Primary Structure of Cytochrome b(5) from Cauliflower (Brassica oleracea L.) Deduced from Peptide and cDNA Sequences

Cytochrome b₅ is a microsomal protein that functions as an intermediate electron donor in fatty acid desaturation and other oxidation/reduction reactions. cDNA clones were isolated from cauliflower (Brassica oleracea L.) by using oligonucleotides based on the partial amino acid sequence of the protein. The deduced amino acid sequence of the polypeptide exhibited approximately 30% sequence identity with the homologous protein from vertebrates.     

Cloning and expression of two autolysin genes, cwlU and cwlV, which are tandemly arranged on the chromosome of Bacillus polymyxa var. colistinus

The cwlV gene, which encodes Bacillus polymyxa var. colistinus autolysin was cloned and sequenced. cwlV comprises a 1497-bp ORF and encodes a polypeptide of 499 amino acid (aa) residues (M_r of 53,707 Da). The N-terminal sequence of the mature 23-kDa CwlV protein is NSXGKKVVVIDAGXGAKD(X, undetermined aa); this processed form corresponds to the C-terminal portion (183?aa, M_r of 20,050?Da) of the cwlV ORF. Sequencing of the flanking region revealed that another putative autolysin gene, cwlU, is located upstream of cwlV. cwlU encodes a polypeptide of 524 aa and its deduced sequence is 34.9% identical to the full-length sequence of CwlV. Downstream of cwlV, the genes for a deduced lipoprotein (OrfW), an endonuclease III homolog (Nth), a non-homologous OrfX, a glutathione peroxidase homolog (Gpx), and the N-terminal region of OrfZ containing a ATP/GTP-binding site motif were found. Northern blotting and primer-extension analyses revealed that cwlU is transcribed as a single cistron, but cwlV is transcribed with orfW. The unprocessed forms of CwlV and CwlU (VΔS and UΔS, respectively) and their predicted mature forms (Vcat and Ucat, respectively) were expressed in, and purified from, Escherichia coli. Enzyme analysis indicated that VΔS and Vcat exhibit low and high cell wall hydrolase activities toward B. polymyxa cell wall, respectively, but UΔS and Ucat exhibit almost no and low cell wall hydrolase activities, respectively.     

Isolation and NH2-terminal sequence of a highly conserved human and porcine pituitary protein belonging to a new superfamily: Immunocytochemical localization in pars distalis and pars nervosa of the pituitary and in the supraoptic nucleus of the hypothalamus

The isolation and purification of a 21,000-Da (pI 4.9) novel protein from porcine anterior pituitary and whole human pituitary is described. Comparison of the NH₂-terminal sequence of the first 77 and 81 residues of the human and porcine homologs shows only one conservative substitution at residue 12, namely an Ala for a Thr between these two species. Such high sequence homology is also reflected in their amino acid composition. A computer data-bank search using a mutation data matrix and comparison with 338,327 segments of proteins revealed that this substance should be classified as belonging to a new protein superfamily. Immunocytochemical staining, using an antibody produced against a synthetic fragment, revealed the presence of immunostainable material in the anterior and posterior lobe of the pituitary and in the supraoptic nucleus of the hypothalamus. No staining was observed in the intermediate lobe of the pituitary. Furthermore, purified neurointermediate lobe secretory granule preparations were also shown to contain this novel polypeptide.     

Chlamydomonas reinhardtii Phosphoribulokinase : Sequence, Purification, and Kinetics

The sequence and kinetic properties of phosphoribulokinase purified from Chlamydomonas reinhardtii were determined and compared with the spinach (Spinacea oleracea) enzyme. Chlamydomonas phosphoribulokinase was purified to apparent homogeneity, with a specific activity of 410 micromoles per minute per milligram. Polyclonal antibodies to the purified protein were used to isolate a Chlamydomonas cDNA clone, which, upon sequencing, was found to contain the entire coding region. The transit peptide cleavage site was determined by Edman analysis of the mature protein. The precursor protein consists of a 31 amino acid transit peptide and a 344 amino acid mature polypeptide. The mature polypeptide has a calculated molecular weight of 38.5 kilodaltons and a pl of 5.75. The V_max of the purified enzyme was 465 micromoles per minute per milligram, with apparent K_m values of 62 micromolar ATP and 56 micromolar ribulose 5-phosphate. Immunoblot analysis indicated antigenic similarity and a similar subunit size for the enzyme from five higher plant species and Chlamydomonas. Southern blot analysis of Chlamydomonas genomic DNA indicated the presence of a single phosphoribulokinase gene. Comparison of the mature proteins from Chlamydomonas and spinach revealed 86 amino acid differences in primary structure (25% of the total) without a major difference in kinetic properties. The transit peptides of the spinach and Chlamydomonas proteins possessed little sequence homology.     

Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M_r of its subunit was 77,000. The cells converted [¹⁴C]-l-phenylalanine into [¹⁴C]-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M_r 77, 137), a 22-bp 5′-noncoding region and a 207-bp 3′-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology.     

Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli

The outer membrane protein OpcP1 of Burkholderia (formerly Pseudomonas) cepacia is one of the subunits forming the porin oligomer OpcPO by non-covalent association. OpcP1 was cleaved with lysyl endopeptidase and the N-terminal amino acid (aa) sequences of the polypeptide fragments were determined. Based on the sequence information, we cloned the opcP gene on a 10-kb EcoRI DNA fragment of the B. cepacia ATCC25416 chromosome. Nucleotide (nt) sequencing revealed a 1086-bp open reading frame (ORF), encoding a 361-aa polypeptide with a signal sequence of 20 residues. The predicted opcP gene encoded a mature protein of M_r 35 696, which agrees well with the value observed previously on SDS-PAGE. The opcP was sub-cloned into pTrc99A and introduced into Escherichia coli. Immunoblot analysis using murine antiserum specific to OpcP1 visualized the protein expressed in the E. coli cells after induction by isopropyl β-d-thiogalactopyranoside (IPTG).     

A new member to the astasin family of metalloendopeptidases: A novel 1,25-dihydroxyvitamin D-3-stimulated mRNA from chorioallantoic membrane of quail

1,25-Dihydroxyvitamin D-3 is essential for the utilization of eggshell calcium by avian embryo through the chorioallantoic membrane (CAM). A cDNA library was constructed from poly(A)⁺ RNA extracted from vitamin D-deficient CAMs given 1,25-dihydroxyvitamin D-3. Screening this library by differential hybridization yielded a full-length (∼ 1.8 kb) cDNA, whose corresponding mRNA is increased 3-fold 2.5 h after a single injection of 1,25-(OH)₂D₃. The complete nucleotide sequence for the full-length cDNA has been determined. An open-reading frame, corresponding to a 310 amino acid, 41 kDa protein was found. Searching protein sequence data bases revealed a strong similarity to the following proteases: astacin, a crayfish digestive protease, Oryzias latipes hatching enzyme constituent protease (Orz), Xenopus laevis developmentally regulated UVS.2 protein secreted by the hatching gland of embryos, the NH₂-terminal domain of human bone morphogenetic protein (BMP-1) and Drosophila dorsal-ventral patterning tolloid. The cDNA has approximately 36% overall identity with astacin and BMP-1, and is more than 60% identical to either Orz or UVS.2. Moreover, multiple alignment analysis indicates that 37 residues, including 3 cysteine residues, are strictly conserved in the complete 200-amino acid astacin sequence. All 6 proteins contain a zinc-binding motif (HEXXH), found at the active site of most metalloendopeptidases. This motif is found within an extended sequence of HEXXHXXGFXHE that is unique to this subgroup of metalloendopeptidases. In addition, the 6 proteins have 50% identity (including the present cDNA) and 79% are conserved in 4 of these proteins in a 24-amino acid sequence that includes the putative active site. The level of mRNA for the new protein reaches a maximum at day 12 of embryonic life and declines thereafter. It is suggested that this clone corresponds to an mRNA encoding for a protease that may play a role in the degradation of eggshell matrix.     

Feeding recombinant E. coli with GST-mBmKTX fusion protein increases the fecundity and lifespan of Caenorhabditis elegans

Scorpion venom could be a useful treatment for a variety of diseases, such as cancer, epilepsy and analgesia. BmKTX is a polypeptide extracts from scorpion venom (PESV), which have attracted much attention from researchers in recent years. mBmKTX is a mutant polypeptide according to the amino acid sequence of BmKTX. We expressed it with the vector pGEX-4T-1 in Escherichia coli, and Caenorhabditis elegans were used as the animal model and fed with the strains. In this study, the expression of pGEX-mBmKTX was analyzed by SDS-PAGE, and GST-mBmKTX purified from pGEX-mBmKTX as a glutathione S-transferase (GST)-tagged fusion protein is approximately 30 kDa. The secondary structure prediction shows that mBmKTX is mainly composed of approximately 13% β-sheet and 86% loop. A food clearance assay and brood size assay indicated that the worms fed pGEX-mBmKTX ate more and had greater fecundity than those fed the empty vector. A lifespan analysis demonstrated that mBmKTX could significantly prolong the lifespan of C. elegans, with an increase of 22.5% compared with the control. Behavioral assays confirmed that mBmKTX had no influence on the locomotion of C. elegans. In addition, microarray analysis and quantitative real-time PCR demonstrated that there are 320 differentially expressed genes, 182 of which are related to reproduction, growth and lifespan. In conclusion, the data suggested that mBmKTX has potential utility for increasing fecundity and animal survival.     

Crotalus adamanteus phospholipase A2-α: Subunit structure,NH2-terminal sequence,and homology with other phospholipases

Automated Edman degradation of reduced and carboxymethylated phospholipase A₂-α from Crotalus adamanteus venom revealed a single amino acid sequence extending 30 residues into the protein from the amino terminus. The singularity of the sequence and the yields of the phenylthiohydantoin amino acids thus obtained indicate that the subunits comprising the phospholipase dimer are identical. Further chemical evidence in support of subunit identity was obtained by cleavage of phospholipase A₂-α with cyanogen bromide. Compositional analysis of the protein revealed one residue of methionine per monomer and the sequence determination placed this amino acid at position 10 in the sequence of 133 amino acids. Cyanogen bromide cleavage of the protein, followed by reduction and carboxymethylation afforded the expected 2 fragments: an NH₂-terminal decapeptide (CNBr-1) and a larger COOH-terminal fragment of 123 residues (CNBr-II). Automated Edman degradation of the latter has extended the sequence analysis to 54 residues in the NH₂-terminal segment of the monomer chain. Comparison of this sequence with those derived for phospholipases from other snake venoms, from bee venom, and from porcine pancreas has revealed striking homologies in this region of the molecules. As expected on the basis of their phylogenetic classification, the phospholipases from the pit vipers C. adamanteus and Agkistrodon halys blomhoffii are more similar to one another in sequence than to the enzyme from the more distantly related viper, Bitis gabonica. Furthermore, the very close similarities in sequence observed among all of these phospholipases in regions corresponding to residues 24 through 53 in the C. adamanteus enzyme suggest that this segment of the polypeptide plays an important role in phospholipase function and probably constitutes part of the active site.     

Synthesis and uptake of cytoplasmically synthesized pyruvate, pi dikinase polypeptide by chloroplasts

Polyadenylated RNA was isolated from maize leaves and translated in vitro. In agreement with a previous report by others, we found among the translation products a 110-kilodalton pyruvate orthophosphate dikinase (PPDK) precursor that is about 16 kilodaltons larger than the polypeptide isolated from cells. This maize PPDK precursor polypeptide was taken up from the translation product mixture by intact spinach chloroplasts and yielded a mature PPDK polypeptide (94 kilodaltons). The uptake and processing support the proposal that the extra 16-kilodalton size of the polypeptide from in vitro translation of maize leaf mRNA represents a transit sequence which is cleaved after its entry into chloroplasts. Moreover, these results provide additional evidence that in vivo in maize leaf cells PPDK polypeptide is synthesized in the cytoplasm and is transported into the chloroplasts.

Location of PPDK in C₃ plant leaves was investigated by immunochemical analysis. Intact chloroplasts were isolated from leaves of spinach, wheat, and maize. A protein blot of stromal protein in each case gave rise to bands corresponding to authentic PPDK polypeptide. This result indicates that PPDK is present in chloroplasts of C₃ plant leaves as it is in the case of C₄ plants.

     

P(700) Chlorophyll a-Protein : Purification, Characterization, and Antibody Preparation

The P₇₀₀ chlorophyll α-protein was purified by preparative sodium dodecyl sulfate (SDS) gel electrophoresis from SDS-solubilized barley (Hordeum vulgare L., cv Himalaya) chloroplast membranes. After elution from the gel in the presence of 0.05 to 0.1% Triton X-100, the recovered protein had a chlorophyll/P₇₀₀ ratio of 50 to 60/1 and contained no chlorophyll b or cytochromes. Analysis of the polypeptide composition of the chlorophyll-protein revealed a 58 to 62 kilodalton (kD) polypeptide component but no lower molecular weight polypeptides. The 58 to 62 kD component was further resolved into two distinct polypeptide bands which were subsequently mapped by partial cyanogen bromide digestion and Staphylococcus aureus proteolysis. Based on results from the mapping experiments and other data, we suggest that the two components are conformational variants of a single polypeptide. Measurement of the chlorophyll to protein ratio by quantitative amino acid analysis and consideration of the yield of P₇₀₀ in the protein isolate suggest that, contrary to previous models (Bengis and Nelson, 1975, 1977), P₇₀₀in vivo is associated with a minimum of four subunits of approximately 60 kD.

Antibodies raised against the photochemically active chlorophyll-protein complex from barley reacted specifically with the 58 to 62 kD apoprotein. The same preparative electrophoresis procedure was used to isolate photochemically active P₇₀₀ chlorophyll a-protein from soybean (Glycine max L.), tobacco (Nicotiana tobacum L.), petunia (Petunia × hybrida), tomato (Lycopersicum esculentum), and Chlamydomonas reinhardti. The isolated complex from all species exhibited identical polypeptide compositions and chlorophyll/P₇₀₀ ratios. Antibodies to the barley protein cross reacted with all species tested demonstrating the highly conserved structure of the apoprotein.

     

DNA sequence analysis of bacterial toxic heavy metal resistances

Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be “linked” together on multiple resistance plasmids. For Cd²⁺, AsO₂ ^?, AsO₄ ^3?, Hg²⁺, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. ThecadA ATPase ofS.aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd²⁺ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K⁺ ATPases ofEscherichia coli andStreptococcus faecalis, the H⁺ ATPases of yeast andNeurospora, the Na⁺/K⁺ ATPases of vertebrate animals, and the Ca²⁺ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein (arsA), an integral membrane protein (arsB gene), and a substrate specificity subunit (arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in themer system include a DNA-binding regulatory protein from themerR gene and a Hg²⁺ transport system consisting of a periplasmic Hg²⁺-binding protein (merP gene) and a membrane protein (merT gene) in gram negative systems.     

The complete amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus

The amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus has been completely determined. The sequence data were mainly obtained by manual sequencing of peptides derived from digestion with trypsin, Staphylococcus aureas protease and pepsin. A few overlaps of tryptic peptides were established by DNA sequence analysis of a chromosomal fragment containing the rpsL gene coding for ribosomal protein S12. The protein contains 138 amino acid residues and has an M_r of 15208. Comparison of this sequence with the sequences of the ribosomal S12 proteins from E. coli as well as from Euglena, tobacco and liverwort chloroplasts shows that 75% of the amino acid residues are identical within the S12 proteins of all four species. Therefore, S12 is the most strongly conserved ribosomal protein known so far.     

Molecular characterization of the sans fille gene in Antheraea pernyi: A highly conserved gene during the evolution of animals

Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B″, and plays an important role in sex determination in Drosophila melanogaster. In this study, the snf gene from Antheraea pernyi (Lepidoptera: Saturniidae), an economically important insect, was isolated and characterized. The obtained 925 bp cDNA sequence contains an open reading frame of 669 bp encoding a polypeptide of 222 amino acids, showing 78% sequence identity to that from D. melanogaster. A database search revealed that SNF protein homologs are present in many animals, including invertebrates and vertebrates, with more than 70% amino acid sequence identities, suggesting that they were highly conserved during the evolution of animals. Phylogenetic analysis revealed that A. pernyi SNF was closely related to Bombyx mori SNF. Quantitative real-time PCR (qRT-PCR) analysis showed that the A. pernyi snf gene was transcribed during five larval developmental stages, and in six tested tissues (ovaries, testes, silk glands, fat body, integument, and hemolymph), with the most abundance determined in the gonads (ovaries or testes). Investigation of expression changes throughout embryonic development indicated that A. pernyi snf mRNA was expressed at a low level from days 0 to 4, and reached a maximum level at day 10, but decreased to a low level before hatching. These results suggest that the product of the snf gene may play important roles in the development of A. pernyi.     

Primary structure of yeast cytochrome c peroxidase. I. Chemical characterization of the polypeptide chain and of tryptic and chymotryptic peptides

The amino acid composition of yeast cytochrome c peroxidase was determined and calculated assuming that the enzyme contained one protoheme per molecule. On the basis of the amino acid composition and heme content the minimum M_r was calculated to be 35,235. Gel electrophoresis in the presence of sodium dodecyl sulfate indicated the presence of a single polypeptide chain with a M_r of approximately 33,000. A single cysteinyl residue present in the molecule was shown to be resistant against reaction with iodoacetic acid in the native form of both holo- and apo-enzymes, but readily modified with the reagent in the denatured form. Automated Edman degradation yielded an aminoterminal sequence of 11 residues beginning with threonine. Twenty-eight tryptic and 47 chymotryptic peptides were isolated from the carboxymethylated apoprotein and subjected to the sequence analysis by the dansyl-Edman method. The results with these peptides confirmed and extended the amino- and carboxyl-terminal sequences and in addition provided a partial sequence covering approximately 90% of the polypeptide chain.     

The distribution of residues in a polypeptide sequence is a determinant of aggregation optimized by evolution

It has been shown that the propensity of a protein to form amyloid-like fibrils can be predicted with high accuracy from the knowledge of its amino acid sequence. It has also been suggested, however, that some regions of the sequences are more important than others in determining the aggregation process. Here, we have addressed this issue by constructing a set of “sequence scrambled” variants of the first 29 residues of horse heart apomyoglobin (apoMb_1-29), in which the sequence was modified while maintaining the same amino acid composition. The clustering of the most amyloidogenic residues in one region of the sequence was found to cause a marked increase of the elongation rate (k_agg) and a remarkable shortening of the lag phase (t_lag) of the fibril growth, as determined by far-UV circular dichroism and thioflavin T fluorescence. We also show that taking explicitly into consideration the presence of aggregation-promoting regions in the predictive methods results in a quantitative agreement between the theoretical and observed k_agg and t_lag values of the apoMb_1-29 variants. These results, together with a comparison between homologous segments from the family of globins, indicate the existence of a negative selection against the clustering of highly amyloidogenic residues in one or few regions of polypeptide sequences.