Covalent Structure of Botulinum Neurotoxin Type E: Location of Sulfhydryl Groups, and Disulfide Bridges and Identification of C-Termini of Light and Heavy Chains

Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an 150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992), Biochem. Biophys. Res. Commun. 183, 107–113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein—at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)—is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419–421 (Gly–Ile–Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1–Lys 418 constitute the 50–kDa light chain, Lys 422–Lys 1251 constitute the 100–kDa heavy chain; Cys 411–Cys 425 and Cys 1196–Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The 150–kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without ^I4C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nucleotide sequence.     

Comparison of the gastric exocrine inhibitory activities and plasma kinetics of somatostatin-28 and somatostatin-14 in cats

The gastric exocrine inhibitory activities of somatostatin-28 (SS-28) and somatostatin-14 (SS-14) were determined in conscious cats prepared with gastric fistulae. Gastric acid and pepsin secretions were stimulated with pentagastrin. Expressed in terms of exogenous doses, SS-14 (ID50: 1.49 nmol . kg-1 . h-1) was 3.4 times more potent than SS-28 (ID50: 5.12 nmol . kg-1 . h-1) as an inhibitor of gastric acid secretion. Similarly SS-14 (ID50: 0.25 nmol . kg-1 . h-1) was 3.8 times more potent than SS-28 (ID50: 0.96 nmol . kg-1 . h-1) as an inhibitor of pepsin secretion. Expressed in terms of circulating plasma concentration measured by radioimmunoassay, SS-14 (ID50: H+, 232 and pepsin 73 pM) was 8-9 times more potent than SS-28 (ID50: H+, 2112 and pepsin, 611 pM) as an inhibitor of gastric exocrine secretions. The plasma immunoreactive half-life of SS-28 (6.1 min) was double that for SS-14 (2.4 min) possibly due to a slower theoretical metabolic clearance rate of the larger peptide (30 and 87 ml . kg-1 . min-1, respectively). Both peptides had similar apparent distribution volumes (SS-14, 306 and SS-28, 263 ml . kg-1). As judged by gel chromatography of plasma samples, there was no evidence for the conversion of SS-28 to SS-14 in vivo. The reduced activity of SS-28, compared with SS-14, against gastric exocrine secretions contrasts with its more potent effects in the pituitary and pancreas.     

Revised 2.3 A structure of porcine pepsin: evidence for a flexible subdomain

A revised three-dimensional crystal structure of ethanol-inhibited porcine pepsin refined to an R-factor of 0.171 at 2.3 A resolution is presented and compared to the refined structures of the fungal aspartic proteinases: penicillopepsin, rhizopuspepsin, and endothiapepsin. Pepsin is composed of two nearly equal N and C domains related by an intra dyad. The overall polypeptide fold and active site structures are homologous for pepsin and the fungal enzymes. The weak inhibition of pepsin by ethanol can be explained by the presence of one or more ethanol molecules, in the vicinity of the active site carboxylates, which slightly alter the hydrogen-bonding network and which may compete with substrate binding in the active site. Structural superposition analysis showed that the N domains aligned better than the C-domains for pepsin and the fungal aspartic proteinases: 107-140 C alpha pairs aligned to 0.72-0.85 A rms for the N domains; 64-95 C alpha pairs aligned to 0.78-1.03 A rms for the C domains. The major structural difference between pepsin and the fungal enzymes concerns a newly described subdomain whose conformation varies markedly among these enzyme structures. The subdomain in pepsin comprises nearly 100 residues and is composed of two contiguous segments within the C domain (residues 192-212 and 223-299). the subdomain is connected, or "hinged," to a mixed beta-sheet that forms one of the structurally invariant, active site psi-loops. Relative subdomain displacements as large as a 21.0 degrees rotation and a 5.9 A translation were observed among the different enzymes. There is some suggestion in pepsin that the subdomain may be flexible and perhaps plays a structural role in mediating substrate binding, determining the substrate specificity, or in the activation of the zymogen.     

Circannual and Circa-Hemiannual Rhythms of Basal and Stimulated Gastric Secretion in Conscious Cats

Conscious cats equipped with a gastric fistula and a denervated Heidenhain pouch were submitted to weekly measurements of the basal and pentagastrin-stimulated gastric secretion for 1 to 14 years. Rhythms of basal secretion were documented in 37 cats for the group studies, in 25 cats only for the individual studies which required at least whole year data. Twelve-month or 6-month rhythms were detected for each variable studied, i.e. volume, acid, pepsin, fucose and uronic acid outputs in the group studies, with peaks for volume, acid and pepsin in Winter, peaks for uronic acid in Spring and Fall indicating different rhythms for oxyntic, chief and mucous cells. Individual studies detected rhythms in 25% of the analyses, and demonstrated male and female and cat to cat differences. Spectral analysis in 3 cats confirmed the differences in the individual rhythms with prominent peaks differing from 365 days in 50% of the cases. Chronopharmacological responses to pentagastrin were documented for volume, acid and pepsin outputs in 5 male and 6 female cats. Group analysis detected a Winter acrophase for volume and acid secretion and a Summer acrophase for pepsin secretion. Analysis of the stimulated response data showed interindividual variation but a higher percentage of detection for rhythms, i.e. 38% for all variables and 50% for pepsin secretion. Different rhythms in acid and pepsin secretion documented in individual studies could provide the basis of a better understanding of the discrepancies reported in the literature concerning the seasonal incidence of peptic ulcer disease.     

凡纳滨对虾虾头内源性蛋白酶同源性分析

以凡纳滨对虾 (Litopenaeus vannamei shrimp) 虾头为原料,采用Q- Sepharose F F和Sephadex G-150对虾头内源碱性蛋白酶进行了纯化,通过SDS-PAGE测定分子量为79.95 kD|采用DEAE-Sepharose F.F和Sephadex G-100对内源酸性蛋白酶进行了纯化,通过SDS-PAGE测定分子量为27.45 kD. 利用HPLC-ESI-MS/MS对虾头内源碱性和酸性蛋白酶同源性进行了初步分析,将检测到内源性蛋白酶的部分氨基酸序列分别与不同物种的胰蛋白酶和胃蛋白酶氨基酸序列于Vector NTI suite 8.0软件上进行序列比对. 结果表明,内源性碱性蛋白酶与猪胰蛋白酶具有很高的同源性,均含有氨基酸序列LSSPATLNSRVATVSLPR|内源性酸性蛋白酶与非洲蟾蜍胃亚蛋白酶具有很高的同源性,均含有氨基酸序列EFGLSETEPGTNF.     

Fractionation of the multiple forms of bovine gastric aspartic proteases by chromatofocusing

By extending the chromatofocusing technique to a very acidic pH range (down to pH 2.0) a method which, in a single-step procedure, allows separation of the three main aspartic proteases secreted by the bovine abomasal mucosa i.e., chymosin (EC 3.4.23.4), gastricsin (EC 3.4.23.3), and pepsin A (EC 3.4.23.1), has been developed. Starting materials for separation were crude commercial milk-clotting extracts or abomasal juices. A multistep procedure, using narrower pH gradients, enabled the fractionation of these proteases into their multiple forms. Chymosins A and B, which are known to differ only by a single amino acid substitution (Asp/Gly), were completely resolved. Their elution pHs, 3.75 and 3.80, respectively, though far from their "normal" pIs (around 4.7 in isoelectric focusing), demonstrate the resolving power of such a technique. Multiple forms of bovine pepsin A, which differ in their organic phosphate content (0-3 phosphate group(s) per molecule of enzyme) and whose pIs are lower than 2.5, were also separated using 15-20 mM glycine buffer, pH 2.0, as eluent. Although many attempts to get a linear gradient remained unsuccessful within this pH range, resolution appeared quite satisfactory, as judged from analytical isoelectric focusing patterns. In particular, the two subcomponents of bpA1, which presumably have a different site of post-translational phosphorylation, were resolved in this way.     

胃内PGE和PGF_(2α)介导胃蛋白酶的适应性细胞保护作用的观察

胃粘膜细胞不断合成和释放的前列腺素(PGs),具有很强的细胞保护作用。我们的前一工作表明,预先用胃蛋白酶灌胃,可防止牛磺胆酸所致的胃粘膜坏死的发生,这一保护作用可被消炎痛所阻断;间接提示这种适应性细胞保护的发生机制可能与内源性PGs有关。本文则用放射免疫方法,直接测定了胃粘膜组织PGE和PGF_(2α)含量在不同情况下的变化。结果表明,单纯胃蛋白酶225U,胃蛋白酶150U溶于 0.1N HCl或75单位溶于 0.2N HCl中,提前 15min灌胃,均可防止由0.2N NaOH、0.6N HCl 和无水乙醇所致的胃粘膜坏死的发生,这种保护作用呈明显的量效关系。上述三种配方的胃蛋白酶溶液灌胃后15min,胃粘膜组织PGE和PGF_(2α)含量明显升高,分别为对照组的2.7—2.9倍和1.9—2.5倍;且以PGE含量的上升占优势。进一步观察不同浓度胃蛋白酶溶于生理浓度盐酸中对胃粘膜PG含量的影响发现,胃蛋白酶与胃粘膜组织PGE和PGF_(2α)含量的增加呈现明显的量效关系。这些结果说明,胃蛋白酶作为弱刺激对强酸、强碱和无水乙醇所致的胃粘膜损伤均有保护作用,其作用机制则是通过诱发内源性PGs的合成和释放而实现的。     

Botulinum type A neurotoxin digested with pepsin yields 132, 97, 72, 45, 42, and 18 kD fragments

Botulinum neurotoxin (NT) serotype A is a dichain protein made of a light and a heavy chain linked by at least one interchain disulfide; based on SDS-polyacrylamide gel electrophoresis their molecular masses appear as 147, 52, and 93 kD, respectively. Digestion of the NT with pepsin under controlledpH (4.3 and 6.0), time (1 and 24 hr), and temperature (25 and 30°C) produced 132, 97, 42, and 18 kD fragments. The three larger fragments were isolated by ionexchange chromatography. The 132 and 97 kD fragments are composed of 52 kD light chain and 72 and 45 kD fragments of the heavy chain, respectively. The sequences of amino terminal residues of these fragments were determined to identify the pepsin cleavage sites in the NT, which based on nucleotide sequence has 1295 amino acid residues (Binzet al., J. Biol. Chem. 265, 9153, 1990). The 42 kD fragment, beginning with residue 866, is the C-terminal half of the heavy chain. The 18 kD fragment, of which the first 72 residues were identified beginning with residue 1147, represents the C-terminal segment of the heavy chain. The 132 kD fragment (residue 1 to 1146) is thus a truncated version of the NT without its 18 kD C-terminal segment. The 97 kD fragment (residue 1 to 865) is also a truncated NT with its 42 kD C-terminal segment excised. These peptic fragments contain one or two of the three functional domains of the NT (binds receptors, forms channels, and intracellularly inhibits exocytosis of the neurotransmitter) that can be used for structure-function studies of the NT. This report also demonstrates for the first time that of the six Cys residues 453, 790, 966, 1059, 1234, and 1279 located in the heavy chain the later four do not form interchain disulfide links with the light chain; however, Cys 1234 and 1279 contained within the 18 kD fragment form intrachain disulfide. The electrophoretic behaviors of type A NT and its fragments in native gels and their comparison with botulinum NT serotypes B and E as well as tetanus NT suggest that each NT forms dimers or other aggregates and the aggregation does not occur when the 42 kD C-terminal half of the heavy chain is excised. Thus, the C-terminal half of the heavy chain appears important in the self-association to form dimers.