Ion chromatography-electrospray mass spectrometry for the identification of low-molecular-weight organic acids during the 2,4-dichlorophenol degradation.

End-product identification of organic pollutants during oxidation water treatments is of environmental concern due to their potential toxicity. In this work the coupling of ion chromatography (IC), equipped with a membrane ion suppressor, and electrospray ionization mass spectrometry (ESI-MS) has been successfully exploited for the identification of novel low molecular weight organic acids formed as final by-products of 2,4-dichloro-phenol (DCP) degradation by Fenton's reagent, an oxidation method widely employed to degrade recalcitrant organic pollutants in industrial wastewater. The IC-ESI-MS analytical set-up not only allowed the detection of known organic acids but also the identification of 13 new low molecular weight organic acids as a result of extensive oxidation of the parent organic pollutant. Some of the novel organic acids were shown to be chlorine containing by-products and, consequently, of environmental concern. The analytical set-up could be used for characterizing the unknown organic carbon fraction arising from oxidation water treatments.

Temperature-dependent expression of a squid Kv1 channel in Sf9 cells and functional comparison with the native delayed rectifier.

SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) alpha-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion. We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings reveal that very few cells display functional potassium current (IK) if cultured at the standard postinfection temperature of 27 degrees C. At 18 degrees C, less SqKv1A protein is produced than at 27 degrees C, but cells with IK currents are much more numerous and can survive for at least 20 days postinfection (vs. approximately 5 days at 27 degrees C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (approximately 3- and 10-fold, respectively) than those of native channels in GFL neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence of conductance and inactivation. Rates of IK inactivation in 20 mM external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA) and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL IK is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A.

The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme.

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated alpha and beta subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus-infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase alpha or beta subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate alpha and beta polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase alpha subunit and a H,K-ATPase beta subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase alpha subunit are fused to cells coexpressing the Na,K-ATPase beta subunit and the H,K-ATPase beta subunit, the Na,K-ATPase alpha subunit selectively assembles with the Na,K-ATPase beta subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase alpha subunit assembles with both beta subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase alpha and beta subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.

The alpha-subunit of the Na,K-ATPase has catalytic activity independent of the beta-subunit.

All catalytic activities of the Na,K-ATPase have been ascribed to the alpha-subunit; however, normal activity requires the presence of the beta-subunit. Using recombinant baculoviruses to infect insect cells, we demonstrate that the alpha-subunit, without the beta-subunit, has catalytic activity. During the normal catalytic cycle of the Na,K-ATPase, the alpha-subunit is transiently phosphorylated by ATP at an aspartate residue. This phosphorylation requires Na+, in the presence of K+ the enzyme undergoes rapid dephosphorylation. In contrast, phosphorylation of the independent alpha-subunit by ATP occurs in the presence of Mg2+, does not require Na+ or K+, and is not affected by ouabain. The phosphorylation is, however, inhibited by EGTA and increasing ionic strength. Chemical properties of the alpha-subunit phosphointermediate are consistent with phosphorylation at the normal aspartyl residue. Membranes from cells infected with the recombinant alpha baculovirus exhibit an EGTA-sensitive Mg(2+)-ATPase activity that is not present in the uninfected cells. The Mg(2+)-ATPase of the alpha-infected cells is reduced under conditions of high ionic strength and completely inhibited by EGTA. Thus the phosphorylation of the unassociated alpha-subunit is representative of the ATPase activity of the enzyme. These results suggest that the alpha-subunit of the Na,K-ATPase can catalyze an activity not normally associated with the enzyme and demonstrate that the bea-subunit plays an important role in conferring normal activity to the enzyme complex.

Expression, targeting, and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells.

Deciphering the roles of the individual subunits of the heteromeric Na,K-ATPase in the structure, function, and assembly of this enzyme has been complicated because most expression systems have endogenous levels of Na,K-ATPase activity. This problem has become even more significant in light of the recent identification of multiple isoforms for both the alpha and beta subunits. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, affords two distinct advantages for the expression of the Na,K-ATPase; some insect cells have little or no levels of Na,K-ATPase, and baculovirus-infected cells produce extremely high levels of foreign protein. We have made two separate recombinant baculoviruses containing the rodent alpha 1 or beta 1 cDNAs and used them to infect the insect cell line Sf-9. The infected Sf-9 cells produce Na,K-ATPase subunit protein on the order of 5-10 micrograms of protein/ml of cultured cells. The rodent alpha 1 polypeptide produced in the Sf-9 cells is indistinguishable electrophoretically and antigenically from the native subunit. The expressed beta 1 subunit is also antigenically identical but has a higher electrophoretic mobility due to differential glycosylation by the infected Sf-9 cell. In contrast to other systems, when expressed alone, each individual Na,K-ATPase subunit is targeted to the infected Sf-9 plasma membrane. In contrast, when infected with a virus that induces the heavy chain of murine IgG, the infected Sf-9 cell retains the polypeptide in the endoplasmic reticulum. However, when both IgG light and heavy chains are expressed, the polypeptides are properly processed and secreted. When the Na,K-ATPase alpha 1 and beta 1 polypeptides are simultaneously expressed, they form detergent-resistant complexes that are functional. Ouabain-sensitive ATPase activity on the order of 5 mumol Pi/mg/h in infected Sf-9 membranes was dependent on the expression of both the alpha 1 and beta 1 subunits. Sodium-dependent phosphorylated intermediates were detected that were potassium- and ouabain-sensitive. No increase in ouabain-sensitive activity or phosphorylated intermediates was detected when either subunit was expressed alone. The alpha 1 beta 1-coinfected cells were also able to transport ions, as detected in 86Rb uptake experiments. Thus, the recombinant Na,K-ATPase expressed in insect cells is biologically active and is suitable for structural and functional analysis.