Immunoassays based on electrochemical detection using microelectrode arrays.

We show that CombiMatrix's VLSI arrays of individually addressable electrodes, using conventional CMOS integrated circuitry, can be used in detecting various analytes via immunoassay protocols. These microarrays provide over 1000 electrodes per square centimeter. The chips are coated with a porous material on which specific affinity tags are synthesized proximate to selected electrode sites. CombiMatrix microarrays are used to develop spatially multiplexed assay formats for biological entities over a wide range of sizes, from small molecules to cells. Antibodies are tagged with coded affinity labels and then allowed to self-assemble on the appropriate electrode assay sites. Each analyte-specific antibody is chaperoned to individual, predetermined locations by the self-assembly process. The resulting chip can perform numerous different analyte-specific immunoassays, simultaneously. We present new detection technologies based upon the use of the active individually addressable microelectrodes on the chip: redox enzyme amplified electrochemical detection. The results for human alpha1 acid glycoprotein, ricin, M13 phage, Bacillus globigii spores, and fluorescein indicate that this method is one of the most sensitive available, with limits of detection in the attomole range. The detection range is 4-5 logs of analyte concentration, with an assay volume of 50 microl or less. The system provides for a host of multiplexed immunoassays because of the large number of electrodes available. We show how the assays can be optimized for maximum performance on the CombiMatrix microarray platform.

Functional characterization of rat gp600/megalin promoter: combination of proximal Sp1 site and JCV repeat is important in rat gp600/megalin promoter activation.

Gp600/megalin is an endocytic receptor belonging to the low-density lipoprotein receptor family. Up or down regulation of this protein were observed in certain disease states. To understand the mechanisms that control gp600/megalin gene expression, we cloned and functionally characterized a 738-bp fragment of the 5'-flanking region of rat gp600/megalin gene. A transcription start site was mapped to 33 bp downstream of TAGAAA sequence (TATA-like box). Multiple transcription factor binding sites were identified. Serial 5' deletions and transient transfection assays showed that the deletion fragment containing the Sp1 site proximal to the TATA-like box and a JCV repeat retained 80% of the promoter activity. Individual mutations of the proximal Sp1 site and JCV repeat reduced the promoter activity by 60 and 34% respectively. Double mutations of the proximal Sp1 site and JCV repeat produced a dramatic 80% reduction in the promoter activity. However, deletions and mutations or double mutations of other transcription factor binding sites in the promoter region had a minor effect on the promoter activity. These results indicate that the combination of proximal Sp1 site and the JCV repeat are necessary for activation of gp600/megalin expression. Moreover, Sp1 and Sp3 proteins interacted with the proximal and the distal Sp1 sites in the nuclear extracts of gp600/megalin expressing cell lines. TCF site seems to be involved in negative regulation of this promoter but no nuclear protein(s) were found to bind to this site. In addition, Ap2 site responsible for 28% promoter activity is able to bind two dominant unknown nuclear proteins. This functional characterization of the regulation of gp600/megalin gene is likely to advance the knowledge of the regulation of this gene in health and disease.

Increased expression of cytoplasmic tail-containing form of gp600/megalin in active Heymann nephritis.

Active Heymann nephritis of rat, an autoimmune glomerular disease, is a model of human membranous glomerulonephropathy. The major target autoantigen of this disease is a 600 kD renal glycoprotein, variously named gp600/gp330/megalin/LRP2. It has been shown previously that there is a marked increase in the gp600/megalin mRNA level in glomeruli of rats with this disease. A rabbit antibody was prepared against a peptide located at the very end of the cytoplasmic tail of rat gp600/megalin. Affinity purified anti-peptide antibody was monospecific and reacted only with gp600/megalin on the immunoblots of total kidney proteins. Immunofluorescence microscopy on frozen kidney sections probed with this antibody revealed the presence of the cytoplasmic tail-containing form of gp600/megalin in both proximal tubules and glomeruli. Expression in glomeruli of normal kidneys was less than in tubules but was clearly detectable. In active Heymann nephritis kidneys, expression of this form was increased most remarkably in glomeruli. These results, showing increased expression of the cytoplasmic tail-containing form in active Heymann nephritis glomeruli, contradict previous reports of its absence. This is the first study to show that there is increased expression of the cytoplasmic tail-containing form of gp600/megalin in the glomeruli of rats with this disease and its role in active Heymann nephritis is discussed.

Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin.

gp600/megalin, an endocytic receptor, belongs to the low-density lipoprotein receptor family. It is most abundant in the renal proximal tubular cells, where it is implicated in the reabsorption of a number of molecules filtered through the glomerulus. The cytoplasmic tail (CT) of gp600/megalin contains a number of sequence similarities, which indicate that gp600/megalin might be involved in signal transduction. To find intracellular proteins that would interact with the gp600/megalin CT, a human kidney cDNA library was screened by using the yeast two-hybrid system. The phosphotyrosine interaction domain (PID) of the Disabled protein 2 (Dab2), a mammalian structural analogue of Drosophila Disabled, was found to bind to the gp600/megalin CT in this system. The interaction between these two proteins was confirmed by a binding assay in vitro and by the co-immunoprecipitation of both proteins from renal cell lysates. The gp600/megalin CT contains three PsiXNPXY motifs (in which Psi represents a hydrophobic residue) that are potentially able to interact with PID. Analysis of the CT deletion and point-mutation variants of gp600/megalin by the two-hybrid system revealed that the third PsiXNPXY motif is most probably involved in this interaction. Dab2 is a mitogen-responsive phosphoprotein thought to be an adaptor molecule involved in signal transduction, and a suggested negative regulator of cell growth. Dab2 is the first intracellular ligand identified for gp600/megalin; gp600/megalin is the first known transmembrane receptor that interacts with the cytosolic protein Dab2. We speculate that their interaction might involve gp600/megalin in signal transduction pathways or might mediate the intracellular trafficking of this receptor.

A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis.

During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7/L12 is an essential component. L7/L12 is present in each 50S subunit in four copies organized as two dimers. Each dimer consists of distinct domains: a single N-terminal ("tail") domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains ("heads") that are required for binding of elongation factors to ribosomes. The two heads are connected by flexible hinge sequences to the N-terminal domain. Important questions concerning the mechanism by which L7/L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties. In an attempt to answer these questions, we constructed a single-headed dimer of L7/L12 by using recombinant DNA techniques and chemical cross-linking. This chimeric molecule was added to inactive core particles lacking wild-type L7/L12 and shown to restore activity to a level approaching that of wild-type two-headed L7/L12.

Localization of two domains of a mutant form of Escherichia coli protein L7/L12 that binds the large ribosomal subunit as a single dimer.

Escherichia coli ribosomal protein L7/L12 occurs on the large subunit as two dimers: one dimer is extended and comprises the stalk, while the second dimer is folded and occupies a site on the subunit body. A variant protein, in which all 18 amino acids of the flexible hinge region that links separate N-terminal and C-terminal domains of L7/L12 has been deleted, binds the subunit as a single dimer and does not generate stalks that are visible in electron micrographs. Monoclonal antibodies directed against each domain of the protein have been used to localize the variant in electron micrographs of 50S subunits. Both C-terminal domains are seen at a shoulder of the subunit, near its edge as viewed in the most common quasisymmetric projection. N-terminal domains are placed on the subunit body, about 50 A from the C-terminal domains. The antibody to the N-terminal domain also causes dissociation of the variant dimer from the particle and the formation of oligomeric antibody-protein dimer complexes. Similar complexes were seen previously (Olson HM et al (1986) J Biol Chem 261, 6924-6936) when this antibody induced dissociation of one dimer of the native protein. We conclude that the shortened variant most probably occupies the lower-affinity site on the subunit that is normally filled by the stalk dimer.

Rotational and conformational dynamics of Escherichia coli ribosomal protein L7/L12.

Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, pyrenemethyl iodoacetate) were attached to these residues. In addition to the full-length proteins, a hinge-deleted variant and variants corresponding to the C-terminal fragment and the N-terminal fragment were also studied. Both steady-state and time-resolved fluorescence measurements were carried out, and the results demonstrated that L7/L12 is not a rigid molecule. Specifically, the two C-terminal domains move freely with respect to one another and with respect to the dimeric N-terminal domain. Removal of the hinge region, however, significantly reduces the mobility of the C-terminal domains. The data also show that the rotational relaxation time monitored by the fluorescent probe-depends upon the probe's excited state lifetime. This observation is interpreted to indicate that a hierarchy of motions exists in the L7/L12 molecule including facile motions of the C-terminal domains and dimeric N-terminal domain, in addition to the overall tumbling of the protein. Probes attached to the N-terminal domain exhibit global rotational relaxation times consistent with the molecular mass of the dimeric N-terminal fragment. Upon reconstitution of labeled L7/L12 with ribosomal cores, however, the motion associated with the dimeric N-terminal domain is greatly diminished while the facile motion of the C-terminal domains is almost unchanged.

Dimer/monomer equilibrium and domain separations of Escherichia coli ribosomal protein L7/L12.

The dimer to monomer equilibrium and interdomain separations of cysteine variants of L7/L12 have been investigated using fluorescence spectroscopy. Steady-state polarization measurements on cysteine containing variants of L7/L12, labeled with 5-(iodoacetamido)fluorescein, demonstrated dimer to monomer dissociation constants near 30 nM for variants labeled at position 33, in the N-terminal domain, and positions 63 and 89, in the C-terminal domain. A dissociation constant near 300 nM was determined for a variant labeled at position 12, in the N-terminal domain. The polarization of a labeled C-terminal fragment did not change over the range of 200 microM to 1 nM, indicating that this construct remains monomeric at these concentrations, whereas a dimer to monomer dissociation constant near 300 nM was observed for an FITC labeled N-terminal fragment. Intersubunit fluorescence resonance energy self-transfer was observed when appropriate probes were attached to cysteines at residues 12 or 33, located in the N-terminal domain. Probes attached to cysteines at positions 63 or 89 in the C-terminal domain, however, did not exhibit intersubunit self-transfer. These results indicate that these residues in the C-terminal domains are, on average, separated by greater than 85 A. Intersubunit self-transfer does occur in a C-89 double mutation variant lacking 11 residues in the putative hinge region, indicating that the loss of the hinge region brings the two C-terminal domains closer together. Rapid subunit exchange between unlabeled wild-type L7/L12 and L7/L12 variants labeled in the N-terminal domain was also demonstrated by the loss of self-transfer upon mixing of the two proteins.

Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers.

The fluorescent probe tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit tetramethylrhodamine dimers form when tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.

Location and domain structure of Escherichia coli ribosomal protein L7/L12: site specific cysteine crosslinking and attachment of fluorescent probes.

Five different variants of L7/L12 containing single cysteine substitutions, two in the N-terminal (NTD) and three in the C-terminal domain (CTD), were produced, modified with [125I]N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio) propionamide ([125I]APDP), a sulfhydryl-specific, heterobifunctional, cleavable photo-cross-linking reagent, and reconstituted into ribosomes. These were irradiated, the total proteins were extracted and reductively cleaved, and the cross-linked proteins were identified. The effect of zero-length disulfide cross-linking on binding and activity was also determined. The same sites in L7/L12 were used to attach a rhodamine dye. The formation of ground-state rhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the free protein and in complexes with L10. The three sites in the CTD, but not the N-terminal sites, cross-linked to L2 and L5 and to 30S proteins S2, S3, S7, S14, and S18 in a manner influenced by elongation factors. Binding to the ribosome and, therefore, function were blocked by zero-length cross-linking within the NTD, but not the CTD. Binding also disrupted rhodamine dimers in the NTD. No rhodamine dimers formed in the CTD.

Different molecular forms of rat kidney gp330, the dominant autoantigen of active Heymann nephritis.

The primary structure, consisting of 1650 amino acid residues, of the C-terminal end of the dominant autoantigen of active Heymann Nephritis, gp330, from rat kidney was obtained by cloning and sequencing of cDNA clones. Comparison of this sequence with the previously published sequences of fragments of the C-terminal end of gp330 [Raychowdhury, Niles, McCluskey and Smith (1989) Science 244, 1163-1165] revealed certain differences in their primary structures. These differences included several single amino acid substitutions, replacement of a stretch of 15 amino acid residues by a different stretch of six amino acid residues, and different lengths of cytoplasmic domain (188 versus 213 amino acid residues). These findings of two different primary structures of gp330 provide direct evidence for the existence of two molecular forms of gp330.

The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis.

A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12: delta 42-52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917-922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12:(Pro)7, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:delta 42-52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro)7, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:delta 42-52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bound in the A site with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro7 sequence retains sufficient flexibility for full activity.

Escherichia coli ribosomal protein L7/L12 dimers remain fully active after interchain crosslinking of the C-terminal domains in two orientations.

Cysteine site-directed mutagenesis was used to create variants of Escherichia coli ribosomal protein L7/L12 that have single cysteine substitutions, at residues 63 or 89, located in different exposed loops in the structure of the globular C-terminal domain indicated by the crystallographic structure. That structure shows a possible dimer interaction in which the two sites of cysteine substitution appear to be too distant for disulfide bond formation. After mild oxidation in solution both of the overexpressed purified cysteine-substituted proteins formed interchain disulfide crosslinked dimers in high yield. Both crosslinked dimers were fully active in restoring activity in poly(U)-directed polyphenylalanine synthesis to ribosomal core particles depleted of wild-type L7/L12. These results show that the two C-terminal domains have independent mobility. The activity of dimeric L7/L12 does not require the independent movement of the two globular C-terminal domains in an L7/L12 dimer; moreover, it appears independent of their mutual orientation when joined by crosslinking at the two loops. A third variant with a cysteine substitution at residue 33 near the junction between the alpha-helical N-terminal domain and the flexible hinge was prepared and tested. This protein was active in the protein synthesis assay in the reduced state. Oxidation produced the interchain crosslinked dimer in high yield, but this crosslinked dimer was inactive in polyphenylalanine synthesis. The inactivation was due to the inability of the Cys33-Cys33 oxidized dimer to bind to the core particle.

Zero-length cross-linking of the C-terminal domain of Escherichia coli ribosomal protein L7/L12 to L10 in the ribosome and in the (L7/L12)4-L10 pentameric complex.

L7/L12Cys89 is a variant of L7/L12 that has a single cysteine residue located in the C-terminal domain in which Cys89 is the only cysteine residue in the protein. A cross-link between this site and the single cysteine in L10, residue 70, was formed with 1,4-di[3'-(2'-pyridyldithio)-propionamido]butane, a sulfhydryl-specific homobifunctional reagent of maximum length 16 A. It is now shown that a zero-length disulfide cross-link between L7/L12Cys89 and L10Cys70 is formed by mild oxidation with Cu2+(phenanthroline)3 of either intact ribosomes or the stable, pentameric complex (L7/L12)4-L10. The formation of the zero-length cross-link defines more closely the contact between the two proteins. Protein L10 is located at the base of the L7/L12 stalk where it provides binding sites for the N-terminal domains of both dimers of L7/L12. The L7/L12Cys89-L10Cys70 cross-link lends further support to our previous model that places at least one of the two dimers of L7/L12 on the surface of the body of the 50S subunit in a bent conformation with the C-terminal domain in close proximity to its N-terminal domain, at the base of the L7/L12 stalk. The L7/L12Cys89-L10Cys70 cross-link in the pentameric L8 complex implies that the protein can exist in this bent conformation there as well as in the ribosome.

Structural and functional domains of Escherichia coli ribosomal protein L7/L12. The hinge region is required for activity.

Variant forms of Escherichia coli ribosomal protein L7/L12 were constructed, overexpressed, and purified. These included proteins that deleted residues 35-52 (delta 35-52) and 42 to 52 (delta 42-52), others that contained single cysteine substitutions at residues 63 and 89, and combinations of the deletions and cysteine substitutions. Chemical modification of the introduced cysteine residues with [14C]iodoacetamide was used to radiolabel the protein variants in order to quantify their binding to the ribosome. Neither of the deletions in the hinge domain, delta 35-52 and delta 42-52, had any effect on L7/L12 dimer formation as detected by cross-linking by dimethyl suberimidate. Perpendicular urea gradient gel electrophoresis showed that both deletion variants retained a compact structural element attributable to the globular C-terminal domain. Reconstitution of core particles depleted of wild type L7/L12 with the deletion proteins showed that delta 42-52 bound normally in 4 copies per particle, whereas delta 35-52 bound in only 2.5 copies following isolation of the particles by high speed centrifugation or gel filtration. Ribosomes mixed with an excess of the deletion variants and assayed directly for polyphenylalanine synthesis were completely inactive. The results suggest that the flexibility conferred by the hinge is required for activity, perhaps by allowing the C-terminal domain to occupy a location near the base of the L7/L12 stalk.

Primary structure of the cDNA 5'-terminal region encoding the N-terminal domain of the rabbit muscle alpha-actinin subunit.

A number of cDNA clones have been obtained in summary encoding the N-terminal domain containing 286 amino acid residues of the rabbit skeletal muscle alpha-actinin subunit. Alpha-Actinin cDNA clones were isolated from specific cDNA libraries using the primer extension method for synthesis of the first cDNA chain. A strong stop signal for AMV reverse transcriptase in the 5'-terminal region of mRNA of alpha-actinin was found. It seems there is a G+C rich region (93% G+C nucleotides) including a continuous sequence of 23 G and C nucleotides encoding 6 glycine residues.

[Cloning and determination of the primary structure of cDNA, coding the central part of elongation factor 2 from the rat liver]. / Klonirovanie i opredelenie pervichnoi struktury kDNK, kodiruiushchei tsentral'nuiu chast' faktora élongatsii 2 iz pecheni krysy.

A number of cDNA clones coding for 417 amino acid residues of the central part of the rat liver elongation factor 2 (EF-2) have been isolated. The oligonucleotides complementary to EF-2 mRNA were used as primers for synthesis of the first strand of cDNA cloned. Structures of these oligonucleotides were determined in course of 3'----5' sequencing coding strand of EF-2 cDNA. This method of synthesis of specific cDNA enabled one to reduce essentially the number of recombinant clones to be screened for EF-2 cDNA. Comparative studies of deduced protein sequences of rat liver EF-2 and hamster EF-2 [1] revealed the only substitution of aspartate residue for glutamate residue (hamster EF-2). The homology between nucleotide sequences of rat and hamster EF-2 cDNA was 89%. Northern-blot analysis of rat liver poly(A)+ mRNA revealed the only species of mRNA 3000 nucleotides long. A strong stop-signal for reverse transcriptase in the 5'-region of rat liver EF-2 mRNA is discovered: probability of dissociation of the enzyme from mRNA is at least 97.5%.

Primary structure of rat liver elongation factor 2 deduced from the cDNA sequence.

A number of cDNA clones in summary encoding 700 amino acid residues from the N-end of rat liver elongation factor 2 (EF-2) and including 49 nucleotides of the 5'-untranslated mRNA region have been obtained. EF-2 cDNA clones were isolated from gradually constructed small (1000-5000 clones) specific cDNA libraries using the primer extension method for synthesis of the first cDNA chain. The complete primary structure of cDNA and protein EF-2 from rat liver was derived taking into account the primary structure of the 3'-terminal region EF-2 cDNA previously reported [(1986) Proc. Natl. Acad. Sci. USA 83, 4978-4982]. Comparison of this cDNA with hamster cDNA has shown that (i) the base sequences had a 89.7% homology while that of the 5'-untranslated region was 73%; (ii) there are two amino acid replacement in rat liver EF-2 as compared with hamster EF-2.

Evidence for the appearance of a reticulocyte population low in lipoxygenase mRNA during the recovery from a phenylhydrazine-induced anemia in rabbits.

It is shown that during recovery from a phenylhydrazine-induced anemia in rabbits a selective decrease in lipoxygenase mRNA takes place with a corresponding shut-off of the synthesis of the enzyme. It is suggested that a new population, 'recovery'-reticulocytes, makes its appearance in the peripheral blood. Their cells are more mature than the stress macroreticulocytes. A cell-free system prepared from the recovery-reticulocytes exhibits low endogenous synthesis of non-globin polypeptides, even without nuclease treatment, but retains full capacity to be stimulated by exogenous mRNA.