Molecular cloning and expression of a new alpha-subunit of soluble guanylyl cyclase. Interchangeability of the alpha-subunits of the enzyme.

A cDNA coding for a new subunit of soluble guanylyl cyclase with a calculated molecular mass of 81.7 kDa was cloned and sequenced. On the basis of sequence homology, the new subunit appears to be an isoform of the alpha 1-subunit and was designated alpha 2 as the new subunit is very similar to the alpha 1-subunit in the middle and C-terminal part; it is quite diverse in the N-terminal part. Preceding experiments had shown that coexpression of the alpha 1- and beta 1-subunits is necessary to obtain a catalytically active guanylyl cyclase in COS cells [(1990) FEBS Lett. 272, 221-223]. The finding that the alpha 2-subunit was able to replace the alpha 1- but not the beta 1-subunit in expression experiments demonstrates the interchangeability of the alpha-subunit isoforms of soluble guanylyl cyclase.     

Atypical human liver alcohol dehydrogenase: the beta 2-Bern subunit has an amino acid exchange that is identical to the one in the beta 2-Oriental chain

The "atypical' human liver alcohol dehydrogenase dimer, homogeneous for beta 2-Bern chains, was isolated from human liver of Caucasian individuals. It is derived from an allelic variant at the ADH2 gene locus and exhibits a considerably higher specific activity and lower pH optimum than its "typical' counterpart (isoenzyme beta 1 beta 1) from the beta 1-chain predominant in Caucasians. Peptides were prepared by trypsin or CNBr cleavage, and were purified by exclusion chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Structural analysis of the peptides showed that beta 2-Bern differs at one position from beta 1. Thus, Arg-47 in beta 1 is substituted by His in beta 2-Bern. This exchange, compatible with a one-base mutation, explains all functional differences by altered interactions with the pyrophosphate moiety of the coenzyme. The difference is also structurally identical to that found for another atypical beta 2-subunit, the beta 2-Oriental type of major Asian occurrence, linking these two atypical forms of human alcohol dehydrogenase.     

GABAA receptors display association of gamma 2-subunit with alpha 1- and beta 2/3-subunits.

Recombinant GABAA (gamma-aminobutyrate-Type A) receptors that are sensitive to benzodiazepine receptor ligands can be generated by coexpression of alpha-, beta-, and gamma 2-subunit cDNAs (Pritchett, D. B., Sontheimer, H., Shivers, B. D., Ymer S., Kettenmann, H., Schofield, P. R., and Seeburg, P. H. (1989) Nature 338, 582-585; Pritchett, D. B., Lüddens, H., and Seeburg, P. H. (1989) Science 245, 1389-1392; Malherbe, P., Sigel, E., Baur, R., Perssohn, E., Richards, J. G., and Mohler, H. (1990) J. Neurosci. 10, 2330-2337). However, in brain tissue, only alpha- and beta-subunit proteins have so far been detected. To identify the size and distribution of the gamma 2-subunit protein in brain tissue, polyclonal antibodies were prepared against two synthetic peptides corresponding to amino acids 1-15 and 336-350 of the cDNA-derived rat gamma 2-subunit sequence. On Western blots, both anti-gamma 2-subunit antisera selectively labeled a 43-kDa protein. gamma 2-Subunit immunoreactivity was detected immunohistochemically in various brain regions, e.g. in the olfactory bulb, cerebral cortex, islands of Calleja, hippocampus, substantia nigra, and cerebellum. Immunoprecipitation with both antisera identified the gamma 2-subunit immunoreactivity in 40 and 50% of the native GABAA receptors purified from bovine and rat brains, respectively. Monoclonal antibody bd24 selectively recognizes the alpha 1-subunit, whereas bd17 recognizes both the beta 2- and beta 3-subunits (Ewert, M., Shivers, B. D., Lüddens, H., Mohler, H., and Seeburg, P. H. (1990) J. Cell Biol. 110, 2043-2048). Since either of these monoclonal antibodies (bd17 and bd24) precipitated approximately 90% of the GABAA receptors, the gamma 2-subunit is frequently associated with the alpha 1-subunit and the beta 2- and/or beta 3-subunit in vivo.     

The Amino Acid Sequence of the Tryptophan-Containing Subunit (α-Subunit) of Bovine Brain S-100 Protein

Abstract: The tryptophan-containing subunit (α-subunit) of bovine brain S-100 protein was purified from a S -aminoethyl derivative of S-100a protein, and its amino acid sequence was determined. The α-subunit contained 93 residues, including one tryptophan, and had a molecular weight of 10,400. The sequence shows an extensive homology (58% identity) to the sequence of another "tryptophan-free" subunit (β-subunit) found in both S-100a and S-100b protein, and has a calcium binding site characteristic of the "E-F hand" proteins, such as calmodulin or troponin C. The tryptophan residue is located at position 90 which is presumably adjacent to the C-terminal end of the α-helix following the calcium binding loop, and thus appears likely to serve as a specific probe in structure-function studies of S-100a protein.     

Cerebral expression of the alpha2-subunit of soluble guanylyl cyclase is linked to cerebral maturation and sensory pathway refinement during postnatal development

Soluble guanylyl cylase (sGC) has been identified for being a receptor for the gaseous transmitters nitric oxide and carbon monoxide. Currently four subunits alpha1, alpha2, beta1, and beta2 have been characterized. Heterodimers of alpha and beta-subunits as well as homodimers of the beta2-subunit are known to constitute functional sGC which use GTP to form cGMP a potent signal molecule in a multitude of second messenger cascades. Since NO-cGMP signaling plays a pivotal role in neuronal development we analyzed the maturational expression pattern of the newly characterized alpha2-subunit of sGC within the brain of Wistar rats by means of RNase protection assay and immunohistochemistry. alpha2-subunit mRNA as well as immunoreactive alpha2-protein increased during postnatal cerebral development. Topographical analysis revealed a selective high expression of the alpha2-subunit in the choroid plexus and within developing sensory systems involving the olfactory and somatosensory system of the forebrain as well as parts of the auditory and visual system within the hindbrain. In cultured cortical neurons the alpha2-subunit was localized to the cell membrane, especially along neuronal processes. During the first 11 days of postnatal development several cerebral regions showed a distinct expression of the alpha2-subunit which was not paralleled by the alpha1/beta1-subunits especially within the developing thalamo-cortical circuitries of the somatosensory system. However, at later developmental stages all three subunits became more homogenously distributed among most cerebral regions, indicating that functional alpha1/beta1 and alpha2/beta1 heterodimers of sGC could be formed. Our findings indicate that the alpha2-subunit is an essential developmentally regulated constituent of cerebral sensory systems during maturation. In addition the alpha2-subunit may serve other functions than forming a functional heterodimer of sGC during the early phases of sensory pathway refinement.     

The complete amino-acid sequence and the phylogenetic origin of phycocyanin-645 from the cryptophytan alga Chroomonas sp

The first complete amino-acid sequence of the cryptomonad phycobiliprotein phycocyanin-645 from Chroomonas sp. is presented. The alpha 1-subunit contains 70 amino-acid residues and the alpha 2-subunit 80 residues. In each of the alpha-subunits a green, 697-nm absorbing chromophore is covalently bound to Cys18. Both alpha-subunits contain a high number of charged residues. The phycocyanin-645 beta-subunit consists of 177 amino-acid residues. Two phycocyanobilin chromophores are singly bound to Cys beta 82 and Cys beta 158. A purple cryptoviolin-like chromophore is doubly bound to Cys beta 50 and Cys beta 61. Sequence comparisons revealed that the phycocyanin-645 beta-subunit is closely related to red algal phycoerythrin (73% identical amino-acid residues) and not so close to C-phycocyanin (55% identical amino-acid residues). The phycocyanin-645 alpha-subunits represent a special type of phycobiliprotein and a direct relationship to other phycobiliproteins or any light-harvesting polypeptide-pigment complexes could not be derived by sequence comparisons.     

Tissue distribution of beta 1- and beta 2-subunits of regulatory guanine nucleotide-binding proteins

The beta-subunit of G-proteins occurs in two forms (beta 1 and beta 2), which differ in their primary structure as derived from cDNA clones and in their mobilities on SDS gels (36 and 35 kDa, respectively). To assess the tissue distribution of the two forms of beta-subunits, we synthesized peptides corresponding to defined regions of beta 1- and beta 2-subunits and injected them into rabbits; the antisera obtained reacted either with both beta-subunits or specifically with the beta 1- or the beta 2-subunit. They were used to identify the two beta-subunits in membranes prepared from various rat tissues and from human placenta. The concentration of total beta-subunits was high in rat brain and lung, human placenta, rat kidney, liver and spleen; it was much lower in rat erythrocytes, cardiac and skeletal muscle. In all tissues studied, both beta 1- and beta 2-subunits were detectable. In most tested tissues, the two forms were about equally distributed, whereas in the placenta, the beta 2-subunit was found to occur in approx. 2-fold excess over the beta 1-subunit. Our results demonstrate that both beta-subunits are widely distributed. In the majority of tissues, levels of beta 2-subunits are very similar to those of beta 1-subunits. Thus, the abundance of beta 2-subunits as compared to that of the beta 1-subunit is considerably higher than was previously estimated by measuring the respective mRNA levels.     

BK channel beta1-subunit regulation of calcium handling and constriction in tracheal smooth muscle

The large-conductance, Ca2+-activated K+ (BK) channels are regulators of voltage-dependent Ca2+ entry in many cell types. The BK channel accessory beta1-subunit promotes channel activation in smooth muscle and is required for proper tone in the vasculature and bladder. However, although BK channels have also been implicated in airway smooth muscle function, their regulation by the beta1-subunit has not been investigated. Utilizing the gene-targeted mice for the beta1-subunit gene, we have investigated the role of the beta1-subunit in tracheal smooth muscle. In mice with the beta1-subunit-knockout allele, BK channel activity was significantly reduced in excised tracheal smooth muscle patches and spontaneous BK currents were reduced in whole tracheal smooth muscle cells. Knockout of the beta1-subunit resulted in an increase in resting Ca2+ levels and an increase in the sustained component of Ca2+ influx after cholinergic signaling. Tracheal constriction studies demonstrate that the level of constriction is the same with knockout of the beta1-subunit and BK channel block with paxillin, indicating that BK channels contribute little to airway relaxation in the absence of the beta1-subunit. Utilizing nifedipine, we found that the increased constriction caused by knockout of the beta1-subunit could be accounted for by an increased recruitment of L-type voltage-dependent Ca2+ channels. These results indicate that the beta1-subunit is required in airway smooth muscle for control of voltage-dependent Ca2+ influx during rest and after cholinergic signaling in BK channels.     

Effects of amino acid substitutions at beta 131 on the structure and properties of hemoglobin: evidence for communication between alpha 1 beta 1- and alpha 1 beta 2-subunit interfaces

Substitutions of Asn, Glu, and Leu for Gln at the beta131 position of the hemoglobin molecule result in recombinant hemoglobins (rHbs) with moderately lowered oxygen affinity and high cooperativity compared to human normal adult hemoglobin (Hb A). The mutation site affects the hydrogen bonds present at the alpha(1)beta(1)-subunit interface between alpha103His and beta131Gln as well as that between alpha122His and beta35Tyr. NMR spectroscopy shows that the hydrogen bonds are indeed perturbed; in the case of rHb (beta131Gln --> Asn) and rHb (beta131Gln --> Leu), the perturbations are propagated to the other alpha(1)beta(1)-interface H-bond involving alpha122His and beta35Tyr. Proton exchange measurements also detect faster exchange rates for both alpha(1)beta(1)-interface histidine side chains of the mutant rHbs in 0.1 M sodium phosphate buffer at pH 7.0 than for those of Hb A under the same conditions. In addition, the same measurements in 0.1 M Tris buffer at pH 7.0 show a much slower exchange rate for mutant rHbs and Hb A. One of the mutants, rHb (beta131Gln --> Asn), shows the conformational exchange of its interface histidines, and exchange rate measurements have been attempted. We have also conducted studies on the reactivity of the SH group of beta93Cys (a residue located in the region of the alpha(1)beta(2)-subunit interface) toward p-mercuribenzoate, and our results show that low-oxygen-affinity rHbs have a more reactive beta93Cys than Hb A in the CO form. Our results indicate that there is communication between the alpha(1)beta(1)- and alpha(1)beta(2)-subunit interfaces, and a possible communication pathway for the cooperative oxygenation of Hb A that allows the alpha(1)beta(1)-subunit interface to modulate the functional properties in conjunction with the alpha(1)beta(2) interface is proposed.     

Folding of active calcium channel beta(1b) -subunit by size-exclusion chromatography and its role on channel function

Voltage-gated calcium channels mediate the influx of Ca(2+) ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming alpha(1)-subunit and the auxiliary beta- and alpha(2)delta-subunits. The beta-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four beta-subunit genes have been cloned, beta(1-4), with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The beta(1b)-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming alpha(1)- and the regulatory beta-subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the beta(1b)-subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The beta(1b)-subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the alpha(1)-subunit (the alpha-interaction domain). Using the cut-open oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac alpha(1)-subunit (alpha(1C)) co-injected with folded-beta(1b)-protein or beta(1b)-cRNA. We demonstrate that the co-expression of the alpha(1C)-subunit with either folded-beta(1b)-protein or beta(1b)-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the beta(1b)-subunit primarily modulates channel activity, rather than expression.     

A newly characterized HLA-DP beta-chain allele. Evidence for DP beta heterogeneity within the DPw4 specificity

cDNA clones corresponding to the DPw4 alpha- and DPw4 beta-chains were isolated from a cDNA library prepared from a DPw4 homozygous cell line, their nucleotide sequences were determined, and the corresponding amino acid sequences were deduced. This DPw4 alpha-chain is identical to the conserved DP alpha-chains from DPw4 and DPw2 haplotypes, although the DPw4 beta-chain (referred to as DPw4b beta) differs from all reported DP beta-chain sequences. The DPw4b beta-chain differs from the reported DPw4 beta sequence (referred to as DPw4a beta) at three amino acid positions in the first domain (36, 55, and 56). The DPw4b beta-chain sequence differs from the DPw2 beta-chain sequence only at position 69 in the first domain, suggesting that the lysine at position 69 in DPw4b beta and the glutamic acid at position 69 in DPw2 beta contribute to the epitopes that define "DPw4-ness" and "DPw2-ness," respectively. In addition, the patterns of sequence identities and differences among the DPw4b beta-, DPw4a beta-, DPw2 beta-, and DPw3 beta-chains suggest that the DPw4b beta sequence arose via a gene conversion event or a point mutation. The I-LR1 mAb, which was previously found to bind only to DPw2, DPw3, and DR5 molecules, binds to an L cell transfectant expressing the DPw4 alpha:DPw4b beta molecule. The DPw4b beta sequence provides the first evidence for structural heterogeneity within the DPw4 specificity.     

Downregulation of the voltage-dependent calcium channel (VDCC) beta-subunit mRNAs in pancreatic islets of type 2 diabetic rats

The present study was undertaken to determine whether altered expression of the VDCC beta-subunits in pancreatic beta-cells could play a role in the changes in beta-cell sensitivity to glucose that occur with diabetes. Application of competitive RT-PCR procedure revealed that in normal Wistar rats, LETO and prediabetic OLETF rats, the beta(2)-subunit mRNA levels were 60-200-fold greater than the levels for the beta(3)-subunit. These findings suggest that the beta(2)-subunit as well as the beta-cell type VDCC1 alpha(1)-subunit may be the predominant form of the VDCC expressed in pancreatic beta-cells. The levels of mRNA encoding the beta-subunits and the beta-cell type alpha(1)-subunit as well as insulin were significantly reduced in diabetic rats. Perfusion experiments revealed that diabetic rats showed the higher basal insulin secretion and profoundly impaired insulin secretory responses to glucose compared with non-diabetic rats. Alternatively, impaired insulin secretory responses to glucose in high dose glucose-infused rats were recovered partly with the elevation of mRNA levels of the VDCC beta(2)- and beta(3)-subunits as well as the alpha(1)-subunit by the treatment with diazoxide. Thus, considering the possibility that the most striking effect of the VDCC alpha(1) beta-subunit coexpression in pancreatic beta-cells might occur on activation kinetics like the skeletal muscle, the impairment of further activation of the VDCCs to acute glucose challenge caused by the reduced expressions of the alpha(1) beta-subunits mRNAs in type 2 diabetic animals might be at least partly associated with the alterations in beta-cell sensitivity to glucose.     

The amino acid sequence of the β-subunit: Of porcine enterotoxigenic Escherichia coli enterotoxin — Analysis and comparison with literature data

Abstract The amino acid composition and sequence of the β-subunit of heat-labile enterotoxin (LT) purified from a porcine (LT_p) strain, WT-1, of enterotoxigenic Escherichia coli was analysed, and the result was compared with that reported by Dallas and Falkow [Nature 288 (1980) 499-501] who deduced the amino acid sequence of LT_p from data on the DNA sequence of a porcine strain, EWD299. The purified β-subunit of the LT_p of WT-1 was carboxymethylated, succinylated, digested with chymotrypsin and subjected to high performance liquid chromatography (HPLC). The amino acid composition of the peptide peaks from the column were analysed and compared with the data reported by Dallas and Falkow. Only one fraction differed in amino acid composition from that reported, containing lysine instead of methionine. This fraction was found to consist of two peptides with the sequences Lys-Ser-Gly-Glu-Thr-Phe and Arg-Ile-Thr-Tyr. The former peptide is reported to have the sequence Met-Ser-Gly-Glu-Thr-Phe. Thus, the amino acid at position 43 from the N terminus of the β-subunit of LT_p is lysine, not methionine as reported. This is the first report which studied the amino acid sequence of LT_p analysed by protein toxin itself, not by DNA sequence analysis.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

Phylogenetic relationships ofBacteria based on comparative sequence analysis of elongation factor Tu and ATP-synthase β-subunit genes

Comparative sequence analyses were performed on 14 genes encoding bacterial elongation factors EF-Tu and 7 genes encoding the -subunit of bacterial F₁F₀ type ATP-synthases. The corresponding predicted amino acid sequences were compared with published primary structures of homologous molecules. Phylogenetic trees were reconstructed from both data sets of aligned protein sequences and from an equivalent selection of 16S rRNA sequences by applying distance matrix and maximum parsimony methods. The EF-Tu data were in very good agreement with the rRNA data, although the resolution within the EF-Tu tree was reduced at certain phylogenetic levels. The resolution power of the ATPase -subunit sequence data were more reduced than those of the EF-Tu data. In comparison with the 16S rRNA tree there are minor differences in the order of adjacent branchings within the ATPase -subunit tree.     

Clathrin-independent endocytosis of GABA(A) receptors in HEK 293 cells.

The endocytosis of GABA(A) receptors was investigated in HEK 293 cells expressing receptor alpha1beta2- and alpha1beta2gamma2-subunit combinations. For assessment of internalized receptors by radioimmunoassay or immunofluorescence, a triple c-myc epitope was introduced into the amino terminus of the beta2 subunit. An assay based on biotin inaccessibility was used for alpha1 subunits. GABA(A) alpha1beta2- and alpha1beta2gamma2-subunit receptors were internalized with a t(1/2) of 5.5 min at 37 degrees C. With both subunit combinations, phorbol 12-myristate 3-acetate enhanced internalization by nearly 100%. Treatment of the cells with hypertonic sucrose prevented both the basal and phorbol ester-induced endocytosis of GABA(A) receptors. GF 109203X, an inhibitor of protein kinase C, blocked the stimulation by phorbol ester but had no detectable effect on basal receptor endocytosis. Coexpression with a dominant-negative mutant of dynamin (K44A) led to a 100% enhancement of GABA(A) receptor internalization, while the endocytosis of beta(2)-adrenergic receptors was completely prevented. The results indicate that the endocytosis of GABA(A) alpha1beta2-subunit receptors in HEK cells is constitutive, positively modulated by activation of protein kinase C, and occurs by a mechanism that requires neither the participation of a GABA(A) receptor gamma2 subunit nor a clathrin-mediated pathway.