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PubMed1989

The cation-dependent mannose 6-phosphate receptor. Structural requirements for mannose 6-phosphate binding and oligomerization.

The structural requirements for oligomerization and the generation of a functional mannose 6-phosphate (Man-6-P) binding site of the cation-dependent mannose 6-phosphate receptor (CD-MPR) were analyzed. Chemical cross-linking studies on affinity-purified CD-MPR and on solubilized membranes containing the receptor indicate that the CD-MPR exists as a homodimer. To determine whether dimer formation is necessary for the generation of a Man-6-P binding site, a cDNA coding for a truncated receptor consisting of only the signal sequence and the extracytoplasmic domain was constructed and expressed in Xenopus laevis oocytes. The expressed protein was completely soluble, monomeric in structure, and capable of binding phosphomannosyl residues. Like the dimeric native receptor, the truncated receptor can release its ligand at low pH. Ligand blot analysis using bovine testes beta-galactosidase showed that the monomeric form of the CD-MPR from bovine liver and testes is capable of binding Man-6-P. These results indicate that the extracytoplasmic domain of the receptor contains all the information necessary for ligand binding as well as for acid-dependent ligand dissociation and that oligomerization is not required for the formation of a functional Man-6-P binding site. Several different mutant CD-MPRs were generated and expressed in X. laevis oocytes to determine what region of the receptor is involved in oligomerization. Chemical cross-linking analyses of these mutant proteins indicate that the transmembrane domain is important for establishing the quaternary structure of the CD-MPR.

Amino Acid Sequence↗
PubMedDate not supplied

[The effect of the oligomerization of pBR322 on the level of transformation in Escherichia coli].

The ability of the known Escherichia coli strain JC3881 recB recC recF sbc15 to produce oligomeric and multimeric forms of pBR322 underlies the study presented. The individual oligomeric forms of pBR322 were isolated from the agarose gel. The plasmid forms were used for electron microscopic control and also introduced into the system of E. coli competent cells. The E. coli transformation level of different forms of plasmid DNA rose from monomers to pentamers. CCC forms of the plasmid possessed high efficiency of the E. coli cell transformation. The systems of the host recombination are to be significant in the process of plasmid oligomerization.

DNA, Bacterial↗
PubMed1988

Activation of the epidermal growth factor receptor tyrosine protein kinase in the absence of receptor oligomerization.

The epidermal growth factor (EGF) receptor exists in a monomeric (170 kDa) form and in several aggregated states (360 kDa, greater than 500 kDa). The hypothesis that the oligomerization of the receptor is required for the stimulation of the kinase was tested by correlating the oligomeric state of the receptor with the protein kinase activity. EGF and sphingosine stimulate the phosphorylation of an exogenous peptide substrate by the receptor to an equal extent. Chemical cross-linking using disuccinimidyl suberate and the analysis of EGF receptor complexes by Western blotting demonstrated that EGF caused the aggregation of receptors. Similar results were obtained when [32P]phosphate-labeled receptors were cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. These results were confirmed by sucrose density gradient sedimentation analysis. In contrast to the effects of EGF, incubation of EGF receptors with sphingosine did not cause the oligomerization of the receptors. These data demonstrate that the EGF receptor kinase can be stimulated independently of the aggregation of the receptors.

Cell Line↗
PubMed1987

Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity.

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure.

Aminohydrolases↗
PubMed1988

[Effect of oligomerization on the properties of essential SH-groups of mitochondrial creatine kinase].

The properties of creatine kinase isolated from bovine heart mitochondria in dimeric (Mr = 84 +/- 6 kD) and octameric (Mr = 340 +/- 17 kD) forms were compared with those of the earlier described hexameric form of the enzyme (Mr = 240 +/- 12 kD). The kinetics of SH-group modification by DTNB, the inactivation kinetics as well as the number of modified SH-groups point to significant differences between the three oligomeric forms of the enzyme. Each subunit of creatine kinase was found to possess one "fast" essential cysteine residue whose modification by DTNB and iodoacetamide led to enzyme inactivation. The formation of an analog of the transition state complex (E--MgADP--NO3--creatine) was paralleled with partial protection of only the "fast" cysteine residue which manifested itself in the decrease of the rate of its interaction with DTNB in all the three oligomeric forms. Dimer association into a hexamer and octamer occurred in parallel with a decrease of the affinity of essential SH-groups of cysteine for DTNB in 50% of the oligomeric molecule subunits. Thus, in the dimer two essential SH-groups were rapidly modified by DTNB at the same rate: k1 = k2 = (23.9 +/- 5.6).10(4) M-1 min-1. Within the hexamer, the rate of modification of 3 out of 6 SH-groups was practically unchanged: k1 = (10.6 +/- 2.3).10(4) M-1 min-1. Another 3 SH-groups in the remaining 50% of the subunits were partly masked, which manifested itself in a 10-fold decrease of their modification rate: k2 = (1.12 +/- 0.28).10(4) M-1 min-1. Within the octamer, the SH-groups rapidly interacted with DTNB only on 4 subunits: k1 = (20.7 +/- 2.2).10(4) M-1 min-1, whereas in the remaining 4 octamer subunits a practically complete masking of essential SH-groups was observed, as a result of which these groups became inaccessible to DTNB. This manifested itself in a 1000-fold decrease of the rate of SH-group modification by DTNB which reached that of non-essential SH-group modification. In has been found that a complete loss of the octamer activity is due to the modification of only 4 SH-groups which interact with DTNB at a high rate. A model for subunit association into a dimer, hexamer and octamer has been proposed. Presumably, 50% of the active centers in the mitochondrial creatine kinase octamer are not involved in the catalytic act.

Animals↗
PubMed1984

Insulin receptor regulation and desensitization in rat hepatoma cells. The loss of the oligomeric forms of the receptor correlates with the change in receptor affinity.

We have previously reported that prolonged incubations of Fao cells, a cell line derived from the well-differentiated Reuber H35 rat hepatoma, with 10(-6) M insulin, induced a decrease in receptor number (down-regulation), an increase in receptor affinity for insulin, and a loss of insulin's biological effect (desensitization). In the present study, we have investigated the relationship between these changes in insulin binding and action and changes in the structure of the insulin receptor. Intact cells were surface labeled with Na125I and lactoperoxidase, and the 125I-labeled insulin receptor was immunoprecipitated using specific antibodies and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of gels done under reducing conditions demonstrated the alpha (Mr = 135,000) and the beta (Mr = 95,000) subunits of the receptor. In nonreduced gels, free insulin receptor subunits were observed as well as four higher molecular weight bands with Mr = 210,000, 270,000, 350,000, and 520,000. Two-dimensional gel electrophoresis revealed that these bands correspond to alpha-beta heterodimer, alpha 2 homodimer, and two alpha-beta oligomers of high molecular weights, respectively. Cross-linking of 125I-insulin to intact cells with disuccinimidyl suberate revealed bands of Mr = 125,000, 210,000, 250,000 and 320,000, indicating that most of the forms of the receptor could bind insulin. After incubation with 10(-6) M insulin for 24 h, Fao cells revealed a marked decrease of the four oligomeric forms of the receptor, with little change in the level of the free alpha and beta subunits. A similar decrease of the oligomeric forms of the insulin receptor and an increase in the free subunits was observed when normal Fao cells are treated with 7 mM dithiothreitol. In dithiothreitol-treated cells, 125I-insulin binding was increased and this increase was accounted for by a change in affinity. In contrast to Fao cells, down-regulation of the insulin receptor in IM-9 lymphocytes occurs without a change in receptor affinity. In these cells, surface labeling revealed a decrease in total receptors after down-regulation, but not change in the proportion of the oligomeric forms to the free subunits of the receptor. These data suggest the following in Fao hepatoma cells. In the native state, the insulin receptor consists of free alpha and beta subunits and several kinds of disulfide-linked oligomers of these subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Subunit composition of oligomeric human von Willebrand factor.

The oligomerization of human endothelial cell-synthesized von Willebrand factor (vWf) has been studied by gel chromatography in columns of Sephacryl S-500 and by discontinuous agarose gel electrophoresis. A quantitative recovery of high Mr vWf oligomers has been obtained after binding to a monoclonal anti-vWf-Sepharose adduct. This reagent has been used to analyze gel filtration chromatographic elution profiles of [35S]methionine-labeled culture medium and cell lysate. It was determined that high Mr oligomers are present in endothelial cell lysates as well as in the medium overlying these cells and are composed of Mr 225,000 subunits. When vWf oligomers were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of a reducing agent, the Mr 240,000 subunit (provWf) was not observed to oligomerize beyond the dimer stage to a significant degree. Therefore, vWf oligomerization appears to be facilitated by conversion of provWf subunits to mature vWf subunits, most likely by proteolytic removal of sequences unique to the intracellular precursor.

Blood Coagulation Factors↗
PubMed1995

Molecular basis of altered red blood cell membrane properties in Southeast Asian ovalocytosis: role of the mutant band 3 protein in band 3 oligomerization and retention by the membrane skeleton.

Southeast Asian ovalocytosis (SAO) is an asymptomatic trait characterized by rigid, poorly deformable red cells that resist invasion by several strains of malaria parasites. The underlying molecular genetic defect involves simple heterozygous state for a mutant band 3 protein, which contains a deletion of amino acids 400 through 408, linked with a Lys 56-to-Glu substitution (band 3-Memphis polymorphism). To elucidate the contribution of the mutant SAO band 3 protein to increased SAO red blood cell (RBC) rigidity, we examined the participation of the mutant SAO band 3 protein in increased band 3 attachment to the skeleton and band 3 oligomerization. We found first that SAO RBC skeletons retained more band 3 than normal cells and that this increased retention preferentially involved the mutant SAO band 3 protein. Second, SAO RBCs contained a higher percentage of band 3 oligomer-ankyrin complexes than normal cells, and these oligomers were preferentially enriched by the mutant SAO protein. At the ultrastructural level, the increased oligomer formation of SAO RBCs was reflected by stacking of band 3-containing intramembrane particles (IMP) into longitudinal strands. The IMP stacking was not reversed by treating SAO RBCs in alkaline pH (pH 11), which is known to weaken ankyrin-band 3 interactions, or by removing the cytoplasmic domain of band 3 from SAO membranes with trypsin. Finally, we found that band 3 protein in intact SAO RBCs exhibited a markedly decreased rotational mobility, presumably reflecting the increased oligomerization and the membrane skeletal association of the SAO band 3 protein. We propose that the mutant SAO band 3 has an increased propensity to form oligomers, which appear as longitudinal strands of IMP and exhibit increased association with membrane skeleton. This band 3 oligomerization underlies the increase in membrane rigidity by precluding membrane skeletal extension, which is necessary for membrane deformation.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1994

Oligomeric forms of skate erythrocyte band 3. Effect of volume expansion.

Volume expansion of little skate (Raja erinacea) erythrocytes leads to the formation of oligomeric forms of the band 3 protein. The oligomers are formed following incubation in hypotonic media or inclusion of a permeant solute such as ethylene glycol. Oligomers were detected by specifically labeling the skate erythrocyte band 3 homolog with the radiolabeled stilbene [3H]4,4'-diisothiocyano-1,2 diphenylethane-2,2'-disulfonic acid and cross-linking membrane proteins with the homobifunctional agent bis(sulfosuccinimidyl) suberate. Under isoosmotic conditions, the distribution of band 3 in the monomer, dimer, and tetramer forms was 38, 54, and 8%. Medium of 0.5 osmolarity caused the distribution to change to 22% monomer, 31% dimer, and 47% tetramer. Upon return to isoosmotic conditions, cell volume, as well as the distribution of band 3 in the monomer, dimer, and tetrameric forms, returned to control values. Return of hypotonically swollen cells to isoosmotic conditions also returned taurine efflux back to control values. Hyperosmolarity caused a 20% shrinkage of cell volume but did not have any effect on either taurine efflux or the distribution of band 3 in the oligomeric forms. Pyridoxal 5'-phosphate and dinitrostilbene disulfonate, two agents that interact with and cause the formation of the tetrameric form of human band 3, also caused a shift toward to tetrameric peak in skate band 3. To determine whether the cross-linked oligomers had proteins that closely associate with band 3 in the intact cell, Western blots were performed to detect ankyrin and band 4.1. Neither protein appeared with the dimeric or tetrameric forms of band 3, suggesting that the mobility shift was in fact due to association of band 3 monomers. This is the first demonstration that oligomerization of band 3 occurs during a physiologic effect that may be mediated by the band 3 protein.

Animals↗
PubMed1993

Oligomerization of immunoglobulin G heavy and light chains in vitro. A cell-free assay to study the assembly of the endoplasmic reticulum.

A biochemical assay to study the assembly of the endoplasmic reticulum (ER) in a cell-free system is introduced. Incubation in vitro of ER vesicles containing only immunoglobulin gamma 1 heavy (H) chains with ER vesicles containing only K light (L) chains results in fusion and oligomerization of the H and L chains to form the H2L2 complex (immunoglobulin G). ER fusion/H2L2 oligomerization is time and temperature dependent and requires energy in the form of ATP. It is stimulated by the addition of cytosol and requires protease-sensitive components present on the membranes. The addition of guanosine 5'-O-(thiotriphosphate) inhibits membrane fusion and subsequent H2L2 oligomerization without affecting the assembly of H2L2 from detergent-solubilized pools, suggesting an important role for GTPases in vesicle recognition or fusion. The development of a rapid and quantitative assay to study the assembly of the ER in a cell-free system will allow us to identify components involved in the recognition, fusion, and post-fusion events critical for ER function in vivo.

Adenosine Triphosphate↗
PubMed1993

The oligomeric nature of the murine Fc epsilon RII/CD23. Implications for function.

The low affinity receptor for IgE (Fc epsilon RII/CD23) is a type II integral membrane protein with an extracellular C-terminal region homologous to C-type animal lectins. Immediately adjacent to this lectin homology region is a sequence that is predicted to form an alpha-helical coiled-coil stalk leading to dimer or trimer formation. This provides an explanation for the known self-associative capacity for the Fc epsilon RII. In this study the self-association to a trimer or tetramer is shown with rFc epsilon RII by chemical cross-linking and affinity purification on IgE columns. The data indicate that only the oligomeric form of Fc epsilon RII has sufficient affinity/avidity to bind to an IgE adsorbent. In contrast, Fc epsilon RII that is purified using anti-Fc epsilon RII mAb adsorbents has largely lost its capacity to bind IgE, as well as its capacity to self-associate, indicating that IgE recognizes the oligomeric form of the Fc epsilon RII. This phenomenon was further examined by performing detailed binding analysis of the mouse IgE/Fc epsilon RII interaction. A biphasic binding curve with high (2-7 x 10(7) M-1) and low (2-7 x 10(6) M-1) affinity binding was seen. Fc epsilon RII mutants were prepared that lack one or more of the 21 amino acid homologous repeat domains in the stalk region of the molecule. These mutant Fc epsilon RII molecules bound IgE with only a single low affinity (5-10 x 10(6) M-1). In addition, cross-linking analysis of one of these mutants demonstrated that it does not exhibit the receptor self-association seen for the intact Fc epsilon RII. Two chimeric Fc epsilon RII molecules were prepared having the mouse Fc epsilon RII lectin homology (carboxyl-terminal) region and the stalk region of either the related human Fc epsilon RII or the corresponding domain of Ly-49. Chimeric molecules using the former (alpha-helical coiled-coil) stalk supported normal binding of IgE although the Ly-49/Fc epsilon RII chimera failed to bind IgE. Taken together, the results indicate that high (approximately 10(8) M-1) affinity IgE binding results from interaction of multiple lectin domains with (presumably) symmetrical sites on the IgE molecule. Specificity for IgE is determined by the lectin domain although the binding avidity is determined by oligomerization through the coiled coil stalk.

Animals↗
PubMed1998

Human lung surfactant protein A exists in several different oligomeric states: oligomer size distribution varies between patient groups.

BACKGROUND: Lung surfactant protein A (SP-A) is a complex molecule composed of up to 18 polypeptide chains. In vivo, SP-A probably binds to a wide range of inhaled materials via the interaction of surface carbohydrates with the lectin domains of SP-A and mediates their interaction with cells as part of a natural defense system. Multiplicity of lectin domains gives high-affinity binding to carbohydrate-bearing surfaces. MATERIALS AND METHODS: Gel filtration analyses were performed on bronchoalveolar lavage (BAL) fluid samples from three patient groups: pulmonary alveolar proteinosis (n = 12), birch pollen allergy (n = 11), and healthy volunteers (n = 4). Sucrose density gradient centrifugation was employed to determine molecular weights of SP-A oligomers. SP-A was solubilized from the lipid phase to compare oligomeric state with that of water soluble SP-A. RESULTS: SP-A exists as fully assembled complexes with 18 polypeptide chains, but it is also consistently found in smaller oligomeric forms. This is true for both the water- and lipid-soluble fractions of SP-A. CONCLUSION: The three patient groups analyzed show a shift towards lower oligomeric forms of SP-A in the following sequence: healthy-pulmonary alveolar proteinosis-pollen allergy. Depolymerization would be expected to lead to loss of binding affinity for carbohydrate-rich surfaces, with loss or alteration of biological function. While there are many complex factors involved in the establishment of an allergy, it is possible that reduced participation of SP-A in clearing a potential allergen from the lungs could be an early step in the chain of events.

Allergens↗
PubMed1998

Transglutaminase-mediated oligomerization of galectin-3 modulates human melanoma cell interactions with laminin.

Tumor cell adhesion and migration to laminin are important events during invasion and metastatic spread. Galectin-3, a multifunctional member of the galectin family, binds specifically the poly-N-acetyllactosamine residues of laminin and has been implicated in tumor invasion and metastasis. Galectin-3 is multimerized by transglutaminase, an enzyme that catalyzes cross-linking between glutamine and other aminoacid residues. In this study, we examined the consequences of transglutaminase-mediated galectin-3 oligomerization on the interactions between cancer cells and laminin. We first demonstrated that human galectin-3 is cross-linked by guinea pig liver transglutaminase, forms oligomers, and incorporates the marker 5-(biotinamido) pentylamine. Expression of transglutaminase activity in the A375 and A2058 human melanoma cell extracts was revealed by its ability to induce galectin-3 oligomerization and 5-(biotinamido) pentylamine incorporation. Transglutaminase-treated galectin-3 did not affect adhesion or migration of the melanoma cells to laminin but consistently induced a significant increase of the percentage of cell spreading compared to the control (23.5 +/- 2.3%, vs. 10.6 +/- 1.9% at 180 min, p < 0.05), or to untreated galectin-3 or transglutaminase alone. Our study is the first demonstration that human galectin-3 is oligomerized by transglutaminase with, as a consequence, a specific effect of melanoma cell spreading on laminin. This phenomenon could be of significance in the modulation of cancer cell interactions with laminin during tumor invasion and metastasis.

Amines↗
PubMed1997

Characterization of monoclonal antibodies recognizing different fragments of cartilage oligomeric matrix protein in human body fluids.

Cartilage oligomeric matrix protein (COMP) is a high-molecular-weight glycoprotein found at a high concentration in articular cartilage. Recent studies have shown that the joint fluid and serum levels of antigenic COMP, measured by an enzyme-linked immunosorbent assay (ELISA) which uses a polyclonal antiserum raised against bovine COMP, provide important information about metabolic changes occurring in the cartilage matrix in joint disease. In this report, we describe the specificity of three monoclonal antibodies (mAbs) to human COMP and their usefulness in quantifying antigenic COMP fragments in body fluids. Two of the mAbs (16-F12 and 18-G3) recognized both oligomeric and monomeric forms of COMP, but the third (17-C10) reacted positively only with the former. Immunoblots of human COMP, predigested with trypsin for up to 6 h, showed that the three mAbs are directed against different epitopes identified on small tryptic fragments of 30 kDa (16-F12), 25 kDa (17-C10), and 40 kDa as well as 30 kDa (18-G3), respectively. The antibodies also recognized a different pattern of fragments in human pathological synovial fluids. This was particularly striking in the case of the medium size fragments (16-F12: 90 and 110 kDa; 17-C10: 70 and 90 kDa; 18-G3: up to five bands from 70 to 130 kDa). Competitive indirect inhibition ELISAs developed with mAbs 16-F12 and 17-C10 revealed further differences in the specificities of these antibodies. Thus, while mAb 16-F12 can be used only to quantify antigenic COMP in human synovial fluid and serum, mAb 17-C10 is useful in addition when analyzing canine and horse synovial fluid as well as canine serum. The results of analyses of synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis provided preliminary evidence in support of the contention that measurement of the different COMP epitopes recognized by these mAbs in body fluids could prove useful in the clinical assessment of patients with joint disease.

Animals↗
PubMed2004

Interactions between the cartilage oligomeric matrix protein and matrilins. Implications for matrix assembly and the pathogenesis of chondrodysplasias.

The cartilage oligomeric matrix protein (COMP) and matrilins are abundant non-collagenous proteins in the cartilage extracellular matrix. In the presence of calcium, COMP and matrilin-1 elute together in the gel filtration of cartilage extracts and can be co-immunoprecipitated. In a screen for ligands of matrilin-1, -3, and -4 using an ELISA-style binding assay, COMP was identified as a prominent binding partner for all three, indicating a conservation of the COMP interaction among matrilins. The interaction of COMP and matrilin-4 is saturable, and an apparent K(D) of 1 nm was determined. However, only the full-length COMP and the full-length matrilin-4 proteins showed a strong interaction, indicating that the oligomeric structures markedly increase the affinity. Mutations in COMP or matrilin-3 cause related forms of human chondrodysplasia, and the COMP mutation D469Delta, which is found in patients with pseudoachondroplasia, has been shown to cause a reduced calcium binding. Despite this, the mutation causes only a slight decrease in matrilin-4 binding. This indicates that impaired binding of COMP to matrilins does not cause the pseudoachondroplasia phenotype but rather that matrilins may be coretained in the rough endoplasmatic reticulum where COMP accumulates in the chondrocytes of patients.

Animals↗
PubMed1996

Modeling of a five-stranded coiled coil structure for the assembly domain of the cartilage oligomeric matrix protein.

The three-dimensional structure of the assembly domain of the cartilage oligomeric matrix protein (COMP) has been modeled. The model demonstrates a parallel five-stranded coiled coil and fits well with a large amount of experimental data that describe the oligomerization state, the alpha-helical conformation, the helix directionality and the properties of the (abcdefg)n repeat sequence containing apolar residues at (a) and (d) positions. Comparison of the pentamer model with the known dimer, trimer, and tetramer coiled coils revealed interactions that could mediate the switch to the formation of the pentamer coiled coil. The most distinctive feature of the pentamer model involves ion pair interactions. Charged side chains of the pentamer can form (f-g), (f-b'), and (e-c') interhelical ion pairs, which are neither experimentally observed nor modeled in the di-, tri-, and tetramers. A polar glutamine residue could be adopted at an interior (d) position of the modeled structure due to the formation of a symmetrical network of buried hydrogen bonds between five such glutamines. The pentamer model contains an axial cavity that can accept water molecules. Conformational analysis was carried out in an attempt to determine the three-dimensional structure of the disulfide bonded C-terminal region of the pentamer. Recent data on crystallization of the COMP assembly domain (Efimov et al., Proteins 24:259-262, 1996) indicate that the prediction can be tested experimentally in the near future.

Cartilage↗
PubMed1997

Oligomerization of smooth muscle myosin light chain kinase and its modifications by melittin and calmodulin.

The catalytic activity of smooth muscle myosin light chain kinase (MLCKase) requires the presence of calcium and calmodulin [CM; J. T. Stull et al. (1993) Molecular and Cellular Biochemistry, Vols. 127/128, pp. 229-237] and can also be modified through its own oligomerization [E. B. Babiychuk et al. (1995) Biochemistry, Vol. 34, pp. 6366-6372]. In the present report we demonstrate that melittin, one of the most potent CM antagonists, interacted reversibly with the MLCKase apoenzyme with affinities comparable to those of CM and influenced the oligomeric state of the kinase. At low melittin to kinase ratios the kinase formed insoluble oligomers (aggregates) while at higher melittin concentrations it existed predominantly as soluble oligomers revealed by cross-linking as octamers and hexamers. The kinase alone exhibited similar biphasic solubility within a 5-30 microM range and its solubility was strongly influenced by the ionic strength of the medium. Melittin was also found to promote both the aggregation of the purified 24-kDa C-terminal fragment of the kinase and its analogue telokin, as well as of myosin light chains, but had no effect on the solubility of bovine serum albumin, caldesmon, or calmodulin. These data and our cross-linkage experiments indicate that the insoluble kinase oligomers arose via melittin-induced aggregation of the kinase dimers in which the relokin-like domain played a main role. The soluble oligomers, in turn, were formed after saturation of the kinase with melittin, which resulted in a weakening of the interaction between the protomers with an increase of the long-range order within the oligomers. This interpretation is consistent with the observed effects of melittin on MLCKase catalytic and autocatalytic activities. At concentrations of melittin required to produce soluble oligomers, the binding of the kinase to myosin filaments was considerably enhanced. A plausible mechanism for the formation of the soluble oligomers and aggregates is suggested and its relation to the possible MLCKase assemblies discussed in terms of a model.

Animals↗
PubMed2000

Anionic oligomerization as a route to chain clusters.

The combination of two old concepts (the "sigma-donor" capability of M-H bonds and the propensity of unsaturated complexes for addition reactions) has led to the first example of addition oligomerization of an organometallic analogue of olefins: chains containing cis-[Re(CO)4] units joined by Re-H-Re interactions have been obtained through the oligomerization of the unsaturated complex [Re2(micro-H)2(CO)8] promoted by anionic "initiators". The limits of the method and the possible extension to other substrates are discussed.

Journal Article↗