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PubMed1986

Synthesis and biological properties of antiparallel and parallel dimers of alpha-human atrial natriuretic peptide.

To obtain antiparallel and parallel dimers of alpha-human atrial natriuretic peptide (alpha-hANP), two fully protected peptides I and II having the same amino acid sequence as alpha-hANP with different protective groups at the cysteinyl residues were synthesized, the former having Acm and Npys and the latter MeBzl and Acm. Equivalent amounts of peptides I and II were mixed and subjected to HF deprotection. Next, the first disulfide bond was linked between the remaining Npys group in I and the liberated SH group in II to form a monodisulfide dimer. The second disulfide bond was formed within the newly formed dimer between the remaining Acm groups by treatment with iodine, giving an antiparallel dimer. The parallel dimer of alpha-hANP was synthesized similarly starting from the protected peptide II. These dimers could be clearly segregated on HPLC. The retention time on HPLC of the antiparallel dimer was identical with that of natural beta-hANP. Both dimers showed biological activities as high as one third to one sixth of alpha-hANP in smooth muscle spasmolytic activity, and almost the same level of natriuretic activity as alpha-hANP at a high dose (10 nmol/kg) but about one fifth the activity at a low dose (1 nmol/kg). In these assay systems, the antiparallel dimer showed a slower onset and a tendency of longer duration than alpha-hANP.

Amino Acid Sequence↗
PubMed1986

Dimeric form of diphtheria toxin: purification and characterization.

Many preparations of diphtheria toxin were found to contain dimeric and multimeric toxin forms. The monomeric and dimeric forms were fractionated to greater than 98% purity, and their properties were compared. Dimeric toxin slowly dissociated to native monomers in solution at neutral pH and could be rapidly dissociated with dimethyl sulfoxide. In cell culture assays and rabbit skin tests, the dimer exhibited no significant toxic activity, except for that attributable to trace contamination by monomer, or partial dissociation to monomer during the incubation period. In guinea pig lethality tests, however, toxic activity varied depending upon the dose. At least 7-fold greater amounts of dimer than monomer (161 ng vs. 22 ng, respectively) were required to cause death at 18 h, whereas similar weights of the two toxin forms (22 ng) caused death at 120 h. This variability probably reflected slow dissociation of dimer to monomer in the animal. The dimer was unable to bind toxin receptors on the surface of susceptible cells, whereas it retained full activity in the ADP-ribosyltransferase, NAD-glycohydrolase, or ligand-binding assays. Thus, the lack of toxicity of the dimeric toxin may have resulted from distortion or occlusion of the receptor binding site on the B moiety. We propose that the dimer contains two monomeric units bound by hydrophobic interactions and that the points of contact involve regions of the B moieties that are normally buried in the native monomer.

Animals↗
PubMed1992

Amino acids necessary for DNA contact and dimerization imply novel motifs in the papillomavirus E2 trans-activator.

The bovine papillomavirus E2 protein regulates viral transcription by binding as a dimer to the DNA sequence ACCGN4CGGT. The dimerization and DNA-binding properties are localized within its carboxy-terminal 85 amino acids (325-410). Utilizing random mutagenesis coupled with phenotypic selection in yeast, functionally important amino acids in the DNA-binding domain were identified. Four trans-activation defective point mutants within a short segment (amino acids 337-344) were DNA binding defective but dimeric. The mutation of a conserved tryptophan to serine also eliminated DNA binding, but loss of dimerization was implicated because addition of dimeric monoclonal antibody complemented this defect. A simple assay for E2 dimerization was developed using UV irradiation to produce an interchain cross-link within a dimer. No heterodimeric complexes were formed when pools of E2 of varying lengths were mixed, and only proteins with tryptophan at position 360 could be UV cross-linked. Peptide mapping of irradiated E2 protein localized the cross-link to an 18-amino-acid region bracketing this tryptophan. Substitutions for this tryptophan demonstrated the requirement for a hydrophobic residue at this position, but surprisingly, even alanine was functional. Replacement of this tryptophan with three polar amino acids or glycine eliminated DNA-binding activity, but addition of dimeric monoclonal antibody restored this function. The amino acids that were identified as being involved in DNA contact and dimerization imply that these functions are mediated by novel binding motifs.

Amino Acid Sequence↗
PubMed1991

Inhibitor stabilization of human immunodeficiency virus type-2 proteinase dimer formation.

We report the first direct observation of the subunit self-association behavior of highly purified recombinant human immunodeficiency virus type-2 (HIV-2) proteinase. Multiple samples of enzyme were subjected to sedimentation equilibrium analytical ultracentrifugation sequentially at 8.8 degrees C and two pH values in the presence and absence of a C2 symmetric, peptidomimetic inhibitor. At both pH values the enzyme exhibited sedimentation equilibrium behavior which fit a monomer-dimer-tetramer model. In the absence of inhibitor, the apparent Kd for dimer formation was less than approximately 100 microM and the apparent Kd for the weaker dimer-tetramer association was greater than approximately 100 microM. In the presence of inhibitor, at either pH, dimer formation was more strongly favored as indicated by a approximately 5-14-fold decrease in the apparent Kd for dimer formation and a approximately 1.2-4-fold increase in the apparent Kd for tetramer formation. The enhanced formation of dimer and decrease in higher order self-associated forms in the presence of an inhibitor is consistent with inhibitor stabilization of an active dimer. The inhibitor-induced stabilization of the dimeric species is consistent with a model for substrate-induced formation of active proteinase dimers in virion assembly.

HIV Protease Inhibitors↗
PubMed1991

Reconstitution of C5 convertase of the alternative complement pathway with isolated C3b dimer and factors B and D.

C5 convertase of the alternative complement pathway is a trimolecular complex consisting of two molecules of C3b and one molecule of Bb. We previously proposed a model of the alternative pathway C5 convertase in which the second C3b molecule binds covalently to the first C3b molecule bearing Bb, and the C5 molecule binds to each C3b molecule of the covalently linked C3b dimer, resulting in its appropriate presentation to the catalytic site on Bb. In the present study, we purified the covalently linked C3b dimer and reconstituted the C5 convertase with the C3b dimer and factors B and D to obtain evidence in support of this model. An insoluble glucan, OMZ-176, was incubated with human serum to activate the alternative pathway and to allow formation of the alternative C5 convertase on the surface of the glucan, and the glucan bearing the C5 convertase was then solubilized by incubation with glucosidases. In this way, the covalently linked C3b dimer was obtained in solution without using a detergent. The C3b dimer was then separated from enzymes, C3b monomer, C3b oligomer, and other materials by chromatographies. SDS-PAGE analysis demonstrated that the purified C3b dimer had intact alpha'-chains. Alternative pathway C5 convertase was reconstituted when the isolated C3b dimer was incubated with factors B and D. The presence of P enhanced C5 convertase formation threefold. These results support the notions that the formation of the covalently linked C3b dimer is a general phenomenon associated with activation of the alternative pathway and that the C3b dimer acts as a part of the C5 convertase.

Complement C3-C5 Convertases↗
PubMed1989

Dimeric dermorphin analogues as mu-receptor probes on rat brain membranes. Correlation between central mu-receptor potency and suppression of gastric acid secretion.

The opioid receptor preference for dermorphin and several dimerized structural analogues was investigated using rat brain synaptosomes and correlated with the potencies of intracerebroventricularly administered dimeric dermorphin peptides to inhibit gastric acid secretion. The carboxyl terminus of dermorphin or amino-terminal dermorphin analogues was bridged by dihydrazide or (poly)ethylenediamine structures. Synaptosomal membranes were prepared for radioligand binding assay in the presence of soybean trypsin inhibitor and preincubated to remove endogenously bound opioid peptides before storage at -70 degrees C. Specific radiolabeled agonists used in the radioligand binding assays were [D-Ala2,N-methyl-Phe4,Gly-ol5] [3H] enkephalin for mu-receptors and [D-Ala2,D-Leu5] [3H]enkephalin for delta-receptors. delta-Receptor binding assays were conducted in the presence of 2.6 microM [N-Me-Phe3,D-Pro4]morphiceptin to suppress peptide binding to mu-receptors. [D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin and dermorphin had affinities of 1.39 and 1.22 nM for mu-receptors and 355.8 and 178.6 nM for delta-receptors, respectively. Affinities of dimeric-dermorphin0 for mu- and delta-receptors, and the mu-selectivity ratio, exceeded values characteristic of dermorphin. The dimerized amino-terminal dermorphin analogues are peptides whose receptor binding differed from the parent molecule; e.g. the affinity of dimeric tetrapeptides toward mu-receptors was reduced but was increased for delta-receptors relative to monomeric dermorphin-(1-4)-amide. Dimeric tetradermorphin linked by a bridge containing 12 methylene units (di-tetra-dermorphin12), exhibited a dramatic loss in the mu-selectivity ratio as a result of diminished mu-affinity. On the other hand, substitution of Gly4 by Sar in di-tetra-dermorphin2 enhanced binding to mu-receptors: substitution of D-Arg2 for D-Ala resulted in an increased binding to mu-receptors while decreasing binding to delta-receptors, yielding a peptide with the highest mu-selectivity ratio. These substitutions of D-Arg2 and Sar4 in dimeric amino-terminal dermorphin pentapeptides enhanced binding to both mu- and delta-receptors relative to dermorphin-(1-5)-amide, but led to a decrease in its mu-selectivity ratio. Several dimeric dermorphin analogues exhibited an enhanced mu-selectivity ratio relative to their monomeric analogues. Dimeric peptides, which had a relatively high affinity for mu-receptors, were effective in the suppression of gastric acid secretion.

Analgesics, Opioid↗
PubMed1989

1H and 31P nuclear magnetic resonance studies of the differences in DNA deformation induced by anti-tumoral 7H-pyrido[4,3-c]carbazole dimers.

Ditercalinium (2,2'-[( 4,4'-bipiperidine]-1,1'-diyldi-2,1-ethane-diyl) bis-[10-methoxy-7H pyrido[4,3-c]carbazolium)tetramethane sulfonate (NSC 366241], a DNA bis-intercalating compound, is a potent anti-tumoral rigid dimer. Previous studies have shown that a reduced flexibility of the linking chain of such a dimer is essential for its biological activity. In order to understand, at the molecular level, the mechanism of action and the structure-activity relationships of this series of DNA intercalators, new dimers with additional methylene groups between the two piperidine rings have been synthesized. Addition of one methylene group in the chain preserved the activity, whereas addition of two methylene groups reduced the cytotoxicity, which finally disappeared when three methylene groups were inserted. Therefore, the study of the interaction of dimers bearing no (202), two (222) and three (232) methylene groups with the self-complementary hexanucleotide d(CGATCG)2 have been investigated by 1H and 31P nuclear magnetic resonance studies. The results reported here indicate that all dimers bis-intercalate into the minihelix. The intermolecular nuclear Overhauser effects (NOEs) between the dimers and the nucleotide lead to the conclusion that the three dimers intercalate with their rigid bis-ethyl bipiperidine chain fitting the major groove of the helix. Inter-residue nuclear Overhauser effects at the DNA level, as well as induced shifts, are discussed in relation to the conformational changes induced in DNA upon intercalation and to the different activity of the dimers.

Antineoplastic Agents↗
PubMed1992

In vitro evidence that UV-induced frameshift and substitution mutations at T tracts are the result of misalignment-mediated replication past a specific thymine dimer.

A previous study of UV-induced (254 nm) mutations in the lacI gene of Escherichia coli found that frameshift mutations accounted for about 35% of the observed mutations and that these mutations occurred predominantly at An.Tn sequences [Miller, J.H. (1985) J. Mol. Biol. 182, 48-65]. Because An.Tn sequences are hotspots for cis-syn thymine dimer formation [Brash, D.E., & Haseltine, W. A. (1982) Nature 298, 189-192], it would appear that UV-induced frameshift mutations are the result of an error during replicative bypass of a thymine dimer within such a sequence. To test the validity of such a proposal, replication experiments were carried out on templates containing cis-syn thymine dimers at each of the five possible sites of a T6 tract. The 59-mer templates were prepared by ligating oligonucleotides containing an EcoRI site to the 5'-end of decamers containing the cis-syn thymine dimer and oligonucleotides containing the primer site to the 3'-end. Primer-extension reactions were then carried out on these templates with a 3'----5' exonuclease-deficient (exo-) Klenow fragment of E. coli polymerase I and an exo-T7 polymerase (Sequenase Version 2.0). The replicative bypass products were cleaved with EcoRI to rigorously establish and quantify the presence of frameshift mutations. Both polymerases were able to bypass dimers at all sites, but only the exo-T7 polymerase led to detectable frameshifts, both -1 (approximately 30%) and -2 (approximately 5%), and only with the template containing a cyclobutane dimer at the second site from the 5'-end of the T6 tract. Sequencing of the T7 polymerase-catalyzed bypass products of all templates demonstrated that within the limits of discrimination only As were introduced opposite the dimer-containing T tracts. The only exception was for the template with the dimer at the second site which led to a readily detectable amount of a substitution mutation (approximately 30%) opposite the 5'-thymine of the T6 tract. A mechanism involving a competition between reversible misalignment and realignment steps and irreversible elongation steps is proposed to explain the origin of both the frameshift and the substitution mutations. The implications of this work to the mechanism of UV-induced frameshift and substitution mutations at T tracts in vivo are discussed.

Autoradiography↗
PubMed1990

cis-syn thymine dimers are not absolute blocks to replication by DNA polymerase I of Escherichia coli in vitro.

Both Escherichia coli DNA polymerase I (pol I) and the large fragment of pol I (Klenow) were found to bypass a site-specific cis-syn thymine dimer, in vitro, under standard conditions. A template was constructed by ligating d(pCGTAT[c,s]TATGC), synthesized via a cis-syn thymine dimer phosphoramidite building block, to a 12-mer and 19-mer. The site and integrity of the dimer were verified by use of T4 denV endonuclease V. Extension of a 15-mer on the dimer-containing template by either pol I or Klenow led to dNTP and polymerase concentration dependent formation of termination and bypass products. At approximately 0.15 unit/microL and 1-10 microM in each dNTP, termination one prior to the 3'-T of the dimer predominated. At 100 microM in each dNTP termination opposite the 3'-T of the dimer predominated and bypass occurred. Bypass at 100 microM in each dNTP depended on polymerase concentration, reaching a maximum of 20% in 1 h at approximately 0.2 unit/microL, underscoring the importance of polymerase binding affinity for damaged primer-templates on bypass. Seven percent bypass in 1 h occurred under conditions of 100:10 microM dATP:dNTP bias, 1% under dTTP bias, and an undetectable amount under either dGTP or dCTP bias. At 100 microM in each dNTP, the ratio of pdA:pdG:pdC:pdT terminating opposite the 3'-T of the dimer was estimated to be 37:25:10:28. Sequencing of the bypass product produced under these conditions demonstrated that greater than 95% pdA was incorporated opposite both Ts of the dimer and that little or no frame shifting took place.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Excision repair of pyrimidine dimers induced by simulated solar radiation in the skin of patients with basal cell carcinoma.

One prominent lesion induced in DNA by ultraviolet (UV) radiation is the cyclobutyl pyrimidine dimer formed between adjacent pyrimidines on the same DNA strand. We investigated whether people who have developed basal cell carcinoma on sun-exposed skin have an altered ability to repair UV-induced pyrimidine dimers in DNA. Twenty-two patients with at least one basal cell carcinoma, aged 31-84 years, and 19 healthy volunteers, aged 25-61 years, took part in the study. Both groups were given one minimal erythema dose (MED) of simulated solar radiation on the lower back. DNA was extracted from the irradiated skin 0 to 6 h later, and the number of UV-induced pyrimidine dimers was determined using a dimer-specific endonuclease. At time 0, the average number of dimers per unit of DNA was similar in the two groups. After 6 h, an average of 22 +/- 4% of the dimers were removed in the group with basal cell carcinoma compared to 33 +/- 4% in the cancer-free group. In the basal cell carcinoma group, only 23% of the patients repaired more than 30% of the dimers after 6 h, compared with 53% of the cancer-free subjects (p less than 0.05). We conclude that patients who develop basal cell carcinoma on sun-exposed skin may have a decreased ability to repair pyrimidine dimers induced in skin exposed to simulated solar radiation.

Adult↗
PubMed1988

Distribution of thymine dimers induced in mouse skin by ultraviolet radiation.

The cellular and tissue distributions of thymine dimers induced in mouse skin by UV radiation were compared in 3 haired mouse strains: Sencar, BALB/c, and C3H. These strains differ markedly in their overall susceptibility to photocarcinogenesis, and they develop UV-induced skin tumors of different histological types and in different anatomical locations. The purpose of this study was to determine whether the differences in strain and site susceptibility are due to inequalities in the distribution of UV radiation-induced DNA damage in the target tissues. An indirect immuno-fluorescence assay was used to examine the location of thymine dimers induced by 254-nm UV radiation (UVC) or 280-400 nm UV radiation (UVAB) in ears and dorsal skin. No differences in the distribution of dimers in skin cells or tissues were found among mouse strains or between anatomical sites (ear vs. dorsum). Thus, these results do not support the hypothesis that differences in the optical properties of skin, which govern penetration and absorption of UV radiation, contribute to site or strain susceptibility in photocarcinogenesis. However, two distinct differences in the cellular and tissue distribution of dimers were found when UVC and UVAB-treated tissues were compared: a) UVAB produced dimers in both dermal and epidermal cells, whereas UVC induced a detectable number of dimers only in epidermal cells; b) UVAB-induced dimers were observed in both the nuclei and cytoplasm of affected cells, whereas dimers induced by UVC were confined to nuclei.

Animals↗
PubMed1989

Dissociation and reconstitution of bovine seminal RNAase: construction of a hyperactive hybrid dimer.

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we described the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.

Amino Acids↗
PubMed1990

Oligomerization of herpes simplex virus glycoprotein B occurs in the endoplasmic reticulum and a 102 amino acid cytosolic domain is dispensable for dimer assembly.

Glycoprotein B (gB) is an essential protein specified by herpes simplex virus and a major envelope component of the virions. It is known to assemble into noncovalently associated dimers. The aim of this study was to investigate the role of topogenic domains of gB in dimer assembly and the intracellular location at which gB dimers are assembled. Therefore, dimer analyses were performed on intact gB and its three COOH-terminus-truncated gB derivatives encoding NH2-terminal 772, 586, and 477 amino acids (aa) of the mature gB, using SDS-polyacrylamide gel electrophoresis and sucrose gradient assays. Dimers were detected in gB and in tgB(772 aa), but were absent from tgB(586 aa) and from tgB(477 aa). These results showed that a 102 aa cytosolic domain (aa 773-874) is not required for the assembly of gB dimers. In addition, using endoglycosidase H treatment and dimer analysis of gB synthesized during 7 min pulse-labeling period, we have demonstrated that ER is the subcellular organelle at which gB monomers are assembled into dimeric forms.

Amino Acids↗
PubMed1985

The interconversion between monomeric and dimeric bovine heart cytochrome c oxidase.

Monomers and dimers of bovine heart cytochrome c oxidase (EC 1.9.3.1.) were separated by gel filtration chromatography on Ultrogel AcA 34 or by sucrose gradient centrifugation. Factors influencing the interconversion of the two aggregation states of this enzyme were analyzed. At very low ionic strength, in the presence of dodecyl maltoside, monomers were the main species. Salts appeared to stabilize the dimeric form, divalent cations being more efficient than monovalent. High enzyme concentrations favoured the formation of dimers, also at low ionic strength. The type of detergent had a strong influence on the monomer-dimer interconversion; in Triton X-100 and dodecyl maltoside (at high ionic strength) cytochrome c oxidase was homogenously dispersed in its dimeric form, while in Tween-80 gel filtration showed only large particles eluting in the void volume. In cholate monomers and aggregates were observed but no dimers. The aggregation state had an influence on the steady state kinetics of the ferrocytochrome c oxidase activity. Monomers showed linear Eadie-Hofstee plots, whilst the dimeric and aggregated enzyme gave nonlinear Eadie-Hofstee plots. Ionic strength, enzyme concentration and type of detergent were affecting the enzyme's kinetics in a way consistent with the molecular form obtained by the gel filtration or sedimentation analysis. The data support a negative cooperative mechanism for the interaction of cytochrome c with the dimeric enzyme, as proposed earlier (K.A. Nałecz et al., (1983) Biochem. Biophys. Res. Commun., 114, 822-828).

Animals↗
PubMed1986

Elevated serum D dimer: a degradation product of cross-linked fibrin (XDP) after intravenous streptokinase during acute myocardial infarction.

D dimer, a degradation product of cross-linked fibrin, is generated by lysis of fibrin but not by lysis of fibrinogen and can be reliably detected by specific monoclonal antibody techniques. The generation of D dimer after intravenous streptokinase in acute myocardial infarction was studied with the use of a semiquantitative latex agglutination immunoassay. This assay utilizes the monoclonal antibody DD-3B6/22, raised by conventional hybridoma technology, against a highly purified preparation of human D dimer and is adjusted to give a positive agglutination at a D dimer serum concentration of greater than 200 ng/ml (upper limit of normal). Twenty-one patients with acute transmural myocardial infarction of less than 3 hours' duration were studied. Fifteen patients received 0.75 to 1.5 million U intravenous streptokinase and 6 patients were treated conventionally without thrombolytic therapy. An elevated serum level of D dimer was detected before treatment in only 1 of 15 patients receiving intravenous streptokinase and within 2 hours of treatment in the remaining 14 patients who received streptokinase. In contrast, an elevated serum D dimer level was not detected during the first 24 hours in any of the six conventionally treated patients, including two patients who manifested the clinical syndrome of spontaneous reperfusion. The data suggest that in patients with acute myocardial infarction, an elevated serum level of D dimer, a cross-linked fibrin degradation product occurs early after administration of a large dose of streptokinase, but is infrequent during the first 24 hours in conventionally treated patients with acute infarction. Measurement of D dimer may be potentially useful for monitoring thrombolysis in patients with acute myocardial infarction.

Aged↗
PubMed1988

Reaggregation behavior of different types of collagen in vitro: variations in the occurrence and structure of dimeric segment long-spacing collagen.

Segment long-spacing collagen (SLS) can be precipitated from solutions of collagen using ATP as the inducing agent. Dimeric SLS aggregates have been observed in addition to monomeric SLS. We have compared collagen types I, II, III, and V with respect to their ability to form dimeric SLS in vitro. These collagen types were isolated from bovine tissues and characterized by polyacrylamide slab gel electrophoresis of the respective alpha-chains. Only monomeric SLS can be detected in preparations of collagen types I and III. Dimeric SLS, on the other hand, accounts for the majority of the crystallites seen in preparations of collagen types II and V. Dimeric SLS from both collagen types II and V reveal overlap zones at the carboxy-terminal ends of the collagen molecules. However, dimeric SLS from collagen types II and V differ with respect to their overlap distances. Significant portions of the triple helical domains of collagen molecules are occupied by the overlap region of dimeric SLS from type II collagen. On the other hand, dimeric SLS from type V collagen is composed of molecules overlapping only at their short nonhelical telopeptides. It is concluded that the ability of collagen molecules to aggregate into dimeric SLS under defined experimental conditions is collagen type dependent.

Adenosine Triphosphate↗
PubMed1992

Characterization of the tetramer-dimer-monomer equilibrium of the enzymatically active subunits of pigeon liver malic enzyme.

The tetrameric malic enzyme from pigeon liver was reversibly dissociated in the sequence of tetramer-dimer-monomer in an acidic environment (pH 4.5) or when the ionic strength or temperature of the solution was perturbed (0.2 M ammonium sulfate or < 10 degrees C). The dissociated monomer was enzymatically active according to the following criteria: (a) separation and direct activity staining of the monomer in the native gradient polyacrylamide gel, (b) activity staining of the monomer at its pI region in the isoelectric focusing gel, and (c) the enzyme showing lower but definite enzyme activity under conditions where only monomer existed in the solution. The catalytic constant (kcat) and specificity constant (kcat/KmMal) for the monomer were found to be 19 +/- 6 s-1 and 58 x 10(3) s-1.M-1, respectively, only one-seventh and one-seventeenth of those for the tetramer. Different types of interactions are involved in the monomer-monomer and dimer-dimer associations: (a) Two dissociation processes showed different pH dependences. The monomer-monomer interactions involve an amino acid with a side chain pKa value around 5.7, and an amino acid with a side chain pKa value of 7.2 is involved in the dimer-dimer association. (b) Ammonium sulfate up to 0.2 M only affects the monomer-monomer but not the dimer-dimer interactions. The Gibb's free energy, enthalpy, and entropy all have negative values for the above subunits' dissociations. The overall dissociation is an enthalpy-driven process. Association of the subunits to form dimers and tetramers involves salt-bridge, van der Waals, and hydrogen-bonding interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Interaction of the alpha beta dimers of the insulin-like growth factor I receptor is required for receptor autophosphorylation.

We have recently found that association of the two alpha beta dimers of the insulin-like growth factor I (IGF I) receptor is required for formation of a high-affinity binding site for IGF I [Tollefsen, S. E., & Thompson, K. (1988) J. Biol. Chem. 263, 16267-16273]. To determine the structural requirements for IGF I activated kinase activity, we have examined the effect of dissociation of the two alpha beta dimers of the IGF I receptor on beta subunit autophosphorylation. The alpha beta dimers formed after treatment with 2 mM dithiothreitol (DTT) at pH 8.75 for 5 min were separated from IGF I receptor remaining as tetramers after DTT treatment by fast protein liquid chromatography on a Superose 6 gel filtration column. Purification of the alpha beta dimers was confirmed by Western blot analysis using 125I-labeled alpha IR-3, a monoclonal antibody to the IGF I receptor. Autophosphorylation of the IGF I receptor (alpha beta)2 tetramer, treated without DTT or remaining after DTT treatment, is stimulated 1.6-2.9-fold by IGF I. In contrast, autophosphorylation of the alpha beta dimers incubated in the presence or absence of IGF I (100 ng/mL) does not occur. Both IGF I receptor dimers and tetramers exhibit similar kinase activities using the synthetic substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, indicating that the failure to detect autophosphorylation of the IGF I receptor dimers does not result from inactivation of the kinase by DTT treatment. We conclude that autophosphorylation of the IGF I receptor depends upon the interaction of the two alpha beta dimers.

Adenosine Triphosphate↗