Homobifunctional Cross-linking Agent Research Articles

Cross-Linking-Based Flexibility and Proximity Relationships between the TM Segments of the Escherichia coli YidC

The YidC family members function to insert proteins into membranes in bacteria, chloroplasts, and mitochondria, and they can also act as a platform to fold and assemble proteins into higher-order complexes. Here, we provide information about the proximity relationships and dynamics of the five conserved C-terminal transmembrane (TM) regions within Escherichia coli YidC. By using a YidC construct with tandem thrombin protease sites introduced into the cytoplasmic loop C1, cross-linking between paired-Cys residues located within TM segments or in the membrane border regions was studied using thio-specific homobifunctional cross-linking agents with different spanner lengths or by iodine-catalyzed disulfide formation. These in vivo cross-linking studies that can detect transient interactions and different conformational states of the protein show that TM3, TM4, TM5, and TM6 each have a face oriented toward TM2 of the in vivo expressed YidC. The studies also reveal that YidC is a dynamic protein, as cross-linking was observed between cytoplasmic Cys residues with a variety of cross-linkers. A large number of conserved proline residues on the cytoplasmic side of the five conserved core TM segments could explain the observed flexibility, and the structural fluctuations of the TM segments could provide an explanation for how YidC is able to recognize a variety of different substrates.

Role of arginine in chemical cross-linking with N-hydroxysuccinimide esters

In order to clarify whether arginine has a promoting effect on the acylation of hydroxyl groups of serine, threonine, or tyrosine by homobifunctional cross-linking agents in aqueous solution, we carried out systematic experiments with model peptides, comparing relative reaction yields with covalently protected and unprotected arginines by MALDI-MS. The guanidinium group could be demonstrated to contribute to the reactivity of hydroxyl groups toward N-hydroxysuccinimide esters and catalyze the nucleophilic substitution, probably via hydrogen bonds.

A Three-dimensional Homology Model of Lipid-free Apolipoprotein A-IV Using Cross-linking and Mass Spectrometry

Human apolipoprotein A-IV (apoA-IV) is a 46-kDa exchangeable plasma protein with many proposed functions. It is involved in chylomicron assembly and secretion, protection from atherosclerosis through a variety of mechanisms, and inhibition of food intake. There is little structural basis for these proposed functions due to the lack of a solved three-dimensional structure of the protein by x-ray crystallography or NMR. Based on previous studies, we hypothesized that lipid-free apoA-IV exists in a helical bundle, like other apolipoprotein family members and that regions near the N and C termini may interact. Utilizing a homobifunctional lysine cross-linking agent, we identified 21 intramolecular cross-links by mass spectrometry. These cross-links were used to constrain the building of a sequence threaded homology model using the I-TASSER server. Our results indicate that lipid-free apoA-IV does indeed exist as a complex helical bundle with the N and C termini in close proximity. This first structural model of lipid-free apoA-IV should prove useful for designing studies aimed at understanding how apoA-IV interacts with lipids and possibly with unknown protein partners.

Enhancing angiogenesis in collagen matrices by covalent incorporation of VEGF

Since the survival of ingrowing cells in biomaterials for regenerative processes largely depends on the supply of nutrients and oxygen, angiogenesis plays an important role in the development of new materials for tissue engineering. In this study we investigated the possibility of enhancing the angiogenic properties of collagen matrices by covalent incorporation of the vascular endothelial growth factor (VEGF). In a previous paper we already reported the use of homo- and heterobifunctional cross-linking agents for modifying collagen matrices [1]. In the present work the angiogenic growth factor was linked to the collagen with the homobifunctional cross-linker disuccinimidyldisuccinatepolyethyleneglycol (SS-PEG-SS) in a two step procedure. The efficiency of the first reaction step-the reaction of SS-PEG-SS with VEGF--was evaluated by western blot analysis. After 10 minutes virtually all of the dimeric molecules VEGF were on average modified by conjugation with 1 cross-linking molecule. The biological activity of the conjugate was investigated by exposing endothelial cells to non-modified VEGF and to VEGF conjugated to the cross-linker. The conjugation only had a limited effect on the mitogenic activity of VEGF. We therefore applied the cross-linking reaction to the VEGF-collagen system. In a first approach the changes were evaluated by the in vitro exposure of HUVECs to non-modified matrices, to matrices in which the VEGF was simply admixed and to matrices in which the VEGF was covalently incorporated. The angiogenic properties were evaluated in vivo with the chorioallantois membrane model. In this assay the chorioallantois membrane of the chicken embryo was exposed to the same set of matrices. The covalent incorporation of VEGF has a small but significant effect both on the formation of microvessels in the chorioallantois membrane and the tissue ingrowth into the implant. The covalent incorporation of angiogenic growth factors may thus be considered as a promising approach for enhancing the angiogenic capabilities of collagen matrices. Also the cross-linking with the homobifunctional cross-linking agent has a positive effect on the angiogenic potential of the collagen matrices.

Cross-linking of C-terminal Residues of Phospholamban to the Ca2+ Pump of Cardiac Sarcoplasmic Reticulum to Probe Spatial and Functional Interactions within the Transmembrane Domain

Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+-phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all cross-linked strongly to V89C-SERCA2a, coupling to 70-100% of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca2+ for cross-linking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2 x ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2 x TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca2+ pump is complete, not partial, when the pump binds Ca2+ (E1 x Ca2) or adopts the E2P or E2 x TG conformations. V49C of PLB cross-linked to 100% of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2a is presented, showing that Val49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA.

Metabolic Activation-related CD147-CD98 Complex

Cell surface CD147 protein promotes production of matrix metalloproteinases and hyaluronan, associates with monocarboxylate transporters and integrins, and is involved in reproductive, neural, inflammatory, and tumor functions. Here we combined covalent cross-linking, mass spectrometric protein identification, and co-immunoprecipitation to show selective CD147 association with three major types of transporters (CD98 heavy chain (CD98hc)-L-type amino acid transporter, ASCT2, and monocarboxylate transporters) as well as a regulator of cell proliferation (epithelial cell adhesion molecule). In the assembly of these multicomponent complexes, CD147 and CD98hc play a central organizing role. RNA interference knock-down experiments established a strong connection between CD147 and CD98hc expression and a strong positive association of CD147 (and CD98hc) with cell proliferation. As the CD147-CD98hc complex and proliferation diminished, AMP-activated protein kinase (a cellular "fuel gauge") became activated, indicating a disturbance of cellular energy metabolism. Our data point to a CD147-CD98 cell surface supercomplex that plays a critical role in energy metabolism, likely by coordinating transport of lactate and amino acids. Furthermore we showed how covalent cross-linking, together with mass spectrometry, can be used to identify closely associated transmembrane proteins. This approach should also be applicable to many other types of transmembrane proteins besides those associated with CD98hc and CD147.

Covalent Immobilization of Lysozyme on Stainless Steel. Interface Spectroscopic Characterization and Measurement of Enzymatic Activity

A new strategy aiming at the protection of metallic surfaces against the growth of biofilms is presented here. This work reports the grafting of primary amines by aminosilanization of oxidized stainless steel followed by chemical coupling of the glycosidase lysozyme from hen egg white using glutaraldehyde as homobifunctional cross-linking agent. Controlled characterization of a stainless steel surface by X-ray photoelectron spectroscopy and Fourier transform infrared reflection-absorption spectroscopy at each step enabled the mode of binding, coverage, and orientation of the grafted molecules to be addressed. As a result, the stainless steel samples covered with a covalently immobilized layer of lysozyme showed some lytic activity on a suspension of bacteria Micrococcus lysodeikticus.

Links between CD147 function, glycosylation, and caveolin-1.

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-catalyzed, beta1,6-branched, polylactosamine-type sugars, which account for its excess size. Therefore, CD147, which is itself elevated on invasive tumor cells, may make a major contribution to the abundance of beta1,6-branched polylactosamine sugars that appear on invasive tumor cells. It was shown previously that caveolin-1 associates with CD147, thus inhibiting CD147 self-aggregation and MMP induction; now we show that caveolin-1 associates with LG-CD147 and restricts the biosynthetic conversion of LG-CD147 to HG-CD147. In addition, HG-CD147 (but not LG-CD147) was preferentially captured as a multimer after treatment of cells with a homobifunctional cross-linking agent and was exclusively recognized by monoclonal antibody AAA6, a reagent that selectively recognizes self-associated CD147 and inhibits CD147-mediated MMP induction. In conclusion, we have 1) determined the biochemical basis for the unusual size variation in CD147, 2) established that CD147 is a major carrier of beta1,6-branched polylactosamine sugars on tumor cells, and 3) determined that caveolin-1 can inhibit the conversion of LG-CD147 to HG-CD147. Because it is HG-CD147 that self-aggregates and stimulates MMP induction, we now have a mechanism to explain how caveolin-1 inhibits these processes. These results help explain the previously established tumor suppressor functions of caveolin-1.

Proximity of cytoplasmic and periplasmic loops in NhaA Na+/H+ antiporter of Escherichia coli as determined by site-directed thiol cross-linking.

The unique trypsin cleavable site of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, was exploited to detect a change in mobility of cross-linked products of NhaA by polyacrylamide gel electrophoresis. Double-Cys replacements were introduced into loops, one on each side of the trypsin cleavage site (Lys 249). The proximity of paired Cys residues was assessed by disulfide cross-linking of the two tryptic fragments, using three homobifunctional cross-linking agents: 1,6-bis(maleimido)hexane (BMH), N,N'-o-phenylenedimaleimide (o-PDM), and N,N'-p-phenylenedimaleimide (p-PDM). The interloop cross-linking was found to be very specific, indicating that the loops are not merely random coils that interact randomly. In the periplasmic side of NhaA, two patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently between the double-Cys replacements A118C/S286C, N177C/S352C, and H225C/S352C; (b) only BMH cross-links the double-Cys replacements A118C/S352C, N177C/S286C, and H225C/S286C. In the cytoplasmic side of NhaA, three patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently the pairs of Cys replacements L4C/E252C, S146C/L316C, S146C/R383C, and E241C/E252C; (b) BMH and p-PDM cross-link efficiently the pairs of Cys replacements S87C/E252C, S87C/L316C, and S146C/E252C; (c) none of the reagents cross-links the double-Cys replacements L4C/L316C, L4C/R383C, S87C/R383C, A202C/E252C, A202C/L316C, A202C/R383C, E241C/L316C, and E241C/R383C. The data reveal that the N-terminus and loop VIII-IX that have previously been shown to change conformation with pH are in close proximity within the NhaA protein. The data also suggest close proximity between N-terminal and C-terminal helices at both the cytoplasmic and the periplasmic face of NhaA.

Close Proximity between Residue 30 of Phospholamban and Cysteine 318 of the Cardiac Ca2+ Pump Revealed by Intermolecular Thiol Cross-linking

Phospholamban (PLB) is a 52-amino acid inhibitor of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SERCA2a), which acts by decreasing the apparent affinity of the enzyme for Ca(2+). To localize binding sites of SERCA2a for PLB, we performed Cys-scanning mutagenesis of PLB, co-expressed the PLB mutants with SERCA2a in insect cell microsomes, and tested for cross-linking of the mutated PLB molecules to SERCA2a using 1,6-bismaleimidohexane, a 10-A-long, homobifunctional thiol cross-linking agent. Of several mutants tested, only PLB with a Cys replacement at position 30 (N30C-PLB) cross-linked to SERCA2a. Cross-linking occurred specifically and with high efficiency. The process was abolished by micromolar Ca(2+) or by an anti-PLB monoclonal antibody and was inhibited 50% by phosphorylation of PLB by cAMP-dependent protein kinase. The SERCA2a inhibitors thapsigargin and cyclopiazonic acid also completely prevented cross-linking. The two essential requirements for cross-linking of N30C-PLB to SERCA2a were a Ca(2+)-free enzyme and, unexpectedly, a micromolar concentration of ATP or ADP, demonstrating that N30C-PLB cross-links preferentially to the nucleotide-bound, E2 state of SERCA2a. Sequencing of a purified proteolytic fragment in combination with SERCA2a mutagenesis identified Cys(318) of SERCA2a as the sole amino acid cross-linked to N30C-PLB. The proximity of residue 30 of PLB to Cys(318) of SERCA2a suggests that PLB may interfere with Ca(2+) activation of SERCA2a by a protein interaction occurring near transmembrane helix M4.

Helix packing in the lactose permease of Escherichia coli: localization of helix VI

Plasmids encoding “split” lactose permease constructs with discontinuities in either the periplasmic loop between helices V and VI (N5/C7) or between helices VI and VII (N6/C6) were used to localize helix VI within the tertiary structure by site-directed thiol cross-linking. A total of 57 double-Cys pairs, with one Cys residue in helix VI and another in helix V or VIII, were studied with homobifunctional cross-linking agents. Significant cross-linking is observed between the periplasmic ends of helices V (position 158 or 161) and VI (position 170) with rigid 6 or 10 Å reagents. Furthermore, the Cys residue at position 170 (helix VI) also cross-links to a Cys residue at either position 264 or 265 (helix VIII) with a 21 Å cross-linking agent. The data indicate that helices V, VI and VIII are in close proximity at the periplasmic face of the membrane, with helix VI significantly closer to helix V. In addition, β,d-galactopyranosyl 1-thio-β,d-galactopyranoside induces a significant increase in cross-linking efficiency between helices VI and VIII and between helices V and VIII, with no significant change in cross-linking between helices V and VI.

Cross-linked hemoglobin as a potential membrane for an artificial red blood cell.

A cross-linked hemoglobin membrane has been created with discerning permeability between dissolved hemoglobin and small molecules. Such a membrane could be used to enclose a sphere of hemoglobin solution thereby allowing the entire "cell" to transport oxygen. The hemoglobin membrane was cross-linked on a polycarbonate support; the mechanical support was necessary for diffusion experiments in this study and would not be used during any sphere preparation. A 30% methemoglobin solution in phosphate buffer was used to fill the pores of the 10 microm polycarbonate support, then cross-linked with a homobifunctional cross-linking agent. The cross-linked hemoglobin within the support was evaluated for hemoglobin and benzoic acid permeability in a side-by-side diffusion cell. Disuccinimidyl glutarate, disuccinimidyl suberate and disuccinimidyl tartrate were used as cross-linking agents. Disuccinimidyl glutarate, 4.65 mM, created a hemoglobin-impermeable membrane after cross-linking for 20 minutes, reducing the concentration to 0.46 mM required a cross-linking time to 60 minutes. Benzoic acid, representing a typical small molecule, was capable of diffusing through the disuccinimidyl glutarate cross-linked hemoglobin membrane at 87.2% of its diffusion through buffer.

Thiol cross-linking of cytoplasmic loops in the lactose permease of Escherichia coli.

The N- and C-terminal halves of lactose permease, each with a single-Cys residue in a cytoplasmic loop, were coexpressed, and cross-linking was studied in the absence or presence of ligand. Out of the 68 paired-Cys mutants in cytoplasmic loops IV/V and VIII/IX or X/XI, three pairs in loop IV/V and X/XI, (i) Arg135 --> Cys/Thr338 --> Cys, (ii) Arg134 --> Cys/Val343 --> Cys, and (iii) Arg134 --> Cys/Phe345 --> Cys, form a spontaneous disulfide bond, indicating that loops IV/V and X/XI are in close proximity. In addition, specific paired-Cys residues in loop IV/V (132-138) and loop VIII/IX (282-290) or loop X/XI (335-345) cross-link with iodine and/or the homobifunctional cross-linking agents N, N'-o-phenylenedimaleimide, N,N'-p-phenylenedimaleimide, and 1, 6-bis(maleimido)hexane. The results demonstrate that loop IV/V is close to both loop VIII/IX and loop X/XI. On the other hand, similar though less extensive cross-linking studies indicate that neither the N terminus nor loop II/III appear to be close to loops VIII/IX or X/XI. The findings suggest that the longer cytoplasmic loops are highly flexible and interact in a largely random fashion. However, although a Cys residue at position 134 in loop IV/V, for example, is able to cross-link with a Cys residue at each position in loop VIII/IX or loop X/XI, Cys residues at other positions in loop IV/V exhibit markedly different cross-linking patterns. Therefore, although the domains appear to be very flexible, the interactions are not completely random, suggesting that there are probably at least some structural constraints that limit the degree of flexibility. In addition, evidence is presented suggesting that ligand binding induces conformational alterations between loop IV/V and loop VIII/IX or X/XI.

Transmembrane helix tilting and ligand-induced conformational changes in the lactose permease determined by site-directed chemical crosslinking in situ

The N-terminal six transmenbrane helices (N 6) and the C-terminal six transmembrane helices (C 6) of the lactose permease of Escherichia coli, each with a Cys residue, were co-expressed independently, and crosslinking was studied. Proximity of paired Cys residues in helices II (position 49, 52, 53, 56, 57, 60, 63 or 67) and VII (position 227, 230, 231, 234, 238, 241, 242 or 245) or XI (position 350, 353, 354, 357, 361 or 364) was examined by using two homobifunctional thiol-specific crosslinking agents of different lengths (6 or 10 Å). The results demonstrate that a Cys residue placed in the periplasmic half of helix II (position 49, 52, 53 or 57) crosslinks to Cys residues in the periplasmic half of helix VII (position 241, 242 or 245). In contrast, no crosslinking is evident with paired-Cys residues in the cytoplasmic halves of helices II (position 60, 63 or 67) and VII (position 227, 230, 231, 234 or 238). Remarkably, a Cys residue in the cytoplasmic half of helix II (position 60, 63 or 67) crosslinks with a Cys residue in the cytoplasmic half of helix XI (position 350, 353 or 354), while paired-Cys residues at positions in the periplasmic halves of the two helices do not crosslink. Therefore, helix II is tilted in such a manner that the periplasmic end is close to helix VII, and the cytoplasmic end is close to helix XI. Furthermore, ligand-binding alters the crosslinking efficiency of paired-Cys residues in helices II and VII or XI, indicating that both interfaces are conformationally active. The results are consistent with the conclusion that ligand-binding induces a scissors-like movement of helices II and VII that increases interhelical distance by 3 to 4 Å at the periplasmic ends and decreases the distance by 3 to 4 Å at the approximate middle of the two transmembrane helices.

The formation of a crosslinked carboxyhemoglobin membrane at an organic-aqueous interface.

A 30% carboxyhemoglobin solution was encapsulated with a membrane composed of crosslinked hemoglobin as a potential artificial red blood cell by adding a 25 microL droplet of 30% carboxyhemoglobin solution in phosphate buffer to a buffer-saturated toluene solution of a homobifunctional crosslinking agent. Disuccinimidyl glutarate, disuccinimidyl suberate, and terephthaldicarboxaldehyde proved successful in crosslinking hemoglobin at concentrations of 0.05%, 0.08%, and 4.3%, respectively (saturated levels in buffer-saturated toluene). Disuccinimidyl glutarate exhibited maximal crosslinking of 11.0% in approximately 30 minutes. Disuccinimidyl suberate and terephthaldicarboxaldehyde required at least 12 hours to form a viable sphere and crosslinked 39.4% and 37.8% of the hemoglobin in 18 hours. Membrane integrity was assessed by determining the permeability of uncrosslinked hemoglobin through the membrane as a function of time.

Thermostabilization of penicillin G acylase obtained from a mutant of Escherichia coli ATCC 11105 by bisimidoesters as homobifunctional cross-linking agents

We investigated the effects of three different bisimidoesters as homobifunctional cross-linking agents on the thermostabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105. Cross-linkers were dimethyladipimidate (DMA), dimethylsuberimidate (DMS), and dimethyl-3,3′-dithiobispropionimidate (DTBP). The thermal inactivation mechanisms of the native and cross-linked PGA were both considered to obey first-order inactivation kinetics during prolonged heat treatment, forming fully active, susceptible transient states. The efficacy of the cross-linkers on the thermostabilization of PGA was estimated to be DMA > DMS > DTBP. Optimal concentrations of DMA, DMS, and DTBP for cross-linking of PGA were found to be 0.5, 0.4, and 0.3% (w/v), respectively. The greatest enhancement of the thermostabilities was observed during DMA cross-linking, as a nearly 15-fold increase at temperatures above 50°C. Cross-linking by DMA did not cause much change in the parameters V m, K m, and the optimal temperature values of PGA, but the activation energy of the enzyme was slightly decreased and k cat value slightly increased after cross-linking.

Expression and Characterization of Functional Bovine Cation-Dependent Mannose 6-Phosphate Receptors in Baculovirus-Infected Insect Cells

A recombinant baculovirus containing the cDNA for the full-length bovine cation-dependent mannose 8-phosphate receptor (CD-MPR) was generated by homologous recombination. Spodoptera frugiperda (Sf9) and Trichoplusia ni 5B1-4 (High Five) insect cells, which do not contain endogenous MPRs as determined by Western blot analyses and pentamannosyl phosphate-agarose affinity chromatography, were infected with recombinant baculovirus. The infected cells expressed 26-, 29-, 32-, 35-, and 39-kDa species that were immunologically reactive with antisera raised against the native bovine CD-MPR and quantitative Western analysis demonstrated 1−2 × 106 CD-MPR molecules/cell, a level which is comparable to that expressed normally in a variety of cell lines and tissues. Digestion with endo-β-N-acetylglucosaminidase H indicated that these multiple species were due to the presence of either 0, 1, 2, 3, or 4 N-linked oligosaccharide chains, respectively, and that the majority (87%) of these carbohydrates were of the high-mannose type. The receptor produced was biologically active since it bound specifically to a pentamannosyl phosphate-agarose column and exhibited acid-dependent ligand dissociation. In addition, studies using a homobifunctional cross-linking agent on the purified CD-MPR suggested that, like the receptor expressed in mammalian cells, the insect-produced CD-MPR existed as a dimer in the membrane.

The bovine mannose 6-phosphate/insulin-like growth factor II receptor. Localization of the insulin-like growth factor II binding site to domains 5-11.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) binds two distinct ligands, mannose 6-phosphate (Man-6-P) and insulin-like growth factor II (IGF-II). The extracytoplasmic region of the receptor is composed of 15 homologous repeating domains and domains 1-3 and 7-9 have been shown to contain the two Man-6-P binding sites. To determine the location of the single IGF-II binding site, truncated forms of the M6P/IGF-II receptor were expressed transiently in COS-1 cells and assayed for their ability to bind iodinated human recombinant IGF-II. The binding of [125I]IGF-II to the receptors in the presence or absence of excess unlabeled IGF-II, IGF-I, or insulin was determined by incubation with homobifunctional cross-linking agents followed by SDS-polyacrylamide gel electrophoresis. These binding studies demonstrated that a construct encoding domains 5-11 bound 0.9 mol of IGF-II/mol of receptor, whereas a construct encoding domains 5-10 exhibited no detectable binding to IGF-II. These results indicate that the IGF-II binding site of the M6P/IGF-II receptor is contained within domains 5-11 and that residues in domain 11 play an important role in IGF-II binding.

Diadenosine tetraphosphate binding protein from human HeLa cells: purification and characterization

The ubiquitous dinucleotide P1,P4-di(adenosine-5') tetraphosphate (Ap4A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding specifically to Ap4A is associated with a multiprotein form of DNA polymerase alpha (pol alpha 2) in HeLa cells. The Ap4A binding protein from HeLa cells has been purified to homogeneity starting from pol alpha 2 complex. The Ap4A binding protein is hydrophobic and is resolved from the pol alpha 2 complex by hydrophobic interaction chromatography on butyl-Sepharose and subsequently purified to homogeneity by chromatography on Mono-Q and Superose-12 FPLC columns. The Ap4A binding activity elutes as a single symmetrical peak upon gel filtration with a molecular mass of 200 kDa. Upon polyacrylamide gel electrophoresis under nondenaturing conditions, the purified protein migrates as a single protein of 200 kDa. Upon electrophoresis under denaturing conditions, the binding activity is resolved into two polypeptides of 45 and 22 kDa, designated as A1 and A2, respectively. A1 and A2 can be cross-linked using the homobifunctional cross-linking agent disuccinimidyl suberate. The cross-linked protein migrates as a single protein of 210 kDa on polyacrylamide gels under denaturing conditions, suggesting that these two polypeptides are subunits of a single protein. The purified protein binds Ap4A efficiently, and by Scatchard analysis, we have determined a dissociation constant of 0.25 microM, indicating high affinity of Ap4A binding protein to its ligand. ATP is not required for the binding activity. The nonionic detergent Triton X-100 is necessary for stabilizing the purified protein. Amino acid composition analysis indicates that A1 and A2 are distinct.

The brain corticotropin-releasing factor (CRF) receptor is of lower apparent molecular weight than the CRF receptor in anterior pituitary. Evidence from chemical cross-linking studies.

The ligand binding subunits of the corticotropin-releasing factor (CRF) receptors in brain and anterior pituitary of a number of species have been identified by chemical affinity cross-linking using the homobifunctional cross-linking agent disuccinimidyl suberate and 125I-Tyr0-oCRF (ovine CRF). In homogenates of rat, monkey, and human cerebral cortex, 125I-Tyr0-oCRF was covalently incorporated into a protein of Mr = 58,000. Under identical conditions in the anterior pituitary of rat, monkey, cow, and pig, 125I-Tyr0-oCRF was incorporated into a protein of apparent Mr = 75,000. The specificity of the labeling was typical of the CRF binding site since both the cerebral cortex- and pituitary-labeled proteins exhibited the appropriate pharmacological rank order profile characteristic of the CRF receptor (Nle21,Tyr32-oCRF approximately equal to rat/human CRF approximately equal to ovine CRF approximately equal to alpha-helical CRF(6-41) greater than alpha-helical oCRF(9-41) greater than or equal to oCRF(7-41) greater than rat/human CRF(1-20) approximately equal to vasoactive intestinal peptide). In addition to the major labeled proteins, 125I-Tyr0-oCRF was incorporated into higher molecular weight peptides which may represent precursors and into lower molecular weight components which may represent fragments of the major labeled proteins or altered forms of the CRF binding subunit. In summary, these data indicate a heterogeneity between brain and pituitary CRF receptors with the ligand binding subunit of the brain CRF receptor residing on a Mr = 58,000 protein, while in the anterior pituitary, the identical binding subunit resides on a protein of apparent Mr = 75,000.