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PubMed1998

Control of protein splicing by intein fragment reassembly.

Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein.

Bacterial Proteins↗
PubMed1998

Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos.

In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media. ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2. A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme. The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit. The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos. Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.

Apoptosis↗
PubMed2001

A microscopic and biochemical study of fragmentation phenotypes in stage-appropriate human embryos.

The occurrence of a pleiomorphic population of cytoplasmic fragments is a common characteristic of early human embryos fertilized in vitro. Here, temporal, spatial, fine structural, and biochemical aspects of fragmentation were examined in fragmented monospermic and dispermic pronuclear to early cleavage stages human embryos classified as stage-appropriate during the first 3.5 days of culture. The morphodynamics of certain common patterns of fragmentation and the movement and composition of fragments were analysed by time-lapse video, mitochondrial fluorescent probes, and transmission electron microscopy. Plasma membrane and nuclear DNA integrity were assessed by annexin V staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and single-cell alkaline gel electrophoresis ('comet') assays respectively. Developmental competence for affected embryos was related to outcome after embryo transfer. The results demonstrate that certain common forms of spontaneous fragmentation affecting early human embryos are not lethal, and that clusters of apparent fragments are often transient structures, which disappear by resorption or lysis. The findings suggest that the occurrence and fate of fragments characteristic of these phenotypes may be related to oncosis-like processes associated with transient and focal ATP deficiencies in blastomeres and mitochondrial deficiencies or absence in extracellular fragments.

Blastomeres↗
PubMed1998

Radiocontrast-induced DNA fragmentation of renal tubular cells in vitro: role of hypertonicity.

BACKGROUND: Radiocontrast-induced nephropathy is a clinically important complication of invasive cardiological procedures. It has been associated with DNA fragmentation of renal tubular cells, which is a hallmark feature of programmed cell death (apoptosis). We investigated the mechanism of this DNA fragmentation in an in vitro model of radiocontrast cytotoxicity on renal epithelial cells. METHODS: Madin Darby canine kidney (MDCK) cell monolayers were incubated (for 2-8 h) with isoiodine doses (37-111 mg iodine/ml) of the highly hyperosmolal, ionic radiocontrast agent diatrizoate or of the less hyperosmolal, non-ionic substance iopamidol. Mannitol, urea, and NaCl control media of corresponding hyperosmolality were used to evaluate the contribution of hypertonicity, hyperosmolality and/or ionic strength to radiocontrast toxicity. DNA fragmentation was assessed using fluorescence-activated cell sorting (FACS), agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labelling (TUNEL), cell morphology was analysed in Giemsa-stained cytospins. RESULTS: Diatrizoate induced concentration- and time-dependent DNA fragmentation of MDCK cells which was associated with morphological signs of apoptosis. Cycloheximide (1 microg/ml) did not prevent diatrizoate-induced DNA fragmentation, indicating that it is not dependent on protein synthesis. Diatrizoate-mediated cell death was associated with cell detachment from the tissue culture matrix. However, the DNA fragmentation is not a consequence of cell detachment since the prevention of cell attachment on agarose-coated dishes induced significantly less DNA fragmentation than diatrizoate. Iopamidol caused no detectable DNA breakdown. In contrast, hypertonic mannitol and sodium chloride, but not hyperosmolal urea, induced DNA fragmentation in MDCK cells, albeit less than diatrizoate. CONCLUSIONS: The DNA fragmentation of MDCK cells induced by diatrizoate is related to its hypertonicity in this in vitro model of radiocontrast cytotoxicity. Nuclear disintegration with subsequent cell death may contribute to the pathophysiology of radiocontrast-induced nephropathy, particularly in the hypertonic/hypoxic environment of the renal medulla. The present results underscore the importance of avoiding hyperosmolal urine states in patients at high risk of radiocontrast-induced nephropathy.

Animals↗
PubMed2000

Differential scanning calorimetry of light meromyosin fragments having various lengths of carp fast skeletal muscle isoforms.

Various recombinant light meromyosin (LMM) fragments were prepared from cDNAs encoding the 10 degrees C and 30 degrees C types of myosin heavy chain isoforms predominantly expressed in fast skeletal muscles of the 10 degrees C- and 30 degrees C-acclimated carp, respectively. These included three kinds of quarter fragments, 1/4-, 2/4-, and 4/4-quarter, composed of residues 1-130, 131-270, and 401-563 from the N-terminus, respectively, as well as three halves, N-, M-, and C-half fragments, containing residues 1-301, 131-400, and 302-563, respectively, and 69K fragments of residues 1-525. Unfortunately, in spite of extensive efforts, the 3/4-quarter fragment was not expressed for both 10 degrees C and 30 degrees C types in our expression system using Escherichia coli. All the LMM fragments except for the 10- and 30-2/4 quarters for the 10 degrees C and 30 degrees C types, respectively, exhibited a typical pattern of a-helix in CD spectrometry. When these were subjected to differential scanning calorimetry (DSC), 30 degrees C-type LMM fragments were all found to be more thermostable than the 10 degrees C-type counterparts. To identify amino acid substitutions responsible for different thermostabilities between the 10 degrees C- and 30 degrees C-type LMMs, six mutant proteins were prepared, mainly focusing on substitutions in the C-terminal half of LMM, and subjected to DSC and CD analyses. For three mutants in which two residues of the 10 degrees C type were replaced by those of the 30 degrees C type, 10-S355T/T361A, 10-M415L/L417V, and 10-S535A/H536Q, the endothermic peaks in DSC increased by 1.4-2.0 degrees C from that of the original 10 degrees C type. The T(m) values for two single-residue substitutions, 10-H449R and 10-T491I, shifted 0.8 and 1.3 degrees C higher than that for the 10 degrees C-type LMM, respectively, whereas the last mutant, 10-G61V, showed no change in thermostability. The finding that the difference in T(m) values for major endothermic peaks from the 10-69K and 30-69K fragments was 4.6 degrees C, which roughly corresponds to that between the original 10 degrees C and 30 degrees C types, suggested that the eight substitutions located in the C-terminal region of the 69K fragments (residues 302-525) are major candidates for the residues responsible for the difference in thermostability between the 10 degrees C- and 30 degrees C-type LMMs.

Amino Acid Sequence↗
PubMed1982

Behavior of fragmented band 3 from chymotrypsin-treated bovine erythrocyte membrane in nonionic detergent solution.

Bovine band 3 which had been cleaved into two membrane intercalated fragments by extracellular chymotryptic digestion was partially purified and the behavior of fragmented band 3 was compared with that of intact band 3 in nonionic detergent, C12E9, solution. The two fragments were always co-eluted with nearly a 1:1 molar ratio from ion-exchange and gel filtration columns, confirming the observation of Reithmeier ((1979) J. Biol. Chem. 254, 3054-3060) that the fragments are bound together. However, as shown by the molecular size of fragmented band 3 and its reactivity with dimethylmaleic anhydride and/or a cross-linking reagent, the fragmentation caused some change in gross conformation of the protein. Dissociation of the two fragments, though not complete, was found to occur on treatment of fragmented band 3 with dimethylmaleic anhydride, which is a dissociating reagent for band 3 oligomers into the monomers. The disruption of self-associated band 3 was accompanied with a disruption of helical structure. The results suggest that a mutual interaction between the chymotryptic fragments is of importance to form the folded structure maintaining a band 3 dimer.

Animals↗
PubMed1997

Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment.

By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the variable domains are taken out of their natural context in the Fab fragment, where they are associated with the constant domains of the light (CL) and heavy chain (CH1). As a consequence, all residues of the former variable/constant domain interface become solvent exposed. In an analysis of 30 non-redundant Fab structures it was found that at the former variable/constant domain interface of the Fv fragment the frequency of exposed hydrophobic residues is much higher than in the rest of the Fv fragment surface. We investigated the importance of these residues for different properties such as folding in vivo and in vitro, thermodynamic stability, solubility of the native protein and antigen affinity. The experimental model system was the scFv fragment of the anti-fluorescein antibody 4-4-20, of which only 2% is native when expressed in the periplasm of Escherichia coli. To improve its in vivo folding, a mutagenesis study of three newly exposed interfacial residues in various combinations was carried out. The replacement of one of the residues (V84D in VH) led to a 25-fold increase of the functional periplasmic expression yield of the scFv fragment of the antibody 4-4-20. With the purified scFv fragment it was shown that the thermodynamic stability and the antigen binding constant are not influenced by these mutations, but the rate of the thermally induced aggregation reaction is decreased. Only a minor effect on the solubility of the native protein was observed, demonstrating that the mutations prevent aggregation during folding and not of the native protein. Since the construction of all scFv fragments leads to the exposure of these residues at the former variable/constant domain interface, this strategy should be generally applicable for improving the in vivo folding of scFv fragments and, by analogy, also the in vivo folding of other engineered protein domains.

Amino Acid Sequence↗
PubMed1995

Bacterial secretion of the Fab fragment of a mouse monoclonal IgM that reacts with IgG variable regions.

In this report, we describe the expression system that enabled us to produce in Escherichia coli the Fab fragment of a mouse IgM that has previously been shown to inhibit the binding of IgG to autoantigens by interacting with their variable regions. In our system, both light chain and heavy chain fragments were put under the control of the malE promoter. The light chain was fused to the MalE signal sequence, while the heavy chain variable and first constant region were fused to the alkaline phosphatase signal sequence. In this system, after induction of the promoter with maltose, the Fab fragment could be detected in a periplasmic extract of the bacteria by Western blotting and also by ELISA. This Fab fragment was purified on a goat anti-mouse immunoglobulin immunoadsorbent and biotinylated. The Fab fragment produced by E.coli reacted with the trinitrophenyl (TNP) hapten and F(ab')2 fragments of mouse IgG and these reactivities could be specifically inhibited by the corresponding soluble antigens. The dissociation constants of this Fab were 1.65 x 10(-6) M for TNP and 5 x 10(-6) M for IgG F(ab')2 fragments, indicating that the affinity of the Fab fragment compared with that of the whole IgM molecule was similar for TNP but was lower for IgG F(ab')2 fragments.

Animals↗
PubMed2005

Carboxyl-terminal parathyroid hormone fragments: role in parathyroid hormone physiopathology.

PURPOSE OF REVIEW: Carboxyl-terminal parathyroid hormone (C-PTH) fragments constitute 80% of circulating PTH. Since the first 34 amino acids of the PTH structure are sufficient to explain PTH classical biological effects on the type I PTH/PTHrP receptor and since C-PTH fragments do not bind to this receptor, they have long been considered inactive. Recent data suggest the existence of a C-PTH receptor through which C-PTH fragments exert biological effects opposite to those of human PTH(1-84) on the type I PTH/PTHrP receptor. This is why a lot of attention has been paid to these fragments recently. RECENT FINDINGS: In vivo, synthetic C-PTH fragments are able to decrease calcium concentration, to antagonize the calcemic response to human PTH(1-34) and human PTH(1-84) and to decrease the high bone turnover rate induced by human PTH(1-84). In vitro, they inhibit bone resorption, promote osteocyte apoptosis and exert a variety of effects on bone and cartilaginous cells. These effects are opposite to those of human PTH(1-84) on the PTH/PTHrP type I receptor. This suggests that the molecular forms of circulating PTH may control bone participation in calcium homeostasis via two different receptors. Clinically, the accumulation of C-PTH fragments in renal failure patients may cause PTH resistance and may be associated with adynamic bone disease. Rare parathyroid tumors, without a set point error, overproduce C-PTH fragments. The implication of C-PTH fragments in osteoporosis is still to be explored. SUMMARY: C-PTH fragments represent a new field of investigation in PTH biology. More studies are necessary to disclose their real importance in calcium and bone homeostasis in health and disease.

Animals↗
PubMed2003

Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments.

Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9-kD and 5-kD peptide fragments formed by limited proteolytic digestion of the N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3'-kinase fold into an active native-like structure on interaction with one another. The corresponding 5-kD fragment of the homologous Src protein, however, was not capable of structurally complementing the p85 9-kD fragment, indicating that fragment complementation among these SH2 domains is sensitive to the sequence differences between the Src and p85 domains. Partial complementation and folding activity could be recovered with hybrid sequences of these SH2 domains. Complete alanine scanning of the 5-kD p85 fragment was used to identify the sequence recognition elements required for complex formation. The alanine substitutions in the p85 5-kD fragment that abolished binding affinity with the cognate 9-kD fragment correlate well with highly conserved residues among SH2 domains that are either integrally involved in core packing or found at the interface between fragments. Surprisingly, however, mutation of a nonconserved surface-exposed aspartic acid to alanine was found to have a significant effect on complementation. A single additional mutation of arginine to aspartic acid allowed for recovery of native structure and increased the thermal stability of the designed Src-p85 chimera by 18 degrees C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability.

Alanine↗
PubMed2001

Molecular dynamics simulation of Escherichia coli dihydrofolate reductase and its protein fragments: relative stabilities in experiment and simulations.

We have carried out molecular dynamics simulations of the native dihydrofolate reductase from Escherichia coli and several of its folded protein fragments at standard temperature. The simulations have shown fragments 1--36, 37--88, and 89--159 to be unstable, with a C(alpha)RMSD (C(alpha) root mean squared deviation) >5 A after 3.0 nsec of simulation. The unfolding of fragment 1--36 was immediate, whereas fragments 37--88 and 89--159 gradually unfolded because of the presence of the beta-sheet core structure. In the absence of residues 1--36, the two distinct domains comprising fragment 39--159 associated with each other, resulting in a stable conformation. This conformation retained most of its native structural elements. We have further simulated fragments derived from computational protein cutting. These were also found to be unstable, with the exception of fragment 104--159. In the absence of alpha(4), the loose loop region of residues 120--127 exhibited a beta-strand-like behavior, associating itself with the beta-sheet core of the protein fragment. The current study suggests that the folding of dihydrofolate reductase involves cooperative folding of distinct domains which otherwise would have been unstable as independent folded units in solution. Finally, the critical role of residues 1--36 in allowing the two distinct domains of fragment 104--159 to fold into the final native conformation is discussed.

Computer Simulation↗
PubMed1995

Characterization of the aminopeptidase activity of epidermal leukotriene A4 hydrolase against the opioid dynorphin fragment 1-7.

Leukotriene A4 hydrolase is a bifunctional cytosolic enzyme, which both hydrolyses leukotriene A4 (LTA4) into leukotriene B4 (LTB4) and exerts aminopeptidase activity against opioid peptides. In the present study we have investigated whether the peptides angiotensin I and II, bradykinin, kallidine, histamine, dynorphin fragment 1-7 and substance P can act as substrates for epidermal and neutrophil LTA4 hydrolase. Among the tested substrates, dynorphin fragment 1-7 was found to be the best substrate for the enzyme. The aminopeptidase activity of epidermal and neutrophil LTA4 hydrolase against dynorphin fragment 1-7 was further characterized. The enzyme was purified from human epidermis and human neutrophils by anion exchange chromatography (Q-Sepharose) and affinity chromatography on a column with the LTA4 hydrolase inhibitor bestatin coupled to AH-Sepharose. The incubation of the dynorphin fragment 1-7 with LTA4 hydrolase resulted in the formation of tyrosine. The presence of the N-terminal amino acid tyrosine is essential for the interaction of opioids with their receptors, and this finding indicates that the LTA4 hydrolase can inactivate dynorphin fragment 1-7. After the two purification steps no other aminopeptidases acting at the N-terminal tyrosine of dynorphin fragment 1-7 was present in the preparation. This was demonstrated by the abolishment of the degradation at the N-terminal end of dynorphin fragment 1-7 when preincubating the enzyme preparation with LTA4 before the incubation with the dynorphin fragment 1-7. The abolishment of the aminopeptidase activity shows that activation of the hydrolase part of the enzyme, with conversion of LTA4 into the potent proinflammatory compound LTB4, results in an inhibition of the aminopeptidase activity of the enzyme. As a result, the catabolism of dynorphin fragment 1-7 and probably of other opioid peptides is inhibited, resulting in sustained biological effects of these opioids. This phenomenon may be important for the maintenance of inflammation in skin conditions, such as psoriasis and atopic dermatitis, in which LTB4 is formed.

Amino Acid Sequence↗
PubMed2004

Elevated leptin fragments in renal failure correlate with BMI and haematopoiesis and are normalized by haemodialysis.

OBJECTIVE: Leptin is an adipocyte hormone important in appetite, energy homeostasis, neuroendocrine and haematopoietic function. Patients with renal failure often have elevated total and free leptin levels. Biologically active leptin fragments (leptin(22-56) and leptin(57-92)) have been identified, but whether these fragments are affected by renal failure and/or haemodialysis (HD) is not known. RESEARCH METHODS: Leptin, leptin(22-56) and leptin(57-92) levels were measured in 28 HD patients [14 men, 14 women, age 45.5 +/- 16.4 years, body mass index (BMI) 23.8 +/- 3.6 kg/m2] and 25 healthy controls with similar age and BMI. In 18 HD patients, leptin and fragment levels were measured before and after 2 consecutive dialysis treatments and on the intermediate, dialysis-free day. RESULTS: Baseline leptin levels were higher in HD patients vs. controls (69.3 +/- 62.2 ng/ml vs. 30.4 +/- 32.7, P < 0.02) as were leptin(22-56) (7.14 +/- 7.04 ng/ml vs. 1.86 +/- 1.84, P < 0.02) and leptin(57-92) (5.94 +/- 6.08 ng/ml vs. 1.58 +/- 1.98, P < 0.02). Baseline leptin and fragment levels correlated significantly and independently with BMI in HD patients (r = 0.70, r = 0.59, r = 0.72, respectively, P < 0.001). After each HD session, leptin levels were reduced to levels not different from controls, but increased on the intermediate, dialysis-free day. Similar to and independently from total leptin, both leptin fragments were reduced after the first HD session to levels not different from controls. The reduction in leptin levels was higher with synthetic vs. cellulosic membrane types (37.7 +/- 23.5%vs. 18.1 +/- 21.8%, P < 0.03). Leptin correlated weakly with the erythropoietin to haematocrit ratio (T = -0.20, P = 0.14), while leptin(22-56) had a significant negative correlation with this index (T = -0.42, P < 0.01), suggesting that this fragment may favour haematopoiesis. DISCUSSION: Leptin fragments are detected in human serum, and both leptin and leptin fragments correlate with BMI, are significantly elevated in HD patients compared to controls, and are significantly decreased by haemodialysis. The elevated leptin fragments may have important physiological significance for the anorexia, hypogonadism, and anaemia commonly seen in HD patients, but this remains to be conclusively shown by interventional trials.

Adult↗
PubMed2006

Hypoallergenic fragment of Par j 2 increases functional expression of Toll-like receptors in atopic children.

BACKGROUND: Parietaria judaica (Par j) is one of the main causes of allergy in the Mediterranean countries. The activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) inhibits nasal inflammation of atopic children. OBJECTIVE: To examine, in vivo and in vitro, the effect of recombinant Par j 2 (rPar j 2) and of its fragments (1-55 and 52-102) on atopic children. METHODS: We used skin prick test for in vivo evaluations. We assessed, in vitro, in peripheral blood mononuclear cells (PBMC), the effect of rPar j 2 and of the two fragments on neutrophil chemotaxis, on CD45RO, on TLR2 and TLR4 expression, on LPS binding and on interferon (IFN)-gamma release, by a microchemotaxis chamber, by flow cytometry and by enzyme-linked immunosorbent assay, respectively. RESULTS: In vivo while rPar j 2 induced a positive skin reaction, 1-55 and 52-102 fragments did not. In vitro, while rPar j 2 increased both CD45RO expression and neutrophils chemotaxis in PBMC, both Par j 2 fragments did not. 1-55 fragment of Par j 2 upregulated both TLR2 and TLR4 expression and LPS binding, while the rPar j 2 and 52-102 fragment did not. Finally, 1-55 fragment of Par j 2 induced IFNgamma release, while the rPar j 2 and 52-102 fragment did not. CONCLUSIONS: Hypoallergenic 1-55 fragment, upregulating innate immunity receptors and increasing IFNgamma, might re-orientate, in atopics, the immune system toward a physiologic balance between Th1 and Th2 responses.

Adolescent↗
PubMed1983

Exchange of histones H1 and H5 between chromatin fragments. A preference of H5 for higher-order structures.

Histones H1 and H5 can exchange between an H1-containing chromatin fragment from rat liver and an H1, H5-containing fragment from chicken erythrocytes at ionic strengths from about 35 mM to 105 mM. The redistribution has reached equilibrium by ionic strength 75 mM in 1 h or less at 4 degrees C. After exchange at ionic strength 75 mM, long fragments, whether of rat liver or chicken erythrocyte chromatin, are recovered with a higher H5:H1 ratio than short fragments, suggesting a stronger preference of H5 than of H1 for higher-order structures which exist for long fragments at ionic strength 75 mM. Competition experiments between occupied H1 or H5 binding sites on chromatin fragments from rat liver or chicken erythrocytes and empty sites on H1-depleted rat chromatin show that rat H1 does not distinguish between the two types of site, whereas H5 discriminates in favour of sites on native chromatin, even when the chicken fragments are too short to form higher-order structures. (The behavior of the chicken H1, which may be bound less tightly than rat H1, depends on the length of the chicken fragment, in a manner suggesting that fragments of 15 nucleosomes and longer can form stable higher-order structures which have high-affinity binding sites for both H5 and H1.) We conclude that the affinity of sites for H5 is in the order:higher-order structures greater than nucleosome filament much greater than H1-depleted chromatin. The same relative order of affinities may well apply for H1 but the discrimination is much lower. This difference between H1 and H5 seems likely to be relevant to the greater stability of H5-containing chromatin, and in turn its transcriptional inactivity, and indeed to the mechanism of replacement of H1 by H5 during the terminal stages of erythropoiesis.

Animals↗
PubMed1990

Use of fragments of hirudin to investigate thrombin-hirudin interaction.

Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methionine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg-NH-Ph). The N-terminal fragment (residues 1-52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C-terminal fragment protected alpha-thrombin against cleavage by trypsin, the N-terminal fragment did not. Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.

Antithrombin III↗
PubMed1990

Localization of the peptidase activity of human serum butyrylcholinesterase in a approximately 50-kDa fragment obtained by limited alpha-chymotrypsin digestion.

Purified human serum butyrylcholinesterase (approximately 90-kDa subunit) is known to exhibit aryl acylamidase and peptidase activity. Limited alpha-chymotrypsin digestion of the purified butyrylcholinesterase gave three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa. In our earlier studies [Rao and Balasubramanian (1989) Eur. J. Biochem. 179, 639-644] we characterized the approximately 20-kDa fragment and showed that it exhibited both butyrylcholinesterase and aryl acylamidase activities. In the present studies the approximately 50-kDa fragment is characterized. This fragment, after isolation by Sephadex G-75 chromatography from a chymotryptic digest of purified butyrylcholinesterase, exhibited only peptidase activity and was devoid of cholinesterase and aryl acylamidase activities. It could bind to a column of Ricinus communis agglutinin bound to Sepharose, indicating its glycosylated nature and the presence of galactose. The peptidase activity in the approximately 50-kDa fragment could be immuno-precipitated by a polyclonal antibody raised against purified butyrylcholinesterase. SDS-gel electrophoresis of this fragment isolated by R. communis agglutinin-Sepharose and Sephadex G-75 chromatography showed a protein band of approximately 50 kDa by silver staining. Amino-terminal sequence analysis of the approximately 50-kDa fragment gave the sequence of Gly-Pro-Thr-Val-Asp which corresponded to amino acid residues 291-295 in the butyrylcholinesterase sequence [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. The combined results suggested that alpha-chymotrypsin digestion of human serum butyrylcholinesterase resulted in the formation of a approximately 20-kDa fragment exhibiting both cholinesterase and aryl acylamidase activities and a approximately 50-kDa fragment exhibiting only peptidase activity.

Amino Acid Sequence↗
PubMed1995

Structural and functional characterization of proteolytic fragments derived from the C-terminal regions of bovine fibrinogen.

A number of new as well as previously described fragments derived from the D region of bovine fibrinogen by limited proteolysis have been characterized by sequence analysis, differential scanning calorimetry and circular dichroism. Determination of the extremities of the polypeptide chains forming individual fragments allowed the scheme of proteolysis and the borders between domains in the D region of fibrinogen to be established. It was also found that the most thermostable region of the D fragment (TSD) can be substantially reduced in size without loss of its compact structure. The alpha-helical content of the newly prepared 21-kDa TSD2 and 16-kDa TSD3 fragments were 82% and 75%, respectively, strongly supporting a coiled-coil structure for this region of the fibrinogen molecule. The DX and DZ fragments, prepared from a chymotryptic digest of the DLA fragment, were found to be similar to the DL and DY fragments, respectively, except for an internal cleavage at K393-T394 in their beta chains. This cleavage leads to destabilization of all thermolabile domains, indicating interaction between them. The DL and DY fragments, containing only one polymerization site in their beta chains, were able to inhibit fibrin polymerization at high concentration. However, these same fragments failed to bind to fibrin-Sepharose under conditions where their structural analogues, DX and DZ, were tightly bound, indicating that cleavage after K393 substantially increases the affinity of this site.

Amino Acid Sequence↗