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PubMed1998

An NMR-based identification of peptide fragments mimicking the interactions of the cathepsin B propeptide.

Selected fragments of the 62-residue proregion (or residues 1p-62p) of the cysteine protease cathepsin B were synthesized and their interactions with cathepsin B studied by use of proton NMR spectroscopy. Peptide fragments 16p-51p and 26p-51p exhibited differential perturbations of their proton resonances in the presence of cathepsin B. These resonance perturbations were lost for the further truncated 36p-51p fragment, but remained in the 26p-43p and 28p-43p peptide fragments. Residues 23p-26p or TWQ25A in the N-terminal 1p-29p fragment did not show cathepsin B-induced resonance perturbations although the same residues had strongly perturbed proton resonances within the 16p-51p peptide. Both the 1p-29p and 36p-51p fragments lack a common set of hydrophobic residues 30p-35p or F30YNVDI35 from the proregion. The presence of residues F30YNVDI35 appears to confer a conformational preference in peptide fragments 16p-51p, 26p-51p, 28p-43p and 26p-43p, but the same residues induce the aggregation of peptides 16p-36p and 1p-36p. The peptide fragment 26p-43p binds to the active site, as indicated by its inhibition of the catalytic activity of cathepsin B. The cathepsin B prosegment can therefore be reduced into smaller, but functional subunits 28p-43p or 26p-43p that retain specific binding interactions with cathepsin B. These results also suggest that residues F30YNVDI35 may constitute an essential element for the selective inhibition of cathepsin B by the full-length cathepsin B proregion.

Cathepsin B↗

PubMed1997

Microvascular permeability to the F(ab')2 fragment of IgG in the male rat reproductive tract at puberty.

Development of contraceptive vaccines has recently raised much interest following the cloning of the sperm and oocyte components involved in the sperm-oocyte interaction. The main difficulty of immunocontraception in the male is the poor access of antibodies to the luminal compartment. As recent literature suggests that many substances are transported to the testis by receptor-mediated or fluid-phase transcytosis, the dependence of the transport of IgG on the Fc receptor was studied in the present investigation by comparing the penetration of whole IgG and the F(ab')2 fragment of IgG to the testis and epididymis. The maximum volume of distribution (Veq) for the F(ab')2 fragment was significantly higher than that for whole IgG in the testis of 30-60-day old rats, in the caput and cauda of 30- and 45-day old rats and the corpus of 45-day old rats. The speeds at which equilibrium between tissue extracellular fluid and serum was reached (K) for the F(ab')2 fragment and whole IgG were significantly different in the testicular capsule of the 60-day old, in the caput and corpus of the 45- and 60-day old and in the cauda of the 45-day old rats. The microvascular permeabilities (PE) to the F(ab')2 fragment were more than 2-fold higher than those to whole IgG in the testis of the 20-, 45- and 60-day old, in the testicular capsule of the 20- and 45-day old, in the caput of 20-, 30- and 60-day old and in the corpus of 20-day old rats. The PE to whole IgG was more than 2-fold higher than that to the F(ab')2 fragment in the cauda of the 45-day-old rats. The PE to the F(ab')2 fragment increased steadily from 20 to 60 days of age in the testis and caput, but in the corpus there was a more abrupt increase between 30 and 45 days of age. In the cauda, PE remained in the same range of magnitude throughout pubertal development. These results suggest that the F(ab')2 fragment reaches the lumen of the reproductive tract more easily than whole IgG from 30 days of age onwards in the testis, whereas in the caput, corpus and cauda epididymidis the rate at which F(ab')2 fragment reaches the lumen increases only temporarily at the time of appearance of spermatozoa in the lumen. Transport of IgG to the male reproductive tract is thus unlikely to be mediated by Fc receptors.

Aging↗

PubMed2003

Differential binding characteristics of protein G and protein A for Fc fragments of papain-digested mouse IgG.

It has been previously reported that staphylococcal protein A (SPA) bound only to the Fc region of mouse immunoglobulin G (IgG) and streptococcal protein G (SPG) bound to both Fab and Fc regions of mouse IgG and the binding sites for SPG and SPA on Fc were overlapped. In this study the binding characteristics of SPG and SPA for papain-digested mouse IgG were analysed. Papain digestion of mouse IgG purified from CAy-IFNg99C hybridoma (secreting IgG1 monoclonal antibody specific for human interferon gamma)-induced ascites resulted in Fab and two major Fc fragments referred to as the high molecular weight (HMW) and the low molecular weight (LMW) Fc fragments. SPG bound to Fab and the LMW Fc fragments of the papain-digested IgG. However SPG did not bind to the HMW Fc fragment. SPA showed practically no reactivity with the Fab and the LMW Fc fragments of the papain-digested mouse IgG but only to the HMW Fc fragment. SPG and SPA binding assays showed that papain digestion discriminated the SPG and SPA binding sites in the Fc fragment of mouse IgG. These results demonstrated a clear evidence for the presence of two independent SPG and SPA binding sites in the Fc fragment of mouse IgG.

Animals↗

PubMed1997

Appearance of 1-2 Mbp giant DNA fragments as an early common response leading to cell death induced by various substances that cause oxidative stress.

The effects of oxidative stress on double strand DNA breakage were examined in T-24 human bladder tumor cells using various active oxygen producing agents such as hydrogen peroxide (H2O2), bleomycin (BLM), neocarzinostatin (NCS), and x-ray irradiation. Analysis of the DNA by pulsed-field gel electrophoresis (PFGE) revealed that discrete giant DNA fragments of 1-2 Mbp and 200-800 kbp had accumulated in the nuclei of the treated cells. The 1-2 Mbp giant DNA fragments were first observed 2 h after the T-24 cells were exposed to the active oxygen producing agents, or irradiated with x-ray. The appearance and the amounts of 1-2 Mbp and 200-800 kbp giant DNA fragments seemed to depend on the concentration and the type of reagents used or the dose of x-ray. Following the accumulation of giant DNA fragments, another type of DNA fragmentation was detected and DNA fragments smaller than 100 kbp accumulated in the nuclei of the cells irradiated with x-ray or treated with NCS. In addition, DNA ladder formation, which is characteristic of apoptosis, was observed. The giant DNA fragments appeared to arise as a consequence of double-stranded DNA breakage, which occurred earlier than cell lysis, as assessed by 51Cr release. These findings indicate that the formation of giant DNA fragments is a specific characteristic of cells responding to oxidative stress, and it may be an initial event that leads to cell death.

Bleomycin↗

PubMed2000

De novo peptide sequencing by two-dimensional fragment correlation mass spectrometry.

A novel concept of two-dimensional fragment correlation mass spectrometry and its application to peptide sequencing is described. The daughter ion (MS2) spectrum of a peptide contains the sequence information of the peptide. However, deciphering the MS2 spectrum, and thus deriving the peptide sequence is complex because of the difficulty in distinguishing the N-terminal fragments (e.g., b series) from the C-terminal fragments (e.g., y series). By taking a granddaughter ion (MS3) spectrum of a particular daughter ion, all fragment ions of the opposite terminus are eliminated in the MS3 spectrum. However, some internal fragments of the peptide will appear in the MS3 spectrum. Because internal fragments are rarely present in the MS2 spectrum, the intersection (a spectrum containing peaks that are present in both spectra) of the MS2 and MS3 spectra should contain only fragments of the same terminal type. A two-dimensional plot of the MS2 spectrum versus the intersection spectra (2-D fragment correlation mass spectrum) often gives enough information to derive the complete sequence of a peptide. This paper describes this novel technique and its application in sequencing cytochrome c and apomyoglobin. For a tryptic digest of cytochrome c, approximately 78% of the protein sequence was determined. For the Glu-C/tryptic digest of apomyoglobin, approximately 66% of the protein sequence was determined.

Algorithms↗

PubMed2005

Self-assembled monolayer of a pepstatin fragment as a sensing element for aspartyl proteases.

A novel disulfide, which carried two pepstatin fragments at both ends, was prepared by the coupling of 11,11'-dithiobisundecanoic acid (DTUA) with a fragment (Val-Val-Sta) carrying a n-hexyl end (Pepsta(h)). The compound obtained (DTUA-Pepsta(h)) formed a self-assembled monolayer (SAM) on a gold electrode and vacuum-evaporated gold thin film as proven by cyclic voltammetry and reflection absorption infrared spectroscopy, respectively. When the SAM-modified gold electrode was incubated with a solution of aspartyl protease, pepsin, a decrease in both anodic and cathodic peak currents and an increase in potential difference were observed in the cyclic voltamogram of hydroquinone as a probe, whereas a coexistence of free pepstatin fragment inhibited these phenomena, indicating the specific binding of pepsin to the fragment at the exterior of the SAM. The binding rate of the enzyme to the SAM was largely dependent on the surface density of the fragment moiety in the SAM. Furthermore, when the SAM of DTUA-Pepsta(h) on a gold colloid array deposited on an amino group-modified glass plate was immersed in a pepsin solution, absorption of the glass plate at 550 nm corresponding to a localized surface plasmon resonance of the gold colloid abruptly increased and slightly red-shifted, and a further addition of pepstatin A gradually decreased the absorbance. From the increasing and decreasing profiles of absorbance, the association constant (K(assoc)) for pepsin with the fragment on the SAM was determined. Similar phenomena were observed upon immersion of the fragment-modified SAM in a solution of HIV-1 protease, suggesting a usability of the pepstatin fragment SAM for the detection and removal of the enzyme from biological fluids.

Aspartic Acid Endopeptidases↗

PubMedDate not supplied

Renal metabolism of 3'-iodohippuryl N(epsilon)-maleoyl-L-lysine (HML)-conjugated Fab fragments.

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. Recently, we reported use of a novel radioiodination reagent, 3'-[131I]iodohippuryl N(epsilon)-maleoyl-L-lysine (HML), that liberates m-iodohippuric acid before antibody fragments are incorporated into renal cells. In mice, HML-conjugated Fab demonstrated low renal radioactivity levels from early postinjection times. In this study, renal metabolism of HML-conjugated Fab fragments prepared by different thiolation chemistries and by direct radioiodination were investigated to determine the mechanisms responsible for the low renal radioactivity levels. Fab fragments were thiolated by 2-iminothiolane modification or by reduction of disulfide bonds in the Fab fragments, followed by conjugation with radioiodinated HML to prepare [131I]HML-IT-Fab and [125I]HML-Fab, respectively. In biodistribution studies in mice, both [131I]HML-IT-Fab and [125I]HML-Fab demonstrated significantly lower renal radioactivity levels than those of [125I]Fab. In subcellular distribution studies, [125I]Fab showed migration of radioactivity from the membrane to the lysosomal fraction of the renal cells from 10 to 30 min postinjection. On the other hand, the majority of the radioactivity was detected only in the membrane fraction at the same time points after injection of both [131I]HML-IT-Fab and [125I]HML-Fab. In metabolic studies, while [125I]Fab remained intact at 10 min postinjection, both HML-conjugated Fab fragments generated m-iodohippuric acid as a radiometabolite at the same postinjection time. [131I]HML-IT-Fab registered two radiometabolites (intact [131I]HML-IT-Fab and m-iodohippuric acid), whereas additional radiometabolites were observed with [125I]HML-Fab. This suggested that metabolism of both HML-conjugated Fab fragments would occur in the membrane fractions of the renal cells. The findings of this study reinforced our previous hypothesis that radiochemical design of antibody fragments that liberate radiometabolites that are excreted into the urine by the action of brush border enzymes would constitute a useful strategy to reduce renal radioactivity levels from early postinjection times.

Animals↗

PubMedDate not supplied

Tumor targeting of radiometal labeled anti-CEA recombinant T84.66 diabody and t84.66 minibody: comparison to radioiodinated fragments.

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. Due to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv dimer) and the T84.66 minibody (scFv-CH3 dimer), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 kDa) exhibited very rapid tumor targeting with 12.5 +/- 0.4% injected dose per gram (% ID g(-1) +/- standard error) at 2 h and reached a maximum of 13.3 +/- 0.9% ID g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- 21.0% ID g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of D-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.2 +/- 1.2% ID g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 kDa), displayed rapid tumor targeting of 14.2 +/- 6.1% ID g(-1) at 2 h, and reached a maximum activity of 24.5 +/- 6.1% ID g(-1) by 12 h. Renal uptake achieved a plateau of 12-13% ID g(-1) which cleared to 7.2% ID g(-1) at 72 h. However, hepatic uptake was elevated and reached a maximum of 26.0 +/- 1.0% ID g(-1) at 12 h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 12 h to 16.6 +/- 1.5% ID g(-1), indicative of an intrinsic hepatic accumulation of the [111In]DOTA-T84.66 minibody or metabolites. While the anti-CEA [111In]DOTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]DOTA forms appeared problematic for RIS and RIT applications. Development of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.

Animals↗

PubMed1995

Farnesylation of CaaX-tagged diphtheria toxin A-fragment as a measure of transfer to the cytosol.

Diphtheria toxin binds to receptor-positive cells through its B-fragment, the toxin is then endocytosed, and the low pH in endosomes triggers the translocation of the enzymatically active A-fragment to the cytosol. A synchronous release of A-fragments into the cytosol can be induced by exposing cells with surface-bound toxin to low pH. We have used this protein translocation system to develop a novel method to study whether or not a protein is exposed to the cytosol. Protein farnesylation is a cytosolic modification signaled by a C-terminal CaaX motif, and to visualize the translocation process, we added a farnesylation signal to the toxin A-fragment. The A-fragment with an added CaaX motif was farnesylated within 1 h after exposure of cells with surface-bound toxin to low pH, and also A-fragment translocated from endosomes was quantitatively farnesylated. The results indicate that all cell-mediated reduction of the toxin implicates translocation of the A-fragment to the cytosol. The farnesylation was inhibited by lovastatin, the alkylating agent NEM, and the peptidomimetic farnesylation inhibitor B581. Farnesylated A-fragment partitioned preferentially into the detergent phase upon extraction with Triton X-114. Our data suggest that farnesylation of a CaaX tag is generally applicable as a cytosolic marker, and this strategy for monitoring protein transfer to the cytosol may have considerable potential for studying the transport to the cytosol of proteins added externally to cells.

Amino Acid Sequence↗

PubMed1993

Calcium binding and translocation properties of glucagon and its fragments.

Earlier studies have indicated that the N- and C-terminal regions of glucagon are functionally and structurally different. We have sought to understand this distinction in terms of the interaction of glucagon and its N- and C-terminal fragments with Ca2+, Mg2+, and Zn2+ in a nonpolar milieu. CD spectral data, in 98% (v/v) trifluoroethanol in water, reveal two binding sites for Ca2+ and Mg2+ and one site for Zn2+ in the intact hormone as well as in the C-terminal 19-29 fragment. The 1-6 fragment did not bind Zn2+ and formed a 2:1 peptide-Ca2+ or -Mg2+ complex. With glucagon and the 19-29 fragment, cation binding caused changes in the peptide's helix content. Fluorescence spectral changes involving Trp-25 in the 19-29 fragment and Trp-25 and Tyr-10 and/or Tyr-13 in glucagon were seen on Ca2+ binding to one of the two sites, while Zn2+ binding produced no change in fluorescence. The spectral data suggest that Ca2+ and Zn2+ binding sites (with Kd in the micromolar range in 98% trifluoroethanol) are distinct and are contained in the C-terminal domain of glucagon. Glucagon and the 19-29 fragment, but not the 1-6 fragment, caused an influx of Ca2+ (as monitored by spectral changes in arsenazo III) in unilamellar vesicles made of dimyristoyllecithin. Leakage of vesicle contents induced by the 19-29 fragment was minimal but was significant (approximately 10%) in the case of glucagon. The transport data suggest an interaction of the C-terminal domain of glucagon with Ca2+ at the lipid-water interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed2000

Cooperative assembly of a nativelike ubiquitin structure through peptide fragment complexation: energetics of peptide association and folding.

Peptide fragments corresponding to the N- and C-terminal portions of bovine ubiquitin, U(1-35) and U(36-76), are shown by NMR to associate in solution to form a complex of modest stability (Kassn approximately 1.4 x 10(5) M(-1) at pH 7.0), with NMR features characteristic of a nativelike structure. The complex undergoes cold denaturation, with temperature-dependent estimates of stability from NMR indicating a DeltaC(p) degrees for fragment complexation in good agreement with that determined for native ubiquitin, suggesting that fragment association results in the burial of a similar hydrophobic surface area. The stability of the complex shows appreciable pH dependence, suggesting that ionic interactions on the surface of the protein contribute significantly. However, denaturation studies of native ubiquitin in the presence of guanidine hydrochloride (Gdn.HCl) show little pH dependence, suggesting that ionic interactions may be "screened" by the denaturant, as recently suggested. Examination of the conformation of the isolated peptide fragments has shown evidence for a low population of nativelike structure in the N-terminal beta-hairpin (residues 1-17) and weak nascent helical propensity in the helical fragment (residues 21-35). In contrast, the C-terminal peptide (36-76) shows evidence in aqueous solution, from some Halpha chemical shifts, for nonnative phi and psi angles; nonnative alpha-helical structure is readily induced in the presence of organic cosolvents, indicating that tertiary interactions in both native ubiquitin and the folded fragment complex strongly dictate its structural preference. The data suggest that the N-terminal fragment (1-35), where interaction between the helix and hairpin requires the minimum loss of conformational entropy, may provide the nucleation site for fragment complexation.

Animals↗

PubMed1991

Cell-binding domain of endothelial cell thrombospondin: localization to the 70-kDa core fragment and determination of binding characteristics.

Endothelial and other cell types synthesize thrombospondin (TSP), secrete it into their culture medium, and incorporate it into their extracellular matrix. TSP is a large multifunctional protein capable of specific interactions with other matrix components, as well as with cell surfaces, and can modulate cell adhesion to the extracellular matrix. With the aim of understanding the mechanism by which TSP exerts its effect on cell adhesion, we studied the interaction of endothelial cell TSP (EC-TSP) with three different cell types: endothelial cells, granulosa cells, and myoblasts. We find that endothelial cells specifically bind radiolabeled EC-TSP with a Kd of 25 nM, and the number of binding sites is 2.6 X 10(6)/cell. Binding is not inhibitable by the cell-adhesion peptide GRGDS, indicating that the cell-binding site of EC-TSP is not in the RGD-containing domain. Localization of the cell-binding site was achieved by testing two chymotryptic fragments representing different regions of the TSP molecule, the 70-kDa core fragment and the 27-kDa N-terminal fragment, for their ability to bind to the cells. Cell-binding capacity was demonstrated by the 70-kDa fragment but not by the 27-kDa fragment. Binding of both intact [125I]EC-TSP and of the 125I-labeled 70-kDa fragment was inhibited by unlabeled TSP, heparin, fibronectin (FN), monoclonal anti-TSP antibody directed against the 70-kDa fragment (B7-3), and by full serum, but not by heparin-absorbed serum or the cell-adhesion peptide GRGDS. The 70-kDa fragment binds to endothelial cells with a Kd of 47 nM, and the number of binding sites is 5.0 x 10(6)/cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Stable substructures of eightfold beta alpha-barrel proteins: fragment complementation of phosphoribosylanthranilate isomerase.

The (beta alpha)8 (or "TIM")-barrel protein phosphoribosylanthranilate isomerase from Saccharomyces cerevisiae was cleaved between the sixth and seventh beta alpha module to test the capacity of the resulting fragments to adopt native format autonomously. The fragments, which were expressed from separate coding sequences, were soluble and monomeric. The amino-terminal fragment p1 was compact, possessed an almost nativelike far-UV but a strongly reduced near-UV CD spectrum, and unfolded cooperativity with guanidinium chloride. In contrast, the carboxyl-terminal fragment p2 was less compact than fragment p1, possessed only a weak far-UV and no detectable near-UV CD spectrum, and unfolded noncooperatively. The fragments assembled stoichiometrically to a complex with Kd = 0.2 microM, which was enzymically almost fully active. The rate of assembly was limited by a first-order process, probably the isomerization of the carboxyl-terminal fragment p2 to an assembly-competent structure. These results support a folding mechanism that comprises an intermediate with the first six beta alpha units folded in roughly native format and the last two beta alpha units partially unfolded. The similar behavior of the analogous fragments of the alpha subunit of tryptophan synthease supports the hypothesis that these two (beta alpha)8-barrel proteins have evolved from a common ancestor.

Aldose-Ketose Isomerases↗

PubMed1994

Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains.

The lactose permease of Escherichia coli is a membrane transport protein containing 12 transmembrane hydrophobic domains connected by hydrophilic loops. Coexpression of lacY gene fragments encoding contiguous polypeptides corresponding to the first and second halves of the permease [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325-4329] or the first two transmembrane domains and the remainder of the molecule [Wrubel, W., Stochaj, U., Sonnewald, U., Theres, C., & Ehring, R. (1990) J. Bacteriol. 172, 5374-5381] leads to active lactose transport. It is shown here that contiguous permease fragments with discontinuities in loop 1 (periplasmic), loop 6 (cytoplasmic), or loop 7 (periplasmic) exhibit transport activity; however, fragments with discontinuities in transmembrane domains III or VII fail to do so. The results are consistent with the interpretation that contiguous permease fragments with discontinuities in hydrophilic loops form functional duplexes, while fragments with discontinuities in transmembrane alpha-helical domains do not. On the basis of this notion, a series of contiguous, nonoverlapping permease fragments with discontinuities at various positions in loop 6, putative helix VII, and loop 7 were coexpressed to approximate the boundaries of putative transmembrane domain VII. Contiguous fragments with a discontinuity between Leu222 and Trp223 or between Gly254 and Glu255 are functional, but fragments with a discontinuity between Cys234 and Thr235, between Gln241 and Gln242, or between Phe247 and Thr248 are inactive. Therefore, it is likely that Leu222 and Gly254 are located in hydrophilic loops 6 and 7, respectively, while Cys234, Gln241, and Phe247 are probably located within transmembrane domain VII.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1984

Independent folding of the carboxyl-terminal fragment 228-316 of thermolysin.

The COOH-terminal fragment 206-316 of thermolysin was shown previously to maintain a stable folded structure in aqueous solution comparable to that of the corresponding region in native thermolysin and thus to possess protein domain characteristics [Fontana, A., Vita, C., & Chaiken, I. M. (1983) Biopolymers 22, 69-78]. In order to study the effect of polypeptide chain length on folding and stability of an isolated domain, the 111 amino acid residue fragment was shortened on the NH2-terminal side by removal of a 22-residue segment. Treatment of fragment 206-316 with hydroxylamine under alkaline conditions permitted selective cleavage of the Asn227-Gly228 peptide bond, and from the reaction mixture fragment 228-316 was isolated in homogeneous form. This fragment appeared to attain in aqueous solution the folding properties of the corresponding segment in the intact protein, as indicated by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunological properties. Thus, double-immunodiffusion analyses showed that fragment 228-316 is able to recognize and precipitate anti-thermolysin antibodies raised in rabbits with native thermolysin as immunogen. The fragment displayed fully reversible and cooperative conformational transitions mediated by pH, heat, and guanidine hydrochloride (Gdn.HCl), as expected for a globular protein species. Thermal denaturation of the fragment in aqueous solution at pH 7.8 showed a Tm of 66 degrees C and the Gdn.HCl-mediated unfolding a midpoint transition at 2.2 M denaturant concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗

PubMed1976

Characterization of the cyanogen bromide fragments of the beta chain of human haptoglobin.

Characterization of the cyanogen bromide (CNBr) fragments of the beta chain of human haptoglobin revealed five major fragments resulting from cleavage of four methionyl residues. The fragments were isolated by gel filtration in guanidine-HCl on Sepharose 6B and Bio-Gel P10 and P60. Compositional analyses of the five cyanogen bromide fragments accounted for 248-253 amino acid residues in agreement with the number of residues determined for the intact beta chain. Most of the carbohydrate was attached to CNBr II. Automated amino-terminal sequence analysis and carboxyl-terminal hydrolysis with carboxypeptidase of the haptoglobin beta chain and cyanogen bromide fragments identified 139 residues, or about 55% of the beta-chain molecule. The placement of the fragments within the beta-chain molecule was established by sequence analysis of whole beta chain and a plasmin cleavage fragment. The position of CNBr V was confirmed by the absence of homoserine or homoserine lactone. Cyanogen bromide reaction of intact haptoglobin 1-1 resulted in the isolation of a beta-chain fragment, CNBr III, covalently attached to the intact alpha1 chain by a single disulfide bond. The beta chain was shown to have primary structural similarities to the chymotrypsin family of serin eproteases. Partial sequence analysis of CNBr V established the region which is comparable to the serine-195 active-site region: /Asp-Thr-Cys-Tyr-Gly-Asp-Ala-Gly-Ser-Ala-Phe/ (residues 189-199, chymotrypsinogen A numbering). The active-site serine-195 is replaced by alanine; however, the specificity residue of the trypsin-like enzymes, Asp-189, is preserved. Several minor cyanogen bromide cleavage products were also identified in yields of up to 15%. These minor cleavage products give evidence that tryptophanyl residues in proteins, or glycoproteins, are also susceptible to cyanogen bromide cleavage.

Amino Acid Sequence↗

PubMed2005

Diversity of human insulin-like growth factor (IGF) binding protein-2 fragments in plasma: primary structure, IGF-binding properties, and disulfide bonding pattern.

The insulin-like growth factor binding proteins (IGFBPs) play a major role in the regulation of the effects and the bioavailability of the insulin-like growth factors (IGFs). IGFs are released from IGFBP-IGF complexes by proteolysis of IGFBPs generating fragments with reduced ligand-binding properties. To identify naturally occurring fragments of IGFBP-2, a peptide library generated from human hemofiltrate was immunologically screened. Purification of immunoreactive IGFBP-2 fragments was performed by consecutive chromatographic steps. A total of 18 different IGFBP-2 fragments was isolated and characterized. The peptides exhibited different N-terminal amino acid residues that were located in the variable midregion of IGFBP-2. Four major cleavage sites were determined to be between Tyr103 and Gly104, Leu152 and Ala153, Arg156 and Glu157, and Gln165 and Met166. The resulting fragments were further processed by amino and/or carboxy peptidases and comprised 37-185 amino acid residues. Ligand blotting, solution binding assays, and BIAcore analyses revealed that all tested fragments retained low IGF-binding capacity. The most abundant fragment IGFBP-2 (167-279) showed 10% of IGF-II binding compared to recombinant human (rh)IGFBP-2. Furthermore, the disulfide bonding pattern of the C-terminal domain of rhIGFBP-2 was defined, indicating linkages between cysteine residues 191-225, 236-247, and 249-270. This study provides the most comprehensive molecular characterization of human IGFBP-2 fragments formed in vivo, exhibiting both residual IGF-binding capacities and the integrin-binding sequence.

Adult↗

PubMed1998

Characterization of the overproduced NADH dehydrogenase fragment of the NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.

The proton-pumping NADH:ubiquinone oxidoreductase of Escherichia coli is composed of 14 different subunits and contains one FMN and up to nine iron-sulfur clusters as prosthetic groups. By use of salt treatment, the complex can be split into an NADH dehydrogenase fragment, a connecting fragment and a membrane fragment. The water-soluble NADH dehydrogenase fragment has a molecular mass of approximately 170,000 Da and consists of the subunits NuoE, F, and G. The fragment harbors the FMN and probably six iron-sulfur clusters, four of them being observable by EPR spectroscopy. Here, we report that the fully assembled fragment can be overproduced in E. coli when the genes nuoE, F, and G were simultaneously overexpressed with the genes nuoB, C, and D. Furthermore, riboflavin, sodium sulfide, and ferric ammonium citrate have to be added to the culture medium. The fragment was purified from the cytoplasm by means of ammonium sulfate fractionation and chromatographic steps. The preparation contains one noncovalently bound FMN per molecule. Two binuclear (N1b and N1c) and two tetranuclear (N3 and N4) iron-sulfur clusters were detected by EPR in the NADH reduced preparation with spectral characteristics identical with those of the corresponding clusters in complex I. The preparation fulfills all prerequisites for crystallization of the fragment.

Electron Transport↗