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PubMed2001

Delta 3,5,delta 2,4-dienoyl-CoA isomerase is a multifunctional isomerase. A structural and mechanistic study.

Delta(3,5),Delta(2,4)-Dienoyl-CoA isomerase (DI), an auxiliary enzyme of unsaturated fatty acid beta-oxidation, was purified from rat mitochondria and peroxisomes and subjected to N-terminal sequencing to facilitate a mechanistic study of this enzyme. The mature mitochondrial DI from rat heart was lacking its 34 N-terminal amino acid residues that have the properties of a mitochondrial targeting sequence. The peroxisomal isomerase was identified as a product of the same gene with a truncated and ragged N terminus. Expression of the cDNA coding for the mature mitochondrial DI in Escherichia coli yielded an enzyme preparation that was as active as the native DI. Because the recombinant DI also exhibited Delta(3,5,7),Delta(2,4,6)-trienoyl-CoA isomerase (TI) activity, both isomerases reside on the same protein. Mutations of any of the 3 acidic amino acid residues located at the active site (Modis, Y., Filppula, S. A., Novikov, D. K., Norledge, B., Hiltunen, J. K., and Wierenga, R. K. (1998) Structure 6, 957-970) caused activity losses. In contrast to only a 10-fold decrease in activity upon replacement of Asp(176) by Ala, substitutions of Asp(204) by Asn and of Glu(196) by Gln resulted in 10(5)-fold lower activities. Such activity losses are consistent with the direct involvement of these latter two residues in the proposed proton transfers at carbons 2 and 6 or 8 of the substrates. Probing of the wild-type and mutants forms of the enzyme with 2,5-octadienoyl-CoA as substrate revealed low Delta(2),Delta(3)-enoyl-CoA isomerase and Delta(5),Delta(4)-enoyl-CoA isomerase activities catalyzed by Glu(196) and Asp(204), respectively. Altogether, these data reveal that positional isomerizations of the diene and triene are facilitated by simultaneous proton transfers involving Glu(196) and Asp(204), whereas each residue alone can catalyze, albeit less efficiently, a monoene isomerization.

Acyl Coenzyme A↗
PubMed1987

Molecular biology of the alpha-L-fucosidase gene and fucosidosis.

Human alpha-L-fucosidase, a lysosomal enzyme, hydrolyzes alpha-L-fucose from glycolipids and glycoproteins. Its activity is deficient in human fucosidosis an autosomal recessive disease. In order to understand the molecular basis of this lysosomal storage disorder we have cloned several cDNAs coding for human alpha-L-fucosidase from a human hepatoma and a human liver cDNA library constructed in lambda gt11. Compiling the cDNA sequences of these clones we have identified 1,829 base pairs (bp) encoding human alpha-L-fucosidase. This includes an open reading frame of 1,172 bp, a consensus polyadenylation signal AAT AAA and a poly(A)+ tail. The sequence is incomplete at the 5'-end, and clones encoding the amino terminus of the native protein, the propeptide and leader signal have not yet been isolated. The open reading frame encodes for 390 amino acids with a calculated Mr of 45,557. This represents 78-95% of the mature processed alpha-L-fucosidase. The availability of these cDNA clones has enabled us to map the structural gene for alpha-L-fucosidase to chromosome 1p34.1-1p36.1 by Southern blot analysis of DNA from human-rodent somatic cell hybrids and by in situ hybridization. Furthermore, a Pvu II restriction fragment length polymorphism (RFLP) has been identified at the human alpha-L-fucosidase gene locus. Analysis of mRNA by Northern blotting gives a major species of 2.25 kb. In 4 patients with fucosidosis no mRNA signal was detected and Western blots gave no immunoreactive enzyme. Southern blotting after Eco RI digestion in two fucosidosis families revealed a banding abnormality (extra 6-kb band).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

An isolated class II aminoacyl-tRNA synthetase insertion domain is functional in amino acid editing.

Aminoacyl-tRNA synthetases are responsible for activating specific amino acids and transferring them onto cognate tRNA molecules. Due to the similarity in many amino acid side chains, certain synthetases misactivate non-cognate amino acids to an extent that would be detrimental to protein synthesis if left uncorrected. To ensure accurate translation of the genetic code, some synthetases therefore utilize editing mechanisms to hydrolyze non-cognate products. Previously class II Escherichia coli proline-tRNA synthetase (ProRS) was shown to exhibit pre- and post-transfer editing activity, hydrolyzing a misactivated alanine-adenylate (Ala-AMP) and a mischarged Ala-tRNAPro variant, respectively. Residues critical for the editing activity (Asp-350 and Lys-279) are found in a novel insertion domain (INS) positioned between motifs 2 and 3 of the class defining aminoacylation active site. In this work, we present further evidence that INS is responsible for editing in ProRS. We deleted the INS from wild-type E. coli ProRS to yield DeltaINS-ProRS. While DeltaINS-ProRS was still capable of misactivating alanine, the truncated construct was defective in hydrolyzing non-cognate Ala-AMP. When the INS domain was cloned and expressed as an independent protein, it was capable of deacylating a mischarged Ala-microhelixPro variant. Similar to full-length ProRS, post-transfer editing was abolished in a K279A mutant INS. We also show that YbaK, a protein of unknown function from Haemophilus influenzae with high sequence homology to the prokaryotic INS domain, was capable of deacylating Ala-tRNAPro and Ala-microhelixPro variants but not cognate Pro-tRNAPro. Thus, we demonstrate for the first time that an independently folded class II synthetase editing domain and a previously identified homolog can catalyze a hydrolytic editing reaction.

Alanine↗
PubMed1996

An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8.

The aspartate aminotransferase gene (AspAT, EC 2.6.1.1) of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced, and its gene product was overproduced. The purified T. thermophilus AspAT was stable up to about 80 degrees C at neutral pH. T. thermophilus AspAT was strictly specific for acidic amino acid substrates, such as aspartate, glutamate, and the respective keto acids. The gene coding for T. thermophilus AspAT showed that it comprised 1,155 bp with a high G+C content (70 mol%), and encoded a 385-residue protein with a molecular weight of 42,050. The amino acid sequence of T. thermophilus AspAT deduced from its gene showed about 15, 46, and 29% homology with those from Escherichia coli, Bacillus sp. YM-2, and Sulfolobus solfataricus, respectively. When the amino acid sequence of T. thermophilus AspAT was compared with that of E. coli AspAT, the number of Cys was found to have decreased from 5 to 1, that of Asn from 23 to 9, that of Gln from 16 to 8, and that of Asp from 20 to 13, all of which are known to be relatively labile at high temperatures. Conversely, the number of Pro was increased from 15 to 25, Arg from 22 to 32, and Glu 27 to 37. As shown by the E. coli AspAT structure, there was a marked tendency for the extra prolyl residues to be located around the surface of the molecule. This was quite different from that in the case of RecA protein, which shows an increased number of prolyl residues in the interior of its molecule. Different strategies of different proteins as to prolyl contribution to thermostability have been suggested. Despite the high degree of conservation of active-site residues, Arg292 in E. coli AspAT, which interacts with the distal carboxylate of the substrate, was not found in T. thermophilus AspAT. Arg89 may complement the function of Arg292.

Amino Acid Sequence↗
PubMed1998

cDNA sequence and heterologous expression of monomeric spinach pullulanase: multiple isomeric forms arise from the same polypeptide.

The spinach pullulanase gene was cloned and sequenced using peptide sequences of the purified enzyme as a starting point and employing PCR techniques and cDNA library screening. Its open reading frame codes for a protein of 964 amino acids which represents a precursor of the pullulanase. The N-terminal transit peptide consists of 65 amino acids, and the mature protein, comprising 899 amino acids, has a calculated molecular mass of 99kDa. Pullulanase is a member of the alpha-amylase family. In addition to a characteristic catalytic (beta/alpha)8-barrel domain, it contains a domain, F, that is specific for branching and debranching enzymes. Pullulanase cDNA was expressed in Escherichia coli, and the purified protein was compared with the enzyme from spinach leaves. Identity of the two proteins was confirmed in terms of catalytic properties, N-terminal amino acid sequences and molecular masses. The pullulanase produced by E. coli showed the same microheterogeneity as the spinach leaf enzyme: it could be resolved into two substrate-induced forms by electrophoresis in amylopectin-containing polyacrylamide gels, and, in the absence of substrate, into several free forms (charge isomers) by isoelectric focusing or chromatofocusing. Rechromatofocusing of single free forms resulted in the originally observed pattern of molecular forms. However, heterogeneity of the protein disappeared on isoelectric focusing under completely denaturing conditions when only one protein band was observed. Post-translational modifications such as glycosylation and phosphorylation could be excluded as potential explanations for the protein heterogeneity. Therefore the microheterogeneity of spinach leaf pullulanase results from neither genetic variation nor post-translational modifications, but is a property of the single unmodified gene product. The different interconvertible forms of the pullulanase represent protein populations of different tertiary structure of the same polypeptide.

Amino Acid Sequence↗
PubMed1994

Identification of serine-143 as the most likely precursor of dehydroalanine in the active site of histidine ammonia-lyase. A study of the overexpressed enzyme by site-directed mutagenesis.

The gene coding for histidase (histidine ammonia-lyase, HAL, EC 4.3.1.3) was isolated from a lambda-EMBL3 genomic library from Pseudomonas putida nicII and subcloned into the expression vector pT7-7. Transformation of Escherichia coli BL21 (DE3) cells with the recombinant vector led to the expression of catalytically active histidase amounting to 20-30% of the total soluble protein in the crude cell extract. A new rapid and highly efficient isolation procedure is described leading to electrophoretically homogeneous histidase within 1.5 days. Six grams of E. coli BL21 (DE3) cells (wet weight) gives approximately 100 mg of homogeneous histidase with a specific activity of 27 IU/mg. To investigate the possible role of serine as a precursor of dehydroalanine in the active site of histidase, each of the four serines, conserved in all known histidases and phenylalanine ammonia-lyases, was consecutively changed to alanine by site-directed mutagenesis. The resulting mutant genes were subcloned into the expression vector pT7-7 and were assayed for histidase activity. The catalytic activities of the four mutants and of wild-type histidase were compared. The Km and Vmax values of the overexpressed mutants S112A, S393A, and S418A and wild-type histidase did not show any significant differences. Mutant S143A, however, was devoid of catalytic activity (< 0.01%), pointing to the outstanding importance of this serine for the formation of an active enzyme. We conclude that serine-143 is the most probable precursor of the active-site dehydroalanine. The role of serine-143 in the biosynthesis of active histidase is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed2003

Recombinant water-soluble chlorophyll protein from Brassica oleracea var. Botrys binds various chlorophyll derivatives.

A gene coding for water-soluble chlorophyll-binding protein (WSCP) from Brassica oleracea var. Botrys has been used to express the protein, extended by a hexahistidyl tag, in Escherichia coli. The protein has been refolded in vitro to study its pigment binding behavior. Recombinant WSCP was found to bind two chlorophylls (Chls) per tetrameric protein complex but no carotenoids in accordance with previous observations with the native protein [Satoh, H., Nakayama, K., Okada, M. (1998) J. Biol. Chem. 273, 30568-30575]. WSCP binds Chl a, Chl b, bacteriochlorophyll a, and the Zn derivative of Chl a but not pheophytin a, indicating that the central metal ion in Chl is essential for binding. WSCP also binds chlorophyllides a and b and even the more distant Chl precursor Mg-protoporphyrin IX; however, these pigments fail to induce oligomerization of the protein. We conclude that the phytol group in bound Chl plays a role in the formation of tetrameric WSCP complexes. If WSCP in fact binds Chl or its derivative(s) in vivo, the lack of carotenoids in pigmented WSCP raises the question of how photooxidation, mediated by triplet-excited Chl and singlet oxygen, is prohibited. We show by spin-trap electron-paramagnetic resonance that the light-induced singlet-oxygen formation of WSCP-bound Chl is lower by a factor of about 4 than that of unbound Chl. This as-yet-unknown mechanism of WSCP to protect its bound Chl against photooxidation supports the notion that WSCP may function as a transient carrier of Chl or its derivatives.

Brassica↗
PubMed2002

Purification and characterization of two polymorphic variants of short chain acyl-CoA dehydrogenase reveal reduction of catalytic activity and stability of the Gly185Ser enzyme.

Short chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric flavoenzyme that catalyzes the first intramitochondrial step in the beta-oxidation of fatty acids. Two polymorphisms in the coding region of the SCAD gene, 511C>T (R147W) and 625G>A (G185S), have been shown to be associated with an increased level of ethylmalonic acid excretion in urine, a clinical characteristic of SCAD deficiency. To characterize the biochemical consequences of these variations, in vitro site-directed mutagenesis and prokaryotic expression were used to produce the corresponding SCAD variant proteins. Both variant proteins were unstable when produced in Escherichia coli, but could be rescued and subsequently purified by coexpressing them with the bacterial chaperonin GroEL/ES. The k(cat)/K(m) values of the green wild-type, R147W, and G185S SCAD enzymes coexpressed with GroEL/ES were 33, 30, and 10 microM(-)(1) s(-)(1), respectively. There were minimal differences in the kinetic parameters measured for the green, degreened, and wild-type enzymes coexpressed with GroEL/ES, and the R147W variant when butyryl-CoA was used as a substrate. The catalytic efficiency of the G185S variant enzyme, however, was reduced compared to that of the wild-type enzyme. The thermal and guanidine HCl stability of the purified enzymes as determined by fluorescence, far-UV CD spectroscopy, and incubation-induced rest activity showed the following order of relative stability: wild-type enzyme > R147W > G185S. Near-UV CD spectroscopy indicated that these impairments are caused by decreased flexibility in the tertiary conformation of the two mutant enzymes. The common SCAD polymorphisms may lead to clinically relevant alterations in enzyme function.

Acyl-CoA Dehydrogenase↗
PubMed2026

Comparative transcriptome analysis reveals ncRNA-mediated regulatory networks associated with muscle crispiness in grass carp.

Non-coding RNAs (ncRNAs) have been demonstrated to be involved in muscle development and to function as key regulators. However, the molecular mechanism underlying muscle crispiness in grass carp (GC) remains poorly understood, and whether these ncRNAs are involved in its regulation is still unknown. In the current investigation, differentially expressed (DE) RNAs (including lncRNAs, circRNAs, miRNAs, and mRNAs) were identified; concomitantly, target genes prediction was conducted, and functional and signaling pathway enrichment analyses were performed. Pathways related to muscle crispiness were identified, and the competitive endogenous RNA (ceRNA) (lncRNA/circRNA-miRNA-mRNA) regulatory network was further constructed. The results showed that a total of 126 DE-lncRNAs, 17 DE-circRNAs, 329 DE-miRNAs, and 442 DE-mRNAs were identified in muscle tissues of both the GC and crisp grass carp (CGC). GO and KEGG enrichment analyses revealed that target genes of DE-ncRNAs were significantly enriched in signaling pathways, including structural constituents of muscle, apoptosis, oxidative phosphorylation, and regulation of actin cytoskeleton, suggesting that these pathways may be involved in muscle texture remodeling. Subsequently, DE-RNAs enriched in related pathways were identified, and a core ceRNA regulation network comprising 3 lncRNAs, 4 circRNAs, 3 miRNAs, and 17 mRNAs was constructed. Additionally, 10 DE-RNAs from randomly selected groups were validated by qRT-PCR. Our findings not only provide scientific evidence elucidating the molecular mechanisms underlying muscle crispiness in GC but also establish a foundation for studying changes in muscle textural qualities across other fish species.

Animals↗
PubMed1993

Synthesis and conformational studies on peptides corresponding to a putative autophosphorylation site of abl TPK.

The transforming gene of Abelson murine leukaemia virus (v-abl) codes for a membrane-associated tyrosine-specific protein kinase (abl TPK). Analysis of the v-abl gene has shown that both the fibroblast-transforming and tyrosine-protein kinase activities reside within a minimal region encoding a protein of 43 kDa (p43v-abl), which represents the most active, isolated form of this enzyme. Since the cellular substrates for p43v-abl are yet to be identified, we synthesized by classical solution methods the octapeptide H-Gly-Asp-Thr-Tyr-Thr-Ala-His-Ala-OH, corresponding to the structural sequence of the main putative autophosphorylation site (Tyr 515) of the abl TPK, as well as some of its analogs modified in positions -2, -1, +1 and +3. The synthetic peptides were tested as substrates for the p43v-abl. The kinetic data obtained indicate that the rates of their phosphorylation vary considerably depending on the sequence of the peptide, as expected. As a rule, no significant increment of the efficiency results from each substitution in the parent sequence. While the replacement of the two charged residues, namely Asp-2 and His-7, with neutral Ala is well tolerated, the substitution with amino acids bearing opposite charges is detrimental. The correlation between secondary structure of our synthetic octapeptides and their substrate recognition by p43v-abl was studied using CD and fluorescence spectroscopy in 5 mM Tris, in 98% TFE/Tris and in 30 mM SDS solutions. The comparison of the spectroscopic data with the kinetic parameters does not confirm a close relationship between the conformational properties of these peptides and their enzymatic role.

Amino Acid Sequence↗
PubMed2001

Molecular, antigenic, and functional analyses of Omp2b porin size variants of Brucella spp.

Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various Brucella species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of Brucella melitensis reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus Brucella seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of Brucella suis biovar 3), the porin variability does not result in major antigenic variability of the Brucella surface that could help the bacteria during the reinfection of a host. Porin variation in Brucella seems to result mainly in porin conductivity modifications.

Amino Acid Sequence↗
PubMed2003

The protein complex composed of nickel-binding SrnQ and DNA binding motif-bearing SrnR of Streptomyces griseus represses sodF transcription in the presence of nickel.

Nickel-responsive transcriptional repression of sodF, which codes for iron- and zinc-containing superoxide dismutase of Streptomyces griseus, was mediated through an operator (-2 to +15) spanning over the 5' end (+1) of the transcript. Two open reading frames, SrnR (12,343 Da) and SrnQ (12,486 Da), with overlapping stop-start codons were identified downstream from sodF and found responsible for the repression of sodF. The deduced amino acid sequence of SrnR revealed a DNA binding motif and showed homology to the transcriptional regulators of ArsR family, whereas SrnQ did not show any similarity to any known proteins. When srnRQ DNA was maintained in trans in S. griseus on a multicopy plasmid, sodF transcription was highly repressed by nickel, but neither srnR nor srnQ alone showed the effect. Consistently, the sodF transcription of srnR-interrupted mutant was no longer repressed by nickel, which was complemented only with srnRQ DNA. Nickel-dependent binding of SrnR and SrnQ to the sodF operator DNA was observed only when the two proteins were provided together. The maximum protein-DNA interaction was shown when SrnR and SrnQ were present in one-to-one stoichiometric ratio. The two proteins appear to constitute an octamer composed of four subunits of each protein. SrnR directly interacted with SrnQ, and the protein interaction did not require nickel. The conformation of SrnQ was changed upon nickel binding, which was in the ratio of one Ni(2+) ion per protein molecule. A model is proposed in which SrnQ of the protein complex senses nickel and subsequently enhances the DNA binding activity of SrnR through the protein-protein interaction.

Amino Acid Motifs↗
PubMed1994

Complete sequence of the mitochondrial DNA of the chlorophyte alga Prototheca wickerhamii. Gene content and genome organization.

The complete nucleotide sequence of the circular mitochondrial (mt) DNA of the chlorophyte alga Prototheca wickerhamii has been determined (55,328 base-pairs, A+T content 74.2%). The genes identified encode three subunits of the cytochome oxidase, apocytochrome b, nine subunits of the NADH dehydrogenase complex (nad1 to 7, nad4L and nad9), three ATPase subunits (atp6, atp9, atp1 (also referred to as atpA)), three ribosomal RNAs (5 S (rrn5), small subunit (srn) and large subunit (lrn) RNA), 26 tRNAs, and 13 ribosomal proteins. A total of five group I introns reside in lrn and cox1, two of which include intronic open reading frames (ORFs). Five free-standing ORFs longer than 60 codons are present. Three of these ORFs are counterparts to genes encoding proteins of unknown function in plant mitochondria (orf25 and orfB of angiosperms and orf244 of liverwort), whereas two of them are unique. Mitochondrial genes are encoded on both DNA strands in a way that suggests the existence of two transcription units, each including approximately one half of the mitochondrial genome. The two intergenic regions in which transcription is believed to initiate and terminate are about ten times longer than the other intergenic regions (1118 and 1993 nt versus 100 to 150 nt). A total of 29 recurring sequence motifs (30 to 200 nt long) have been found in intergenic regions. Nine different types of motifs are present, most of them arranged as tandem repeats. These motifs may be implicated in transcription, e.g. as signals for initiation, termination and/or processing. Phylogenetic analysis on the basis of the cox1 gene strongly suggested that P. wickerhamii and plant mitochondrial genomes are monophyletic. The finding of plant-specific mitochondrial genes such as orf25, orf244, orfB and rrn5 in P. wickerhamii mitochondria corroborates this idea.

Amino Acid Sequence↗
PubMed1996

Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes.

L-Aromatic amino acid decarboxylase (dopa decarboxylase; DDC) is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L-dopa and other L-aromatic amino acids. To advance structure-function studies with the enzyme, a cDNA that codes for the protein from pig kidney has been cloned by joining a partial cDNA obtained by library screening with a synthetic portion constructed by the annealing and extension of long oligonucleotides. The hybrid cDNA was then expressed in Escherichia coli to produce recombinant protein. During characterization of the recombinant enzyme it was unexpectedly observed that it possesses certain differences from the enzyme purified from pig kidney. Whereas the later protein binds 1 molecule of PLP per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer. Moreover, the Vmax was twice that of the protein purified from tissue. On addition of substrate, the absorbance changes accompanying transaldimination were likewise 2-fold greater in the recombinant enzyme. Examination of the respective apoenzymes by absorbance, CD and fluorescence spectroscopy revealed distinct differences. The recombinant apoprotein has no significant absorbance at 335 nm, unlike the pig kidney apoenzyme; in the latter case this residual absorbance is associated with a positive dichroic signal. When excited at 335 nm the pig kidney apoenzyme has a pronounced emission maximum at 385 nm, in contrast with its recombinant counterpart, which shows a weak broad emission at about 400 nm. However, the holoenzyme-apoenzyme transition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm. Taken together, these findings indicate that recombinant pig kidney DDC has two active-site PLP molecules and therefore displays structural characteristics typical of PLP-dependent homodimeric enzymes. The natural enzyme contains one active-site PLP molecule whereas the remaining PLP binding site is most probably occupied by an inactive covalently bound coenzyme derivative; some speculations are made about its origin. The coenzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm. A putative working model is presented to explain this behaviour.

Amino Acid Sequence↗
PubMed1988

Identification of a transcriptionally active pseudogene in the chorion locus of the silkmoth Bombyx mori. Regional sequence conservation and biological function.

We have determined the primary structure of a 3500 base-pair part of the silkmoth chorion locus mapping in a region containing genes of late developmental specificity. This part of the locus was found to harbour a pseudogene related to one of the families of chorion genes encoding high cysteine proteins, HcB. The pseudogene exhibits an overall sequence identity of 84% to the consensus coding region of HcB chorion genes. A 95% identity was observed over a length of 190 base-pairs of its immediate 5' upstream sequences and the corresponding part of the consensus 5'-intergenic sequences of Hc gene pairs, normally encompassing 270 base-pairs. Thus, the pseudogene has retained part of the promoter region that includes sequence elements whose presence is thought to be necessary for transcriptional competence of HcB genes. The pseudogene is also characterized by the elimination of part of its first exon containing most of the 5' untranslated region, the ATG translation initiation codon and part of the signal peptide sequences. Its intron is longer than that of other HcB genes due to the insertion of a copy of a repetitive element that appears to be transcribed by RNA polymerase III. A previously characterized chorion cDNA clone, m2282, representing a rare mRNA sequence of late developmental specificity, was found to be identical to the pseudogene over its entirety spanning 65% of the pseudogene's second exon. Hybridizations of clones spanning a 260,000 base-pair domain of the chorion locus of Bombyx mori and of total genomic DNA to a subfragment of the cDNA clone containing relatively unique sequences, coupled to primer extension experiments, have demonstrated that m2282 mRNA originated from the pseudogene and that the pseudogene transcripts are initiated at the chorion cap site consensus sequence. We conclude that the 5'-flanking sequences retained by the pseudogene encompass elements necessary and adequate for correct transcriptional activation, but may not include those required for quantitative expression of the promoter. Possible reasons for the observed lack of random drift in the 5'-upstream sequences of the pseudogene and the maintenance of a functional promoter in a non-functional gene are discussed on the basis of the observation that elements resembling scaffold attachment sites are present in these sequences.

Amino Acid Sequence↗
PubMed1996

Functional characterisation of an ovine endometrial oxytocin receptor cDNA transiently expressed in Cos-7 cells.

The entire coding region of an ovine endometrial oxytocin receptor (OTR) cDNA was generated by PCR, subcloned into the SV40 major late promoter expression vector pSVLJ and transiently expressed in Cos-7 cells. A specific OTR antagonist, 125I-labelled d(CH2)5 [Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin (OTA), was used to describe the binding kinetics of the expressed receptor which had a Kd of 4.5 nM and Bmax of 2.4 nM/mg protein (6.8 x 10(5) receptor molecules/transfected cell). The functional properties of the expressed OTR were determined by measuring oxytocin-induced phosphoinositide (PI) hydrolysis. Oxytocin increased PI turnover in OTR transfected cells fourfold in excess of residual endogenous activity, and stimulated phospholipase C (PLC) activity in a dose- and time-dependent manner, confirming that the expressed OTR cDNA was functional. Arginine vasopressin also stimulated PI turnover in a dose-dependent manner; thresholds of responses to oxytocin and arginine vasopressin were 10(-9) M and 10(-7) M respectively. OTA did not increase PI turnover and competitively inhibited the oxytocin-induced response. Direct activation of the pathway by aluminium fluoride and guanosine (3'-O-thio)-triphosphate (GTP gamma S) confirmed that the OTR was G-protein linked. Co-incubation of GTP gamma S with oxytocin shifted the PI-response threshold from 10(-7) M to 10(-9) M and significantly increased the level of response, suggesting that maximum PI turnover was agonist-dependent. The G-protein involved in mediating the signal transduction pathway was pertussis toxin-insensitive and, therefore, probably a member of the Gq subfamily. The PLC inhibitor, U73122, had no effect on oxytocin-induced PI turnover, consistent with the response in endometrial tissue. These data suggest that the signalling pathway mediated by expressed OTR is similar to that attributed to OTR occupancy in ovine endometrium.

Animals↗
PubMed1999

Expression, purification and copper-binding studies of the first metal-binding domain of Menkes protein.

The cDNA, coding for the first metal-binding domain (MBD1) of Menkes protein, was cloned into the T7-system based vector, pCA. The T7 lysozyme-encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg.L-1. Menkes protein, a P-type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N-terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)-MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding. A copper-bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 microM. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo-MBD1. Given the same total copper concentration CuCl43- was superior to Cu(I)-thiourea (structural analogue of metallothionein) and Cu(I)-glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo-MBD1. Cu(II) was not able to reconstitute apo-MBD1, presumably due to Cu(II)-induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.

Adenosine Triphosphatases↗
PubMed2002

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands.

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).

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