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After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudornonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: [This equation can not be represent in ASCII.TXT]. [ABSTRACT FROM AUTHOR]

A purified glycoprotein with blood-group-A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide-bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N-acetylgalactosaminyl-peptide were hydrolysed with 0.2 M hydrocholoric acid for 24 h at 110 °C. fractionation of the products on Sephadex G-10, followed by preparative separation on amino-acid analyzer and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N-terminal sequence Thr-Thr-Ser, three tetrapeptides with the sequences, Thr-Ser-Thr-Ser, Thr-Pro-Thr-Ser and Pro-Thr-Thr-Ser, one pentapeptide with the sequence Thr-Pro-Thr-Ser, one hexapeptide with the sequence Pro-Thr-Thr-Thr-Pro-Ser and one heptapeptide with the sequence Ala-Pro-Thr-Thr-Ser-Gly-Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated. The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino-acid substituent at only one position in the chain; Pro-Thr-Ala-ser, Thr-Thr Pro-Ser, thr-Thr-Ala-Ser, Ser-Pro-Thr-Ser, and Ser-Ala-Thr-Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro-Thr-Ser, Pro-thr-Ala. These peptides do not indicate a clearly defined amino-acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood-group-specific glycoproteins there is close packing of the threonine in the region of the peptide moiety carrying the carbohydrate chains. [ABSTRACT FROM AUTHOR]

Two c-type cytochromes were isolated from cells of the gram-negative bacterium Aquaspirillum itersonii grown under low aeration in the presence of nitrate. The major component, cytochrome c-550, was equated with the (single) c-type cytochrome previously reported to be present in this organism [Clark-Walker, G. D. & Lascelles, J. (1970) Arch. Biochem. Biophys. 136, 153-159], although a significantly higher molecular mass was apparent in the present work. The complete amino acid sequence of this cytochrome is reported in the accompanying paper. A second soluble c-type cytochrome, designated c-556, was also isolated. The molecular mass, isoelectric point, spectrum, midpoint oxidation reduction potential and amino acid composition of this monoheam cytochrome are reported. The possible relationship of this cytochrome to other cytochromes c-556 is discussed. [ABSTRACT FROM AUTHOR]

The DNA-binding protein HU from Escherichia coil is a heterodimer constituted of two poly- peptide chains termed HU-1 and HU-2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino-terminal and carboxy-terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116–120]. The amino acid sequences of proteins HU-1 and HU-2 are identical to those of proteins NS-1 and NS-2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395–398]. The amino acid sequences of proteins HU-1 and HU-2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy-terminal part of the molecule. No sequence homology could be established between the proteins HU-1 and HU-2 and any one of the five histones from different eukaryotes. [ABSTRACT FROM AUTHOR]

1. Leghemoglobin component a from Phaseolus vulgaris (kidney bean)was digested with trypsin; 15 tryptic peptides and free lysine were purified and the amino acid sequences of the peptides determined. 2. The internal order of the tryptic peptides was determined by the bridge peptides obtained from the thermolytic digest and the dilute acid hydrolyzate of kidney bean leghemoglobin α; 12 thermolytic peptides and two acid hydrolysis peptides were purified and the sequences were partially or completely determined. 3. The complete amino acid sequence of kidney bean leghemoglobin a is compared to that of leghemoglobin a from soybean (Glycine max) and to some animal globins. As regards sequence, the kidney bean globin has 79% identity with the soybean globin and 21 % identity with human hemoglobin γ-chain. Seven of the 14 amino acid residues common to most globins are found in the kidney bean globin. Trp-15 and Tyr-145 are evolutionarily conserved in this globin, which confirms the concept of a common origin of animal and plant globins. [ABSTRACT FROM AUTHOR]

The globin of sheep heart myoglobin was first cleaved by cyanogen bromide and the resulting fragments were fractionated by gel filtration and preparative paper electrochromatography; each fragment was submitted to endopeptidase digestion and the complete amino acid sequence has been determined. The intact globin and the median cyanogen bromide peptide were analysed by the protein sequanator (Edman). This analysis confirmed the sequence of the first 46 N- terminal residues of the intact globin and established the sequence of the first 39 N-terminal residues of the median cyanogen bromide peptide. Between sheep and beef myoglobin six differences over 153 residues can be noticed; among the six amino acid replacements, two correspond to the exchange of two bases and four to the exchange of one base in the coding triplets. [ABSTRACT FROM AUTHOR]

Degradation of protein antigens by cellular proteases is a crucial step in the initiation of a T-cell-mediated immune response. But still little is known about the enzymes responsible for the processing of antigens, including their specificity. In this paper, we show that the combination of automated N-terminal sequencing with a newly developed method for C-terminal sequencing of peptide pools generated by the aspartic proteases cathepsins D and E is a fast and/easy method to obtain detailed information of the substrate specificity of these endopeptidases. Using a 15-residue synthetic peptide library and a native protein as substrates, we confirm and extend the knowledge about the cleavage motif of cathepsin E where positions P1 and P1′ of the substrate must be occupied exclusively by hydrophobic amino acids with aromatic or aliphatic side chains. However, Val and Ile residues are not allowed m position P1. Position P2" accepts a broad range of amino acids, including charged and polar ones. Additional requirements concerning the substrate positions P3′ and P4′ were also defined by pool sequencing. Furthermore. pool sequencing analysis of melittin digests with the aspartic proteases cathepsin D and E provided evidence that both enzymes share the same cleavage motif, identical to the one derived from the peptide library, and the native protein. Therefore, pool sequencing analysis is a valuable and fast tool to determine the substrate specificity of any endopeptidase. [ABSTRACT FROM AUTHOR]

According to the signal hypothesis, a signal sequence, once having initiated export of a growing protein chain across the rough endoplasmic reticulum, is cleaved from the mature protein at a specific site. It has long been known that some part of the cleavage specificity resides in the last residue of the signal sequence, which invariably is one with a small, uncharged side-chain, but no further specific patterns of amino acids near the point of cleavage have been discovered so far. In this paper, some such patterns, based on a sample of 78 eukaryotic signal sequences, are presented and discussed, and a first attempt at formulating rules for the prediction of cleavage sites is made. [ABSTRACT FROM AUTHOR]

We have developed a semi-empirical method to determine the subunit molecular weight of a protein. The method is a minor modification of the Edman degradation and is based on a simple chemical procedure to modify specifically the N-terminal NH2 group. Initially, all NH2 groups, including the ε-NH2 groups of lysine residues and the N-terminal α-NH2 group, are reacted with phenylisothiocyanate and the protein derivative is subjected to one step of the Edman degradation. The newly exposed N terminus is then reacted with radioactively labelled phenylisothiocyanate. A value for the subunit molecular weight can be obtained from an analysis of the incorporated radioactivity and the amount of the protein in the sample. The molecular weight of five different proteins have been determined by this method. Our method is particularly useful for proteins containing lipid or sugar components and also for relatively small peptides. The procedure described in this paper for the specific modification of the N terminus has been found to be a powerful tool for protein sequencing. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of mouse pancreatic ribonuclease has been determined by analysis of tryptic, chymotryptic, thermolytic and CNBr peptides and by automatic sequence analysis of the intact protein. The sequence of mouse RNase differs in 20–30% of the positions from other RNase sequences. Three unique or nearly unique substitutions were found, viz. Gly-68 → Arg-68, Arg-85 → His-85 and Ser-123 → Thr-123. All these three residues might be involved in interactions with substrate molecules. A most parsimonious tree of the myomorph rodent RNase shows that after the divergence of rat and mouse, the ribonuclease of rat accumulated substitutions at a rate 2.5–4.3 times as high as the rates in other branches of the tree and 23 times as high as the average rate in the Bovidae ribonuclease evolution. These extreme fluctuations in substitution rate are difficult to reconcile with the hypothesis of the evolutionary clock. The high evolution rate of rat ribonuclease is thought to be caused by positive selection, leading to new functional properties of the enzyme. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the light chain of the mouse monoclonal MOPC 173 immunoglobulin molecule (IgG2a,χ) is presented. This kappa chain contains 214 residues. Comparisons of this sequence with murine kappa chains already published by other workers bring a confirmation of the large size of the murine Vχ chain pool. A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214. [ABSTRACT FROM AUTHOR]

The αA2 chain of bovine α-crystallin was fragmented by means of cyanogen bromide treatment and by tryptic, chymotryptic and thermolytic digestions. Twenty tryptic peptides were obtained from the S-aminoethylated αA2 chain, accounting together for the complete sequence. The direct Edman degradation and the dansyl-Edman technique were used to determine the sequences of the tryptic peptides. The order of the tryptic peptides was deduced from overlapping peptides obtained by cyanogen bromide treatment, tryptic digestion of the maleylated chain and chymotryptic and thermolytic digestion of the S-aminoethylated chain. The sequence of the αA2 chain comprises 173 residues and corresponds to a molecular weight of 19832. [ABSTRACT FROM AUTHOR]

Hydrolysis of the Bordetella pertussis endotoxin, extracted from both ‘hase I’ and ‘hase IV’ bacteria, with 4 M HCl for 1 h at 100 °C, released the disaccharide named in the title; it was isolated by paper electrophoresis or by ion-exchange chromatography in about 1% yield (w/w). The structure of the heptose could be rigorously established by chemical degradation; the facts that the glucosaminidic linkage was hydrolysed by an enzyme preparation containing both, α and β-N-acetylglucosaminidase activities, whereas it was resistant to cleavage by pure β-N-acetyl- glucosaminidase strongly support the assumption that the disaccharide contains an α-D-glucosaminide linkage. [ABSTRACT FROM AUTHOR]

As a final step in the elucidation of the primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, the amino acid sequences of a CNBr peptide (CB1, positions 111-279), that accounts for the middle part of the sequence, and the C-terminal CNBr peptide (CB2, positions 277-394) from the enzyme were determined. Important sequence information was obtained from two subfragments that were formed by the cleavage with CNBr of the Met-Thr sequence (positions 346-347) in peptide CB2. The alignment of the two subfragments from peptide CB2 and three one-residue overlaps between peptides from one of these subfragments were confirmed by investigation of well-resolved parts of a 0.25-nm electron-density map. The sequence of residue 343-346 could not be determined with chemical methods and was assigned from the size and shape of the amino acids in the electron-density map. An important tool in the analysis of the amino acid sequence of peptide CB1 was the proteinase Lys-C fro Lysobacter enzymogenes, which preferentially cleaves at lysine residues. [ABSTRACT FROM AUTHOR]

Fatty acid synthetase was covalently labelled with [14C]palmitic acid from [14C]palmityl-CoA. Tryptic and peptic digestion of the [14C]palmityl enzyme resulted in the formation of radioactive palmityl peptides carrying the long-chain acyl residue both in oxygen-ester and thio-ester linkage. The lipophilic palmityl peptides were purified by column and thin-layer chromatography using organic solvent systems. Peptides arising from the acyl carrier protein, the condensing enzyme and the palmityl transferase were identified and characterized. The amino acid sequence of a 4'-phosphopantetheine- containing peptide was established. It comprises 13 residues and shows a high degree of homology with the acyl carrier protein from Escherichia coli. A heptapeptide and an octapeptide from the palmityl transferase active site were partially sequenced. The identical amino acid composition of palmityl transferase and malonyl transferase core peptides is briefly discussed. [ABSTRACT FROM AUTHOR]

After digestion of protein S4 with trypsin, all 32 tryptic peptides were isolated. Their amino acid compositions were analyzed and the sequence of the amino acids within the tryptic peptides was determined by means of a solid-phase peptide sequenator and by exopeptidases. Alignment of the tryptic peptides was established by analyzing and partially sequencing peptides isolated after digestion of the $4 protein with chymotrypsin, thermolysin and a glutamic-acid-specific protease. Further information about the alignment of peptides came from treatment of S4 with CNBr and with a lysine-modifying reagent. Protein S4 consists of 203 amino acids (Asp8, Asn8, Thr6, Ser10, Glu16, Gln10, Pros, Gly15, Ala18, Va116, Cys1, Met3, Ile8, Leu20, Tyr8, Phe4, His3, Lys20, Arg22 and Trp1) and has a molecular weight of 22550. The basic amino acids are clustered in five regions. Many short repetitions mainly with charged amino acids occur. A prediction is made for regions with or α-helices and with β-sheets. [ABSTRACT FROM AUTHOR]

The protein part of phospholipase A2 from the venom of the common European honey-bee consists of a single chain of 128 amino acid residues [l], eight of which are half-cystine. The absence of free sulphydryl groups indicates that the enzyme contains four disulphide bridges. Digestion of the native enzyme with pepsin resulted in formation of a fragment containing all four disulphide bridges. Further digestion of this fragment with trypsin allowed the isolation of two singly cross-linked peptides, one containing half-cystine residues 37 and 107, and the other containing residues 59 and 99. A fragment containing the remaining two disulphide bridges was digested with dilute sulphuric acid and a peptide was isolated from the digest in which half-cystine residues 9 and 30 were linked. The remaining disulphide bridge is between residues 31 and 89. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of neurotoxin I of the North African scorpion Androctonus australis Hector collected in Algeria (area of Chellala) is described. S-Methylation of cysteine residues formed by reduction of disulphide bonds was found useful for their identification in sequence determination using the phenylisothiocyanate method. From the N-terminal sequence (38 residues) established in the protein sequenator, and from the sequence of chymotryptic peptides the complete sequence of the protein has been deduced. The molecule consists of a single polypeptide chain cross-linked by four disulphide bridges. An isoneurotoxin (I') has been found in the venom of A. australis collected in Tunisia (area of Tozeur). It differs from neurotoxin I in the replacement, of the valine residue in position 17 by an isoleucine residue. [ABSTRACT FROM AUTHOR]

One Bence Jones protein and two Bence Jones fragments (fragmentary chains) of K type, and one Bence Jones protein and two fragments of L type have been analyzed. These proteins have been aminoethylated and digested with proteolytic enzymes. The resulting peptides were separated by different procedures. The peptides obtained were analyzed and could be located in most. cases in the amino acid sequence of the corresponding protein by homology with published sequences. These tentative sequences were particularly satisfactory in the case of the three K type proteins. These belonged to subtype ICE and showed considerable homologies with other KI proteins. It was more difficult to position peptides of the three L type proteins on the basis of homology with published sequences. Several peptides of one protein (BJ 98) which were completely sequenced by Edman degradation combined with proteolytic digestion could not be positioned. Amino acid analysis of peptides obtained from Bence Jones fragments confirmed that these correspond to the N-terminal variable region of Bence Jones proteins for K type fragments, whereas they extend for three residues over the C-terminal constant region for some L type fragments. In any case, no peptide from the constant region of light chains has been isolated from tryptic digests of Bence Jones fragments. [ABSTRACT FROM AUTHOR]

The λ-chains of normal pig γG-immunoglobulin were digested with pepsin in 1 M formic acid. Cystine-containing peptides were isolated by gel filtration on Sephadex G-25 and by chromatography on Dowex 50 and Dowex 1. The following structures were found:[this equation cannot be presented in ASCII text] In addition to these peptides obtained in a high yield similar peptides were found but their complete structures were not determined in view of their low yield. The structures found in the λ-chains display a high degree of similarity with corresponding structures known to exist in human Bence­Jones proteins of the L-type. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of neurotoxin II of the North African scorpion Androctonus auatratis Hector has been determined. From the N-terminal sequence (43 residues) obtained by automatic phenylisothiocyanate degradation in the protein sequenator, and from the sequence of tryptic peptides obtained by digestion of the reduced and methylated toxin of which amino groups had been previously masked by reaction with ethylthioltrifluoroacetate the complete amino acid sequence of the protein has been inferred. It is composed of a single polypeptide chain of 64 amino acid residues, including 8 half-cystines, ended by an histidinamide residue at the C-terminus. [ABSTRACT FROM AUTHOR]

The outstanding feature of a diatom is the species-specific design and ornamentation of the silica-based cell wall, termed frustulum. A new frustulum is shaped in a specialized organelle (silica deposition vesicle) and secreted. Proteins in the lumen of this organelle may control the biomineralization process and are likely to remain associated with the mature cell wall. Therefore a study of the structures of proteins associated with the diatom cell wall was initiated. The complete primary structures of three cell wall proteins (denoted as frustulins) have been determined. In addition, partial amino acid sequences from two more cell wall components were obtained. From these data, a highly conserved domain has been identified as a common building block of diatom cell wall proteins that is repeated several times per polypeptide chain together with polyproline/hydroxyproline or polyglycine spacers. All frustulins characterized so far, are synthesized as preproteins with a novel type of N-terminal presequence. [ABSTRACT FROM AUTHOR]

Little or no endopeptidase activity was detected in extracts from storage organs of dark-grown rice seeds until day 6 of post-imbibition, and the activity expressed per seed increased notably after day 9, reached a maximum on day 18, then decreased. Two major endopeptidases, REP-1 and REP-2, were present in the 40-75% saturated ammonium sulfate fraction from day-9 germinated seeds, and could be separated by hydrophobic column chromatography. REP-1 was further purified to a single polypeptide of 36 kDa. REP-1 digested in vitro both the acidic and basic subunits of rice glutelin, the major seed storage protein of rice. Determination of the N-terminal amino acid sequence and experiments with protease inhibitors indicated that REP-1 is a cysteme endopeptidase. Tile nucleotide sequence of a full-length REP-1 cDNA was determined by a combination of screening of cDNA libraries from rice seeds and the 5′ rapid amplification of cDNA ends technique. [ABSTRACT FROM AUTHOR]

To study the molecular structure and function of pyruvate decarboxylase (PDC) from plants the protein was isolated from pea seeds and partially characterised. The active enzyme which occurs in the form of higher oligomers consists of two different subunits appearing in SDS/PAGE and mass spectroscopy experiments. For further experiments, like X-ray crystallography. It was necessary to elucidate the protein sequence. Partial cDNA clones encoding pyruvate decarboxylase from seeds of Pisum sativum cv. Miko have been obtained by means of polymerase chain reaction techniques. The first sequence were found using degenerate oligonucleotide primers designated according to conserved amino acid sequences of known pyruvate decarboxylases. The missing parts of one cDNA were amplified applying the 3'- and 5' -rapid amplification of cDNA ends systems. The amino acid sequence deduced from the entire cDNA sequence displays strong similarity to pyruvate decarboxylases from other organisms, especially from plants. A molecular mass of 64 kDa was calculated for this protein correlating with estimations for the smaller subunit of the oligomeric enzymes. The PCR experiments led to at least three different clones representing the middle part of the PDC cDNA indicating the existence of three isozymes. Two of these isoforms could be confirmed on the protein level by sequencing tryptic peptides. Only anaerobically treated roots showed a positive signal for PDC mRNA in Northern analysis although the cDNA from imbibed seeds was successfully used for PCR. [ABSTRACT FROM AUTHOR]

Examines the cartography of ribosomal proteins of the 3OS subunit from the halophilic Haloarcula marismortui and complete sequence analysis of protein HS26. Analysis using protein sequence; Encoding of the protein HmaS2 by the opening reading frame orfMSG; Sequence data of a further protein.

Reports on the bacterial expression, characterization and DNA binding studies on Drosophila melanogaster c-MyB DNA-binding protein. Use of polymerase chain reaction amplification and T7 expression vector to overproduce trytophan repeat domain of Drosophila Myb in Escherichia coli; Indication of circular dichroic measurements; Study of the DNA binding properties of the protein using fluorescence spectroscopy.

Discusses a new fungal immunomodulatory protein, FIP-five isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Determination of the apparent molecular mass; Use of protein sequencing techniques to elucidate the complete amino acid sequence; Enhancement of the transcriptional expression of interleukin and interferon.

The total protein mixture of the 30S subunit (TP-30) of the bacterium Thermus thermophilus has been purified using reverse-phase HPLC and the proteins obtained were identified both by means of two-dimensional polyacrylamide gel electrophoresis as well as by amino-terminal amino acid microsequence analysis. The proteins are numbered according to their primary structural similarity with known prokaryotic ribosomal proteins. Eight of them, namely proteins S6, S7, S9, S10, S14, S15, S16, S17 run at different positions in the two-dimensional gel electrophoresis system to those suggested [Sedelnikova, S.C., Agalarov, M.B., Garber, M. & Yusupov, M.M. (1987) FEBS Lett. 220, 227-230]. All characterized proteins are homologous to known ribosomal proteins from other species, except for a small basic protein which shows homology only to a ribosomal protein from spinach chloroplasts [Choli, T., Franceschi, F., Yonath, A. & Wittmann-Liebold, B. (1993) Biol. Chem. Hoppe-Seyler 374, 377-383' Subramanian, A.-R. (1984) Trends Biochem. Sci. 9, 491-494]. [ABSTRACT FROM AUTHOR]

A genomic library from Lactobacillus delbrueckii subsp, bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and tiros unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD+-dependent D-2-hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD+-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp, bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-oxoacid substrates except those having a branched β-carbon. [ABSTRACT FROM AUTHOR]

Human interleukin-4 (IL-4) is a small four-helix-bundly protein which is essential for organizing defense reactions against macroparasites, in particular helminthes. Human IL-4 also appears to exert a pathophysiological role during various IgE-mediated allergic diseases. Seven different monoclonal antibodies neutralizing the activity of human IL-4 were studied in order to identify functionally important epitopes. A collection of 41 purified IL-4 variants was used to analyse how defined amino acid replacements affect binding and thus inhibited IL-4 function. However, other mAbs also inhibiting IL-4 function recognized an epitope on helix C of IL-4 and did not inhibit IL-4 binding to the receptor protein. One mAB, recognizing N-terminal and C-terminal residues, partially competed for binding to the receptor. The results of these mAb epitope analyses confirm and extend previous data on the functional consequences of the amino acid replacements which showed that amino acid residues in helices A and C of IL-4 provide a binding site for the cloned IL-4 receptor and that a signaling site in helix D interacts with a further receptor protein. [ABSTRACT FROM AUTHOR]

An acidic endoprotease (MAEP) secreted during vegetative growth by Myxococcus xanthus Dk101 was purified to homogeneity by a series of chromatographic procedures. The endoprotease cleaved the Phe-Met bond of κ-casein under acidic conditions (pH 5.9). Its apparent molecular mass and its isoelectric point have been estimated to be 12kDa and 4.5, respectively. From the N-terminal amino acid sequence, a set of two primers for polymerase chain reaction have been designed. Amplification of the corresponding DNA fragment (84 bp) generated a probe, then used to screen an expression DNA library of M. xanthus and to isolate a recombinant plasmid which contained a 2127-bp insert. The nucleotide sequence included an open reading frame (ORF) of 585 nulceotides, encoding 195 amino acids, that exhibited a high degree of similarity with the N-terminal amino acid sequence of the purified MAEP. The polypeptide sequence inferred from this ORF revealed that the mature enzyme should contain 131 amino acids arising from a 195-amino-acid precursor protein. [ABSTRACT FROM AUTHOR]

Pyridoxal-5'-phosphate-dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio-specificity. In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the imine linkage with the coenzyme. This family was thus named a family. It comprises glycine hydroxymethyltransferase, glycine C-acetyltransferase, 5-aminolevulinate synthase, 8-amino-7-oxononanoate synthase, all aminotransferases (with the possible exception of subgroup III), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol-lyase which, however, catalyze β-elimination reactions. The β family includes [- and Dserine dehydratase, threonine dehydratase, the β subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze β-replacement or β-elimination reactions. The ), family incorporates O-succinylhomoserine (thiol)-lyase, O-acetylhomoserine (thiol)-lyase, and cystathionine γ-1yase, which catalyze γ-replacement or γ-elimination reactions, as well as cystathionine β-lyase. The a and γ family might be distantly related with one another, but are clearly not homologous with the β family. Apparently, the primordial pyridoxal-5'-phosphate-dependent enzymes were reglo-specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal-5'-phosphate-dependent enzymes seem to be unrelated with the α, β or γ family by the criterion of profile analysis: alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B[SUB6] enzymes. [ABSTRACT FROM AUTHOR]

Salivary proline-rich proteins have a repetitive primary structure particularly rich in the amino acids proline, glutamine and glycine. One of the biological roles of these proteins is to bind and precipitate polyphenois (vegetable tannins) present in the diet (e.g. tea, coffee, fruit, chocolate) neutralising their harmful actions which include nutritional loss, inhibition of gut enzymes and oesophageal cancer. Two peptides overlapping in sequence, corresponding to the mouse salivary proline-rich protein MP5 repeat sequence: QGPPPQGGPQQRPPQPGNQ and GPQQRPPQPGNQQGPPPQGGPQ have been synthesised and studied in H[SUB2],O/([SUP2]H[SUB6])dimethyl sulphoxide (9:1, by vol.) using [SUP1]H-NMR spectroscopy. Low-temperature far-ultraviolet CD spectroscopy and NMR conformational parameters indicate that the peptides adopt an extended random coil conformation in solution. There is no evidence for a defined polyproline type II helix in the peptides, despite the high proline content. NMR data show that the trans-proline isomer predominates to at least 90%. [ABSTRACT FROM AUTHOR]

We have isolated the basement membrane component nicein and performed rotary-shadow analyses using electron microscopy that showed the presence of two forms (I and II) of the protein. Molecular cloning of the cDNA that codes for the 100-kDa chain of the protein revealed that the sequence matches those independently identified for the 105–155-kDa subunit of kalinin, a recently identified basement membrane component. These data demonstrate that nicein and kalinin contain an identical chain. The length of the open reading frame in the cDNA (∼5200 nucleotides) and amino acid sequences obtained from the N-terminus of the 105-kDa kalinin chain showed the occurrence of a precursor polypeptide. This immature polypeptide is probably related to form I, observed by rotary shadowing, while the mature form is related to form II. It is noteworthy that nicein/kalinin subunits share discrete sequence similarities with the B2 chain of human laminin, but with a cleavage occurring within domain III that eliminates domains IV and V from the final product. The sequence of this subunit is nearly identical to that of B2t, a recently described polypeptide supposed to be related to a new laminin variant. Since nicein/kalinin expression is specifically impaired in the severe genodermatosis Herlitz junctional epidermolysis bullosa, the role and structure of this tissue-restricted laminin variant is crucial for the understanding of epidermal-demal adhesion. [ABSTRACT FROM AUTHOR]

Determines the primary structures of two basic low-molecular-mass proteins from the pharate cuticle of the migratory locust, Locusta migratoria. Combined use of plasma-desorption mass spectrometry and automatic Edman degradation in the sequencing strategy; Strong similarity shown by both proteins to other exocuticular proteins from L. migratoria.

The primary structure of an intracellular ribonuclease (RNase LX) from cultured tomato (Lycopersicon esculentum) cells has been determined. Previous studies have shown that the protein is located inside the tomato cells but outside the vacuoles and that its synthesis is induced alter depleting thc cells for phosphate [Löffler, A., Abel, S., Jost. W., Beintema, J. J., Glund. K. (1992) Plant Physiol. 98. 1472–14781]. Sequence analysis was carried out by analysis of peptides isolated after enzymatic and chemical cleavage of the protein. RNase LX consists of 213 amino acids and has a molecular mass of 24300 Da and an isoelectric point of 5.33. The enzyme contains 10 half-cystines and there are no potential N-glycosylation sites detectable in the sequence. RNase LX, as compared to an extracellular tomato RNase (RNase LE), which is also phosphate regulated and the amino acid sequence of which was recently established [Jost, W., Bak, H., Glund, K., Terpstra, P. & Beintema, J. J. (1991) Eur. J. Biochem. 198. 1–6] has 60% of all amino acids identical and in identical positions, revealing a high degree of similarity between both proteins. In contrast to RNase LE, RNasc LX has a C-terminal extension of nine amino acids. The C-terminal tetrapeptide HDEF may be a retention signal of the protein in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

The 80-kDa actin-fragmin kinase (AFK) was purified from Physarum polycephalum microplasmodia to apparent homogeneity through a procedure involving six chromatographic steps. Taking the activity at the first purification step as 100%, the kinase was purified more than 1500-fold, with an overall yield of 8%. The specific activity of the purified enzyme was 700 U/mg. The total amount of AFK present could be estimated as 34 ng/mg extracted protein. One of the polycional antibodies raised against four tryptic peptides of the purified 80-kDa AFK recognized the 80-kDa band in an immunoblot and could inhibit the AFK activity up to 100%. A minor 78-kDa protein, also displaying AFK activity, appeared to be derived from the 80-kDa AFK. A partial amino acid sequence analysis. covering up to 20% of the protein (150 amino acids), was performed and confirmed the lack of similarity with any of the sequenced kinases. These data are in agreement with the unique physical and enzymatic properties of the AFK. [ABSTRACT FROM AUTHOR]

A protein responsible for clot formation was isolated from plasma of the crayfish Pacifastacus leniusculus, by repeated precipitation at low ionic strength, pH 6.0. The protein, here named clotting protein (CP), is a lipoglycoprotein, which consists of two 210-kDa subunits, covalently associated by disulfide bonds. Preparations of the CP can form stable clots in the presence of crayfish haemocyte lysate supernatant, which contains endogenous, Ca(2+)-dependent transglutaminase (TGase) activity. The covalent, TGase-mediated polymerization of CP could clearly be visualized in SDS/PAGE under reducing conditions, where the 210-kDa subunit is covalently cross-linked into dimeric, trimeric and higher polymeric forms. The CP was shown to be a substrate for transglutaminases, since two different fluorescent TGase substrates, namely dansylcadaverine and a dansylated glutamine-containing peptide, were incorporated into the CP subunit by active TGase. This indicates the presence of both glutamine and lysine residues in the CP, accessible for TGase cross-linking. The amino acid composition and the N-terminal amino acid sequence of the clotting protein was determined. [ABSTRACT FROM AUTHOR]

A purification procedure consisting of DEAE-Sephacel chromatography, heparin-Sepharose CL-6B chromatography and Mono-S chromatography led to the isolation of three proteins endowed with RNase activity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. They were referred to as p1, p2 and p3, according to their elution order from the Mono-S column. Complete amino acid sequence of p2 and partial sequence of p3 displayed high sequence similarity to the 7-kDa DNA-binding proteins previously isolated in Sulfolobus strains [Choli, T., Wittman-Liebold, B. & Reinhardt, R. (1988) J. Biol. Chem. 263, 7087-7093]. The molecular mass of p2, calculated from sequence data, was 7.02 kDa, which compares fairly well with the value of 7.4 kDa determined by SDS/PAGE. Gel filtration of the molecule under native conditions displayed, however, a largely prevailing form with an assessed molecular mass of 13.0 kDa, which points to a dimeric structure. Kinetic characterization of protein p2 showed a broad pH optimum in the range 6.7 - 7.6 using yeast RNA as substrate; also, it was shown that activity was unaffected by EDTA, Mg2+ and phosphate. The enzyme did not accept as substrate any homopolyribonucleotide, which points to a rather narrow substrate specificity. This was also confirmed by incubating p2 with tRNAfMet Met (fMet, N-formylmethionine) from Escherichia coli: the hydrolysis products were thus identified as 3'-phosphooligonucleotides. [ABSTRACT FROM AUTHOR]

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB- encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182–191 ). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coil is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coil using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays. [ABSTRACT FROM AUTHOR]

Three tryptic-peptide sequences of an 88-kDa pair of phosphoproteins of the malaria parasite Plasmodium falciparum were determined. They exhibited a striking similarity to corresponding sequences of the 89-kDa domain of human erythrocyte ankyrin. [35S]Methionine labeling of the two proteins demonstrated their parasitic origin. Using an appropriate oligonucleotide probe, Southern-blot analysis of genomic malaria DNA and Northern-blot analysis of malaria RNA suggest the existence of ankyrin-related sequences in the parasite genome and the presence of an ankyrin-related transcript of about 3.2 kb. Our studies provide further evidence of malaria-specific analogues of host-cells proteins, implying an unusual kind of parasite/host interaction. [ABSTRACT FROM AUTHOR]

Resident proteins of the endoplasmic reticulum lumen are continuously retrieved from an early Golgi compartment by a receptor-mediated mechanism. The sorting or retention sequence on the endoplasmic reticulum proteins is located at the C-terminus and was initially shown to be the tetrapeptide KDEL in mammalian cells and HDEL in Saccharomyces cerevisiae. The carboxylesterases are a large family of enzymes primarily localized to the lumen of the endoplasmic reticulum. Retention sequences in these proteins have been difficult to identify due to atypical and heterogeneous C-terminal sequences. Utilizing the polymerase chain reaction with degenerate primers, we have identified and characterized the C-termini of four members of the carboxylesterase family from rat liver. Three of the carboxylesterases sequences contained C-terminal sequences (HVEL, HNEL or HTEL) resembling the yeast sorting signal which were reported to be non-functional in mammalian cells. A fourth carboxylesterase contained a distinct C-terminal sequence, TEHT. A full-length esterase cDNA clone, terminating in the sequence HVEL, was isolated and was used to assess the retention capabilities of the various esterase C-terminal sequences. This esterase was retained in COS1 cells, but was secreted when its C-terminal tetrapeptide, HVEL, was deleted. Addition of C-terminal sequences containing HNEL and HTEL resulted in efficient retention. However, the C-terminal sequence containing TEHT was not a functional retention signal. Both HDEL, the authentic yeast retention signal, and KDEL were efficient retention sequences for the esterase. These studies show that some members of the rat liver carboxylesterase family contain novel C-terminal retention sequences that resemble the yeast signal. At least one member of the family does not contain a Cterminal retention signal and probably represents a secretory form. [ABSTRACT FROM AUTHOR]

The nucleotide sequence encoding the chloroplast enzyme, sedoheptulose-1,7-bisphosphatase [Sed(1,7)P2ase], was obtained from wheat cDNA and genomic clones. The transcribed region of the Sed(1,7)P2ase gene has eight exons (72–507 bp) and seven introns (85–626 bp) and encodes a precursor polypeptide of 393 amino acids. Comparison of the deduced amino acid sequence of Sed(1,7)P2ase with those of fructose-1,6-bisphosphatase (Fru(1,6)P2ase] enzymes from a variety of sources reveals 19% identity, rising to 42% if conservative changes are considered. Most importantly, the amino acid residues which form the active site of Fru(1,6)P2ase are highly conserved in the Sed(1,7)P2ase molecule, indicating a common catalytic mechanism. Interestingly, although the activities of both Sed(1,7)P2ase and chloroplast Fru(1,6)P2ase are modulated by light via the thioredoxin system, the amino acid sequence motif identified as having a role in this regulation in chloroplast Fru(1,6)P2ase is not found in the Sed(1,7)P2ase enzyme. [ABSTRACT FROM AUTHOR]

We have cloned and sequenced mouse brain AP47, the medium chain of the trans-Golgi network clathrin-associated protein complex AP-1. The predicted protein sequence of AP47 is closely related to rat and calf brain AP50, the corresponding medium chain of the plasma-membrane clathrin-associated protein complex AP-2. We have also identified in the yeast genome an open reading frame encoding a protein of previously unknown function. Referred to here as YAP54, its predicted protein sequence displays a striking homology to AP47. We therefore propose that Yap54 is the medium chain subunit of a putative AP-1 complex in yeast. From the analyses of the optimized sequence alignments of AP47, AP50 and Yap54p, we suggest a model for the domain organization of the medium chains. [ABSTRACT FROM AUTHOR]

The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C-terminus of the molecule. Since MIP is though to function as a channel protein in lens plasma membranes, possibly as a cell-to-cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termed in vivo phosphorylation. Purified membranes were also phosphorylated with cAMP-dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP-derived peptides on different HPLC columns and to determine possible cAMP-dependent protein kinase phosphorylation sites. Lens membranes, which contain 50-60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C-terminal region of MIP and a major product of 21-22 kDa remained membrane-associated. Separation of the lysylendopeptidase-C-released peptides on C8 reversed-phase HPLC demonstrated that one of these fragments, corresponding to residues 239-259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC. In vivo phosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with cAMP-dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for the in vivo phosphorylation demonstrated at serine 245. [ABSTRACT FROM AUTHOR]

A 3133-bp nucleotide sequence of the gene Paz1 on chromosome 4 of barley, encoding endosperm protein Z4, has been determined. The sequence includes 1079 bp 5' upstream and 523 bp 3' downstream of the coding region. The 1079-bp 5' upstream region of the gene shows little similarity to 5' regions of other sequenced genes expressed in the developing cereal endosperm. The coding sequence is interrupted by one 334-bp-long intron (bases 1497-1830). The deduced amino acid sequence, which was corroborated by peptide sequences, consists of 399 amino acids and has a molecular mass of 43 128 Da. This sequence confirms protein Z4 to be a member of the serpin superfamily of proteins. The similarity with other members of the family expressed as amino acids in identical positions is in the order of 25-30% and pronounced in the carboxy-terminal half of the molecule. Sequence residues assumed to form clusters stabilizing the tertiary structure are highly conserved. Protein Z4 is synthesized in the developing endosperm without a signal peptide and protein Z4 mRNA was evenly distributed among the free and membrane-bound polyribosomes of the endosperm cell. An internal hydrophobic region of 21 amino acids (residues 36-56) may serve as a signal for targeting the polypeptide into the lumen of the endoplasmic reticulum. The gene for protein Z4 could not be detected in the barley variety Maskin and some of its descendants. The 'high-lysine' allees, lysl (Hiproly barley) and lys3a (Bomi mutant 1508) on chromosome 7, enhance and repress, respectively, the expression of the protein Z4. Also, 1554 bp of another 8-kbp fragment of the barley genome PazΨ, similar to the protein-Z4-coding region, have been determined. Small insertions and deletions and the presence of an internal stop codon identify this fragment as part of a pseudogene related to the protein Z4 gene. [ABSTRACT FROM AUTHOR]

The first two steps of de novo pyrimidine synthesis in Saccharomyces cerevisiae are catalyzed by a multifunctional protein, coded by the URA2 gene and which has the carbamoyl-phosphate (CPSase) synthetase and aspartate transcarbamylase (ATCase) activities. The native enzyme purified from protease-B-deficient URA2- transformed cells, was phosphorylated in vitro using catalytic subunits of pure cAMP-dependent protein kinase. After electrophoresis under denaturing conditions, a single 240-kDa species was found to be phosphorylated. Trypsin digestion of this species gave a single, very acidic phosphopeptide upon isoelectric focussing. Purification by HPLC followed by amino acid sequencing of this peptide, showed a phosphoserine at the expected consensus sequence Arg-Arg-Phe-Ser. Knowledge of the URA2 gene sequence allowed the site to be located in the peptide link between dihydroorotase-like and ATCase domains. Such a location may explain why phosphorylation of the URA2 protein changed neither CPSase and ATCase activities nor their sensitivity to UTP, their common specific inhibitor. [ABSTRACT FROM AUTHOR]

Attenuated total reflection Fourier-transform infrared spectroscopy of thin hydrated films of soluble and membrane protein included in a phospholipid bilayer is shown to provide useful information as to the secondary structure of the protein. The analysis of the amide I band of deuterated samples by Fourier self-deconvolution followed by a curve fitting was performed by a new procedure in which all the input parameters are generated by the computer rather than by the investigator. The results of this analysis provide a correct estimation of the α-helix and β-sheet structure content with a standard deviation of 8.6% when X-ray structures are taken as a reference. We also show that the orientation of the different secondary structures resolved by the Fourier self-deconvolution/ curve-fitting procedure and of the phospholipid acyl chains can be simultaneously evaluated for membrane proteins reconstituted in a lipid bilayer. Of special interest for reconstitution of membrane proteins, the lipid/ protein ratio can be accurately and quickly determined from the infrared spectrum. [ABSTRACT FROM AUTHOR]

EF-Tu is often referred to as a model for guanine-nucleotide-binding regulatory proteins (G-proteins), since X-ray diffraction analysis of its GTP-binding domain shows a detailed location of the ‘consensus’ amino acid sequences involved in nucleotide binding. Fluoroaluminates are thought to mimick the γ-phosphate in the GTPase centre on account of their activating effect on a variety of GTP binding proteins. In the case of EF-Tu, we could find no such effects on the basis of at least three independent functional assays. We did notice, however, complicating interactions between free nucleotides, fluoroaluminates and other ligands. By consequence, if indeed A1F4- behaves as a γ-phosphate analogue in G-proteins, then EF-Tu must have a different GDP/GTP binding site, despite of the conserved consensus sequences. [ABSTRACT FROM AUTHOR]

Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. Ca2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228–239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins. [ABSTRACT FROM AUTHOR]