Amino Acid Sequence of Natural Tumor Necrosis Factor α Inhibitor Purified from Human Urine

Tumor necrosis factor α inhibitor (TNF–INH) was purified from human urine and it was composed of 161 amino acid residues. The complete amino acid sequence of TNF–INH found by sequence analysis agreed with that predicted from the cDNA structure for the extracellular domain (1–161 portion) of 55-kDa TNF receptor and its processing site at the C-terminal was Asn-161.     

Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (K _i) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride K _i by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn²⁺ ions, in particular the αS583C mutant showing a K _i for Zn²⁺of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride K _i, the later mutation generating a Ca²⁺blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na⁺ions at an external Na⁺binding site preventing ion permeation through the channel pore.     

Amino Acid Sequence of αkSubstance,a Mating Pheromone of Saccharomyces kluyveri

A fraction containing IgA (IgA-rich fraction) was prepared from bovine colostrum by anion exchange chromatography using DEAE-Sephadex A-50 and gel filtration on Sephadex G-200. A large amount of IgG1-dimer was found in this fraction, which could not be separated from IgA by repeated gel filtration.

The Fc fragment of bovine colostral IgG (IgG-Fc) was prepared from papain digestion mixtures. IgG-Fc was found to be heterogeneous on DEAE-cellulose column chromatography. Two IgG-Fc fractions were obtained, but no antigenic difference was found between them. Anti-IgG-Fc antibodies raised in rabbits by injection of these Fc preparations reacted only with IgG1 and IgG2. An immunoadsorbent (anti-IgG-Fc-Sepharose) was prepared by coupling these anti-IgG-Fc antibodies to CNBr-activated Sepharose 4B.

IgA was purified from the IgA-rich fraction by affinity chromatography on anti-IgG-Fc-Sepharose adsorbent. IgG1-dimer was effectively removed by this treatment. The purified sample gave only one precipitin arc characteristic of IgA on immunoelectrophoresis with multiple anti-bovine colostral whey antiserum. A small amount of IgA was found to be adsorbed to the affinity column nonspecifically.

When a rabbit was immunized with the purified IgA, besides anti-IgA antibodies, antibodies against the secretory component (SC) were found in the antiserum. This finding leads us to expect that the purified IgA is secretory IgA containing SC.     

Unique Combinations of βαβ-Units and Π-Like Modules in Proteins and Specific Features of Their Amino Acid Sequences

Possible combinations of βαβ-units and Π-like modules in proteins in both right- and left-handed forms have been analyzed in detail. The correlation between the mutual arrangement of the structural elements in the polypeptide chain and their handedness has been shown. In the βαβΠ combinations, which is encountered most frequently in proteins, the Π-module follows the βαβ unit along the chain and both elements are right-handed. In the Πβαβ combinations, where the Π-module is located at the N end and the βαβ-unit follows it, the former is left-handed and the latter is right-handed. In relatively rare combinations of the left-handed βαβ-units and right-handed Π-modules, the βαβ-unit follows Π-module in the chain. The combinations of left-handed Π-modules and the left-handed βαβ-units are unobservable in proteins. It has also been shown that the Π-modules with a β-strand—α-helix—arch—β-strand structure are observed in proteins only in a right-handed form and half of them (51%) contains cis-prolines in their arches. These arches of nonhomologous proteins, as well as the positions of cis-prolines, nearly coincide when superimposed. The superimposed Π-modules also demonstrate that their overall folds are very similar. Structural alignment of their amino acid sequences has shown that the Π-modules have very similar sequence patterns of the key hydrophobic, hydrophilic, glycine, and cis-proline residues.     

Identification and Characterisation of the α and β Subunits of Succinyl CoA Ligase of Tomato

Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding its constituent enzymes have been functionally identified. In yeast, the heterodimeric protein succinyl CoA ligase is encoded for by two single-copy genes. Here we report the isolation of two tomato cDNAs coding for α- and one coding for the β-subunit of succinyl CoA ligase. These three cDNAs were used to complement the respective Saccharomyces cerevisiae mutants deficient in the α- and β-subunit, demonstrating that they encode functionally active polypeptides. The genes encoding for the subunits were expressed in all tissues, but most strongly in floral and leaf tissues, with equivalent expression of the two α-subunit genes being expressed to equivalent levels in all tissues. In all instances GFP fusion expression studies confirmed an expected mitochondrial location of the proteins encoded. Following the development of a novel assay to measure succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximal catalytic activities of the enzyme in a range of tissues revealed that these paralleled those of mRNA levels. We also utilized this assay to perform a preliminary characterisation of the regulatory properties of the enzyme suggesting allosteric control of this enzyme which may regulate flux through the TCA cycle in a manner consistent with its position therein.     

Features of Anabaena PCC 7120ΔHUP Mutants with Amino Acid Substitutions in Nitrogenase

Russian Journal of Plant Physiology - Mutant strains of the filamentous cyanobacterium Anabaena strain PCC 7120 ΔHup (dc-Q193S and dc-R284H) with amino acid substitutions located in the...     

Amino Acid Sequence of Kalinowaski's Tinamou (Nothoprocta Kalinowskii) Hemoglobin and the Rate of Evolution of Bird αD-Globin

Two hemoglobin components are recognized in erythrocytes of the adult Tinamou. We determined the amino acid sequences of Tinamou α^D-, α^A-, and β-globins from intact globin chains and several chemically cleaved fragments. A remarkable feature of Tinamou hemoglobin was a deletion in the α^D-globin chain. This has not been reported in the literature, except in pigeon embryonic α^D-globin. The amino acid sequences of Tinamou globin were highly similar to those of Ostrich and Rhea hemoglobin. Comparison between Tinamou, Ostrich, and Rhea that suggested the evolution speed of globin, α^D = α^A > β, was related with the early appearance birds. The important residues in Tinamou hemoglobin as the heme contact and oxygen binding regions were highly conserved in other species.     

α-Aminoisobutyric Acid as the Constituent Amino Acid of Protein

α-Aminoisobutyric acid is the only tertiary amino acid which is reported to occur in the proteins. Nevertheless, this amino acid has not been yet isolated from the proteins. Recently we succeeded in isolating this amino acid as white prismy crystalline substance from both acid and pepsin hydrolysate of horse hind leg muscle proteins, and this crystal was identified to be α-amino-isobutyric acid by elementary analysis, properties of this derivates, etc.     

The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase

The crystal structure of mitochondrial F₁-ATPase indicatesthat the and subunits fold into a structure defined by threedomains: the top -barrel domain, the middle nucleotide-binding domain,and the C-terminal -helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The -barrel domains of the and subunits form a crown structure at the top ofF₁, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the -barrel domain in the and subunits of the yeast Saccharomyces cerevisiae F₁,with regard to its folding and assembly, was investigated. The -barreldomains of yeast F₁ and subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the -barrel domain of the subunit formed a largeaggregate structure, while the domain of the subunit waspredominately a monomer or dimer. However, coexpression of the -barreldomain of subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the -barrel domain of the and subunits interact specifically with each other and thatthese interactions prevent the aggregation of the -barrel domain of the subunit. These results mimic in vivo results and suggest thatthe interactions of the -barrel domains may be critical during thefolding and assembly of F₁.     

Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel

The L-type Ca²⁺ channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α_1s or Ca_V1.1, α₂, β_1a, δ1, and γ), we created transgenic mice expressing a recombinant β_1a subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α₂-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca_V1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the β subunit in a single docking possibility that defines the α1-β interaction. The β subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α_1s subunit to the membrane and its suggested role in excitation-contraction coupling.     

Complete Amino Acid Sequence of Crystalline (α–Glucosidase from Aspergillus niger

Rhodanine (1), rhodanine-3-acetic acid (2), methylrhodanine (3), and aminorhodanine (4) inhibited the growth of plants. Among them, 4 was the strongest inhibitor of the roots of all the tested plants. On the other hand, N-acetylaminorhodanine (5) and N-benzoylaminorhodanine (6) greatly decreased the inhibitory activity. The results suggest that the free amino group at N-3 of 4 is essential to the greater inhibitory activity of rhodanine derivatives. The plant-growth inhibition of 1–6 is related to the chlorophyll content of the plant treated with them and their acute toxicities in mice.     

Isolation of α and β Brain Tubulin Subunits after Alkaline Treatment of the Protein

We demonstrate here that brain purified tubulin can be dissociated into and subunits at pH > 10 and that the subunits can be separated by using the Triton X-114 phase separation system. After phase partition at pH > 10, tubulin but not tubulin behaves as a hydrophobic compound appearing in the detergent rich phase. After three extractions of the alkaline aqueous phase with Triton X-114, about 90% of the tubulin was recovered in the detergent rich phase. The hydrophobic behavior observed for tubulin after its dissociation at pH 11.5 was not due to an irreversible change of the protein, because when the detergent rich phase containing tubulin was diluted with a buffer solution at pH 7.3 and the solution allowed to partition again, -tubulin is recovered in the aqueous phase. The detergent in the aqueous phase of the and tubulin preparations can be removed up to 90% by 12 h dialysis. The and subunits of tubulin from kidney and liver behave, in this phase separation system, like those of brain tubulin.     

Do Pheromone Binding Proteins Converge in Amino Acid Sequence When Pheromones Converge?

Convergence in amino acid sequences between proteins can be strong evidence for selection. Here, I look for evidence of convergence in the amino acid sequences of pheromone binding protein (PBP) in response to convergence in pheromones. PBPs are involved in sex pheromone reception by the antennae of male moths. In this role PBPs may selectively bind pheromone components and experience convergent selection in response to convergence in pheromone components. However, examination of the PBPs of the taxa that have converged upon the use of (E)- or (Z)-11-tetradecenyl acetate as their major pheromone component reveals little evidence for convergence in the PBPs identified from these taxa. A few sites show a pattern consistent with convergence or parallelism; however, it cannot be ruled out that these sites share the ancestral state. Two of these sites fall within the proposed binding region of PBPs. These results suggest that PBPs either have not converged in sequence or have converged at very few sites in response to convergence on the same pheromone component. Received: 29 July 1999 / Accepted: 8 November 1999     

Evolution of Phosphagen Kinase VII. Isolation of Glycocyamine Kinase from the Polychaete Neanthes diversicolor and the cDNA-Derived Amino Acid Sequences of α and β Chains

Glycocyamine kinase (GK) was isolated from the marine polychaete Neanthes diversicolor by gel filtration, DEAE-cellulose chromatography, butyl-Toyopearl hydrophobic chromatography, and chromatofocusing. The GK was eluted as a single peak on the latter three chromatographies, and the molecular mass for the native GK was estimated to be about 80 kDa. The SDS–PAGE showed that the isolated GK consists of two distinct subunits in equal proportion, α and β chains, with molecular masses of 42.2 and 43.8 kDa, respectively. The present results suggest that the Neanthes GK has a heterodimeric structure. The cDNAs for α and β chains of Neanthes GK were amplified by PCR and their cDNA-derived amino acid sequences were determined. The α and β chains are composed of 374 and 390 amino acids, and the molecular masses were calculated to be 42,392 and 43,966 Da, respectively, in good agreement with the apparent masses on SDS–PAGE. The β chain has a characteristic N-terminal extension of 15 amino acids, and all of the sequence differences between α and β chains were restricted in the N-terminal region of 50 residues. The overall sequence identity was 92%. The occurrence of heterodimeric nature in Neanthes GK is of great interest from the evolutionary point of view, because the heterodimeric structure is only known for creatine kinase MB-isozyme specific for mammalian heart muscle among phosphagen kinases.     

Improved Large-Scale Purification of Transducin,and Its α and βγ Subunits from Frozen Retinas

Abstract

The transducin heterotrimer and its α- and βγ-subunits have been purified from frozen bovine rod outer segments by modifying existing procedures. The methods described here are relatively simple and fast. The yield (ca. 8 mgs/100 retinas) and purity of the transducin heterotrimer and subunits from frozen retinas is equal to or larger than those previously obtained from fresh or frozen retinas.     

Na,K-ATPase and the role of α isoforms in behavior

The Na,K-ATPase is composed of multiple isoforms and the isoform distribution varies with the tissue and during development. The α1 isoform for example, is the major isoform in the kidney and many other tissues, while the α2 isoform is the predominate one in skeletal muscle. All three isoforms are found in the brain although in adult rodent brain, the α3 isoform is located essentially in neurons while the α2 isoform is found in astrocytes and some limited neuronal populations. Interestingly the α4 isoform is found exclusively in the mid region of the sperm tail. The distribution of the isoforms of the Na,K-ATPase has been extensively studied in many tissues and during development. The examples cited above provide some indication to the diversity of Na,K-ATPase isoform expression. In order to understand the significance of this distribution, we have developed animals which lack the α1, α2, and α3 isoforms. It is anticipated that these studies will provide insight into the role that these isoforms play in driving various biological processes in specific tissues. Here we describe some of our studies which deal with the behavioral aspects of the α1, α2, and α3 deficient mice, particularly those that are haploinsufficient in one isoform i.e. lacking one functional gene for the α1, α2, or α3 isoforms. Such studies are important as two human diseases are associated with deficiency in the α2 and α3 isoforms. These are Familial Hemiplegic Migraine type 2 and Rapid-Onset Dystonia Parkinsonism, these diseases result from α2 and α3 isoform haploinsufficiency, respectively. We find that the haploinsufficiency of both α2 and α3 isoforms result in behavioral defects.     

Formation of N-Carboxymethyl Amino Acid from the Reaction of α-Amino Acid with Glyoxal

Enrichment cultures in a medium containing 0.1% methanol and 0.1% bicarbonate at pH 7.0 under anaerobic conditions in the light became mainly green in color. Forty-four enrichment cultures, which showed abundant growth, were obtained from 46 different sources and found to contain cells of methanol-utilizing bacteria and green algae as predominant members. From these enrichment cultures, two strains of bacteria and two strains of algae were isolated. The microorganisms isolated were designated as bacterium No. 7, bacterium No. 8, Chlorella sp. A-1 and Chlorella sp. B-1, respectively. Stable mixed cultures were easily formed by mixing the isolated cultures of bacteria and algae. Both methanol and bicarbonate were necessary for the growth of the mixed cultures under anaerobic-light conditions. Growth behavior of the mixed cultures was examined on a medium containing 0.1% methanol and 0.1 % bicarbonate at 30°C in the light (about 6000 lx). The maximum specific growth rate for the cultures, µ_max, was 0.092 hr^?1 (doubling time, 7.5 hr). The maximum cell yield was 0.87 g dry-cell weight per g of methanol used. The protein content of the biomass was 65%.     

Amino Acid Sequence of Tremerogen A–10, a Peptidal Hormone,Inducing Conjugation Tube Formation in Tremella mesenterica Fr.

κ-Casein components having various carbohydrate contents were prepared by diethylaminoethyl-cellulose chromatography and the interactions of each κ-casein component both with α_s1-casein and with β-casein were examined by Sepharose 4B gel chromatography, ultra-centrifugal experiments and viscosity measurements. Each κ-casein component could form complex with α_s1- and β-casein in the absence and presence of CaCl₂. Molecular weight of complexes of unfractionated κ-casein both with α_s1-casein and with κ-casein were about 70 × 10⁴ at 37°C in the absence of CaCl₂, while those of complexes of each κ-casein component with α_s1 and β-casein were about 50 × 10⁴. Stokes radii of complexes increased with increasing calcium ion. While sedimentation coefficient at 37°C of complex with β-casein had almost the same value, those of complexes with α_s1-casein decreased with increase of carbohydrate content of κ-casein components. Intrinsic viscosity of complex of κ-casein component having much carbohydrate was almost the same among tested temperatures. It is suggested that heterogeneity of κ-casein is necessary to form large complex and that the carbohydrate moiety of κ-casein contributes the stability of casein complex.     

Mutations in Elongation Factor Ef-1α Affect the Frequency of Frameshifting and Amino Acid Misincorporation in Saccharomyces Cerevisiae

A mutational analysis of the eukaryotic elongation factor EF-1 alpha indicates that this protein functions to limit the frequency of errors during genetic code translation. We found that both amino acid misincorporation and reading frame errors are controlled by EF-1 alpha. In order to examine the function of this protein, the TEF2 gene, which encodes EF-1 alpha in Saccharomyces cerevisiae, was mutagenized in vitro with hydroxylamine. Sixteen independent TEF2 alleles were isolated by their ability to suppress frameshift mutations. DNA sequence analysis identified eight different sites in the EF-1 alpha protein that elevate the frequency of mistranslation when mutated. These sites are located in two different regions of the protein. Amino acid substitutions located in or near the GTP-binding and hydrolysis domain of the protein cause suppression of frameshift and nonsense mutations. These mutations may effect mistranslation by altering the binding or hydrolysis of GTP. Amino acid substitutions located adjacent to a putative aminoacyl-tRNA binding region also suppress frameshift and nonsense mutations. These mutations may alter the binding of aminoacyl-tRNA by EF-1 alpha. The identification of frameshift and nonsense suppressor mutations in EF-1 alpha indicates a role for this protein in limiting amino acid misincorporation and reading frame errors. We suggest that these types of errors are controlled by a common mechanism or closely related mechanisms.