The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.     

Oligomeric and subunit structure of the Helicobacter pylori vacuolating cytotoxin

Disease-associated strains of Helicobacter pylori produce a potent toxin that is believed to play a key role in peptic ulcer disease in man. In vitro the toxin causes severe vacuolar degeneration in target cells and has thus been termed VacA (for vacuolating cytotoxin A). Cytotoxic activity is associated with a > 600-kD protein consisting of several copies of a 95-kD polypeptide that undergoes specific proteolytic cleavage after release from the bacteria to produce 37- and 58-kD fragments. Quick freeze, deep etch electron microscopy has revealed that the native cytotoxin is formed as regular oligomers with either six- or seven-fold radial symmetry. Within each monomer, two domains can clearly be distinguished, suggesting that the 37- and 58-kD fragments derive from proteolytic cleavage between discrete subunits of the monomer. Analysis of preparations of the toxin that had undergone extensive cleavage into the 37- and 58-kD subunits supports this interpretation and reveals that after cleavage the subunits remain associated in the oligomeric structure. The data suggest a structural similarity with AB-type toxins.     

Primary structure of the peptidoglycan-derived tracheal cytotoxin of Bordetella pertussis

The etiological agent of whooping cough, Bordetella pertussis, destroys the ciliated epithelial cells lining the large airways of infected individuals. This cytopathology can be reproduced in respiratory epithelium by tracheal cytotoxin (TCT), a small peptidoglycan-related molecule purified from the culture supernatant of growing B. pertussis organisms. Using fast atom bombardment mass spectrometry, we analyzed the positive- and negative-ion spectra of the purified, biologically active material and assigned a mass of 921 daltons to TCT. Analysis of fragment ions in these spectra as well as the spectra of the methyl ester and acetylated derivatives of TCT unambiguously defined the primary structure of TCT as N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramylalanyl-gamma- glutamyldiaminopimelylalanine. TCT is therefore identical with the ciliostatic anhydropeptidoglycan monomer released by Neisseria gonorrhoeae and with the neurologically active slow-wave sleep-promoting factor FSu. These and other structurally related glycopeptides containing muramic acid thus form a family of molecules with remarkably diverse biological activities.     

The primary structure of aphrodisin

Aphrodisin is a protein which is secreted in hamster vaginal discharge and acts via the vomeronasal organ of the accessory olfactory system to elicit copulatory behavior in male hamsters. The complete primary structure of aphrodisin was determined by sequence analysis of intact aphrodisin after unblocking the amino terminus with pyroglutamate aminopeptidase and from peptides generated by trypsin and Lys-C digests. Alignment of the peptides was obtained from sequence analysis of peptides from cyanogen bromide and hydroxylamine cleavages. The protein consists of 151 residues of Mr = 17,000. It has disulfide bonds linking cysteine residues at positions 38 and 42 and at 57 and 149. N-acetylglucosamine residues are linked to asparagines at positions 41 and 69. Based on its similarity to the major urinary proteins in rats and mice, aphrodisin is a putative member of the alpha 2u-globulin superfamily of extracellular proteins.     

The primary structure of alamethicin

Alamethicin, an antibiotic that can transport cations and induce action potentials in synthetic membranes, is shown to be a cyclic peptide with 18 residues including 7-alpha-aminoisobutyric acid residues, two glutamine residues and one free carboxyl group. The composition indicates microheterogeneity. Alamethicin itself and many peptides derived from it are immune to enzymic digestion, but specific partial acid cleavages have allowed determination of the complete sequence. Diborane reduction has shown that the alpha-carboxyl group of glutamine-18 is free, but the ring is formed by a peptide bond between the imino group of proline-1 and the gamma-carboxyl group of glutamic acid-17. The structure is contrasted with that of other cation-transporting antibiotics. Model building allows a structure that could stack to form a tunnel with a lipophilic exterior and hydrophilic interior and flexible internal arms formed by the pendant C-terminal glutamine residue.     

The symplectic structure of the primary visual cortex

We propose to model the functional architecture of the primary visual cortex V1 as a principal fiber bundle where the two-dimensional retinal plane is the base manifold and the secondary variables of orientation and scale constitute the vertical fibers over each point as a rotation–dilation group. The total space is endowed with a natural symplectic structure neurally implemented by long range horizontal connections. The model shows what could be the deep structure for both boundary and figure completion and for morphological structures, such as the medial axis of a shape.     

Production and localization of restrictocin in Aspergillus restrictus.

The production and secretion of restrictocin (a cytotoxin that cleaves ribosomal RNA) by cultures of the fungus Aspergillus restrictus was investigated. Previous studies have indicated that restrictocin production in liquid culture coincides with the appearance of differentiated cell structures. A study of the correlation between the appearance of differentiated structures and restrictocin production was conducted with A. restrictus grown on agar medium. Restrictocin was found to be associated with the cell mass of the agar-grown culture (in contrast to liquid cultures), and was first observed when aerial hyphae emerged. Restrictocin levels increased until the time of conidiation, after which they fell off sharply. No restrictocin could be found in the agar medium. The presence of restrictocin upon and within various cell structures was determined by immunofluorescent laser microscopy. This study showed that restrictocin became localized to the conidiophores and phialides during the process of conidiation. Prior to this, restrictocin was found within the hyphae in localized concentrations that may correspond to secretory vesicles.     

The primary structure of the ovine beta-caseins.

Ovine whole casein contains 2 multiphosphorylated beta-casein components designated as beta 1 and beta 2-caseins. The complete sequence of beta 1-casein and the partial sequence of beta 2-casein have been determined from cyanogen bromide and tryptic digests. The ovine beta 1 and beta 2-caseins have the same polypeptide chain and appear to differ only in that they contain 6 and 5 phosphates respectively. The amino acid composition of ovine beta 1-casein can be written as: Asp4, Asn4, Thr10, ThrP1, Ser9, SerP5, Glu19, Gn21, Pro34, Gly5, Ala4, Val21, Met6, Ile9, Leu22, Tyr3, Phe9, Trp1, Lys12, His5, Arg3. Compared to bovine beta-casein A2, which is made up of 209 residues, ovine beta 1-casein has a deletion of 2 residues (either Pro-179--Tyr-180 or Tyr-180--Pro-181) and 20 largely conservative amino acid substitutions. Although 20% of the substitutions involve proline residues, the proline contents of ovine beta 1 and bovine beta A2-caseins are very similar, around 16%. The average hydrophobicity, calculated according to Bigelow, is 5.51 kJ/residue, which is similar to that calculated for bovine beta-casein A2. The cluster of 4 phosphorylated serine residues and the highly charged nature of the amino terminal region observed for bovine beta-casein are conserved in the ovine beta-caseins. The substitution from Ile-12 (bovine) to Thr-12 (ovine) results in a new phosphorylation site, according to the phosphorylation code proposed for caseins. This site is only partially phosphorylated hence the occurrence of both beta 1 and beta 2-caseins in ovine milk.     

幽门螺杆菌空泡毒素研究进展

幽门螺杆菌空泡毒素是该菌产生的已知其它细菌毒素无明显源性的唯一蛋白毒素。该毒素是幽门螺杆菌重要的毒力致病因子,它的产生与感染胃肠上皮损伤和溃疡形成密切相关。本就幽门螺杆菌空泡毒素的结构与功能研究进展以及在未来免疫预防与免疫治疗中的作用进行了阐述。     

The vacuolating cytotoxin of Helicobacter pylori

Helicobacter pylori, the causative agent of chronic superficial gastritis and duodenal ulcer disease in humans, produces a unique cytotoxin (VacA) that induces cytoplasmic vacuolation in eukaryotic cells. The structural organization and processing of the vacuolating cytotoxin are characteristic of a family of proteins exemplified by Neisseria gonorrhoeae IgA protease. Although only 50% of H. pylori isolates produce detectable cytotoxin activity in vitro, vacA homologues are present in virtually all isolates. Several families of vacA alleles have been identified, and there is a strong correlation between presence of specific vacA genotypes, cytotoxin activity, and peptic ulceration. Experiments in a mouse model of H. pylori-induced gastric damage indicate that the cytotoxin plays an important role in inducing gastric epithelial necrosis.     

The primary structure of myohemerythrin.

The complete amino acid sequence of muscle hemerythrin (myohemerythrin) from the sipunculid Themiste (syn. Dendrostomum) pyroides has been determined by analysis of tryptic, chymotryptic, and cyanogen bromide peptides. The primary structure of myohemerythrin differs substantially from that of coelomic hemerythrins of Phascolopsis (syn. Golfingia) gouldii and Themiste pyroides, the amino acid sequence of the muscle protein being only 46 and 45% homologous with the respective coelomic hemerythrins. The most extensive regions of homology between muscle and coelomic proteins occur near the terminii. These and other shorter regions of homology are interpreted in terms of the essential iron ligand residues of the active center.     

Role of individual cysteine residues and disulfide bonds in the structure and function of Aspergillus ribonucleolytic toxin restrictocin.

Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins. It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis. Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147. These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131. In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides. The mutants were expressed and purified from Escherichia coli. Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro. However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities. The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion. Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin. For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required.     

Regulation of restrictocin production in Aspergillus restrictus.

The production of restrictocin (a cytotoxin that specifically cleaves ribosomal RNA) by cultures of Aspergillus restrictus grown in liquid medium was investigated. The function of restrictocin, the method of its accumulation and the mode of resistance to restrictocin in A. restrictus are unknown. Previous studies have indicated that restrictocin accumulates in the medium with culture age. These observations have been extended in this study by cloning the cDNA of the res gene and using this cDNA clone to probe the onset of messenger RNA synthesis in the cells. The results of the Northern analysis were compared to the production and accumulation of restrictocin and morphological differentiation of the cells in culture. Restrictocin was found in the medium at the same time that mRNA was detected in the cells. This suggests that the leader sequence encoded by the cDNA provides an efficient secretion system for the protein. Both the protein and the mRNA were detected coincident with the formation of differentiated cell structures. These structures develop into conidiophores with one layer of sterigmata and conidia forming from the sterigmata. These results suggest that restrictocin is either involved in the process of conidiation or is coordinately regulated with differentiation leading to conidiation.     

The primary structure of the Chloroflexus aurantiacus reaction-center polypeptides

The complete nucleotide sequence of two Chloroflexus aurantiacus reaction-center genes has been obtained. The amino acid sequence deduced from the first gene showed 40% similarity to the L subunit of the Rhodobacter sphaeroides reaction center. This L subunit was 310 amino acids long and had an approximate molecular mass of 35 kDa. The second gene began 17 bases downstream from the first gene. The amino acid sequence deduced from it (307 amino acids; 34950 Da) was 42% similar to the M subunit of the Rhodobacter sphaeroides reaction center. 20% of the deduced primary structure were confirmed through automated Edman degradation of cyanogen bromide peptide fragments or N-chlorosuccinimide peptide fragments isolated from the purified reaction-center complex or from the individual subunits. The peptides were isolated by preparative gel electrophoresis combined with molecular sieve chromatography in the presence of a mixture of formic acid, acetonitrile, 2-propanol and water. This method appeared to be applicable to the isolation of other hydrophobic proteins and their peptides.     

The primary structure of rabbit muscle enolase

The primary amino acid sequence of rabbit muscle enolase has been determined by standard spinning-cup sequencing techniques applied to peptides produced by chemical (cyanogen bromide and mild acid hydrolysis) and enzymatic fragmentation of the enzyme. The 433 amino acid sequence has been compared to other available enolase sequences from eukaryotic and prokaryotic sources, confirming a high degree of conserved sequence identity; the three mammalian muscle sequences (mouse and rat deduced from c-DNA sequences and rabbit) show 94% identity.     

The primary structure of mouse saposin.

The primary structure of mouse sphingolipid activator protein (saposin) was determined by cDNA sequencing. The amino acid sequence predicted by the cDNA sequence revealed that mouse saposin was highly homologous to human saposin and also to rat sertoli cell glycoprotein. Mouse saposin also has four functional domains, which are structurally similar to each other, and each domain has cysteines, prolines, and a potential glycosylation site at an almost identical position. An amino acid comparison between human and mouse saposins revealed that the similarity was approximately 70%, and human saposin lacks thirty-one amino acids between domains C and D. Heterogeneities of mRNA were found in both the coding and noncoding regions.