Expression and substrate specificity of the toluene dioxygenase of Pseudomonas putida NCIMB 11767

Pseudomonas putida NCIMB 11767 oxidized phenol, monochlorophenols, several dichlorophenols and a range of alkylbenzenes (C1-C6) via an inducible toluene dioxygenase enzyme system. Biphenyl and naphthalene were also oxidized by this enzyme. Growth on toluene and phenol induced the meta-ring-fission enzyme, catechol 2,3-oxygenase, whereas growth on benzoate, which did not require expression of toluene dioxygenase, induced the ortho-ring-cleavage enzyme, catechol 1,2-oxygenase. Monochlorobenzoate isomers and 2,3,5-trichlorophenol were gratuitous inducers of toluene dioxygenase, whereas 3,4-dichlorophenol was a fortuitous oxidation substrate of the enzyme. The organism also grew on 2,4- and 2,5-dichloro isomers of both phenol and benzoate, on 2,3,4-trichlorophenol and on 1-phenylheptane. During growth on toluene in nitrogen-limited chemostat culture, expression of both toluene dioxygenase and catechol 2,3-oxygenase was positively correlated with increase in specific growth rate (0.11-0.74 h-1), whereas the biomass yield coefficient decreased. At optimal dilution rates, the predicted performance of a 1-m3 bioreactor supplied with 1 g nitrogen l-1 for removal of toluene was 57 g day-1 and for removal of trichloroethylene was 3.4 g day-1. The work highlights the oxidative versatility of this bacterium with respect to substituted hydrocarbons and shows how growth rate influences the production of competent cells for potential use as bioremediation catalysts.     

Nucleotide-dependent movement of the epsilon subunit between alpha and beta subunits in the Escherichia coli F1F0-type ATPase

Mutants of ECF1-ATPase were generated, containing cysteine residues in one or more of the following positions: alphaSer-411, betaGlu-381, and epsilonSer-108, after which disulfide bridges could be created by CuCl2 induced oxidation in high yield between alpha and epsilon, beta and epsilon, alpha and gamma, beta and gamma (endogenous Cys-87), and alpha and beta. All of these cross-links lead to inhibition of ATP hydrolysis activity. In the two double mutants, containing a cysteine in epsilonSer-108 along with either the DELSEED region of beta (Glu-381) or the homologous region in alpha (Ser-411), there was a clear nucleotide dependence of the cross-link formation with the epsilon subunit. In betaE381C/epsilonS108C the beta-epsilon cross-link was obtained preferentially when Mg2+ and ADP + Pi (addition of MgCl2 + ATP) was present, while the alpha-epsilon cross-link product was strongly favored in the alphaS411C/epsilonS108C mutant in the Mg2+ ATP state (addition of MgCl2 + 5'-adenylyl-beta,gamma-imidodiphosphate). In the triple mutant alphaS411C/betaE381C/epsilonS108C, the epsilon subunit bound to the beta subunit in Mg2+-ADP and to the alpha subunit in Mg2+-ATP, indicating a significant movement of this subunit. The gamma subunit cross-linked to the beta subunit in higher yield in Mg2+-ATP than in Mg2+-ADP, and when possible, i.e. in the triple mutant, always preferred the interaction with the beta over the alpha subunit.     

The Na(+)-K(+)-ATPase beta 1 subunit is associated with the HK alpha 2 protein in the rat kidney

The Na-K-ATPase beta 1 subunit acts as the beta subunit for the HK alpha 2 protein in the rat kidney. The colonic H(+)-K(+)-ATPase is a member of the P-type ATPases, and has been shown to contribute to potassium transport by the mammalian kidney and colon. The P-type ATPases often consist of an alpha subunit that contains the catalytic site and a beta subunit that participates in regulation of enzyme activity and targeting of the enzyme to the plasma membrane. The cDNA of the alpha subunit (HK alpha 2) has been cloned and the HK alpha 2 protein has been isolated from the rat kidney and colon. However, a unique beta subunit for the colonic H(+)-K(+)-ATPase has not been described. To determine if one of the known beta subunits present in the kidney might act as the beta subunit for the colonic H(+)-K(+)-ATPase, microsomes enriched in the colonic H(+)-K(+)-ATPase were isolated using an HK alpha 2-specific antibody (AS 31.7) and the Minimac magnetic separation system. Immunoblots of rat kidney microsomal protein isolated with antibody AS 31.7 were probed with antibodies directed against the gastric HK beta subunit, Na(+)-K(+)-ATPase alpha 1, and Na(+)-K(+)-ATPase beta 1 subunits. A band of the appropriate size was detected with Na(+)-K(+)-ATPase beta 1-specific antibodies, but not those directed against HK beta 1. These data suggest that Na(+)-K(+)-ATPase beta 1 could be the beta subunit for the colonic H(+)-K(+)-ATPase in the kidney.     

The cytochrome c maturation operon is involved in manganese oxidation in Pseudomonas putida GB-1

A Pseudomonas putida strain, strain GB-1, oxidizes Mn2+ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn2+ oxidation and/or secretion of the Mn2+-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn2+ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn2+-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn2+ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.     

Importin beta can mediate the nuclear import of an arginine-rich nuclear localization signal in the absence of importin alpha

The import of proteins into the nucleus is dependent on cis-acting targeting sequences, nuclear localization signals (NLSs), and members of the nuclear transport receptor (importin-beta-like) superfamily. The most extensively characterized import pathway, often termed the classical pathway, is utilized by many basic-type (lysine-rich) NLSs and requires an additional component, importin alpha, to serve as a bridge between the NLS and the import receptor importin beta. More recently, it has become clear that a variety of proteins enter the nucleus via alternative import receptors and that their NLSs bind directly to those receptors. By using the digitonin-permeabilized cell system for protein import in vitro, we have defined the import pathway for the Rex protein of human T-cell leukemia virus type 1. Interestingly, the arginine-rich NLS of Rex uses importin beta for import but does so by a mechanism that is importin alpha independent. Based on the ability of the Rex NLS to inhibit the import of the lysine-rich NLS of T antigen and of both NLSs to be inhibited by the domain of importin alpha that binds importin beta (the IBB domain), we infer that the Rex NLS interacts with importin beta directly. In addition, and in keeping with other receptor-mediated nuclear import pathways, Rex import is dependent on the integrity of the Ran GTPase cycle. Based on these results, we suggest that importin beta can mediate the nuclear import of arginine-rich NLSs directly, or lysine-rich NLSs through the action of importin alpha.     

Reconstitution of F1-ATPase activity from Escherichia coli subunits alpha, beta and subunit gamma tagged with six histidine residues at the C-terminus

OBJECTIVE: To determine the effect of a specific galloping exercise regimen on collagen fibril mass-average diameters (MAD) in the deep digital flexor tendon (DDFT) and suspensory ligament (SL) of young Thoroughbreds. ANIMALS: 12 Thoroughbred fillies, 21 +/- 1 (mean +/- SD) months old. PROCEDURE: 6 horses underwent a specific 18-month treadmill training program involving galloping exercise. The remaining 6 horses served as controls, undertaking low-volume walking exercise over the same period. Sections were excised from the midpoint of the DDFT and SL, and small strips were dissected from central and peripheral locations for each structure. Fibril diameters were measured from micrographs of transverse ultrathin sections, using a computerized image analysis program. An MAD value was calculated for the central and peripheral regions of the DDFT and SL for each horse. Values for both regions were compared between exercised and control horses. RESULTS: The MAD did not change significantly with exercise for either the DDFT or the SL. CONCLUSION: Loading of the DDFT as a result of this exercise regimen was not sufficient to stimulate collagen fibril hypertrophy, in keeping with current data that indicate this tendon, compared with the SL and superficial digital flexor tendon (SDFT), is subjected to low loads. Microtrauma, in terms of reduction in fibril MAD, may have occurred in the SL at a site different from that sampled. Another possibility is that, between the trot and the gallop, loading of the SL does not increase to the same extent as that of the SDFT.     

Multiple modulation pathways of calcium channel activity by a beta subunit. Direct evidence of beta subunit participation in membrane trafficking of the alpha1C subunit

In order to study the precise mechanisms of alpha1 subunit modulation by an auxiliary beta subunit of voltage-dependent calcium channels, a recombinant beta3 subunit fusion protein was produced and introduced into oocytes that express the human alpha1C subunit. Injection of the beta3 subunit protein rapidly modulated the current kinetics and voltage dependence of activation, whereas massive augmentation of peak current amplitude occurred over a longer time scale. Consistent with the latter, a severalfold increase in the amount of the alpha1C subunit in the plasma membrane was detected by quantitative confocal laser-scanning microscopy after beta3 subunit injection. Pretreatment of oocytes with bafilomycin A1, a vacuolar type H+-ATPase inhibitor, abolished the increase of the alpha1C subunit in the plasma membrane, attenuated current increase, but did not affect the modulation of current kinetics and voltage dependence by the beta3 subunit. These results provide clear evidence that the beta subunit modifies the calcium channel complex in a binary fashion; one is an allosteric modulation of the alpha1 subunit function and the other is a chaperoning of the alpha1 subunit to the plasma membrane.     

Post-translational modifications of alpha5beta1 integrin by glycosaminoglycan chains. The alpha5beta1 integrin is a facultative proteoglycan

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.     

The subunit b of the F0F1-type ATPase of the bacterium Mycoplasma pneumoniae is a lipoprotein

The DNA sequence analysis of the F0F1-ATPase operon of the bacterium Mycoplasma pneumoniae predicted that the subunit b, encoded by the gene atpF, is a lipoprotein of the murein lipoprotein type of Escherichia coli. Here we experimentally verify this prediction by metabolic labeling of subunit b with [14C]palmitic acid and by in vivo interfering with the processing of the prolipoprotein form of subunit b by the antibiotic globomycin, a specific inhibitor of the signal peptidase II. Our results suggest that the subunit b of the F0F1-ATPase of M. pneumoniae is anchored at the cytoplasmic membrane by an N-terminal lipid modification in addition to its transmembrane domain. The lipoprotein nature of subunit b and its proposed membrane topology seems to be characteristic for mycoplasmas, since among all sequenced bacterial atpF genes, only those from Mycoplasma gallisepticum and Mycoplasma genitalium code for a conserved lipoprotein consensus sequence.     

Distinct alpha 7A beta 1 and alpha 7B beta 1 integrin expression patterns during mouse development: alpha 7A is restricted to skeletal muscle but alpha 7B is expressed in striated muscle, vasculature, and nervous system

The laminin binding alpha 7 beta 1 integrin has been described as a major integrin in skeletal muscle. The RNA coding for the cytoplasmic domain of alpha 7 integrin undergoes alternative splicing to generate two major forms, denoted alpha 7A and alpha 7B. In the current paper, we have examined the developmental expression patterns of the alpha 7A and alpha 7B splice variants in the mouse. The alpha 7 integrin expression is compared to that of the nonintegrin laminin receptor dystroglycan and to that of laminin-alpha 1 and laminin-alpha 2 chains. Alpha 7A integrin was found by in situ hybridization to be specific to skeletal muscle. Antibodies specific for alpha 7B integrin and in situ hybridization revealed the presence of alpha 7 mRNA and alpha 7B protein in the E10 myotome and later in primary and secondary myotubes. In the heart, alpha 7B integrin was not detectable in the endocardium or myocardium during embryonic and fetal heart development. Northern blot analysis and immunohistochemistry revealed a postnatal induction of alpha 7B in the myocardium. In addition to striated muscle, alpha 7B integrin was localized to previously unreported nonmuscle locations such as a subset of vascular endothelia and restricted sites in the nervous system. Comparison of the alpha 7 integrin expression pattern with that of different laminin isoforms and dystroglycan revealed a coordinated temporal expression of dystroglycan, alpha 7 integrin, and laminin-alpha 2, but not laminin-alpha 1, in the forming skeletal muscle. We conclude that the alpha 7A and alpha 7B integrin variants are expressed in a developmentally regulated, tissue-specific pattern suggesting different functions for the two splice forms.     

Identification and characterization of a novel beta subunit of soluble guanylyl cyclase that is active in the absence of a second subunit and is relatively insensitive to nitric oxide

Previously characterized soluble guanylyl cyclases form alpha-beta heterodimers that can be activated by the gaseous messenger, nitric oxide. In mammals, four subunits have been cloned, named alpha1, alpha2, beta1, and beta2. We have identified a novel soluble guanylyl cyclase isoform from the nervous system of the insect Manduca sexta that we have named M. sexta guanylyl cyclase beta3 (MsGC-beta3). It is most closely related to the mammalian beta subunits but has several features that distinguish it from previously identified soluble cyclases. Most importantly, MsGC-beta3 does not need to form heterodimers to form an active enzyme because guanylyl cyclase activity can be measured when it is expressed alone in COS-7 cells. Moreover, this activity is only weakly enhanced in the presence of the nitric oxide donor, sodium nitroprusside. Several of the amino acids in rat beta1 subunits, previously identified as being important in heme binding or necessary for nitric oxide activation, are substituted with nonsimilar amino acids in MsGC-beta3. There are also an additional 315 amino acids C-terminal to the catalytic domain of MsGC-beta3 that have no sequence similarity to any known protein. Northern blot analysis shows that MsGC-beta3 is primarily expressed in the nervous system of Manduca.     

The Ca2+ channel beta3 subunit differentially modulates G-protein sensitivity of alpha1A and alpha1B Ca2+ channels

We have shown previously that the Ca2+ channel beta3 subunit is capable of modulating tonic G-protein inhibition of alpha1A and alpha1B Ca2+ channels expressed in oocytes. Here we determine the modulatory effect of the Ca2+ channel beta3 subunit on M2 muscarinic receptor-activated G-protein inhibition and whether the beta3 subunit modulates the G-protein sensitivity of alpha1A and alpha1B currents equivalently. To compare the relative inhibition by muscarinic activation, we have used successive ACh applications to remove the large tonic inhibition of these channels. We show that the resulting rebound potentiation results entirely from the loss of tonic G-protein inhibition; although the currents are temporarily relieved of tonic inhibition, they are still capable of undergoing inhibition through the muscarinic pathway. Using this rebound protocol, we demonstrate that the inhibition of peak current amplitude produced by M2 receptor activation is similar for alpha1A and alpha1B calcium currents. However, the contribution of the voltage-dependent component of inhibition, characterized by reduced inhibition at very depolarized voltage steps and the relief of inhibition by depolarizing prepulses, was slightly greater for the alpha1B current than for the alpha1A current. After co-expression of the beta3 subunit, the sensitivity to M2 receptor-induced G-protein inhibition was reduced for both alpha1A and alpha1B currents; however, the reduction was significantly greater for alpha1A currents. Additionally, the difference in the voltage dependence of inhibition of alpha1A and alpha1B currents was heightened after co-expression of the Ca2+ channel beta3 subunit. Such differential modulation of sensitivity to G-protein modulation may be important for fine tuning release in neurons that contain both of these Ca2+ channels.     

Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The alpha1 and alpha3 isoforms of the alpha subunit, and the beta2 and beta3 isoforms of the beta subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found beta2 but not beta1 in reticulocyte single-stranded cDNA, and beta1 but not beta2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that alpha1, alpha2, and alpha3 as well as all three beta isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.     

The GABAA receptor alpha 6 subunit gene (Gabra6) is tightly linked to the alpha 1-gamma 2 subunit cluster on mouse chromosome 11

Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by hyperplasia and neoplasia in several endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a 900-kb region on chromosome 11q13. The human phosphatidylinositol-specific phospholipase C beta 3 (PLC beta 3) gene, which is located within this region, was considered to be a good candidate for the MEN 1 gene. In this study, the structure and expression of the PLC beta 3 gene in MEN 1 patients were investigated in more detail, to determine its potential role in MEN 1 tumorigenesis. Southern blot analysis, using blood and tumor DNA from affected persons from seven different MEN 1 families, did not reveal structural abnormalities in the PLC beta 3 gene. To detect possible point mutations, or other small structural aberrations, direct sequencing of PLC beta 3 cDNAs from two affected persons from two different MEN 1 families was performed, but no MEN 1-specific abnormalities were revealed. Several common nucleotide sequence polymorphisms were detected in these cDNAs, proving that both alleles of the PLC beta 3 gene were expressed and analyzed. In conclusion, these results exclude the PLC beta 3 gene as a candidate gene for MEN 1.     

The gamma 2 subunit of the GABAA receptor is concentrated in synaptic junctions containing the alpha 1 and beta 2/3 subunits in hippocampus, cerebellum and globus pallidus

The gamma 2 subunit is necessary for the expression of the full benzodiazepine pharmacology of GABAA receptors and is one of the major subunits in the brain. In order to determine the location of channels containing the gamma 2 subunit in relation to GABA-releasing terminals on the surface of neurons, a new polyclonal antipeptide antiserum was developed to the gamma 2 subunit and used in high resolution, postembedding, immunoelectron-microscopic procedures. Dual immunogold labelling of the same section for two subunits, and up to three sections of the same synapse reacted for different subunits, were used to characterize the subunit composition of synaptic receptors. The gamma 2 subunit was present in type 2, "symmetrical" synapses in each of the brain areas studied, with the exception of the granule cell layer of the cerebellum. The gamma 2 subunit was frequently co-localized in the same synaptic junction with the alpha 1 and beta 2/3 subunits. The immunolabelling of synapses was coincident with the junctional membrane specialization of the active zone. Immunolabelling for the receptor often occurred in multiple clusters in the synapses. In the hippocampus, the gamma 2 subunit was present in basket cell synapses on the somata and proximal dendrites and in axo-axonic cell synapses on the axon initial segment of pyramidal and granule cells. Some synapses on the dendrites of GABAergic interneurones were densely labelled for the gamma 2, alpha 1 and beta 2/3 subunits. In the cerebellum, the gamma 2 subunit was present in both distal and proximal Purkinje cell dendritic synapses established by stellate and basket cell, respectively. On the soma of Purkinje cells, basket cell synapses were only weakly labelled. Synapses on interneuron dendrites were more densely labelled for the gamma 2, alpha 1 and beta 2/3 subunits than synapses on Purkinje or granule cells. Although immunoperoxidase and immunofluorescence methods show an abundance of the gamma 2 subunit in granule cells, the labelling of Golgi synapses was much weaker with the immunogold method than that of the other cell types. In the globus pallidus, many type 2 synapses were labelled for the gamma 2 subunit together with alpha 1 and beta 2/3 subunits. The results show that gamma 2 and beta 2/3 subunits receptor channels are highly concentrated in GABAergic synapses that also contain the alpha 1 and beta 2/3 subunits. Channels containing the gamma 2 subunit are expressed in synapses on functionally distinct domains of the same neuron receiving GABA from different presynaptic sources. There are quantitative differences in the density of GABAA receptors at synapses on different cell types in the same brain area.     

p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.     

The integrin alpha IIb-beta 3, platelet glycoprotein IIb-IIIa, can form a functionally active heterodimer complex without the cysteine-rich repeats of the beta 3 subunit

Integrin alpha IIb-beta 3 binds fibrinogen via the recognition sequence Arg-Gly-Asp-Ser (RGDS). We have used the baculovirus/insect cell expression system to study the structural requirements for the formation of a functionally active fragment of alpha IIb-beta 3. A tandem baculovirus transfer vector was constructed containing the cDNA coding for the heavy chain of human alpha IIb (alpha IIbH, amino acids 1-874) and the cDNA coding for a truncated form of human beta 3 (t beta 3; amino acids 1-469). Sf9 insect cells were infected with the corresponding baculovirus, and the produced soluble recombinant proteins were purified using an RGD-like affinity column. The bound receptor fragments were specifically eluted with RGDS and existed as a heterodimeric complex (rec alpha IIbH-t beta 3) with an apparent M(r) of 160,000. In an immunocapture assay, the monoclonal antibody pl-55, which only recognizes the functionally active form of alpha IIb-beta 3, bound to the purified complex. Rec alpha IIbH-t beta 3 specifically bound 125I-fibrinogen with an affinity comparable with that of purified platelet alpha IIb-beta 3. Electron micrographs of rotary-shadowed rec alpha IIbH-t beta 3 showed that the complex had the characteristic globular head, but the two rodlike tails were 4-6 nm shorter than those found in intact alpha IIb-beta 3. Thus, the cysteine-rich repeats of beta 3 are not required for assembly, stability, and functional activity of this integrin.     

The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase. The B subunits interact with F1 as a dimer and through the delta subunit

The delta and b subunits are both involved in binding the F1 to the F0 part in the Escherichia coli ATP synthase (ECF1F0). The interaction of the purified delta subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified delta binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1, i.e. in the presence of ECF1-delta, delta, and bsol interact strongly, and a complex of ECF1.bsol can be isolated by native gel electrophoresis. Both delta subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of delta as delta(1-134) cannot replace intact delta in the binding experiments. As purified, bsol is a stable dimer with 80% alpha helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem. 271, 7038-7042). Monomeric bsol has less alpha helix, i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to delta in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.     

The development of experimental autoimmune encephalomyelitis in the mouse requires alpha4-integrin but not alpha4beta7-integrin

Because monoclonal antibodies (mAbs) directed against alpha4-integrin and VCAM-1 inhibit the development of experimental autoimmune encephalomyelitis (EAE) in vivo, it has been concluded that the successful therapeutic effect is due to interference with alpha4beta1/VCAM-1-mediated interaction of autoaggressive T cells with the blood-brain barrier. A possible role for alpha4beta7-integrin, or interference with other T cell mediated events during the pathogenesis of EAE, has not been considered. We have compared the effects of mAb therapy on the development of EAE in the SJL/N mouse, using a large panel of mAbs directed against alpha4, beta7, the alpha4beta7-heterodimer, and against VCAM-1. Although encephalitogenic T cells express both alpha4-integrins, mAbs directed against the alpha4beta7-heterodimer or against the beta7-subunit did not interfere with the development of EAE. In contrast, mAbs directed against alpha4 and VCAM-1 inhibited or diminished clinical or histopathological signs of EAE. Our data demonstrate for the first time that alpha4beta7 is not essential for the development of EAE. Furthermore, our in vitro studies suggest that the therapeutic effect of anti-alpha4-treatment of EAE might also be caused by inhibition of antigen-specific T cell proliferation.