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PubMed1997

Rat MDC family of proteins: sequence analysis, tissue distribution, and expression in prepubertal and adult rat testis.

Increasing number of sequence-related cysteine-rich membrane proteins containing metalloproteinase-like and disintegrin-like domains (the MDC protein family) have been identified in mammalian tissues. Here, we report the cloning and sequence analysis of cDNAs encoding several rat orthologues of this protein family, some of which are found to be expressed exclusively in the male reproductive tract, others exhibiting a broader tissue distribution. We also examine their expression in prepubertal an adult rat testis, which, in conjunction with the data on tissue distribution, form a necessary prelude to further studies aimed at establishing their individual functions.

ADAM Proteins↗
PubMed2004

A Tobacco necrosis virus D isolate from Olea europaea L.: viral characterization and coat protein sequence analysis.

A virus isolated from Olea europaea L. grown in Portugal, was identified as a member of the species Tobacco necrosis virus D (TNV-D, genus Necrovirus, family Tombusviridae), based on the molecular and serological properties of the purified virus particles. The genomic region encoding the coat protein (CP) of this isolate (named GP isolate) was amplified by RT-PCR and the cDNA was cloned and sequenced. The CP gene encodes a predicted protein of 269 amino acids showing high identity (86.2%) to TNV-D coat protein sequence. Phylogenetic analysis based on necroviruses CP sequences, confirmed GP as a TNV-D isolate. The alignment with homologous TNV-D CP sequences revealed four conserved amino acids involved in Ca(2+) binding as well as the plant virus icosahedral capsid protein "S' signature. Based on the determined nucleotide sequence, specific primers were designed and successfully used in RT-PCR for virus diagnosis in naturally infected olive trees.

Amino Acid Sequence↗
PubMed1990

Sequence analysis of proteins separated by polyacrylamide gel electrophoresis: towards an integrated protein database.

Improved technologies or the synergistic use of complementary methods enhance the efficiency of research and permit the exploration of new approaches for the investigation of complex problems. High sensitivity protein sequence analysis and polyacrylamide gel electrophoresis are such complementary methods. Here we summarize the current status of high sensitivity sequence analysis of proteins separated in polyacrylamide gels and discuss strategies by which this technology can enhance biological research by generating new approaches for the solution of complex, multifacetted problems. Finally, we outline imminent technological advances in the area of high sensitivity protein sequence analysis and argue that further technological developments will ultimately lead to the generation of an integrated protein database (containing structural and functional as well as physiological information in an easily accessible form) of all the proteins separated by high resolution two-dimensional gel electrophoresis.

Amino Acid Sequence↗
PubMedDate not supplied

Direct atmospheric pressure coupling of polyacrylamide gel electrophoresis to mass spectrometry for rapid protein sequence analysis.

Using laser desorption-atmospheric pressure chemical ionization we describe a novel approach for coupling mass spectrometry to polyacrylamide gel electrophoresis. In contrast to other approaches, the method allows for the direct sampling of a polyacrylamide gel-embedded protein without the addition of any exogenous matrixes and is performed at atmospheric pressure. After electrophoresis and enzymatic digestion, the gel is analyzed at AP by photons that desorb neutral peptide molecules, followed by corona discharge ionization in the gas-phase, and subsequent mass analysis. Our experimental results demonstrate the method to (1) rapidly identify electrophoresed proteins via "peptide fingerprinting" using protein databases, (2) detect single-amino acid polymorphisms, and (3) has potential for sub-picomole sensitivity while still maintaining in situ gel desorption-ionization at ambient conditions.

Atmospheric Pressure↗
PubMed1994

On-line procedures for alkylation of cysteine residues with 3-bromopropylamine prior to protein sequence analysis.

We have previously shown that 3-bromopropylamine offers several advantages over other alkylating reagents in the modification and subsequent identification of cysteine residues by protein sequencing. We describe here simple on-sequencer procedures for alkylating cysteines in proteins which employ the reduction of cystines in proteins with tri-n-butylphosphine and concomitant alkylation of the resulting cysteines with 3-bromopropylamine. Addition of an aqueous acetone wash to a modified reaction cycle on the Applied Biosystems 477A sequencer removes excess 3-bromopropylamine. As a result, very little background in the first step of the sequence analysis is seen. Under these conditions, cysteines are readily modified and identified during sequencing. Moreover, very little preview of the next amino acid is observed, which indicates that the N-terminal amino acid is not appreciably alkylated by 3-bromopropylamine. On-sequencer methods have been developed for proteins spotted onto glass fiber filters and proteins electroblotted onto polyvinylidene difluoride membranes.

Alkylation↗
PubMed1985

A putative Ca2+-binding protein: structure of the light subunit of porcine calpain elucidated by molecular cloning and protein sequence analysis.

cDNA clones specific for the light subunit of porcine calpain I have been isolated from a porcine kidney cDNA library. The complete primary structure of the light subunit has been revealed by nucleotide sequence analysis of the cDNA clones isolated and amino acid sequence analysis of peptides isolated from the purified mature protein. We found that the light subunit contains two distinct domains. Domain I, the amino-terminal half, has two unusually long, paired polyglycyl sequences and may serve as a binding site to the heavy subunit. Domain II, the carboxyl-terminal half, is a region highly homologous to the putative Ca2+-binding domain of the heavy subunit of chicken calpain elucidated recently. This region has four potential Ca2+-binding sites, each having the "E-F hand" structure. Our results suggest that the Ca2+-mediated proteolytic activity of calpain is controlled through the cooperative and/or sequential actions of multiple Ca2+-binding sites present in both two-subunit molecules, heavy and light subunits of calpain.

Amino Acid Sequence↗
PubMed1994

Mouse liver NAD(P)H:quinone acceptor oxidoreductase: protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis.

The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986, J Biol Chem 261:1372-1378), that the 2 forms--the hydrophilic and hydrophobic forms--of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino-termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification. Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109. The E. coli-expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H-menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Characterization of the Escherichia coli SSB-113 mutant single-stranded DNA-binding protein. Cloning of the gene, DNA and protein sequence analysis, high pressure liquid chromatography peptide mapping, and DNA-binding studies.

The ssb-113 (formerly lexC113) gene encoding a mutant single-stranded DNA binding protein (SSB) has been cloned into plasmid pSC101 resulting in 5- to 10-fold more mutant protein than strains carrying only one (chromosomal) copy of the gene. Analysis of tryptic and chymotryptic peptides of the mutant protein by high pressure liquid chromatography and solid phase protein sequencing has shown that the ssb-113 mutation results in the substitution of serine for proline at residue 176 of SSB. This change could only occur in one step by a C leads to T transition in the DNA sequence. Physicochemical studies of the homogeneous mutant protein have shown that it binds as well as wild type SSB to single-stranded DNA and that it is a slightly better helix-destabilizing protein than wild type SSB as measured by its ability to lower the thermal melting transition of poly[d(A-T)]. In vivo studies of ssb-113 strains carrying the cloned ssb-113 gene in pSC101 have shown that overproduction of the mutant protein does not complement the temperature-sensitive conditional lethality caused by the ssb-113 mutation when present in single gene copy in contrast to effects recently observed in ssb-1 strains overproducing the ssb-1 encoded protein (Chase, J. W., Murphy, J. B., Whittier, R. F., Lorensen, E., and Sninsky, J. J. (1983) J. Mol. Biol. 164, 193-211). Also noted in this report are two corrections to the DNA sequence of wild type SSB, one of which places glycine (codon GGC) at residue 133 rather than serine as previously reported (Sancar, A., Williams, K. R., Chase, J. W., and Rupp, W. D. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4274-4278). The second correction to the DNA sequence is in the serine 39 codon, previously reported to be TCA and now correctly shown to be TCC.

Amino Acid Sequence↗
PubMed1988

Characterization of a benzyladenine binding-site peptide isolated from a wheat cytokinin-binding protein: sequence analysis and identification of a single affinity-labeled histidine residue by mass spectrometry.

A wheat embryo cytokinin-binding protein was covalently modified with the radiolabeled photoaffinity ligand 2-azido-N6-[14C]benzyladenine. A single labeled peptide was obtained after proteolytic digestion and isolation by reversed-phase and anion-exchange HPLC. Sequencing by classical Edman degradation identified 11 of the 12 residues but failed to identify the labeled amino acid. Analysis by laser photodissociation Fourier-transform mass spectrometry of 10 pmol of the peptide independently confirmed the Edman data and also demonstrated that the histidine residue nearest the C terminus (underlined) was modified by the reagent in the sequence Ala-Phe-Leu-Gln-Pro-Ser-His-His-Asp-Ala-Asp-Glu.

Adenine↗
PubMed1988

Chemical characterization by protein sequence analysis of the bovine estrogen receptor.

Tryptic peptides generated from bovine estrogen receptor have been fractionated and purified using microbore column high performance liquid chromatography. Sequence analysis performed on six of these peptides, derived from diverse structural regions of the receptor protein, yielded 73 unique assignments corresponding to approximately 12% of the molecule. The amino acid sequences of these peptides displayed a high degree of similarity with corresponding sequences from estrogen receptors of mammalian origin, but were only moderately conserved in receptors from non-mammalian species. The sequenced residues of one tryptic peptide, positioned in the estrogen binding domain, were fully conserved in all estrogen receptors.

Amino Acid Sequence↗
PubMed2003

Coat protein sequence analysis reveals occurrence of new strains of Sweet potato feathery mottle virus in Uganda and Tanzania.

The 3'-proximal part (1.8 kb) of the Sweet potato feathery mottle virus (SPFMV) genome was studied in four SPFMV isolates collected from farmers' fields in western Uganda (SPFMV-Bny), eastern Uganda (SPFMV-Sor) and Bagamoyo district, Tanzania (SPFMV-TZ1 and SPFMV-TZ2). Unlike the other three SPFMV isolates, SPFMV-Sor was not detected with the polyclonal antisera to SPFMV. It showed moderately high coat protein (CP) nucleotide (93.3-96.7%) and amino acid (93.6-96.8%) sequence identity to the isolates of the SPFMV strain group C. In contrast, identities (78.1-80.1%, and 79.9-83.1%) to isolates of the SPFMV strain groups O, RC, and the East African (EA) strain group were low. Similar to some isolates (SPFMV-CH2 and SPFMV-6) of strain group C, but different from other SPFMV isolates, SPFMV-Sor contained a deletion of 6 nucleotides in the CP-encoding region (CP amino acid positions 62-63). Phylogenetic analysis of the CP sequences indicated that SPFMV-Sor belongs to the SPFMV strain group C that has not been reported from Africa. Sequence data were obtained for the first time from Tanzanian SPFMV isolates in this study, and phylogenetic analysis indicated that they belong to the strain group EA, which is unique to East Africa.

3' Untranslated Regions↗
PubMed1990

A knowledge-based architecture for protein sequence analysis and structure prediction.

Methods for analyzing the amino-acid sequence of a protein for the purposes of predicting its three-dimensional structure were systematically analyzed using knowledge engineering techniques. The resulting entities (data) and relations (processing methods and constraints) have been represented within a generalized dependency network consisting of 29 nodes and over 100 links. It is argued that such a representation meets the requirements of knowledge-based systems in molecular biology. This network is used as the architecture for a prototype knowledge-based system that simulates logically the processes used in protein structure prediction. Although developed specifically for applications in protein structure prediction, the network architecture provides a strategy for tackling the general problem of orchestrating and integrating the diverse sources of knowledge that are characteristic of many areas of science.

Amino Acid Sequence↗
PubMed1989

Purification and protein sequence analysis of rat liver prolactin receptor.

Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1978

Silkmoth chorion proteins: sequence analysis of the products of a multigene family.

Five polypeptide components have been isolated from the eggshell (chorions) of a silkmoth. Two are homogeneous on sodium dodecyl sulfate and isoelectric focusing gels, and three contain predominantly two proteins each. Amino acid analyses show that all five components are similar to each other. These proteins have been sequenced from the amino terminus. Homogeneous components yielded single sequences; heterogeneous components yielded two residues at some positions, consistent with their containing two major electrophoretic components. Striking similarities are apparent among all these sequences. These similarities can be increased dramatically by separating each of the three protein mixtures into two sequences and introducing a small number of gaps or insertions. This is due in part to bringing into register a portion that contains short repeating subunits found in all sequences. All proteins are also characterized by a region of high cysteine content near the amino terminus followed by a longer low-cysteine region. The data suggest that these proteins share a common evolutionary origin and are encoded by a multigene family.

Amino Acid Sequence↗