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1.
Low molecular weight fragments derived from the beta subunit of human lutropin have been frequently observed. These fragments are detected by polyacrylamide gel electrophoresis in sodium dodecyl sulfate following reduction of the disulfide bonds. A sample of human lutropin was identified that had a major portion of its beta subunit showing this proteolytic nick. Over 83% of the subunit was nicked based on reduction, carboxymethylation, and isolation of the low molecular weight fragments. This preparation had 53% of the activity of an intact human lutropin (radioligand assay). The proteolytic nick in the subunit was shown by N-terminal sequencing of the C-terminal fragments to be derived from three clips in a hexapeptide region (residues 44–49) characterized by hydrophobic alkyl side chains. Specific clips were on the amino side of Leu-45 (8%), Val-48 (45%) and Leu-49 (47%). Thus the proteolytic activity, presumably derived from the pituitary during processing, has a substrate specificity reminiscent of the bacterial protease, thermolysin.  相似文献   

2.
The secondary structures of three gastrin analogs, HCl · H-Trp-Nle-Asp(O-tBu)-Phe-NH2 (tetragastrin), pGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (octagastrin), and H-Leu-(Glu)5-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (minigastrin) were studied by 1H-n.m.r. in dimethylsulfoxide and in trifluoroethanol. All three compounds were found to assume a random conformation in the former solvent, while some ordered secondary structure is present in trifluoroethanol even at the tetra-peptide level. This was shown by temperature studies and solvent titrations. At least four amide protons were found to be solvent shielded in the longer hormone.  相似文献   

3.
Abstract: Human neutrophil α‐defensins (HNPs) are small, cationic, Cys‐rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre‐proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro‐peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45‐residue pro‐peptide and the C‐terminal functional domain. Here we described, total chemical synthesis of the 75‐residue human neutrophil pro α‐defensin‐1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met45–Ala46 peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys1–Cys6, Cys2–Cys4 and Cys3–Cys5, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro‐peptide binds to HNP1 intermolecularly with an apparent Kd value of 6.2 μm at pH 7.4, confirming the mode of intramolecular inactivation of human α‐defensin precursors.  相似文献   

4.
The N-terminal pentacosapeptide of Cowpea Chlorotic Mottle Virus (CCMV) was synthesized as the Nα25-acetyl, Cα25-methylamide. The compound contains six arginyl residues, which were incorporated in the peptide chain protected by protonation. The compound was obtained from an azide and a carbodiimide mediated condensation, unifying three fragments. The identity of the compound was proved by amino acid analysis, 13C-n.m.r. and 1H-n.m.r. spectroscopy. It behaved like the native protein in its tendency to interact with viral RNA (fragments).  相似文献   

5.
The 1H-n.m.r. spectra (360 MHz) of 12-(β-(3-pyridyl)-l -Ala) ribonuclease S-peptide (1–14), a tetradecapeptide incorporating (β-3-pyridyl-l -Ala) instead of His at position 12, have been assigned. The shift vs. temperature dependence has been analyzed at three different pD's in terms of a two-state helix (3–13) ± coil equilibrium, and the corresponding values for the thermodynamic quantities ΔH° and ΔS° determined. Helix populations at 0°C have been measured as a function of pD, showing their dependence on two apparent pKa's at ? 3.3 and 5.5, with a maximum at pD ? 4.2. All the obtained results show that the new peptide has very similar folding properties to those shown by S-peptide and particularly to those of C-peptide. The 3–13 helix formed is stabilized by two interactions: a salt-bridge Glu 2-. Arg 10+ and a partial stacking between the aromatic rings of residues Phe 8 and His 12. Calculations involving ring current shifts and potential energies validate the possible existence of this latter interaction, which must present a local geometry defined by χ1X8 180°, χ2X8 100°, χ112 – 60 and χ212 80.  相似文献   

6.
Bovine plasma albumin Fr. V (BPA) has been known to contain small amounts of proteolytic enzyme. Wilson & Foster (1971) found a very limited and specific cleavage of BPA catalyzed by the enzyme with BPA in the F-form near pH 3.8, resulting in the formation of partially hydrolyzed BPA (BPA*). BPA* had a tendency to form a transparent gel at pD 4.0 (pD range of the F-form) above 8%, though proteolytic enzyme-free bovine mercaptalbumin (BMA) was in a transparent solution at pD 4.0 even at 12%. Water structures of the F-form of BMA in the solution state and of BPA* in the gel state were studied by measuring 1 H-n.m.r. spectra, spin-lattice relaxation time (T1) and cross relaxation time (TIS) between irradiated and observed protons. Protein concentration-dependent changes of T1 of water protons indicated that the amount of hydrated water of BPA* in the gel state is far greater than that of the F-form of BMA in the solution state. TIS values from protein protons to water protons also indicated a large amount of hydration of BPA*, strong interaction between water and BPA* and rapid exchange between bound and bulk water in the gel state.  相似文献   

7.
A sensitive assay was developed for human epidermal growth factors (hEGF) 1–48 (dosed), hEGF 1–53 (endogenous), without interference from potential metabolites hEGFs 1–47 or 1–46. Spiked human plasma samples were injected directly, utilizing on-line immunoaffinity HPLC (anti-hEGF) clean-up. No change in capacity was noted after 81 cycles. After release from the immunoaffinity column, the fragments were further resolved by strong cation-exchange (SCX) via a column switching valve. Method development also required interfacing immunoaffinity, ion-exchange, and detection components. Immunoassays on collected fractions yielded a detection limit of 1 μg ml−1, although a detection limit of 75 pg ml−1 appears feasible.  相似文献   

8.
Considering the three-dimensional structure and the native Zn(II)-binding ligands of carboxypeptidase A followed by extensive model building, a cyclic octapeptide, cyclo-(Gly-L-Glu-Gly-Gly-L-His-Gly-L-His-Gly) was designed to mimic the Zn(II)-binding site of carboxypeptidase A. The cyclic octapeptide was prepared by high dilution technique from the corresponding linear octapeptide, N-Boc-Gly-γ-OBut-L-Glu-Gly-Gly-L-His-Gly-L-His-Gly-OBzlNO2 via the azide method. The linear octapeptide was obtained by coupling of the two tetrapeptide fragments: N-Boc-Gly-γ-OBut-L-Glu-Gly-Gly-ONp and L-His-Gly-L-His-Gly-OBzlNO2. The cyclic peptide was purified to homogeneity by the method of countercurrent distribution. The product obtained was both ninhydrin negative and Pauli's reagent positive. Further confirmation of this material was obtained by the proper amino acid ratio of its acid hydrolysate and by the proton magnetic resonance spectrum in which the various kinds of protons of this peptide were accounted for. A detailed 13C- and 1H-n.m.r. investigation was undertaken to determine the Zn(II)-binding ligands of the cyclo-octapeptide. The assignments for all the resonances were attempted by pH titration, by employing homonuclear decoupling experiments and by synthesis of cyclo-octapeptide containing specifically deuterated amino acids cyclo-(Gly-L-Glu-Gly-d2-Gly-d2-L-His-Gly-L-His-Gly). Titration results of Zn(II) bound form of the cyclic peptide showed the presence of a 1:1 complex. Upon Zn(II)-binding, the proton resonances were shifted downfield, the largest change being that of the histidine residues and more particularly C(2)-H protons. The chemical shifts induced on glutamic acid residue were also observed for Glu CH2γ. In the case of 13C resonances, the maximum change in chemical shift was observed in the histidine residues and especially in the imidazole ring upon complexation. Two methylenes of the glutamic acid residue showed a large change in chemical shift upon ligating to the metal ion. The most significant observation was the deshielding effect of the Glu COO- group. The results demonstrate that Zn(II) binds both the imidazoles of the two histidine residues and the carboxyl side chain of the glutamic acid residue of the designed cyclic octapeptide.  相似文献   

9.
Digestion of human growth hormone (hGH) with the Glu-specific protease from Staphylococcus aureus V8 was performed at 20-22°C or 37°C at a 1:20 ratio (by weight) at pH 7.8 with or without 0.2% SDS. There are 14 Glu-residues evenly distributed along the polypeptide chain of hGH as possible sites of proteolytic cleavage of V8-protease. The pattern of fragmentation of hGH was analyzed by electrophoresis and reversed-phase HPLC, and the identity of the proteolytic fragments isolated to homogeneity was established by their partial sequencing and amino acid analysis after acid hydrolysis. Kinetic analysis of the proteolytic digestion process allowed to establish that initial nicking of the protein occurs at Glu33 and subsequently at Glu56 and Glu66. Much slower cleavages occur at G1u30 and Glu186. These cleavage sites are located at chain loops in the hGH molecule, and in particular outside the helical segments of the four-helix bundle of the crystal structure of hGH. Fragments 1-33 and 67-191 comprising entirely the N-terminal helix and the three C-terminal helices of hGH, respectively, were isolated to homogeneity in amounts useful for subsequent conformational and functional studies. The results of this study and of previous ones [Li, C.H. (1982) Mol. Cell. Biochem. 46 , 31-41] describing limited proteolysis of hGH by various proteases have been interpreted on the basis of the three-dimensional structure and dynamics of hGH. Overall, it is shown that proteolytic enzymes preferentially cleave hGH at exposed and flexible loops only, thus emphasizing the fact that proteases can be used as reliable probes of protein structure and dynamics. © Munksgaard 1995.  相似文献   

10.
Boc-L-Asu-L-Ala-Gly-OMe crystallizes in the monoclinic space group P21 with cell dimensions a = 14.315(3) Å, b = 9.280(2) Å, c = 14.358(3) Å, β= 103.63 (1) d?, V= 1853.4(9) Å3, with two molecules in the asymmetric unit. The conformation of the two molecules is characterized by a type II' β-bend, similar to that predicted earlier by potential energy calculations, stabilized by an intramolecular hydrogen bond. I.r. and 1H-n.m.r. data show that the folded conformation is also stable in chloroform solution.  相似文献   

11.
Aqueous Stability of Human Epidermal Growth Factor 1-48   总被引:1,自引:0,他引:1  
Human epidermal growth factor 1-48 (hEGF 1-48, Des(49-53)hEGF) is a single chain polypeptide (48 amino acids; 3 disulfide bonds; 5445 Da) possessing a broad spectrum of biologic activity including the stimulation of cell proliferation and tissue growth. In this study, three primary aqueous degradation products of hEGF 1-48 were isolated using isocratic, reverse phase/ion-pair HPLC. The degradation products were characterized using amino acid sequencing, electrospray ionization mass spectrometry, isoelectric focusing, and degradation kinetics. Results indicate that hEGF 1-48 degrades via oxidation (Met21), deamidation (Asn1), and succinimide formation (Asp11). The relative contribution of each degradation pathway to the overall stability of hEGF 1-48 changes as a function of solution pH and storage condition. Succinimide formation at Asp11 is favored at pH < 6 in which aspartic acid is present mostly in its protonated form. Deamidation of Asn1 is favored at pH > 6. The relative contribution of Met21 oxidation is increased with decreasing temperature, storage as a frozen solution (–20°C), and exposure to fluorescent light.  相似文献   

12.
A novel synthesis of thymosin α1 by classical methods using seven tert. -butyl side chain protected fragments is described. Optimum conditions were found for the final DCC/HOBt coupling of the two key intermediates; decapeptide and octadecapeptide. Thymosin α1 was purified by two stages of preparative HPLC (partial purification with C8 and final purification with C18 reverse phase silica gel) to give a 30% overall yield for the final four stages of synthesis (including catalytic hydrogenation of octadecapeptide, coupling, deprotection and purification). The product was shown to be homogeneous by thin-layer and paper high voltage electrophoresis, isoelectric focusing analysis, thin-layer chromatography and high performance liquid chromatography. Amino acid analysis, optical rotation, 1 H-n.m.r. spectroscopy, FAB mass spectroscopy and peptide mapping after tryptic digestion confirmed the structure of thymosin α1. Three minor stereoisomer contaminants were isolated by HPLC and characterized as [D-Lys14]-thymosin α1, [D-Lys17]-thymosin α1 and [D-Ala3]-thymosin α1 resulting from racemization at Lys14, Lys17 and Ala3 during the coupling of the fragments. A final contaminant, isolated by HPLC, was characterized as Nα-isobutyloxycarbonyl-thymosin α1 (15–28), which results from “wrong way opening” of an activated mixed anhydride.  相似文献   

13.
Abstract

1H-n.m.r. spectroscopy of small unilamellar phospholipid vesicles in the presence of the lanthanide probe ion Dy3+ has been used to study the permeability of these liposomes induced by the bile salts (glycocholate and glycodeoxycholate) and pancreatic phospholipase A2. A marked synergism is demonstrated in the combined effects of these digestive agents in producing permeability of the vesicles to Dy3 +. Changes in the 1H-n.m.r. spectrum of the vesicular phospholipid head-groups before permeability is induced, indicate that the products of the enzymic hydrolysis (lyso lipids and fatty acids) and transmembrane lipid exchange are involved in the permeability mechanism. The results are discussed in terms of the advantages of the use of n.m.r. techniques in the future design of liposomes for oral use.  相似文献   

14.
A cyclic heptapeptide [cyclo-(Gly-L-His-Gly-L-His-Gly-L-His-Gly)] was designed to mimic the Zn(II)-binding site of carbonic anhydrase. The cyclic heptapeptide was synthesized from the linear heptapeptide, Gly-L-His-Gly-L-His-Gly-L-His- Gly-OH, which in turn was obtained by coupling of the fragments, viz. BOC-Gly-L-His-Gly-N3 and L-His-Gly-L-His-Gly-OBzlNO2 followed by deblocking of amino and carboxyl protecting groups. Conversion of the linear heptapeptide to the azide by treatment with diphenylphosphoryl azide was followed by cyclization in high dilution. A homogeneous material was isolated by counter-current distribution followed by gel filtration. It was found to be ninhydrin negative. The n.m.r. spectrum of the material upon integration indicated the proper ratios of various kinds of protons to be expected of the cyclic heptapeptide. A detailed 13C- and 1H-n.m.r. investigation was undertaken to determine the Zn(II)-binding ligands of the cyclic heptapeptide. The assignments for all the resonances were attempted by spin-decoupling method, pH and solvent effects, and by comparison of resonances of similar protons and carbons of model peptides. The n.m.r. titration results of the Zn(II) bound form of the cyclic peptide showed the presence of a 1:1 complex. Upon Zn(II)-binding, the changes in the chemical shift of the imidazole protons were relatively large, indicating that this ring is involved in the complexation. All the peptide -NH- resonances were observable and unaffected; consequently, none of these nitrogens can serve as a ligand. In the case of 13C resonances, addition of 1 equiv. of Zn(II) to the cyclic heptapeptide, the C(2), C(4), and C(5) carbon resonances of this group were dramatically affected and showed a very large change in chemical shift upon complexation. The results demonstrate that Zn(II) binds to all three imidazole residues of the designed cyclic heptapeptide.  相似文献   

15.
The sensitivities of the R25-I26 bond on bovine β-casein and on its N-terminal fragment β(1–105) to trypsin digestion were compared by monitoring the liberation of the β(1–25) product. It was shown that this peptide bond was poorly and slowly hydrolysed on β(1–105), while it is highly susceptible to trypsin attack when whole protein is used as substrate. The marked resistance of β(1–105) is linked to its inhibitory effect on trypsin activity (apparent K′i= 1.2 × 10?6, M), as demonstrated by using a related chromogenic substrate. Indeed, a preincubation step of trypsin with β(1–105) leads to a more pronounced inhibitory effect. The progress curves obtained with and without preincubation show that β(1–105) acts as a slow binding inhibitor on trypsin activity. These findings promise further insight into the action and the regulation of proteolytic enzymes. © Munksgaard 1997.  相似文献   

16.
The synthesis of three collagen model analogs is described: Ac-Ala-Gly-Pro-Ala-Gly-Pro-NHMe, Ac-Ala-Gly-Pro-Ala-Glc-Pro-NHMe, and Ac-Ala-Glc-Pro-Ala-Gly-Pro-NHMe, where Glc stands for glycolic acid. The 1H-n.m.r. properties of these compounds in dimethylsulfoxide-d6 and trifluoroethanol are described. While in DMSO-d6 the compounds are random, in TFE the glycine amide protons seem to be less solvent exposed than the other amide protons. Little difference was found in the behavior of the three compounds.  相似文献   

17.
1H-n.m.r. studies at 500MHz have been performed on a trypsin inhibitor (CMTI-III) found in squash seed (Cucurbita maxima). The sequential resonance assignments have been made using two-dimensional techniques. The chemical shifts for the assigned protons are reported at 30°, pH 2.8 and form a basis for the determination of the solution structure of CMTI-III. Analysis of the NOE data, NH-αCH vicinal coupling constants and pattern of slowly exchanging amide protons indicates that the predominant feature of the solution conformation is a triple stranded β sheet consisting of residues 8-10, 21-23, and 26-29. Residues 12-15 appear to form a β turn.  相似文献   

18.
Abstract— The objective of this study was to compare, in rat small intestinal and colonic enterocytes, subcellular distributions of activities degrading the large peptides, neurotensin, acetylneurotensin (8–13), GRF(1–29)NH2 (human growth hormone releasing factor fragment), (desNH2Tyr1,D-Ala2,Ala15)-GRF(1–29)NH2, insulin, and insulin B-chain. Proteolytic activities degrading individual peptides in the 10000-g pellet, rich in intracellular organelles, 27000-g pellet, rich in brush-border membrane, 100000-g pellet, and 100000-g supernatant, rich in cytosol, were determined and compared for both the small intestine and colon. In colonic fractions, the cytosol had highest activity (g protein)?1 degrading three out of four peptides tested, while in small intestinal fractions, the 27000-g pellet had the highest activity (g protein)?1, degrading four out of five peptides tested. In both small intestine and colon, the cytosol had a higher percentage of total proteolytic activity degrading each of the above polypeptides and the highest insulin-degrading activity (g protein)?1. The results suggest that at pH 7·5, proteolytic activities (g protein)?1 in the fraction of subcellular organelles are much lower than those in cytosol and that cytosolic proteolytic activities degrading polypeptides and analogues are significant.  相似文献   

19.
Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205–316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu302 residue into fragments 205–302 and 303–316. Upon incubation for 2–5 days of fragment 205–302 with a 5–fold excess of peptide 303–316, prepared by solid phase synthesis, with V8-protease in 0.1M ammonium acetate, pH6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to ñ90% (based on fragment 205–302). The same procedure was used to prepare also the thermolysin fragments 205–315 and 205–311 by enzymatic coupling of fragment 205–302 to peptide 303–315 or 303–311, these last prepared by proteolytic digestion of the synthetic peptide 303–316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205–316 of thermolysin at the level of its helical segment encompassing residues 301–312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.  相似文献   

20.
The mode of calcium and europium binding to (4′-amino)-Phe4, Leu5-enkephalin in acetonitrile was investigated by the spectrofluorimetric method. It was found that both these cations evoke the 1:1 complex formation. The complexation is accompanied by an increase of the distance between the aromatic rings (Tyr1 and APhe4) in the peptide. The study of 1 H-n.m.r. of the peptide in water in the presence of increasing amounts of Eu+3 allowed us to identify the cation complexation sites of the peptide as the Leu5-carboxyl group and (probably) the APhe4 amide carbonyl group.  相似文献   

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