micron.     

Cholesterol sensitivity and lipid raft targeting of Kir2.1 channels

This study investigates how changes in the level of cellular cholesterol affect inwardly rectifying K+ channels belonging to a family of strong rectifiers (Kir2). In an earlier study we showed that an increase in cellular cholesterol suppresses endogenous K+ current in vascular endothelial cells, presumably due to effects on underlying Kir2.1 channels. Here we show that, indeed, cholesterol increase strongly suppressed whole-cell Kir2.1 current when the channels were expressed in a null cell line. However, cholesterol level had no effect on the unitary conductance and only little effect on the open probability of the channels. Moreover, no cholesterol effect was observed either on the total level of Kir2.1 protein or on its surface expression. We suggest, therefore, that cholesterol modulates not the total number of Kir2.1 channels in the plasma membrane but rather the transition of the channels between active and silent states. Comparing the effects of cholesterol on members of the Kir2.x family shows that Kir2.1 and Kir2.2 have similar high sensitivity to cholesterol, Kir2.3 is much less sensitive, and Kir2.4 has an intermediate sensitivity. Finally, we show that Kir2.x channels partition virtually exclusively into Triton-insoluble membrane fractions indicating that the channels are targeted into cholesterol-rich lipid rafts.     

Vegetative compatibility groups in indigenous and mass-released strains of the entomopathogenic fungus Beauveria bassiana: likelihood of recombination in the field

Using nitrate non-utilizing (nit) mutants, we determined vegetative compatibility groups (VCG) among strains of Beauveria bassiana representing strains indigenous to North America, isolated from diverse insect hosts, and strains that have been mass released for insect control. Genetic similarity among these strains was analyzed using random amplified polymorphic DNA (RAPD) markers. Our data revealed 23 VCGs among the 34 strains tested, with most of these groups comprised of only a single strain. We also observed a VCG comprised of eight genetically similar strains isolated from Colorado potato beetles (CPB). Co-inoculation studies of CPB larvae with complementary nit mutants from the same or from different VCGs revealed heterokaryosis in four out of five same-VCG pairs, with only 5-17% of the sporulating cadavers generating few parasexual recombinants. In contrast, none of the infected beetles treated with non-compatible pairs generated recombinants. The large number of VCGs observed and the low frequency of in vivo recombination limited to vegetatively compatible strains indicate that this self/non-self recognition system may be an effective barrier preventing genetic exchange between dissimilar strains in the field.