Diversity of light chain variable region sequences among rabbit antibodies elicited by the same antigens.
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We report the complete variable region sequences of three homogeneous rabbit antibody light chains and the partial sequences of five others. Wehn these are compared to other published rabbit light chain sequences, two regions of markedly increased variability are revealed, which are homologous in position to the first and third hypervariable regions of murine and human myeloma light chains. In addition, there is increased variability among the first three residues at the aminoterminal end. A hypervariable region homologous to that identified at positions 50 to 56 in myeloma light chains is not present in these rabbit antibody light chains. The available three-dimensional models of Fab fragments based on x-ray crystallography indicate that neither the amino-terminal portion of the light chain nor the region homologous to positions 50 to 56 forms a part of the combining site. Comparison of the hypervariable regions among six light chains from antibodies to Type III pneumococcal polysaccharide and among four from antibodies to Type VIII pneumococcal polysaccharide suggests that a large number of different sequences may be found in antibodies specific for these relatively simple antigens. Certain residues outside of the hypervariable regions are invariant in the rabbit light chains and correspond to residues that are required for proper chain folding in human and murine myeloma light chains, indicating that the general conformation of myeloma light chains is the same as that of light chains of elicited antibodies. (+info)
Alterations in secretory patterns following antrectomy in rats with Pavlov pouches.
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1. In conscious rats provided with Pavlov pouches, with the antrum retained or resected,the gastric secretory response to various stimuli has been studied. Each acid secretory response was related to that obtained with maximal doses of methacholine and histamine in combination, presumed to reflect the maximal secretory capacity of the mucosa. 2. Three weeks after the operation, the maximal acid secretory capacity was 60 percent lower in the antrectomized than in the intact Pavlov pouch rats; the difference was still larger at 6 weeks and 3-5 months, owing to a gradual increase in the rats with the antrum retained. 3. Antrectomy reduced interdigestive secretion of acid to the same degree as the concomitant reduction in maximal secretory capacity. 4. Acid secretion in response to a maximal infusion of pentagastrin was reduced by about 50 percent at 3 and about 65 percent at 6 weeks after antrectomy. No significant difference was, however, noted between the antrectomized and intact rats when the responses were related to the maximal secretory capacity. The dose response curve to pentagastrin revealed a redcued responsiveness to submaximal doses of this agent following antrectomy. 5. The maximal acid secretory response to histamine was reduced after antrectomy, although the sensitivity to submaximal infusions of histamine appeared to be increased. 6. The mean secretroy output to 2-deoxy-D-glucose was reduced by about 65 percent and that to food by about 85 percent following antrectomy. 7. After antrectomy a background infusion of pentagastrin enhanced the secretory responses to 2-deoxy-D-glucose and to food but did not restore the responses to the levels in the intact rats. The feeding responses as related to the maximal secretory capacity were, however, similar in the two groups on infusing pentagastrin in the antrectomized rats. 8. Interdigestive secretion of pepsin was reduced by about 60 percent after antrectomy, while the peak response to 2-deoxy-Dglucose was about twice the interdigestive level in both groups. Pepsin secretion in response to food showed an increased secretion above the interdigestive level of longer duration in the antrectomized than in the intact Pavlov pouch rats. 9. The irreversibily reduced responsiveness of the gastric mucosa after antrectomy is discussed in relation to known morphological and biochemical changes. (+info)
Primary structure of porcine pepsin. I. Purification and placement of cyanogen bromide fragments and the amino acid sequence of fragment CB5.
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Fragments resulting from the cyanogen bromide cleavage of reduced and aminoethylated porcine pepsin were purified. Only four of the five fragments theoretically present could be accounted for in major yield when the cyanogen bromide reaction was carried out at room temperature. The NH2-terminal fragment, CB2, contained an internal homoserine which was not cleaved to any significant extent. The amino acid sequence around this internal homoserine was determined by isolating and partially determining the sequence of an alpha-chymotryptic peptide. Cleavage at this methionine was increased by 50% when the cyanogen bromide reaction was carried out at 37 degrees. The NH2- and COOH-terminal sequences of five major fragments were determined. The placement of these fragments in the native pepsin molecule was demonstrated. The amino acid sequence of one of the fragments, CB5, was determined. This fragment contains 44 residues with an internal disulfide bridge. The COOH-terminal methionine of this fragment was connected to another 37-residue cyanogen bromide fragment of known sequence. Together these two fragments formed the COOH-terminal 81 residues of porcine pepsin. (+info)
Primary structure of porcine pepsin. II. Amino acid sequence of two cyanogen bromide fragments, CB3 and CB4.
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The amino acid sequences of two cyanogen bromide fragments from porcine pepsin have been determined. Fragment CB3 which represents the NH2-terminal 80 residues of pepsin was assembled from the peptides purified from proteolytic digests of this fragment using alpha-chymotrypsin, thermolysin, and staphylococcal protease. Two chymotryptic peptides were isolated from the NH2-terminal region of this fragment. One of these contains 2 extra residues, Ala-Leu-, at the NH2 terminus. This peptide is apparently derived from a different cleavage site of pepsinogen in its conversion to pepsin. The second cyanogen bromide fragment, CB4, contains 47 residues. The sequence was established from the peptides resulting from proteolytic digests using alpha-chymotrypsin, alpha-lytic protease, and thermolysin. An isoleucyl residue at position 29 of fragment CB4 appears to be absent in some molecules. This represents a structural variant of pepsin. (+info)
Primary structure of porcine pepsin. III. Amino acid sequence of a cyanogen bromide fragment, CB2A, and the complete structure of porcine pepsin.
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The complete amino acid sequence of porcine pepsin (EC 3.4.4.1) was constructed from the sequence of five cyanogen bromide fragments. The sequence of one of these fragments, CB2A, is reported here. The sequences of 4 other fragments are known from previous work. Porcine pepsin contains 327 residues with three structural variants. The active center aspartyl residue, which reacts with 1,2-epoxy-3-(p-nitrophenoxy)propane (Chen, K. C. S., and Tang, J. (1972) J. Biol. Chem. 247, 2566-2574), is located at residue 32. Another active site aspartyl residue, which reacts with diazo inactivators (Bayliss, R. S., Knowles, J. B., and Wybrandt, G. B. (1969) Biochem. J. 113, 377-386, IS LOCATED AT RESIDUE 215. The sequences around these 2 aspartyl residues are apparently homologous to each other. The sequences around the tryptophanyl residues at positions 39, 141, 181, and 300 are also homologous to one another. These homologous sequences could be genetic in origin. Fragment CB2A which contains 119 residues was constructed from the peptide sequences resulting from six proteolytic digestions and chemical cleavage at tryptophanyl bonds. (+info)
Pepsin-mediated processing of the cytoplasmic histone H2A to strong antimicrobial peptide buforin I.
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The intestinal epithelium forms a first line of innate host defense by secretion of proteins with antimicrobial activity against microbial infection. Despite the extensive studies on the antimicrobial host defense in many gastrointestinal tracts, little is known about the antimicrobial defense system of the stomach. The potent antimicrobial peptide buforin I, consisting of 39 aa, was isolated recently from the stomach tissue of an Asian toad, Bufo bufo gargarizans. In this study we examined the mechanism of buforin I production in toad stomach tissue. Buforin I is produced by the action of pepsin isozymes, named pepsin Ca and Cb, cleaving the Tyr39-Ala40 bond of histone H2A. Immunohistochemical analysis revealed that buforin I is present extracellularly on the mucosal surface, and unacetylated histone H2A, a precursor of buforin I, is localized in the cytoplasm of gastric gland cells. Furthermore, Western blot analysis showed that buforin I is also present in the gastric fluids, and immunoelectron microscopy detected localization of the unacetylated histone H2A in the cytoplasmic granules of gastric gland cells. The distinct subcellular distribution of the unacetylated histone H2A and the detection of the unacetylated buforin I both on the mucosal surface and in the lumen suggest that buforin I is produced from the cytoplasmic unacetylated histone H2A secreted into the gastric lumen and subsequently processed by pepsins. Our results indicate that buforin I along with pepsins in the vertebrate stomach may contribute to the innate host defense of the stomach against invading microorganisms. (+info)
Purification and Characterization of two bacteriocins from Streptococcus faecium.
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Two bacteriocins were found in the supernatant fluid and in an extract of Streptococcus faecium strain EI. The small soluble enterocin EIA represented more than 90% of the total activity in the supernatant fluid, and was purified 400-fold by ammonium sulphate fractionation, gel filtration on Sephadex G-75 and chromatography on DEAE-cellulose. Enterocin EIB, with a particle weight greater than 4 x 10(6), was the predominant type in the extract. It was released in appreciable quantities after breakage of the bacteria and was purified 100-fold by differential centrifugation, chromatography on Sepharose 4B and density gradient ultracentrifugation. Enterocin EIA, a basic substance with a molecular weight of about 10000, was resistant to heat and was attacked by trypsin, whereas enterocin EIB was less thermostable and insensitive to proteolytic enzymes. The activity of enterocin EIB was unchanged by treatment with DNAase. Sensitivity to enterocin action was confined to certain strains of various enterococcus species, Streptococcus salivarius and Listeria monocytogenes; all the other Gram-positive and Gram-negative bacteria tested for sensitivity were unaffected by either enterocin. (+info)
Gastric, antral and fundic pouch secretion in sheep.
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1. Fundic secretion of HCl and pepsin was studied in sheep with both fundic and antral pouches. The antral pouches were of the entire pyloric region. Continuity of the alimentary tract was restored by an abomasoduodenal anastomosis. 2. Secretion from fundic pouches was continuous. It was reduced in volume and acidity, was pepsin output, by resection of the antral pouches. 3. Teasing with food, feeding and injection of pentagastrin stimulated fundic acid and pepsin secretion in animals with antral pouches before and after antrectomy. 4. Pouches prepared from the entire pyloric region showed continuous secretion with variations not related to feeding or fasting. Antral secretion was increased after s.c. injections of histamine, carbachol and pentagastrin. 5. It is concluded that although the pyloric antrum contributes to the magnitude of the secretory response of an abomasal fundic pouch, the direction of the secretory response is similar whether or not the pyloric antrum is present as a separated pouch, or is removed. (+info)