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PubMed1982

Binding of an 80 000 dalton trypsin fragment of spectrin to intact spectrin.

A specific and saturable interaction of an 80 000 dalton tryptic fragment of spectrin with intact spectrin has been detected. When spectrin was incubated with 125I-labelled 80 kDa fragment at 37 degrees C in the presence of 0.1 M NaCl, acrylamide gradient gel electrophoresis showed the presence of bands in addition to the usual spectrin dimer and tetramer and the 80 kDa fragment. These bands correspond to 5.6 X 10(5) daltons (dimer + fragment), 1.04 X 10(6) daltons (tetramer + fragment) and 1.52 X 10(6) daltons (hexamer + fragment). Measurement of radioactivity showed that these additional bands contained the labelled fragment. Maximal binding capacity of the 80 kDa fragment was approximately 170 micrograms fragment per mg spectrin, corresponding to 1 mol fragment per mol spectrin dimer.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

The isolated 21 kDa N-terminal fragment of myosin binds to actin in an ATP and ionic strength-dependent manner.

Recently we reported that the isolated 23 kDa N-terminal fragment of myosin heavy chain, which contains the 'consensus' ATP binding site, binds to actin in an ATP-sensitive manner (Muhlrad, A. (1989) Biochemistry 28, 4002). In order to determine whether the 'consensus' ATP site has a role in the ATP-dependent actin binding of the fragment, we isolated a shorter 21 kDa N-terminal fragment, which contains only a part of the 'consensus' site. The 21 kDa fragment was obtained by photocleavage of myosin subfragment-1 in the presence of vanadate (Mocz, G. (1989) Eur. J. Biochem. 179, 373); the cleavage was followed by dissociation of the S-1 heavy chain fragments with guanidine hydrochloride and renaturation. The isolated 21 kDa fragment binds to F-actin, since it cosediments with actin, inhibits the actin-activated ATPase activity of myosin subfragment-1 and shows increase in light scattering upon titration by actin. The affinity of the binding is rather high (Kassoc = 0.83.10(7) M-1). The light scattering increase is reversed, e.g., the 21 kDa-actin complex is dissociated, upon addition of ATP both in the presence and absence of Mg, but less ATP is needed for dissociation when Mg is absent. Other polyphosphates, including inorganic triphosphate, pyrophosphate and ADP, also dissociate both the 21 kDa-actin and 23 kDa-actin complexes but the latter needs a higher concentration of polyphosphates for dissociation. However, these polyphosphates, except ATP, do not dissociate the (subfragment-1)-actin complex in the absence of Mg. The 21 kDa-actin and the 23 kDa-actin complexes are also dissociated by increasing ionic strength or by a low concentration of polyglutamate, which hardly affect the light scattering of the (subfragment-1)-actin complex. The results indicate that the binding of the N-terminal fragments of myosin to actin, unlike that of intact subfragment-1, is essentially of electrostatic nature. The polyanions dissociate the myosin fragment-actin complexes not by reacting with the 'consensus' ATP binding site, but by competing with actin for a positively charged binding site on the 21 kDa fragment. The only positively charged cluster in the amino acid sequence of this fragment is the 143-147 stretch, which may participate in forming the actin binding site.

Actins↗
PubMed1977

Possible evidence for the existence of a growth hormone fragment in porcine pituitary.

In an attempt to search for growth hormone fragments in the pituitary, a radioimmunoassay was developed for a 55 residue S-amino-ethylated CNBr fragment (fragment B) of porcine growth hormone corresponding to residues 126-180 of human growth hormone. The assay was sensitive to 50 pg of fragment B whereas displacement of 125I-labelled fragment B by procine growth hormone required a 10(3) M excess and was non-parallel. In a homogolous porcine growth hormone radioimmunoassay, fragment B was non-reactive. Gel filtration of an extract of porcine pituitary on Sephadex G-75 revealed three peaks of fragment B immunoreactivity: peak I (29% of total immunoreactivity) eluted in the void volume, peak II (49%) eluted in the position of growth hormone, and peak III (12%) was more retarded than fragment B. Nearly all of the growth hormone immunoreactivity eluted as a single peak in the position of 125I-labelled porcine growth hormone. The dilution curve of peak III but not of peaks I or II was parallel to that of fragment B. The results indicate the existence within porcine pituitary of material cross-reactive with a portion of the growth hormone molecule, possible representing a growth hormone fragment.

Animals↗
PubMed1993

Fibronectin fragments bind to and penetrate cartilage tissue resulting in proteinase expression and cartilage damage.

We have reported that fibronectin (Fn) fragments added to bovine articular cartilage slices in culture causes marked cartilage damage by enhancing proteinase expression and resultant degradation and release of proteoglycan (PG). Several different non-overlapping Fn fragments, an amino-terminal 29-kDa, gelatin-binding 50-kDa and integrin-binding 140-kDa Fn fragment, representing nearly all of the polypeptide chain, were compared in terms of ability to cause PG release from cartilage and to bind cartilage. The most active fragment, the 29-kDa fragment, was able to enter cartilage in an intact metacarpophalangeal joint in culture and cause PG release at the same rate as with surgically cut cartilage. Further, when radiolabelled 29-kDa fragment was added to cartilage, a large proportion bound the intact articular surface, while a lesser amount diffused throughout the tissue matrix and concentrated in clusters near the mid-section of full thickness cartilage. The 29-kDa, 50-kDa, 140-kDa Fn fragments and Fn, respectively, showed PG degradation activities 9-, 6-, 2- and 1.1-fold that of control levels and bound cartilage to the extent of 180, 20, 18 and 2 pmol/100 mg cartilage, respectively. Therefore, the PG degradation activities were greatest for the smaller fragments, which bound to the greatest extent. The apparent Kd values for interaction of the 29-kDa, 50-kDa, 140-kDa fragments and Fn for cartilage tissue were about 1.2, 0.3, 0.1 and 0.02 microM, respectively, and the order was inversely related to PG degradation activities. We conclude that the smaller the Fn fragment, the greater the degradation activity and extent of binding to cartilage tissue, but the weaker the affinity.

Animals↗
PubMed1991

High precision immunoscanning electron microscopy using Fab fragments coupled to ultra-small colloidal gold.

Ultra-small colloidal gold (less than 1 nm), bound to Fab fragments provides the shortest practical specific marker system to date and can be used in concert with field emission scanning electron microscopes to precisely locate antigenic sites. An "in-lens" FE-SEM equipped with a highly sensitive single crystal YAG-detector for backscattered electrons, as well as the use of advanced specimen preparation techniques based on cryofixation, are among the indispensible prerequisites. A T-even type Escherichia coli bacteriophage, Tu II*-46, was chosen to study properties of the immunogold labeling system. Distinct regions on the tail fibers of this phage were labeled with Fab fragments derived from antibodies against the related phage Tu II*-6. The tail fibers are composed of pairs of homologous proteins, thus offering two identical antigenic sites at the same locus on the tail fibers. Fab fragments can be visualized in the SEM at high accelerating voltage (30 kV) without any additional marker. This permits comparison of the labeling characteristics of unmarked and colloidal gold-marked Fab fragments. Unmarked Fab fragments often bind by pairs (two singlet Fab fragments bound opposed to each other along the axis of the tail fiber). The labeling efficiency of unmarked Fab fragments was greater than that of ultra-small gold-labeled Fab fragments. Binding by pairs was not seen after labeling with ultra-small gold-Fab fragments. The conjugates used in this study exhibited one colloidal gold per Fab fragment.

Animals↗
PubMed2005

Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test.

OBJECTIVE: To improve the sperm chromatin dispersion (SCD) test and develop it as a simple kit (Halosperm kit) for the accurate determination of sperm DNA fragmentation using conventional bright-field microscopy. DESIGN: Method development, comparison, and validation. SETTING: Medical genetics laboratory, academic biology center, and reproductive medicine centers. PATIENT(S): Male infertility patients attending the Reproductive Medicine Center. A varicocele patient and a group of nine fertile subjects. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): [1] The quality of chromatin staining in relaxed sperm nuclear halos and tail preservation; [2] SCD scoring reproducibility; [3] comparison with the sperm chromatin structure assay in 45 samples; [4] frequency of sperm with DNA fragmentation after incubation with increasing doses of the nitric oxide donor sodium nitroprusside and in sperm samples for 9 fertile men, 46 normozoospermic patients, 23 oligoasthenoteratozoospermic patients, and a subject with varicocele. RESULT(S): The sperm nuclei with DNA fragmentation, either spontaneous or induced, do not produce or show very small halos of DNA loop dispersion after sequential incubation in acid and lysis solution. The improved SCD protocol (Halosperm kit) results in better chromatin preservation, therefore highly contrasted halo images can be accurately assessed using conventional bright-field microscopy after Wright staining. Moreover, unlike in the original SCD procedure, the sperm tails are now preserved, making it possible to unequivocally discriminate sperm from other cell types. The chi2 test did not detect significant differences in the mean number of sperm cells with fragmented DNA as scored by four different observers. The intraobserver coefficient of variation for the estimated percentage of spermatozoa with fragmented DNA ranged from 6% to 12%. There was good correlation between the SCD and the sperm chromatin structure assay DNA fragmentation index (intraclass correlation coefficient R: 0.85; percent DNA fragmentation index mean difference: 2.16 significantly higher for SCD). Using the Halosperm kit, a dose-dependent increase in sperm DNA damage after sodium nitroprusside incubation was detected. The percentage of sperm cells with fragmented DNA in the fertile group was 16.3 +/- 6.0, in the normozoospermic group, 27.3 +/- 11.7, and in the oligoasthenoteratozoospermic group, 47.3 +/- 17.3. In the varicocele sample, an extremely high degree of nuclear disruption was detected in the population of sperm cells with fragmented DNA. CONCLUSION(S): The improved SCD test, developed as the Halosperm kit, is a simple, cost effective, rapid, reliable, and accurate procedure, for routinely assessing human sperm DNA fragmentation in the clinical andrology laboratory.

Chromatin↗
PubMed2006

Sperm nuclear DNA fragmentation in adolescents with varicocele.

OBJECTIVE: To verify if sperm from adolescents with varicocele have an increased rate of DNA fragmentation when compared with adolescents without varicocele. DESIGN: Controlled prospective study. SETTING: Patients in an academic research environment. PATIENT(S): Adolescent patients with a clinical diagnosed bilateral varicocele, grades II and III, and adolescent patients without a varicocele. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Rate of sperm DNA fragmentation as assessed by the Comet assay, graded as class I (no DNA fragmentation), II (little DNA fragmentation), III (meaningful DNA fragmentation), or IV (high DNA fragmentation). RESULT(S): A higher percentage of cells with no DNA fragmentation (class I) was found in the nonvaricocele group (47.62 +/- 7.69) when compared with the varicocele group (27.52 +/- 10.73). A higher percentage of sperm with class III and class IV DNA fragmentation was found in the varicocele group (20.43 +/- 8.97, and 19.57 +/- 10.68) when compared with the nonvaricocele group (11.38 +/- 5.55, and 5.71 +/- 2.35). CONCLUSION(S): Although standard semen analysis showed no difference between the groups, adolescents with varicocele have an increase in sperm nuclear DNA fragmentation. Thus, DNA fragmentation evaluation could be important in deciding treatment options for adolescent patients.

Adolescent↗
PubMed2005

Rho kinase regulates fragmentation and phagocytosis of apoptotic cells.

During the execution phase of apoptosis, a cell undergoes cytoplasmic and nuclear changes that prepare it for death and phagocytosis. The end-point of the execution phase is condensation into a single apoptotic body or fragmentation into multiple apoptotic bodies. Fragmentation is thought to facilitate phagocytosis; however, mechanisms regulating fragmentation are unknown. An isoform of Rho kinase, ROCK-I, drives membrane blebbing through its activation of actin-myosin contraction; this raises the possibility that ROCK-I may regulate other execution phase events, such as cellular fragmentation. Here, we show that COS-7 cells fragment into a number of small apoptotic bodies during apoptosis; treating with ROCK inhibitors (Y-27632 or H-1152) prevents fragmentation. Latrunculin B and blebbistatin, drugs that interfere with actin-myosin contraction, also inhibit fragmentation. During apoptosis, ROCK-I is cleaved and activated by caspases, while ROCK-II is not activated, but rather translocates to a cytoskeletal fraction. siRNA knock-down of ROCK-I but not ROCK-II inhibits fragmentation of dying cells, consistent with ROCK-I being required for apoptotic fragmentation. Finally, cells dying in the presence of the ROCK inhibitor Y-27632 are not efficiently phagocytized. These data show that ROCK plays an essential role in fragmentation and phagocytosis of apoptotic cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2003

High molecular weight DNA fragments are processed by caspase sensitive or caspase independent pathways in cultures of cerebellar granule neurons.

Many recent reports on internucleosomal DNA fragments have appeared, however, little is known about the mechanisms of the generation of their upstream high molecular weight (HMW) fragments. Caspases are a family of proteases with important functions in the execution of apoptotic cell death. The caspase-sensitivity of the formation of HMW fragments was therefore investigated using a specific caspase-3 inhibitor (Ac-DEVD-cmk) and a general caspase inhibitor (boc-D-fmk). Apoptosis inducing factor (AIF) can translocate to the nucleus and generate HMW fragments independently of caspase. Cultures of cerebellar granule neurons (CGNs) were therefore exposed to glutamate (100 micro M) or deprived of potassium and serum to induce apoptosis, or treated with a high concentration of calcium ionophore A23187 (1 micro M) to induce necrosis. Fragmentation of DNA into two classes of HMW fragments (>680 and 50-300 kbp) was observed after treatment with glutamate or A23187. Traces of approximately 50-kbp fragments were detectable after the K(+)/serum-deprivation. The amount of >680-kbp HMW fragments increased (i.e. their further degradation was inhibited) and cell death was reduced in the presence of Ac-DEVD-cmk or boc-D-fmk following glutamate treatment. Only boc-D-fmk treatment resulted in a similar accumulation of >680-kbp HMW fragments and reduced cell death after K(+)/serum-deprivation. No such changes were observed with caspase inhibitors after A23187 treatment. AIF redistribution was observed following glutamate treatment and K(+)/serum-deprivation. Thus, even in a simple cell culture of CGNs, HMW fragments are formed by diverse mechanisms: the degradation of DNA may be sensitive to different caspases or be caspase and AIF independent.

Animals↗
PubMed2001

Characterization of DNA fragmentation events caused by genotoxic and non-genotoxic agents.

Leukemic cells have been shown to generate several classes of DNA fragments after treatment with cytotoxic cancer chemotherapy agents. However, it is unclear which of these fragmentation events are a direct effect of DNA-damaging chemotherapy agents, and which fragmentation events are caused by downstream processes, such as apoptosis. We have performed a detailed analysis of DNA fragmentation events which occur following cytotoxic chemotherapy in four representative leukemic cell lines (HL-60, Jurkat, K562, and Molt-4). We used a DNA topoisomerase II inhibitor (etoposide), an alkylating agent (melphalan), a nucleoside analog (cytosine arabinoside), and a non-genotoxic agent (N-methylformamide) to induce cell death. We studied high molecular weight and low molecular weight DNA fragmentation events, as well as the specific cleavage of the MLL breakpoint cluster region (bcr). The DNA fragments produced at late time points were largely independent of the agents used, while those generated at earlier time points showed clear differences in terms of fragment size and relative abundance, depending on the agent used. In addition, there were clear differences between cell lines in terms of size, relative abundance, and rate at which DNA fragments were produced by treatment with the same agents. We think that this survey documents the importance of studying several different cell lines, time points, and assays before reaching conclusions about the types of DNA fragments produced during treatment with cytotoxic agents, and provides a useful framework for studying a wide range of DNA fragments produced by cytotoxic agents.

Antimetabolites, Antineoplastic↗
PubMed2001

Cu(II) interaction with N-terminal fragments of human and mouse beta-amyloid peptide.

The stoichiometry, stability constants and solution structure of the complexes formed in the reaction of copper(II) with N-terminal fragments of human and mouse beta-amyloid peptide, 1-6, 1-9, 1-10 have been determined by potentiometric, UV/VIS, CD and EPR spectroscopic methods. The fragments 1-9 and 1-10 form complexes with the same coordination modes as the fragments 1-6. The coordination of the metal ion for human and mouse fragments starts from the N-terminal Asp residue which stabilizes significantly the 1N complex as a result of chelation through the beta-carboxylate group. In a wide pH range of 4-10, the imidazole nitrogen of His(6) is coordinated to form a macrochelate. Results show that, in the pH range 5-9 the human fragments form the complex with different coordination mode compared to that of the mouse fragments. The low pK(1)(amide) values (approximately 5) obtained for the mouse fragments may suggest the coordination of the amide nitrogen of His(6) while in case of the human fragments the coordination of the amide nitrogen of Ala(2) is suggested. The replacement of glycine by the arginine residue in the fifth position of the beta-amyloid peptide sequence changes the coordination modes of a peptide to metal ion in the physiological pH range. In a wide pH (including physiological) range the mouse fragments of beta-amyloid peptide are much more effective in Cu(II) binding than the human fragments.

Amino Acid Sequence↗
PubMed2001

Hydrogen peroxide induces rapid digestion of oligodendrocyte chromatin into high molecular weight fragments.

High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H2O2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H2O2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H2O2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50-200 kb fragments. Chromatin digestion could be elicited with H2O2 concentrations as low as 50 microM. After the removal of H2O2, most >400 kb fragments were religated within 2 h; however, digestion into 50-200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H2O2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.

Animals↗
PubMed1996

Fibronectin and fibronectin fragments modulate the expression of proteinases and proteinase inhibitors in human periodontal ligament cells.

Fragments of the matrix molecule fibronectin (FN) have been shown to modulate tissue remodeling activity by inducing matrix metalloproteinases (MMPs) in synovial fibroblasts. These molecules could contribute to the tissue degradation that occurs during periodontal disease if they also modulate the expression of proteinases in cells of the periodontal ligament (PDL). We tested the hypothesis that FN and specific FN fragments induce the expression of specific proteinases in PDL cells. Using substrate zymograms, reverse zymograms and Western immunoblots, we found that PDL cells constitutively express 72 kDa gelatinase, urokinase-type plasminogen activator (uPA) and at least three inhibitors whose molecular masses correspond to those of the tissue inhibitors of metalloproteinases (TIMPs). A fourth, previously uncharacterized, proteinase inhibitor of approximately 22 kDa was also observed in some cell isolates. PDL cells, when exposed to a 120 kDa proteolytic FN fragment containing the cell-binding domain, were induced to express collagenase and stromelysin and also demonstrated an increased secretion of the serine proteinase uPA. Expression of collagenase increased with increasing concentrations (0.001 microM-1 microM) of the 120 kDa FN fragment. This fragment also induced the expression of a 20 kDa inhibitor, but not of the higher-molecular-mass inhibitors, in PDL cells. The observed alterations in proteinases were associated specifically with the 120 kDa FN fragment, since similar responses were not seen when PDL cells were exposed to either a 60 kDa heparin-binding FN fragment or a 45 kDa collagen/gelatin-binding FN fragment. PDL cells exposed to intact FN did not express the proteinases induced by the 120 kDa fragment but did express 92 kDa gelatinase and the 20 kDa proteinase inhibitor. These data suggest that FN and specific FN fragments can differentially induce the expression of proteinases in PDL cells. Thus, functional regions of FN may modulate many of the functions of PDL cells that contribute to periodontal disease, wound healing and maintenance of extracellular matrix in periodontal tissues.

Cells, Cultured↗
PubMed1985

Calcium-dependent and calcium-independent interactions of prothrombin fragment 1 with phosphatidylglycerol/phosphatidylcholine unilamellar vesicles.

We have measured the phase behavior of mixed dipentadecanoylphosphatidylglycerol (DC15PG)/dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUV) in the presence of saturating (greater than 98% occupancy of binding sites) concentrations of bovine prothrombin fragment 1 and 5 mM Ca2+. Binding of fragment 1 in the presence of Ca2+ was verified by an increase in 90 degrees light scattering. Only in the cases of DC15PG/DMPC SUV below their phase transition and of pure DMPC SUV were such light scattering measurements not reversible upon addition of ethylenediaminetetraacetic acid to complex Ca2+. Phase-behavior changes of DC15PG/DMPC SUV as monitored by diphenylhexatriene fluorescence anisotropy occurred in concert with the binding of fragment 1. The major effects of peptide binding on SUV phase behavior were to raise the phase-transition temperature by 2-15 degrees C, depending on vesicle composition, and, in general, to make the phase diagram for these small vesicles closely resemble that of large vesicles. No evidence was obtained for the existence of lateral membrane domains with distinct compositions induced by the binding of prothrombin fragment 1 plus Ca2+. Surprisingly, fragment 1 without Ca2+ also altered the phase behavior of DC15PG/DMPC SUV. Most striking was the effect of fragment 1 (with or without Ca2+) on DMPC SUV phase behavior. Freeze-fracture electron microscopy demonstrated that pure DMPC vesicles were induced to fuse in the presence of fragment 1, while vesicles containing DC15PG remained intact. The rate of DMPC SUV fusion (followed by 90 degrees light scattering) increased with increasing fragment 1 concentration but was not saturable up to 40 microM fragment 1, suggesting a weak, nonspecific interaction between fragment 1 and the neutral phospholipid vesicle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Domain characteristics of the carboxyl-terminal fragment 206-316 of thermolysin: immunochemical studies.

The extent of nativeness of the stable conformation of the thermolysin fragment containing the carboxyl-terminal third of the protein (from residues 206 to 316, denoted fragment FII) was examined by its immunogenic and antigenic characteristics. Antisera elicited in rabbits by either intact thermolysin or fragment FII were fractionated serially on two affinity columns, containing either the isolated fragment or intact protein. Both sera gave rise to substantial antibody populations which recognized the fragment FII region in native thermolysin. The relative affinities of these specific antibodies for isolated fragment FII and intact thermolysin were evaluated by radioimmunoassay, by assessing the relative extents of competition by these for binding of either 14C-labeled thermolysin or 14C-labeled fragment FII to each antibody population. Competition by fragment FII was substantial, though generally weaker than that for intact thermolysin, for antibody binding of both labeled antigens. The data demonstrate that the stable structure of fragment FII as observed spectroscopically likely is one which possesses conformational features similar to those of this region in intact thermolysin, but with perhaps less conformational rigidity. The results support the view that the region of thermolysin composed primarily of residues 206-316 is a conformational domain of the intact protein and that isolated fragment FII retains domain-like characteristics of stable and native-like conformation.

Animals↗
PubMed1979

Apolipoprotein A-II: chemical synthesis and biophysical properties of three peptides corresponding to fragments in the amino-terminal half.

Three peptide fragments of apolipoprotein A-II corresponding to residues 17--31, 12--31, and 7--31 have been synthesized by solid-phase techniques and purified to apparent homogeneity. Each of these fragments contains residues 18--30, a region previously proposed to possess potential amphipathic helical properties. Secondary structural changes of these synthetic fragments accompanying their interaction with phospholipid have been studied by circular dichroism. The magnitude of this interaction has been evaluated from the yields and stoichiometry of lipid-protein complexes isolated by density gradient ultracentrifugation. Fragment 17--31, the smallest peptide containing the proposed amphipathic helix, did not interact with dimyristoylphosphatidylcholine (DMPC) single bilayer vesicles at 24 degrees C; upon addition of DMPC, no ellipticity change could be detected nor could a stable lipid-peptide complex be isolated. However, fragments 12--31 and 7--31 did interact with phosphilipid; in the absence of lipid, both fragments had primarily disordered structures, but when isolated as DMPC-peptide complexes, both fragments possessed increased helical structure. The phospholipid:peptide molar ratio was 14:1 for fragment 12--31 and 27:1 for fragment 7--31. Studies of space-filling models of these fragments suggest that hydrophobicity and/or length are important properties of phospholipid binding apoproteins.

Amino Acid Sequence↗
PubMed1996

DNA polymerase photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site.

The nucleotide photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment. Photolabel [3H]-1 covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 microM and with an EC50 (the effective concentration at 50% maximum photoincorporation) of about 0.74 microM. Saturating concentrations of poly(dA).(T)10 protect the Klenow fragment from [3H]-1 photoincorporation, and TTP at a concentration approximately equal to its KD for the free enzyme form shifts the dose-response curve for photoincorporation of [3H]-1 into the Klenow fragment by a factor of 2, indicating a competitive relationship between TTP and 1. Additionally, the photoincorporation of [3H]-1 into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of 1 up to 10 microM in the absence of magnesium. These results demonstrate that, as designed, photoprobe 1 binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp732 in the peptide fragment Asp732-Ile733-His734-Arg735 as the site of covalent modification. Molecular modeling and complementary NMR analysis of the conformation of 1 indicated preferred C3'-exo and C2'-exo-C3'-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle. Docking studies using Asp732 as an anchor point for the azide alpha-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.

Affinity Labels↗
PubMed1997

Contribution of the prothrombin fragment 2 domain to the function of factor Va in the prothrombinase complex.

The prothrombinase complex assembles through reversible interactions between factor Xa, factor Va and acidic phospholipid-containing membranes in the presence of calcium ions. This complex catalyses the conversion of prothrombin to thrombin through two proteolytic steps. We have used prethrombin 2 as a substrate analog for the first cleavage reaction of prothrombin activation (cleavage at Arg323-Ile324) catalyzed by the prothrombinase complex and have also relied on the known ability of prethrombin 2 to interact tightly but reversibly with fragment 2 or fragment 1.2. The kinetics of cleavage at Arg323-Ile324 have been assessed with these substrate analogs to investigate the contribution of cofactor-substrate interactions mediated by the fragment 2 domain to the ability of factor Va to enhance the catalytic efficiency of factor Xa within the prothrombinase complex. Initial velocity measurements indicated that the rate of prethrombin 2 cleavage by the factor Xa-PCPS binary complex was increased by a factor of approximately 1300 upon the addition of saturating concentrations of factor Va to assemble prothrombinase. Although the measured initial velocity was higher when either fragment 2 or fragment 1.2 was present, the factor Va-dependent enhancement in initial rate (2600- and 1500-fold) was comparable in each case. Steady state kinetic constants were obtained using prethrombin 2, prethrombin 2 plus fragment 2, and prethrombin 2 plus fragment 1.2 as substrates. For each substrate, the addition of saturating concentrations of factor Va to the Xa-PCPS binary complex led to increases in catalytic efficiency of between 1000 and 9000-fold. The kcat/Km for prethrombin 2 cleavage by prothrombinase was essentially identical to that obtained for prethrombin 2 saturated with fragment 2. Thus, comparable accelerating effects of factor Va are observed independent of the presence of the fragment 2 domain in the substrate. The results indicate that interactions between factor Va and the substrate mediated by the fragment 2 domain do not contribute in a dominant way to the ability of factor Va to enhance the catalytic efficiency of factor Xa within the prothrombinase complex.

Animals↗