Amino Acid Sequence Of Fragment Research Articles

Biochemical characterization of an alkaline metallopeptidase secreted by a Pseudomonas fluorescens isolated from soil

The extracellular endopeptidase synthesized by soil bacterium Pseudomonas fluorescens was purified to homogeneity in a four-step procedure. The enzyme was purified 45-fold, with a 20% recovery. The endopeptidase appeared to be a monomer with a molecular mass of approx. 50 kDa. The enzyme was active in the pH range of 7 to 10. The optimal activity was detected at pH 9.0 and at 42 degrees C. Enzyme activity was inhibited by EDTA, EGTA and 1,10 phenanthroline, typical metalloprotease inhibitors. Ca(2+) activated the enzyme while Zn(2+), Co(2+), Cd(2+)(in high concentration) strongly inhibited it. The presence of calcium ions strongly stabilized the enzyme with regard to thermal resistance. The amino acid sequence of fragments of the enzymatic protein determined by MS analysis revealed a high similarity to the sequences of other alkaline metalloendopeptidases of bacteria belonging to the genus Pseudomonas.

Molecular composition of progenitor toxin produced by Clostridium botulinum type C strain 6813.

The molecular composition of the purified progenitor toxin produced by a Clostridium botulinum type C strain 6813 (C-6813) was analyzed. The strain produced two types of progenitor toxins (M and L). Purified L toxin is formed by conjugation of the M toxin (composed of a neurotoxin and a non-toxic nonhemagglutinin) with additional hemagglutinin (HA) components. The dual cleavage sites at loop region of the dichain structure neurotoxin were identified between Arg444-Ser445 and Lys449-Thr450 by the analyses of C-terminal of the light chain and N-terminal of the heavy chain. Analysis of partial amino acid sequences of fragments generated by limited proteolysis of the neurotoxin has shown to that the neurotoxin protein produced by C-6813 was a hybrid molecule composed of type C and D neurotoxins as previously reported. HA components consist of a mixture of several subcomponents with molecular weights of 70-, 55-, 33-, 26 through 21- and 17-kDa. The N-terminal amino acid sequences of 70-, 55-, and 26 through 21-kDa proteins indicated that the 70-kDa protein was intact HA-70 gene product, and other 55- and 26 through 21-kDa proteins were derived from the 70-kDa protein by modification with proteolysis after translation of HA-70 gene. Furthermore, several amino acid differences were exhibited in the amino acid sequence as compared with the deduced sequence from the nucleotide sequence of the HA-70 gene which was common among type C (strains C-St and C-468) and D progenitor toxins (strains D-CB16 and D-1873).

Differentiation between cucurbit yellow stunting disorder virus and beet pseudo‐yellows virus by a reverse transcription‐polymerase chain reaction assay

Two whitefly‐transmitted viruses elicit identical symptoms of interveinal chlorosis (yellowing) in greenhouse‐ and field‐grown cucurbits in Mediterranean countries. The agents of these diseases are partially characterized closteroviruses and are known as beet pseudo‐yellows virus (BPYV), which is transmitted by Trialeurodes vaporariorum, and cucurbit yellow stunting disorder virus (CYSDV), transmitted by Bemisia tabaci. Using previously determined sequence information for fragments of the HSP70 homolog genes of both BPYV and CYSDV, oligonucleotide primers were designed for reverse transcription‐polymerase chain reaction (RT‐PCR) protocols to differentiate between the two viruses. RT‐PCR amplified products for each virus were subsequently cloned and specifically hybridized with the homologous dsRNA (approximately 9 kbp in size for both viruses) following Northern hybridization analysis. Minor dsRNAs of approximately 5 kbp for both viruses and one dsRNA of 3 kbp for CYSDV were also identified by Northern hybridization and might represent defective RNA species. In order to compare the predicted amino acid sequence of fragments of the HSP70 homolog genes of BPYV and CYSDV, oligonucleotide primers were designed for RT‐PCR amplification of a previously unknown segment of the BPYV gene from a Greek isolate and the American isolate of the virus. The RT‐PCR products of both isolates were cloned and found to be identical in sequence. Computer assisted analysis of the deduced amino acid sequence of the assembled HSP70 homolog gene fragments showed that BPYV and CYSDV are closely related to each other and individually as closely related to the corresponding gene of lettuce infectious yellows virus, which is the prototype member of the recently proposed Crinivirus genus of whitefly‐transmitted, bipartite closteroviruses.

A very limited number of keywords (main patterns) describes all sequences of the human variable heavy (VH) and kappa (Vkappa) domains.

Sequences of the variable heavy (VH) and kappa (Vkappa) domains of Ig structures were divided into 21 fragments that correspond to strands, loops, or parts of these structural units of the variable domains. Amino acid sequences of fragments (termed "words") were collected from the 1,172 human heavy and 668 human kappa chains available in the Kabat database. Statistical analysis of words of 17 fragments was performed (fragments that comprise the complementary determining regions' fragments will not be discussed in this paper). The number of different words (those with different residues in at least one position) ranged, for various fragments, from 11 to 75 in the kappa chains, and from 23 to 189 in the heavy chains. The main result of this study is that very few keywords, or main patterns of words, were necessary to describe over 90% of the sequences (no more than two keywords per fragment in the kappa and no more than five per fragment in the heavy chains). No identical keywords were found for different fragments of the variable domains. Keywords of aligned fragments of the VH and Vkappa domains were different in all but two instances. Thus, knowing the keywords, one can determine whether any given small part of a sequence belongs to a heavy or kappa chain and predict its precise localization in the sequence. In addition, by using all of the keywords obtained through analysis of the Kabat database, it was possible to describe completely the sequences of the human VH and Vkappa germ-line segments.

Cloning, nucleotide sequence, and overexpression in Escherichia coli of the ?-tyrosinase gene from an obligately symbiotic thermophile, Symbiobacterium thermophilum

Symbiobacterium thermophilum is an obligately symbiotic thermophile that can grow only in coculture with a specific Bacillus strain. The amino acid sequences of fragments obtained by cyanogen bromide decomposition of the thermostable beta-tyrosinase (tyrosine phenol-lyase, E.C. 4.1.99.2) from this organism resembled that of the tryptophanase produced by the same organism. DNA-probing with the tryptophanase gene as the hybridization probe led to cloning in Escherichia coli of the beta-tyrosinase (tpl) gene. The nucleotide sequence revealed that the beta-tyrosinase of 458 amino acids (relative molecular mass, 52269) showed significant similarity in amino acid sequence to the tryptophanase over the entire sequence. DNA manipulation of the cloned tpl gene in E. coli led to production of 375 times as much beta-tyrosinase as that produced by the original S. thermophilum strain.

Identification of genes encoding two capsid proteins (VP24 and VP26) of herpes simplex virus type 1.

The capsid of herpes simplex virus type 1 is composed of seven proteins. VP5, VP19C, VP22a and VP23 have been shown previously to be the products of genes UL19, UL38, UL26.5 and UL18, respectively. The genes encoding VP21, VP24 and VP26 have not been identified to date. We have determined amino acid sequences of fragments of isolated capsid proteins generated by partial cleavage with CNBr. The results confirm the gene assignments for VP5, VP19C and VP23. They also show that VP26 is the product of gene UL35 and that VP24 contains the protease domain present in the N-terminal portion of the UL26-encoded protein. VP21 was not investigated, but we suggest that it is the C-terminal portion of the UL26-encoded protein remaining after release of VP24 and that it thus corresponds to a form of VP22a extended at the N terminus.

Neuronal Survival Factor from Bovine Brain Is Identical to Neuron‐Specific Enolase

Neuronal survival factors in the central nervous system were investigated by using a primary culture of embryonic rat neocortical neurons. Bovine hippocampus was homogenized, and the supernatant from high-speed centrifugation was used as the starting material. At the step of DE-52 ion-exchange chromatography, neuronal survival activity was recovered in two fractions, fraction 14 (F14) and fraction 23 (F23). Antisera to the crude F14 and F23 fractions were raised in rabbits. These two antisera completely inhibited the neurotrophic activity of both fractions. Western blotting analysis revealed that anti-F14 antiserum recognized mainly a 30-kDa protein in F14 and anti-F23 antiserum recognized mainly a 44-kDa protein in F23. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of F23, the 44-kDa protein was cut out from the gel and partial amino acid sequences of the protein fragments were determined. A GenBank data bank indicated that the amino acid sequence of the fragment was identical to that of neuron-specific enolase (NSE). In our assay system, commercially available NSE itself possessed neuronal survival activity for the cultured neocortical neurons. The effects of NSE and F23 were inhibited completely by anti-NSE polyclonal antibody. Furthermore, highly purified NSE supported the survival of cultured neurons in a dose-dependent manner, and the neurotrophic effect was inhibited by monoclonal antibody to the NSE. These results strongly suggest that NSE is one of the neuronal survival factors in the central nervous system.

Tryptic and chymotryptic cleavage sites in sequence of α-subunit of (Na + + K +)-ATPase from outer medulla of mammalian kidney

Localization of selective proteolytic splits in α-subunit of (Na + + K +)-ATPase is important for understanding the mechanism of active Na +,K +-transport. Proteolytic fragments of α-subunit from pig kidney were purified by chromatography in NaDodSO 4 on TSK 3000 SW columns. NH 2-terminal amino acid sequences of fragments as determined in a gas phase sequenator were unambiguously located within the total sequence of α-subunit from sheep kidney (Shull, C.E., et al. (1985) Nature 316, 691–695) and pig kidney (Ovchinnikov, Y.A., et al. (1985) Proc. Acad. Sci. USSR 285, 1490–1495). The primary chymotryptic split in the E 1-form is located between Leu-266 and Ala-267 while the tryptic cleavage site appears to be between Arg-262 and Ile-263 (Bond 3). Tryptic cleavage in the initial fast phase of inactivation of the E 1-form is located between Lys-30 and Glu-31 (Bond 2). In the E 2-form, primary tryptic cleavage is between Arg-438 and Ala-439 (Bond 1). Chymotryptic cleavage between Leu-266 and Ala-267 stabilizes the E 1-form of the protein without affecting the sites for binding of cations or nucleotides. Titration of fluorescence responses demonstrates the importance of the NH 2-terminal for E 1-E 2 transition. Protonation of His-13 facilitates transition from E 1- to E 2-forms of the protein. Removal of His-13 after cleavage of bond 2 can explain the increase in apparent affinity of the cleaved enzyme for Na + and the shift in poise of E 1-E 2 equilibrium in direction of E 1-forms. The NH 2-terminal sequence in renal α-subunit is not conserved in α + from rat neurolemma or in α-subunit from Torpedo or brine shirmp. A regulatory function of the NH 2-terminal part of the α-subunit may thus be a unique feature of the α-subunit in (Na + + K +)-ATPase from mammalian kidney.

Sequence analysis of the membrane protein V3 of the flavivirus west nile virus and of its gene

Flaviviruses contain a large membrane-associated protein V3, having a mol mass of about 50 kDa which is responsible for hemagglutination. We have isolated the V3 protein from the West Nile (WN) flavivirus and determined its amino-terminal amino acid sequence and amino acid sequences of fragments derived from this protein. We have also transcribed parts of the WN virus genome RNA into cDNA and cloned and sequenced this CDNA. The results of these analyses have allowed us to identify the region of the viral genome coding for the V3 protein. In this report we describe the total nucleotide sequence of the genome region coding for the WN virus V3 protein and the amino acid sequence of the V3 protein derived from these analyses. The exact carboxy terminus of the V3 protein has not been determined in these experiments. These analyses have shown that the V3 protein of WN virus does not contain an Asn-X-Ser/Thr sequence which could allow addition of N-linked carbohydrate chains to this protein. In accordance with this finding, analyses of metabolic labeling of the V3 protein using [ 3H]glucosamine indicate that the WN virus V3 protein is an unglycosylated protein. Together with our earlier analyses these results show that the viral structural proteins are present on the genome RNA in the order 5′-terminuscore protein (V2)-small membrane-associated protein (NV2)-large membrane-associated protein (V3) and describe the nucleotide sequences coding for all WN virus structural proteins identified so far. A hypothesis concerning the processes involved in the synthesis of all viral structural proteins and the probable orientation of these proteins relative to the endoplasmatic reticulum membrane based on the structure of these proteins is discussed.

The complete amino acid sequence of diphtheria toxin fragment B. Correlation with its lipid-binding properties

The complete amino acid sequence of fragment B from diphtheria toxin has been determined. The polypeptide chain was split with cyanogen bromide, o-iodosobenzoic acid, clostripain and trypsin; all amino acid sequence analyses were made by automated Edman degradation. Fragment B, which corresponds to the carboxy terminus of the toxin molecule, contains 342 amino acids and has an M r of 37 240. The proposed amino acid sequence fully confirms the structure recently deduced from the nucleotide sequence of the structural gene. The complete sequence is analyzed in relationship with the role of fragment B in the transfer of diphtheria toxin fragment A from the extracellular medium into the cell cytoplasm.

Amino acid sequence of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli.

We propose a primary structure for the catalytic subunit of aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli based on amino acid sequences of fragments obtained by cyanogen bromide cleavage, by tryptic digestion of the succinylated polypeptide, and by chymotryptic and proteinase C digestion of the intact catalytic chain. The protein contains 310 amino acids and has a calculated molecular weight of 33,944. The negatively and positively charged residues are distributed uniformly, and there is no indication of charge clustering in the linear sequence.

Primary structure of the polypeptide chain elongation factor Tu from E. coli. I. Amino acid sequence of fragment B.

The complete amino acid sequence of Fragment B obtained by the limited tryptic digestion of E. coli polypeptide chain elongation factor Tu (EF-Tu) was determined. Seven peptides formed from Fragment B by cleavage with cyanogen bromide (designated as CB1 to CB7 according to their order of alignment from N- to C-termini of Fragment B) were purified, and six of them were completely sequenced by the manual method of sequential Edman degradation with direct identification of the phenylthiohydantoin-amino acids. The remaining one cyanogen bromide peptide (CB6) containing 109 amino acid residues was further digested with trypsin. Twelve tryptic peptides (designated as T1 to T12 according to their order of alignment from N- to C-termini of CB6) were isolated, and their amino acid sequences were analyzed. The alignment of CB peptides was based on the results of the automated sequence analysis of Fragment B from its N-terminal, and the sequence analysis of the overlapping peptides containing sulfhydryl groups obtained by the complete tryptic digestion of Fragment B. The alignment of peptides T1 to T12 on CB6 was based on the result of the automated sequence analysis of CB6, and the sequence of the overlapping peptide obtained by the chemical cleavage of CB6 at the tryptophan residue using cyanogen bromide in heptafluorobutyric acid. The nucleotide sequence of the tuf A gene was also utilized for the alignment of these peptides. Fragment B comprises amino acid residues 59 to 263 of E. coli EF-Tu, which consists of 393 amino acids. It contains two functional (SH1 and SH2) and one non-functional (SH3) sulfhydryl groups of EF-Tu. All of the five histidine residues in Fragment B were distributed within the first N-terminal quarter, and three of them were found to be clustered around SH2. Although E. coli EF-Tu consists of two gene products (tuf A and tuf B), no microheterogeneity was found in the amino acid sequence of Fragment B.

Structure of rabbit skeletal myosin: Analysis of the amino acid sequences of two fragments from the rod region

The amino acid sequences of fragments from light meromyosin and heavy meromyosin subfragment-2 have been analysed and structural features noted. As with other α-fibrous protein sequences, there is a regular disposition of apolar residues in positions a and d of the heptapeptide-type repeat characteristic of the coiled-coil conformation. The common occurrence of acidic and basic residues in the e and g positions, respectively, give rise to a maximum number of interchain ionic interactions when the two parallel chains of myosin are in axial register. Although the quasi-repeating heptapeptides in the sequences both have two points of discontinuity (unlike that in most other α-fibrous proteins), secondary structure prediction methods indicate that the fragments will be 90 to 100% α-helical. Fast Fourier transform techniques have revealed a significant periodicity of about 27.4 ± 0.3 residues (~41 Å) in the linear disposition of the acidic residues and the basic residues in both of the fragments. This period is compatible with similarly directed myosin molecules in the thick filament being axially staggered with respect to one another by an odd multiple of 143 Å. Preliminary evidence is also presented to show that the sequence of the rod region of myosin may have a 28 residue gene duplication repeat.

Rabbit immunoglobulin lambda chains: Amino acid sequences of variable- and constant-region peptides from normal and allotype-suppressed rabbits

The amino acid sequences of fragments from the variable and constant regions of rabbit IgG λ-chains from different sources were determined and compared. The peptides had considerable homology with known sequences of human and murine λ-chains and, thus, could be readily aligned. Sequences of corresponding peptides from different sources and preparations reveal differences in the variable as well as the constant region. The latter probably reflect the existence of different alleles and/or isotypes at the c-locus, each coding for a different constant half of the λ-chain. Comparison of the rabbit λ-light-chain sequences with those of human and mouse λ-chains reveals a surprising degree of homology. The amino acid sequences of the peptides reported cover approximately one half of the total sequence of the light chains.

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. III. The chymotryptic peptides, the peptides derived by cleavage at tryptophan residues, and the complete sequence of the protein.

Fragment A (21,145 daltons in its longest known form) may be derived from diphtheria toxin (60,000 daltons) by mild tryptic digestion and reduction. Purified Fragment A consists of a mixture of 3 molecules of 190, 192, and 193 residues; the first 190 residues are in common and correspond to the NH2-terminal region the toxin. All three species of Fragment A are active in catalyzing ADP ribosylation of elongation factor 2, an essential component of protein synthesis. This reaction inactivates the factor and is responsible for the toxin's action in inhibiting protein synthesis in animal cells. It is believed that Fragment A or similar enzymically active fragments released into the cytosol of toxin-treated cells mediate this inhibition. The complete amino acid sequence of Fragment A has been determined from 32 chymotryptic peptides, three peptides derived by chemical cleavage of Fragment A at its 2 tryptophan residues, five cyanogen bromide peptides, and six tryptic peptides from the maleylated protein.

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. I. The tryptic peptides from the maleylated protein.

Six tryptic peptides ranging in size from 3 to 126 residues were isolated from maleylated Fragment A of diphtheria toxin after tryptic hydrolysis. These peptides accounted for all 193 residues found by amino acid analysis. After demaleylation, the six peptides were purified by chromatography on Sephadex G-50, coupled with paper chromatography and electrophoresis, and were analyzed by various methods. The compositions and properties of the peptides are reported. Almost 70% of the residues were positioned within these peptides.

Amino acid sequences of fragments I and II obtained by cyanogen bromide cleavage of rat serum albumin.

The amino acid sequences of fragment I, the N-terminal cyanogen bromide fragment, and fragment II, a fragment between the first and second methionine residues, of rat serum albumin were determined by conventional methods in consideration of the sequences of human and bovine serum albumin. These sequences were compared with those of human and bovine serum albumin.

Alignment of the tryptic fragments and location of sulfhydryl groups of the polypeptide chain elongation factor Tu

Summary A limited hydrolysis with trypsin of the polypeptide chain elongation factor Tu (EF-Tu) in the native state has been investigated in detail. The reaction proceeded in two stages. In the first stage, the protein was cleaved into two polypeptides, with molecular weights of 39,000 and 8,000, which were designated as Fragments A and D, respectively. In the second stage, the Fragment A was transformed to Fragments B (M.W. 23,000) and C (M.W. 12,000) which still existed as a complex molecule containing Fragment D. Analyses of N- and C-terminal sequences of the intact EF-Tu as well as these 4 fragments have revealed that Fragments D and C were derived from N- and C-termini of EF-Tu, respectively. Further, the amino acid sequence of Fragment B containing all of the three sulfhydryls of EF-Tu has been investigated. On cleavage of Fragment B with cyanogen bromide, three sulfhydryl-containing polypeptides were obtained, i.e. a 3000-dalton peptide containing the sulfhydryl essential for the binding of guanine nucleotides (SH1), a 5000-dalton peptide containing the sulfhydryl essential for aminoacyl-tRNA binding (SH2), and a 10,000-dalton peptide containing the non-reactive sulfhydryl (SH3). From the partial sequence determination of these three peptides, the location of the sulfhydryls was determined as in the order of SH2, SH1, and SH3 from the N- to C-termini of EF-Tu.

Human fibrinogen--amino acid sequence of fragment E and of adjacent structures in the Aalpha and Bbeta -chains.

Abstract Fragment E from a plasmic digest of human fibrinogen has been studied. Cleavage of Fragment E with cyanogen bromide produced three fragments: a core fragment which contains all disulfide bridges of Fragment E and constitutes (Aα1–51, β 54–118, γ1–58)2 (Kowalska-Loth, B. et al. Thromb. Res. 2 , 423, 1973) and two peptides, one of which constitutes α52–81 and the other β 119–121. Two peptides were isolated from cyanogen bromide cleaved fibrinogen which on the basis on their amino acid sequence proved to commence at Aα52 and Bβ 119. The peptide derived from the Aα-chain was completely sequenced and was found to constitute α52–91. The peptide derived from the Bβ -chain was partly sequenced (24 of the approximately 62 residues, i.e. β 119–142). From the latter sequence it was concluded that the NH2-terminal amino acid of the β-chain derivative of the plasmic Fragment D is β 134.

Primary Structure of Bovine Plasma High-Molecular-Weight Kininogen. The Amino Acid Sequence of a Glycopeptide Portion (Fragment 1) Following the C-Terminus of the Bradykinin Moiety1

On incubation of bovine plasma high-molecular-weight (HMW) kininogen with purified plasma kallikrein [EC 3.4.21.8], a large glycopeptide fragment and the vasoactive peptide, bradykinin, were initially liberated; the former, named fragment 1-2, was subsequently cleaved into fragment 1 (glycopeptide) and the previously established fragment 2 (histidine-rich peptide). The isolated fragment 1-2 contained a total of 108 to 110 amino acid residues and carbohydrates, and the amino-terminal sequence Ser-Val-Gln was established. The other fragment, fragment 1, consisted of a total of 69 amino acid residues with serine and arginine (and lysine) at the amino and carboxyl termini, respectively. It contained eleven residues each of histidine and glycine, together with an oligosaccharide chain consisting of galactosamine, hexose and sialic acid. The complete amino acid sequence of fragment 1 was determined by Edman degradation and standard enzymatic and chemical techniques. These results established the following sequence: H-Ser-Val-Gln-Val-Met-Lys-Thr-Glu-Gly-Ser-Thr-Pro/Thr-Val-Ser(CHO)-Val/Leu-Pro-His-Ser-Ala-Met-Ser-Pro-Val-Gln-Asp-Glu-Glu-Arg-Asp-Ser-Gly-Lys-Glu-Gln-Gly-Pro-Thr-His-Gly-His-Gly-Trp-Asp-His-Gly-Lys-Gln-Ile-Lys-Leu-His-Gly-Leu-Gly-Leu-Gly-His-Lys-His-Lys-His-Asp-Gln-Gly-His-Gly-His-His-Lys/ArgOH.