Cysteine 254 of the 73-kDa A subunit is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by sulfhydryl reagents.

The vacuolar class of (H+)-ATPases are highly sensitive to sulfhydryl reagents, such as N-ethylmaleimide. The cysteine residue which is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by N-ethylmalemide is located in subunit A and is able to form a disulfide bond with the cysteine moiety of cystine through an exchange reaction. This unique property distinguishes this cysteine residue from the remaining cysteine residues of the (H+)-ATPase. Using this reaction, we selectively labeled the cystine-reactive cysteine residue of subunit A with fluorescein-maleimide. After complete digestion of the labeled subunit A by V8 protease, a single labeled fragment of molecular mass 3.9 kDa was isolated and the amino-terminal sequence was determined. This fragment contains 2 cysteine residues, Cys240 and Cys254. Since Cys254 is conserved among all vacuolar (H+)-ATPases whereas Cys240 is not, it is likely that Cys254 is the residue which is responsible for the sensitivity of the vacuolar (H+)-ATPase to sulfhydryl reagents.     

A multifunctional cell surface developmental stage-specific antigen in the cockroach embryo: involvement in pathfinding by CNS pioneer axons

mAb DSS-8 binds to a 164-kD developmental stage-specific cell surface antigen in the nervous system of the cockroach, Periplaneta americana. The antigen is localized to different subsets of cells at various stages of development. The spatial and temporal distributions of DSS-8 binding were determined and are consistent with this antigen playing multiple roles in the development of the nervous system. Direct identification of some of these functions was made by perturbation experiments in which pioneer axon growth occurs in embryos that are cultured in vitro in the presence of mAb DSS-8 or its Fab fragment. Under these conditions the pioneer axons of the median fiber tract grow but follow altered pathways. In a smaller percentage of the ganglia, the immunoreagents additionally produce defasciculation of a subset of DSS-8 labeled axons. Therefore, direct roles for the DSS-8 antigen in both the guidance of pioneer axons and selective fasciculation have been demonstrated.     

A novel mechanism for regulation of vacuolar acidification.

We have recently demonstrated that Cys-254 of the 73-kDa A subunit of the clathrin-coated vesicle (H+)-ATPase is responsible for sensitivity of the enzyme to sulfhydryl reagents (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 5817-5822). In the present study we observe that for the purified enzyme, disulfide bond formation causes inactivation of proton transport which is reversed by dithiothreitol (DTT). DTT also restores activity of the oxidized enzyme following treatment with N-ethylmaleimide (NEM). These results indicate that disulfide bond formation between the NEM-reactive cysteine (Cys-254) and a closely proximal cysteine residue leads to inactivation of the (H+)-ATPase. To test whether sulfhydryl-disulfide bond interchange may play a role in regulating vacuolar acidification in vivo, we have determined what fraction of the (H+)-ATPase is disulfide-bonded in native clathrin-coated vesicles. Vesicles were isolated under conditions that prevent any change in the oxidation state of the sulfhydryl groups. NEM treatment of vesicles causes nearly complete loss of activity while subsequent treatment with DTT restores 50% of the activity of the fully reduced vesicles. By contrast, treatment of fully reduced vesicles with NEM leads to inactivation which is not reversed by DTT. These results indicate that a significant fraction of the clathrin-coated vesicle (H+)-ATPase exists in an inactive, disulfide-bonded state and suggest that sulfhydryl-disulfide bond interconversion may play a role in controlling vacuolar (H+)-ATPase (V-ATPase) activity in vivo.     

Glycosylation and membrane insertion of newly synthesized rat dopamine beta-hydroxylase in a cell-free system without signal cleavage.

Dopamine beta-hydroxylase (DBH, EC 1.14.17.1) is present in both membrane-bound and soluble forms in neurosecretory vesicles. This study was designed to investigate the differences between membrane-bound and soluble DBH and how they may arise from translation of a single mRNA. Antisera to a peptide corresponding to the carboxyl terminus of rat DBH was found to specifically immunoprecipitate the 77- and 73-kDa subunits of newly synthesized DBH in rat brain. Thus, both soluble and membrane-bound forms contain the same carboxyl terminus. To investigate differences at the amino terminus, full-length rat DBH mRNA, translated in a cell-free system, produced a 66-kDa peptide. An additional higher molecular mass product was synthesized upon co-translational addition of microsomal membranes. This product was glycosylated since it bound to concanavalin A-Sepharose and reverted to the 66-kDa polypeptide after treatment with endoglycosidase H. This glycosylated product was resistant to protease digestion and fractionated with microsomal membranes on sucrose gradients, indicating that it is incorporated into the microsomal membranes. Amino-terminal sequencing of the glycosylated translation product indicated that the amino-terminal "signal" sequence was not cleaved. The results indicate that in the cell-free system newly synthesized DBH undergoes glycosylation and incorporation into microsomal membranes without cleavage of the NH2-terminal signal sequence.     

Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters.

DNAs extracted from Vibrio vulnificus seeded into oyster homogenates were evaluated as templates for the polymerase chain reaction. Several extraction procedures were examined, and it was determined that DNA recovered from cells lysed by guanidine isothiocyanate, extracted with chloroform, and precipitated with ethanol was most suitable for use as a polymerase chain reaction template. The region targeted was a 519-bp portion of the cytotoxin-hemolysin gene of V. vulnificus. This region was amplified only when DNA from this species was present in the homogenate. V. vulnificus seeded into oyster homogenates at an initial level of 10(2) CFU/g of oyster meat was consistently observed after 24 h of incubation in alkaline peptone water.     

Effect of heme binding on the structure and stability of Escherichia coli apocytochrome b562

The structure and stability of apocytochrome b562 were explored using absorption and circular dichroism spectroscopic methods. The polypeptide chain retains a well-defined structure when the prosthetic heme group is removed from cytochrome b562. Circular dichroism measurements estimate 60% helicity for apocytochrome b562, compared with 80% helicity found in holocytochrome b562. At low pH, apocytochrome b562 displays a midpoint pH of 2.9, while ferricytochrome b562 displays a midpoint pH of 2.3. The unfolding of the apoprotein by urea and heat can be well approximated by the two-state transition model. The stability of apocytochrome b562 is significantly reduced from that of the holoprotein. The free energy of stabilization (delta G degrees) and the midpoint transition temperature (Tm) for apocytochrome b562 are found to be 3.2 +/- 0.5 kcal/mol and 52.3 +/- 0.9 degrees C, respectively, compared with 6.6 +/- 0.5 kcal/mol and 67.2 +/- 0.5 degrees C for ferricytochrome b562. The smaller heat capacity change upon unfolding of apocytochrome b562 than that of ferricytochrome b562, estimated from the thermodynamic parameters, indicates that apocytochrome b562 possesses a smaller hydrophobic core than holocytochrome b562. Size-exclusion chromatography studies indicate that the apoprotein is slightly more extended in molecular dimension than ferricytochrome b562. The data suggest that apocytochrome b562 resembles a "molten globule" or a "collapsed form" of the holoprotein, in which secondary structure formation is largely complete while the global folding is either only partially complete or dynamically expanded.     

1H and 15N NMR resonance assignments and preliminary structural characterization of Escherichia coli apocytochrome b562

The 1H and 15N resonances of uniformly enriched apocytochrome b562 (106 residues) have been assigned. The assignment work began with the identification of the majority of HN-H alpha-H beta subspin systems in two-dimensional DQF-COSY and TOCSY spectra of unlabeled protein in D2O and in 95% H2O/5% D2O buffer. Intraresidue and interresidue NOE connectivities were then searched for in two-dimensional homonuclear NOESY spectra recorded on unlabeled protein and in the three-dimensional NOESY-HMQC spectrum recorded on uniformly 15N-enriched protein. Those data, combined with the main-chain-directed assignment strategy (MCD), led to the assignment of the main-chain and many side-chain resonances of 103 of the 106 residues. Qualitatively, the helical conformation is found to be the dominant secondary structure in apocytochrome b562 as it is in holocytochrome b562. The helical segments in apocytochrome b562 overlap extensively with the helical regions defined in the crystal structure of ferricytochrome b562. In addition, a number of tertiary NOEs have been identified which indicate that the global fold of the apoprotein at least partially resembles the four-helix bundle of the holoprotein. The results presented here, together with the evidence obtained with other methods [Feng and Sligar (1991) Biochemistry (submitted)], support the notion that the interior of the protein is fluid and may correspond to a molten globule state.     

Chiral resolution of alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene, a novel antiplatelet compound.

alpha,alpha'-Bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, a novel antiplatelet agent, was resolved into three isomers A, B, and C, on a chiral alpha 1-acid glycoprotein analytical column using a mobile phase of 0.025 M phosphate buffer containing 0.025 M tetrabutylammonium hydrogen sulfate, at a pH of 6.5. The effect of molarity, temperature, pH, flow rate, and organic modifiers on the enantioselectivity was examined. Based on circular dichroic spectra at 220 nm, A and C appear to be the (-)- and (+)-enantiomers, respectively, and B the meso diastereomer. Attempts at resolution using Pirkle type columns gave unsatisfactory results. It appears that both hydrophobic and polar interactions between the compound and the stationary phase are important determinants of resolution.     

Spatial patterns and sampling plans for cereal aphids [Hom.: Aphididae] killed by entomophthoralean fungi and hymenopterous parasitoids in spring wheat

Cereal aphids infesting spring wheat in southwestern Idaho were surveyed during 1988 and 1989 for the presence of entomophthoralean fungi and hymenopterous parasitoids. Cereal aphids killed by the fungi (cadavers) and parasitoids (mummies) includedDiuraphis noxia (Mordvilko),Metopolophium dirhodum (Walker),Sitobion avenae (F.), andSchizaphis graminum (Rondani). Taylor's power law was used to describe the relationships between mean densities of cadavers or mummies (number per tiller) and associated variances. Except forS. graminum mummies, which were at low levels throughout the growing seasons and tended to be randomly distributed, Taylor's slopes exceeded 1.0 for all the aphid cadavers and mummies, indicating varying degrees of clumping in spatial pattern. The spatial patterns ofD. noxia andM. dirhodum cadavers were similar, simply reflecting those of their own populations, but more aggregated than were their respective mummies resulting from parasitoid attack. The intercepts and slopes from the power law analysis were used to generate functional relationships between the proportion of wheat tillers bearing cadavers or mummies of each aphid species and the mean density, and develop optimal numerical (direct counting) and binomial (presence or absence) sample size curves for both cadavers and mummies of each aphid species.