Possible involvement of aminotelopeptide in self-assembly and thermal stability of collagen I as revealed by its removal with proteases.
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The functions of aminotelopeptide and N-terminal cross-linking of collagen I were examined. Acetic acid-soluble collagen I (ASC) was purified from neonatal bovine skin and treated with three kinds of proteases. The amino acid sequencing analysis of the N terminus showed that ASC contained a full-length aminotelopeptide. Pepsin and papain cleaved the aminotelopeptide of the alpha1 chain at the same site and the aminotelopeptide of the alpha2 chain at different sites. Proctase-treated ASC lost the whole aminotelopeptide, and the N-terminal sequence began from the tenth residue inside the triple helical region. The rates of fibril formation of pepsin-treated ASC and proctase-treated ASC were the same and were slower than that of ASC. The denaturation temperatures, monitored by CD ellipticity at 221 nm, of ASC, pepsin-treated, or papain-treated collagens were the same at 41.8 degrees C. Proctase-treated ASC showed a lower denaturation temperature of 39.9 degrees C. We also observed the morphology of the collagen fibrils under an electron microscope. The ASC fibrils were straight and thin, whereas the fibrils of pepsin-treated ASC were slightly twisted, and the fibrils from papain- and proctase-treated ASC were highly twisted and thick. When the collagen gel strength was examined by a modified method of viscosity-measurement, ASC was the strongest, followed by pepsin-treated ASC, and papain- and proctase-treated ASCs were the weakest. These results suggest that the aminotelopeptide plays important roles in fibril formation and thermal stability. In addition, the functions of intermolecular cross-linking in aminotelopeptides may contribute to the formation of fibrils in the correct staggered pattern and to strengthening the collagen gel. (+info)
The pepsin residue glycine-76 contributes to active-site loop flexibility and participates in catalysis.
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Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71--80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes. (+info)
Characterization of Alzheimer's beta -secretase protein BACE. A pepsin family member with unusual properties.
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The cerebral deposition of amyloid beta-peptide is an early and critical feature of Alzheimer's disease. Amyloid beta-peptide is released from the amyloid precursor protein by the sequential action of two proteases, beta-secretase and gamma-secretase, and these proteases are prime targets for therapeutic intervention. We have recently cloned a novel aspartic protease, BACE, with all the known properties of beta-secretase. Here we demonstrate that BACE is an N-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We have used a secreted Fc fusion-form of BACE (BACE-IgG) that contains the entire ectodomain for a detailed analysis of posttranslational modifications. This molecule starts at Glu(46) and contains four N-glycosylation sites (Asn(153), Asn(172), Asn(223), and Asn(354)). The six Cys residues in the ectodomain form three intramolecular disulfide linkages (Cys(216)-Cys(420), Cys(278)-Cys(443), and Cys(330)-Cys(380)). Despite the conservation of the active site residues and the 30-37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases. This difference may affect the substrate specificity of the enzyme. Taken together, both the presence of a transmembrane domain and the unusual disulfide bond structure lead us to conclude that BACE is an atypical pepsin family member. (+info)
Fluorescence studies on the active sites of porcine pepsin and Rhizopus-pepsin.
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Fluorescence studies on the interaction, with porcine pepsin, of oligopeptides bearing a mansyl (Mns, 6-(N-methylanilino)-2-naphthalenesulfonyl) or dansyl (Dns, 5-dimethylaminonaphthalene-1-sulfonyl) group at the NH2 or COOH terminus have provided further evidence showing that the probe group is drawn into the extended active site largely as a consequence of the specific binding of the peptide portion of the substrate. The active site does not appear to have appreciable intrinsic affinity for the mansyl or dansyl group, and the principal contribution to the specific peptide-protein interaction is provided by the sensitive L-phenylalanyl-L-phenylalanyl (Phe-Phe) unit of the substrates tested. The pepsin inhibitor pepstatin can displace substrates such as Mns-(Gly)n-Phe-Phe-OR or Gly-Gly-Phe-Phe-NHNH-Mns from the active site of porcine pepsin; in these circumstances the mansyl group is bound weakly at a separate, nonspecific locus, distinct from the active site, which can accept the mansyl group of Mns-Gly-Gly-OR or mansylamide. In the interaction with substrates such as Mns-(Gly)n-Phe-Phe-OR or Dns-(Gly)n-Phe-Phe-OR, the above conclusions for porcine pepsin also apply to Rhizopus-pepsin. With substrates such as Gly-Gly-Phe-Phe-NHNH-Mns, however, the active site of Rhizopus-pepsin shows less affinity for the fluorescent probe group than does that of porcine pepsin, suggesting structural differences between the two acid proteinases in the region of their extended active sites which bind the COOH-terminal portion of small oligopeptide substrates. (+info)
Reactive half-cystine peptides of the secretory component of human exocrine immunoglobulin A.
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On the basis of previous work the two forms of human secretory component, namely that which is covalently bound as a part of the exocrine immunoglobulin A molecule and the free form, are probably different states of the same protein. From autoradiographs of trypic peptide maps of bound and free secretory components which were radioactively alkylated after partial reduction, it was concluded that the same half-cystines in each are sensitive to reduction. in the present work the easily reduced half-cystines of the bound and free secretory components have been studies in more detail. In each form there are two such half-cystines. In the case of bound secretory component they provide the linkage to the remainder of the exocrine immunoglobulin A molecule. Peptides from the sensitive half-cystines were isolated from tryptic-peptic digests of free secretory component and sequenced. By diagonal electrophoresis these two peptides were shown to be joined in an intrachain disulfide bridge. Therefore, it is proposed that the exocrine immunoglobulin A molecule becomes fully assembled when a single, reactive intrachain disulfide bridge in free secretory component rearranges to yeild two interchain bridges with dimeric serum-type immunoglobulin A. This process is thought to occur within the epithelial lining cells of mucous membranes. (+info)
Primary structure of the L chain from a rabbit homogeneous antibody to streptococcal carbohydrate. II. Sequence determination of peptides from tryptic and peptic digests.
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A complete amino acid sequence containing 210 residues of a rabbit light chain of a homogeneous Group C streptococcal antibody has been determined as: See journal for formula. This light chain is allotype b4 and is of the V-K1 subgroup. (+info)
Basement membrane collagen of renal glomerulus.
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Collagen has been prepared from steer glomerular basement membrane by controlled pepsin solubilization. Four collagen polypeptides of potential alpha chain size have been isolated following denaturation and reduction with mercaptoethanol. Purification was obtained through sequential chromatography by gel filtration on agarose and by ion exchange on carboxymethyl- and diethylaminoethylcellulose and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fractions A and B resemble interstitial alpha chains in apparent molecular weight by gel filtration (93,000). Fractions C and D were released from a single high molecular weight fraction (II) by reduction with mercaptoethanol and had a larger apparent molecular weight by gel filtration (140,000). Amino acid composition of all fractions demonstrated that they are closely interrelated by generally distinctive from interstitial collagens. Cysteine was present in all fractions except Fraction A. Prior to reduction, all mercaptan groups were inaccessible to iodoacetate and p-chloromercuribenzoate but became completely titrable after treatment with mercaptoethanol. The starting material and all fractions contained large amounts of hexose. Glucose and galactose predominated; but mannose, glucosamine, and galactosamine were also present in substantial amounts. Small amounts of fucose and sialic acids were found in starting material only. (+info)
Influence of the extent of haemoglobin hydrolysis on the digestive absorption of haem iron in the rat. An in vitro study.
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This study was designed to assess the haem-peptide interactions which occur during progressive haemoglobin hydrolysis by digestive enzymes and their relationship with haem iron digestive absorption. The behaviour of different haemoglobin hydrolysates was studied using the Ussing chamber model. Hydrolysates were produced from enzyme digestion of bovine haemoglobin at pH 3 by pepsin and at pH 10 by subtilisin. Samples with increasing degrees of hydrolysis (0-15 %) were studied. Biochemical assays (pyridine haemochromogen method and UV absorption spectra) were used to follow haem solubility and haem-peptide interactions in samples. Increasing the hydrolysis level of haemoglobin was associated with an enhanced iron uptake; the highest uptake rate was reached between 8 and 11 % of globin hydrolysis, whichever enzyme was used. The mechanisms rendering iron soluble and available differ between the two enzymes. The comparison between biochemical and absorption data suggests that the formation of soluble peptide-haem complexes was not sufficient to enhance haem iron absorption, since globin-bound iron is poorly absorbed; an efficient absorption occurred only when haem was loosely bound to low molecular weight peptides. (+info)