The primary structure of human Rieske iron-sulfur protein of mitochondrial cytochrome bc1 complex deduced from cDNA analysis

We isolated a cDNA encoding human Rieske Fe-S protein of mitochondrial cytochrome bc1 complex from a fibroblast cDNA library by colony hybridization. The cDNA contains the nucleotide sequence encoding all of the amino acids (274 residues) comprising the putative precursor to the protein. Based on the known amino acid sequence of bovine Rieske Fe-S protein, the N-terminal extension sequence is presumed to be composed of 78 amino acids with a molecular weight of 8053. The mature protein consists of the same number of amino acid residues as that of its rat and bovine counterparts, having a homology of about 92% with the latter.     

An osmotin-like cryoprotective protein from the bittersweet nightshade Solanum dulcamara

Cold acclimation in plants is a polygenic phenomenon involving increased expression of several genes. The gene products participate either directly or indirectly towards increasing cold tolerance. Evidence of proteins having a direct effect on cold tolerance is emerging but limited. With isolated protoplasts from warm-grown kale (Brassica oleracea) as a model system, we tested protein fractions from winter bittersweet nightshade, Solanum dulcamara, stems for the presence of proteins that have a cryoprotective effect. Purification of one such fraction resulted in isolation of a 25 kDa protein. N-terminal Edman degradation amino acid sequence analysis showed that it has high homology to osmotin and osmotin-like proteins. When added to warm-grown protoplasts, it increased the cryosurvival of frozen-thawed protoplasts by 24% over untreated or BSA-treated controls at –8 °C. A cDNA library which was made in November from stems and leaves of S. dulcamara was successfully screened for the corresponding cDNA clone. The deduced amino acid sequence indicated that the protein consists of 206 amino acid residues including a N-terminal signal sequence and a putative C-terminal propeptide. The mature protein, without the N-terminal signal sequence, was expressed in Escherichia coli. The partially purified protein in the supernatant fraction of the culture medium had cryoprotective activity.     

Nucleotide sequence of cDNA clones encoding the complete precursor for subunit delta of thylakoid-located ATP synthase from spinach

The nucleotide sequence of the entire nuclear-encoded precursor for subunit delta of the ATP synthase from spinach thylakoid membranes was determined by cDNA sequencing. Appropriate recombinant DNAs were selected from pBR322 and lambda gt11 libraries made from polyadenylated RNA of greening spinach seedlings. The mature protein consists of 187 amino acid residues corresponding to a molecular weight of 20468. The precursor protein (257 amino acid residues; M _r=27676) is probably processed between a Met-Val bond. The predicted secondary structure of the transit sequence (70 residues; 7.2 kDa) resembles that of the Rieske Fe/S polypeptide, but shows little similarity with those of stromal or luminal proteins. The comparison of the chloroplast delta amino acid sequence with the published delta sequences from respiratory ATP synthases of bacterial and mitochondrial sources and from the thylakoid ATP synthase of the cyanobacterium Synechococcus suggests substantial divergence at the genic level although structural elements appear to be remarkably conserved.     

Nucleotide sequence and characterization of a cDNA encoding the acetohydroxy acid isomeroreductase from Arabidopsis thaliana

The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site.     

The b-32 protein from maize endosperm, an albumin regulated by the O2 locus: Nucleic acid (cDNA) and amino acid sequences

Summary The cDNA coding for the b-32 protein, an albumin expressed in maize endosperm cells under the control of the O2 and O6 loci, has been cloned and the complete amino acid sequence of the protein derived. A lambda gt11 cDNA library from mRNA of immature maize endosperm was screened for the expression of the b-32 protein using antibodies against the purified protein. One of the positive clones obtained was used to isolate a full-length cDNA clone. By Northern analysis, the size of the b-32 mRNA was estimated to be 1.2 kb. Hybrid-selected translation assays show that the message codes for a protein with an apparent molecular weight of 30–35 kDa. The nucleotide sequence shows that several internal repeats are present. The protein has a length of 303 amino acid residues (mol. wt. 32430 dalton) and its sequence shows the following features: no signal peptide is observable; it contains seven tryptophan residues, an amino acid absent in maize storage proteins; polar and hydrophobic residues are spread along the sequence; several pairs of basic residues are present in the N-terminal region; the secondary structure allows the prediction of two structural domains for the b-32 protein that would fold up giving rise to a globular shape. The cloning of this gene may help in understanding the role of the O2 and O6 loci in regulating the deposition of zein, the major storage protein of maize endosperm.     

Identification and Characterization of Putative Receptors for Sperm-Activating Peptide I (SAP-I) in Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus1

We characterized putative receptors specific for sperm-activating peptide I (SAP-I: GFDLNGGGVG) in spermatozoa of the sea urchin Hemicentrotus pulcherrimus, using both binding and crosslinking techniques. Analysis of the data obtained from the equilibrium binding of a radioiodinated SAP-I analogue [GGGY(¹²⁵I)-SAP-I] to H. pulcherrimus spermatozoa showed the presence of two classes of receptors specific for SAP-I in the spermatozoa. The incubation of intact spermatozoa as well as sperm tails or sperm membranes prepared from H. pulcherrimus spermatozoa with GGGY(¹²⁵I)-SAP-I and a chemical crosslinking reagent, disuccinimidyl suberate, resulted in the radiolabelling of a 71 kDa protein. The protein appears to be associated with a 220 kDa wheat germ agglutinin (WGA)-binding protein. A cDNA encoding the 71 kDa protein was isolated from a H. pulcherrimus testis cDNA library. The cDNA was 2443 bp long and an open reading frame predicted a protein of 532 amino acids containing a 30-residue amino-terminal signal peptide, followed by the same sequence as the N-terminal sequence of the 71 kDa protein. The amino acid sequence of the matured 71 kDa protein is strikingly similar to the 77 kDa protein of Strongylocentrotus purpuratus (95.5% identical) and also similar to cysteine rich domain of a human macrophage scavenger receptor. Northern blot analysis demonstrated that mRNA of 2.6 kb encoding the 71 kDa protein was expressed only in the testis.     

Complementary DNA sequence of rabbit CAP18--a unique lipopolysaccharide binding protein

CAP18 is a novel 18 kDa cationic protein [pI approximately 10] originally purified from rabbit granulocytes using as an assay the agglutination of lipopolysaccharide (LPS) coated erythrocytes. cDNA clones encoding CAP18 were isolated from a rabbit bone marrow cDNA library using a PCR generated oligonucleotide probe derived from the N-terminal amino acid sequence. The deduced amino acid sequence reveals a putative signal sequence of 29 amino acids and a mature protein of 142 amino acid residues. The predicted size of the encoded protein is 16.6 kDa with a pI of 10. There are no N-linked glycosylation sites. The CAP18 sequence bears no homology with other known LPS-binding proteins including human bacterial permeability increasing protein (BPI)(1) and rabbit LPS binding protein (LBP)(2).     

Isolation,sequencing and expression of cDNA sequences encoding uroporphyrinogen decarboxylase from tobacco and barley

We have cloned and sequenced a full-length cDNA for uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) from tobacco (Nicotiana tabacum L.) and a partial cDNA clone from barley (Hordeum vulgare L.). The cDNA of tobacco encodes a protein of 43 kDa, which has 33% overall similarity to UROD sequences determined from other organisms. We propose that tobacco UROD has an N-terminal extension of 39 amino acid residues. This extension is most likely a chloroplast transit sequence. The in vitro translation product of UROD was imported into pea chloroplasts and processed to ca. 39 kDa. A truncated cDNA, from which the putative transit peptide had been deleted, was used to over-express the mature UROD in Escherichia coli. Purified protein showed UROD activity, thus providing an adequate source for subsequent enzymatic characterization and inhibition studies. Expression of UROD was investigated by northern and western blot analysis during greening of etiolated barley seedlings, and in segments of barley primary leaves grown under day/night cycles. The amount of RNA and protein increased during illumination Maximum UROD-RNA levels were detected in the basal segments relative to the top of the leaf.Abbreviations ALA 5-aminolevulinic acid - copro coproporphyrin - coprogen coproporphyrinogen - protogen IX protoporphyrinogen IX - UROD uroporphyrinogen decarboxylase - uro uroporphyrin - urogen uroporphyrinogen     

A unique EF-hand motif in mnemiopsin photoprotein from Mnemiopsis leidyi: implication for its low calcium sensitivity

Up to now, all reported Ca²⁺-regulated photoproteins, except for mnemiopsin, have been cloned and expressed in Escherichia coli. In this study, the cDNA for an isotype of mnemiopsin, from the ctenophore Mnemiopsis leidyi, has been cloned, sequenced, and functionally expressed. The full length cDNA encoding mnemiopsin of M. leidyi was 624 bp open reading frame encoding a protein of 207 amino acid residues with calculated molecular mass of ∼24 kDa. The deduced amino acid sequence showed 90% and 84% identity to berovine (from ctenophore Beroe abyssicola) and bolinopsin 2 (from the ctenophore Bolinopsis infundibulum) respectively. In contrast to all known EF-hand in photoproteins, a unique EF-hand motif was found in mnemiopsin, in which a conserved glycine is substituted with glutamic acid. According to the results, the optimum pH was 9.0, time course of regeneration was 15 h and its Ca²⁺ sensitivity was lower than aequorin. Results of pK_a calculation for ionizable residues, motif scan and hydrophobic interactions of cavity aromatic residues of mnemiopsin in comparison with aequorin showed different patterns in these two photoproteins. In addition, experimental results are confirmed with the theoretical studies.     

Molecular Characterization of a Novel Family-46 Chitosanase from Pseudomonas sp. A-01

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5–8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.     

Porcine pyridoxal kinase: c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.     

The extrinsic 33 kDa polypeptide of the oxygen-evolving complex of photosystem II is a putative calcium-binding protein and is encoded by a multi-gene family in pea

The extrinsic 33 kDa polypeptide of the water-oxidizing complex has been extracted from pea photosystem II particles by washing with alkaline-Tris and purified by ion-exchange chromatography. The N-terminal amino acid sequence has been determined, and specific antisera have been raised in rabbits and used to screen a pea leaf cDNA library in gt11. Determination of the nucleotide sequence of positive clones revealed an essentially full-length cDNA for the 33 kDa polypeptide, the deduced amino acid sequence showing it to code for a mature protein of 248 amino acids with an N-terminal transit peptide of 81 amino acids. The protein showed a high degree of conservation with previously reported sequences for the 33 kDa protein from other species and the sequence contained a putative Ca²⁺-binding site with homology to mammalian intestinal calcium-binding proteins. Northern analysis of total pea RNA indicated a message of approximately 1.4 kb, in good agreement with the size of the cDNA obtained at 1.3 kbp. Southern blots of genomic DNA probed with the labelled cDNA give rise to several bands suggesting that the 33 kDa polypeptide is coded by a multi-gene family.Abbreviations ATZ - anilinothiazolinone - DITC - p-phenylenediisothiocyanate - PTH - phenylthiohydantoin - TFA - trifluoroacetic acid - Tris - tris (hydroxymethyl) aminomethane - bis-Tris - bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - p.f.u. - plaque-forming units     

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.     

Purification and Characterization of Intracellular Proteinases in Pleurotus ostreatus Fruiting Bodies

A serine proteinase (ProA, EC 3.4.22.9) and two metalloendopeptidases (ProB, EC 3.4.99.32 and ProC, 3.4.24.4), have been purified to homogeneity from the fruiting bodies of Pleurotus ostreatus. ProA is a serine proteinase with a mass of 30 kDa, which has amidolytic and esterolytic activities besides proteolysis and catalyzes preferential cleavage of the peptide bonds involving the carboxyl groups of hydrophobic amino acid residues in oxidized bovine insulin B chain. The N-terminal amino acid sequence was VTQTNAPWGLSRL.

ProB is a zinc-enzyme with a mass of 18 kDa, which is devoid of lysine, and its N-terminal sequence was ATFVGCSATRQ. The enzyme is inactivated completely by EDTA and 1,10-phenanthroline, and Zn²⁺-depleted ProB can regain the activity with Zn²⁺, Co²⁺, or Mn²⁺. Specific cleavage of Pro₂₉-LYS₃₀ in oxidized bovine insulin B chain, preferential generation of lysylpeptides from proteins, and a high susceptibility of polylysine suggest that ProB splits specifically the peptide bonds involving the α-amino group of lysyl residues.

ProC is a metalloendopeptidase of a mass of 42.5 kDa, and Zn²⁺ was the most effective divalent metal ion to activate the EDTA-inactivated enzyme.     

Isolation and characterization of a cDNA clone encoding the anti-viral protein from Phytolacca americana

Phytolacca anti-viral protein (PAP) was purified from Phytolacca leaves and the N-terminal was sequenced. A cDNA library was made from mRNAs isolated from Phytolacca leaves and cDNA clones for PAP were identified using oligonucleotide probes derived from the N-terminal amino acid sequence. The PAP-cDNA clone was sequenced from both directions. The predicted amino acid sequence of PAP was compared with the amino acid sequences of other ribosome-inactivating proteins. The identities of these proteins to PAP ranged from 29 to 38%, and a region was found in each with a sequence similar to the PAP sequence (AIQMVSEAARFKYI). Southern blot analysis indicates that PAP is encoded by a multi-gene family.Abbreviations MAP Mirabilis jalapa anti-viral protein - PAP Phytolacca anti-viral protein - SO6 30 kDa ribosome-inactivating protein from the seeds of Saponaria officinalis     

Molecular cloning and expression analysis of interferon-γ inducible lysosomal thiol reductase (GILT)-like cDNA from disk abalone (Haliotis discus discus)

Interferon Gamma (IFN-γ) Inducible Lysosomal Thiol reductase (GILT) has been described as a key enzyme in processing and presentation of major histocompatibility complex (MHC) class II restricted antigen (Ag) by catalyzing disulfide bond (S–S) reduction in mammals. Abalone GILT-like (AbGILT) full-length cDNA was isolated from the normalized disk abalone cDNA library. The 807-bp AbGILT cDNA consists of an open reading frame of 684-bp, encoding 228 amino acid residues. The predicted AbGILT protein has a molecular weight of 25 kDa and an isoelectric point of 7.8. The N-terminus of the AbGILT was found to have a putative signal peptide with a cleavage site amino acid position at 19–20. AbGILT contains two active site C-XX-C motifs, (²³CLDC²⁶ and ⁴⁶CPYC⁴⁹) which motif is highly conserved in GILT protein family. AbGILT exhibited a characteristic GILT signature sequence ⁹²CQHGX₂ECX₂NX₄C¹⁰⁷ and 12 cysteine residues representing 5% in the mature peptide. Phylogenetic analysis showed that AbGILT has been derived from a common ancestor with other GILT proteins. RT-PCR results showed that AbGILT expression was up-regulated in the gill, mantle and digestive tract 24 h post injection of phytohemagglutinin (PHA) while Vibrio alginolyticus up-regulation appeared in the gill and digestive tract after 48 h. In contrast, AbGILT expression was not up-regulated by poly inosinic–cytidylic acid (poly I:C) during the 48 h induction. However, AbGILT was constitutively expressed in gill, mantle, and digestive tract tissues suggesting that it may maintain first line of innate immune defense at basal level in disk abalone.     

Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts

Using the expression vector gt11 and immunochemical detection, six cDNA clones that encode the entire precursor polypeptides for spinach thioredoxin m were isolated and characterized. The ca. 1.0 kb cDNA sequence of the largest clone hybridizes to an RNA species of 1.1 kb. In each instance the cDNA sequences display single open reading frames encoding polypeptides of 181 amino acid residues corresponding to a molecular mass of 19.8 kDa. The sequences of the independently selected cDNAs fall into two classes that are indicative of at least two (closely related) genes for this protein. The amino acid sequences deduced from the cDNA sequences differ to some extent from the amino acid sequence published for spinach thioredoxin m. The sequences predict identical mature proteins of 112–114 amino acids corresponding to a polypeptide molecular mass of ca. 12.4–12.6 kDa, and include stroma-targeting N-terminal transit peptides of 67 residues which are removed during or after import into the organelle. Precursor protein was made in vitro from each of the different cDNA clones and imported into isolated intact chloroplasts. Independent of the cDNA clone used, two isoforms were detected in the chloroplasts after import in each instance. They comigrated with authentic thioredoxin mb and mc. These results indicate that the size variants observed for this protein in vivo result from post-translational modification and do not originate in different genes.     

Purification and identification of the violaxanthin deepoxidase as a 43 kDa protein

Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH₄)₂SO₄ fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g^–1 s^–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b ₆ and an internal sequence of polyphenol oxidase.Abbreviations DMSO dimethylsulfoxid - HIC hydrophobic interaction chromatography - MGDG monogalactosyl diacylglycerol - VDE violaxanthin deepoxidase A preliminary report of these results was presented at the Xth Int. Congress on Photosynthesis, Montpellier, France, 1995.