Molecular Weight Complexes Research Articles

Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris

Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.

Влияние иммуносупрессии на эффекторы воспаления в бронхоальвеолярном смыве при моделировании хронической обструктивной болезни легких у крыс

The model of chronic obstructive pulmonary disease (COPD) induced in rats by 60-day exposure to nitrogen dioxide was used to assess the effect of immunosuppression on the nature and rate of structural and functional disorders formation in the lungs. In the conditions of immunosuppression induced by cyclophosphamide, an earlier activation of the inflammatory process in the lungs was noted: (i) the influx of neutrophils was increased, (ii) the blood content of highly pathogenic immune complexes of average molecular weight increased, (iii) the content of proinflammatory cytokines in the bronchoalveolar lavage fluid increased. In contrast to the control group, signs of the respiratory epithelium damage and the lung tissue restructuring with the manifestation of characteristic features of the COPD model (emphysema, sclerosis, and fibrosis) were detected after 30 (not 60) days from the beginning of nitrogen dioxide exposure. The results of the study suggest that when exposed to aggressive aerogenic substances that damage the respiratory epithelium, the initial immune dysfunction can contribute to the formation of a pro-inflammatory environment and the phenotype of premature aging of the lungs, predisposing to the development of chronic obstructive pulmonary pathology.

Abstract PR15: BRD9 defines a novel mammalian SWI/SNF (BAF) complex configuration which supports proliferation in AML

Abstract Genes encoding subunits of the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complexes are mutated in over 20% of human cancer. Specific subunits are mutated in specific malignancies, highlighting their tissue-specific protective roles; moreover, synthetic lethal screens have uncovered genetic- and lineage-based features that confer dependence on specific mSWI/SNF subunits. As combinatorial complexity represents a major challenge, identification of specialized mSWI/SNF configurations, subunit-specific functions, binding restrictions, and exclusivity relationships is critical for understanding oncogenic mechanisms and for the selection of appropriate therapeutic agents targeting mSWI/SNF complex subunits. Here, we discover that BRD9, a recently identified mSWI/SNF subunit, defines a novel complex configuration distinct from BAF and PBAF, which we term noncanonical BAF, or ncBAF. We used biochemical methods to isolate BRD9-containing complexes and find that BRD9 selectively marks a sub-stoichiometric group of mSWI/SNF complexes of smaller molecular weight that lack several members of canonical BAF complexes such as BAF47 and ARID1A. Moreover, chemoproteomics using a BRD9 inhibitor (I-BRD9, GSK) isolates only ncBAF and does not resolve BAF-specific or PBAF-specific components, including the highly related bromodomain-containing subunit BRD7. We further identified regions of BRD9 and BRD7 that confer specificity of these subunits to ncBAF and PBAF complexes, respectively, resolving their mSWI/SNF binding domains. Using genome-wide ChIP-seq and RNA-seq experiments, we determined that ncBAF complexes target a subset of all mSWI/SNF complex target genes, and that associated GO terms for these genes are involved in epigenetic regulation, hemopoiesis, and differentiation. Finally, we applied a newly generated approach to deriving functional relationships within and between protein complex families from shRNA and CRISPR-based genetic screening datasets across hundreds of cancer cell lines to explore ncBAF-specific subunits. We find that ncBAF-specific complex subunits form a distinct functional module, supporting biochemical studies and pointing to the specific and divergent functions of the ncBAF configuration. Cancers of hematologic origin collectively exhibit the most significant responses to perturbation of ncBAF subunits, substantiating previous small-molecule screening efforts using BRD9 bromodomain inhibitors. These data demonstrate that ncBAF complexes represent a novel mSWI/SNF complex composition with distinct function in cancer. This abstract is also being presented as Poster 14. Citation Format: Brittany C. Michel, Andrew R. D'Avino, Josh Pan, Robin M. Meyers, Aviad Tsherniak, Paola Grandi, Phillip Humphreys, Neil Garton, Rab K. Prinja, Cigall Kadoch. BRD9 defines a novel mammalian SWI/SNF (BAF) complex configuration which supports proliferation in AML [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR15.

Some properties of three αB-crystallin mutants carrying point substitutions in the C-terminal domain and associated with congenital diseases

Physico-chemical properties of G154S, R157H and A171T mutants of αB-crystallin (HspB5) associated with congenital human diseases including certain myopathies and cataract were investigated. Oligomers formed by G154S and A171T mutants have the size and apparent molecular weight indistinguishable from those of the wild-type HspB5, whereas the size of oligomers formed by R157H mutant is slightly smaller. All mutants are less thermostable and start to aggregate at a lower temperature than the wild-type protein. All mutants effectively interact with a triple phosphomimicking mutant of HspB1 and form large heterooligomeric complexes of similar composition. All mutants interact with HspB6 forming heterooligomeric complexes with size and composition dependent on the molar ratio of two proteins. The wild-type HspB5 and its G154S and A171T mutants form only high molecular weight (300–450 kDa) heterooligomeric complexes with HspB6, whereas the R157H mutant forms both high and low (∼120 kDa) molecular weight complexes. The wild-type HspB5 and its G154S and A171T mutants form two types of heterooligomers with HspB4, whereas R157H mutant effectively forms only one type of heterooligomers with HspB4. G154S and A171T mutants have lower chaperone-like activity than the wild-type protein when subfragment S1 of myosin or βL-crystallin are used as a model substrates. With these substrates, the R157H mutant shows equal or higher chaperone activity than the wild-type HspB5. We hypothesize that the mutations in the C-terminal region modulate the binding of the IP(I/V) motif to the core α-crystallin domain. The R157H mutation is located in the immediate proximity of this motif. Such modulation could cause altered interaction of HspB5 with partners and substrates and eventually lead to pathological processes.

Abstract 1020: BRD9 defines a novel mammalian SWI/SNF (BAF) complex configuration which supports proliferation in AML

Abstract Genes encoding subunits of the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complexes are mutated in over 20% of human cancer. Specific subunits are mutated in specific malignancies, highlighting their tissue-specific protective roles; moreover, synthetic lethal screens have uncovered genetic- and lineage-based features which confer dependence on specific mSWI/SNF subunits. As combinatorial complexity represents a major challenge, identification of specialized mSWI/SNF configurations, subunit-specific functions, binding restrictions, and exclusivity relationships is critical for understanding oncogenic mechanisms and for the selection of appropriate therapeutic agents targeting mSWI/SNF complex subunits. Here, we discover that BRD9, a recently identified mSWI/SNF subunit, defines a novel complex configuration distinct from BAF and PBAF, which we term non-canonical BAF, or ncBAF. We used biochemical methods to isolate BRD9-containing complexes and find that BRD9 selectively marks a sub-stoichiometric group of mSWI/SNF complexes of smaller molecular weight that lack several members of canonical BAF complexes such as BAF47 and ARID1A. Moreover, chemoproteomics using a BRD9 inhibitor (GSK) isolates only ncBAF and does not resolve BAF-specific or PBAF-specific components, including the highly related bromodomain-containing subunit BRD7. We further identified regions of BRD9 and BRD7 that confer specificity of these subunits to ncBAF and PBAF complexes, respectively, resolving their mSWI/SNF binding domains. Using genome-wide ChIP-seq and RNA-seq experiments, we determined that ncBAF complexes target a distinct subset of all mSWI/SNF complex target genes and, consistent with previous studies, maintain proliferation of AML cells. Finally, we applied a newly- generated approach to deriving functional relationships within and between protein complex families from shRNA and CRISPR-based genetic screening datasets across hundreds of cancer cell lines to explore ncBAF-specific subunits. We find that ncBAF-specific complex subunits form a distinct functional module, supporting biochemical studies and pointing to the specific and divergent functions of the ncBAF configuration. Cancers of hematologic origin collectively exhibit the most significant responses to perturbation of three ncBAF subunits including BRD9, substantiating previous small molecule screening efforts using BRD9 bromodomain inhibitors. These data demonstrate that ncBAF complexes represent a novel BAF complex composition with distinct function in cancer. Citation Format: Brittany C. Michel, Joshua Pan, Robin M. Meyers, Paola Grandi, Phillip G. Humphreys, Neil G. Garton, Rab K. Prinja, Cigall Kadoch. BRD9 defines a novel mammalian SWI/SNF (BAF) complex configuration which supports proliferation in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1020. doi:10.1158/1538-7445.AM2017-1020

Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change.

AtTDX, a thioredoxin-like plant-specific protein present in Arabidospis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW) complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins.

Using light scattering to determine the stoichiometry of protein complexes.

The stoichiometry of a protein complex can be calculated from an accurate measurement of the complex's molecular weight. Multiangle laser light scattering in combination with size exclusion chromatography and interferometric refractometry provides a powerful means for determining the molecular weights of proteins and protein complexes. In contrast to conventional size exclusion chromatography and analytical centrifugation, measurements do not rely on the use of molecular weight standards and are not affected by the shape of the proteins. The technique is based on the direct relationship between the amount of light scattered by a protein in solution, and the product of its concentration and molecular weight. A typical experimental configuration includes a size exclusion column to fractionate the sample, a light scattering detector to measure scattered light, and an interferometric refractometer to measure protein concentration. The determination of the molecular weight of an anthrax toxin complex will be used to illustrate how multiangle laser light scattering can be used to determine the stoichiometry of protein complexes.

Некоторые иммунноэндокринные критерии затяжных форм истерического расстройства

The purpose of this study was to identify prognostically unfavorable immune and endocrine development criteria hysterical disorders. The study involved patients with hysterical personality development, hysterical neurosis and healthy donors. Immunological reactions were performed by standard methods, hormonal - immune enzyme analysis. Changes in cellular and humoral immunity in the form of a reduction in the absolute number of T- and B-lymphocytes, decrease the metabolic activity of neutrophils, the ratio of circulating immune complexes of different molecular weight, and lower cortisol levels indicate the involvement of the immune endocrine reactions in the pathogenetic mechanisms of the development of the protracted forms of conversion disorders, and confirm the need for surveys of the immune and hormonal status of patients suffering from hysterical personality development. Identified by the authors the changes in neuro-immune-endocrine system can be regarded as a prognostically unfavorable in the development of hysterical disorders, and involve the use of immunomodulators in the treatment of these diseases.

Differential Involvement of Atg16L1 in Crohn Disease and Canonical Autophagy

A single nucleotide polymorphism in Atg16L1, an autophagy-related gene (ATG), is a risk factor for Crohn disease, a major form of chronic inflammatory bowel disease. However, it is still unknown how the Atg16L1 variant contributes to disease development. The Atg16L1 protein possesses a C-terminal WD repeat domain whose function is entirely unknown, and the Crohn disease-associated mutation (T300A) is within this domain. To elucidate the function of the WD repeat domain, we established an experimental system in which a WD repeat domain mutant of Atg16L1 is stably expressed in Atg16L1-deficient mouse embryonic fibroblasts. Using the system, we show that the Atg16L1 complex forms a dimeric complex and that the total Atg16L1 protein level is strictly maintained, possibly by the ubiquitin proteasome system. Furthermore, we show that an Atg16L1 WD repeat domain deletion and the T300A mutant have little impact on canonical autophagy and autophagy against Salmonella enterica serovar Typhimurium. Therefore, we propose that Atg16L1 T300A is differentially involved in Crohn disease and canonical autophagy.

Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.

GATA-1 Forms Distinct Activating and Repressive Complexes in Erythroid Cells.

GATA-1 is a key transcription factor essential for the differentiation of the erythroid, megakaryocytic and eosinophilic lineages. GATA-1 functions in erythropoiesis involve lineage-specific gene activation and repression of early hematopoietic transcription programs. GATA-1 is known to interact with other transcription factors, such as FOG-1, TAL-1 and Sp1 and also with CBP/p300 and the SWI/SNF chromatin remodeling complex in vitro. Despite this information the molecular basis of its essential functions in erythropoiesis remains unclear. We show here that GATA-1 is mostly present in a high (> 670kDa) molecular weight complex that appears to be dynamic during erythroid differentiation. In order to characterize the GATA-1 complex(es) from erythroid cells, we employed an in vivo biotinylation tagging approach in mouse erythroleukemic (MEL) cells1. Briefly, this involved the fusion of a small (23aa) peptide tag to GATA-1 and its specific, efficient biotinylation by the bacterial BirA biotin ligase which is co-expressed with tagged GATA-1 in MEL cells. Nuclear extracts expressing biotinylated tagged GATA-1 were bound directly to streptavidin beads and co-purifying proteins were identified by mass spectrometry. In addition to the known GATA-1-interacting transcription factors FOG-1, TAL-1 and Ldb-1, we describe novel interactions with the essential hematopoietic transcription factor Gfi-1b and the chromatin remodeling complexes MeCP1 and ACF/WCRF. Significantly, GATA-1 interaction with the repressive MeCP1 complex requires FOG-1. We also show in erythroid cells that GATA-1, FOG-1 and MeCP1 are stably bound to repressed genes representing early hematopoietic (e.g. GATA-2) or alternative lineage-specific (e.g. eosinophilic) transcription programs, whereas the GATA-1/Gfi1b complex is bound to repressed genes involved in cell proliferation. In contrast, GATA-1 and TAL-1 are bound to the active erythroid-specific EKLF gene. Our findings on GATA-1 complexes provide novel insight as to the critical roles that GATA-1 plays in many aspects of erythropoiesis by revealing the GATA-1 partners in the execution of specific functions.

Using light scattering to determine the stoichiometry of protein complexes.

The stoichiometry of a protein complex can be calculated from an accurate measurement of the complex's molecular weight. Multiangle laser light scattering in combination with size-exclusion chromatography and interferometric refractometry provides a powerful means for determining the molecular weights of proteins and protein complexes. In contrast to conventional size-exclusion chromatography and analytical centrifugation, measurements do not rely on the use of molecular weight standards and are not affected by the shape of the proteins. The technique is based on the direct relationship between the amount of light scattered by a protein in solution and the product of its concentration and molecular weight. A typical experimental configuration includes a size-exclusion column to fractionate the sample, a light-scattering detector to measure scattered light, and an interferometric refractometer to measure protein concentration. The determination of the molecular weight of an anthrax toxin complex will be examined to illustrate how multiangle laser light scattering can be used to determine the stoichiometry of protein complexes.

A protochlorophyllide-dependent protein import complex of chloroplasts

Chloroplasts synthesize an abundance of different tetrapyrrole compounds. Among them are chlorophyll and its precursor protochlorophyllide, which accumulate in light- and dark-grown plants, respectively. Protochlorophyllide is bound to two NADPH:Pchlide oxidoreductases (POR), termed PORA and PORB, which in angiosperms form higher molecular weight complexes in the prolamellar body of etioplasts. Both POR proteins are nuclear gene products which must be imported from the cytosol.. Import of the precursor of the PORA requires Pchlide and is due to the operation of a second, substrate-dependent protein import site in the plastid envelope. Here we report the isolation of components of the Pchlide-dependent import (PIM) complex and show that it is composed of at least 12 different polypeptides. Among them is a Pchlide a oxygenase which provides Pchlide b as the import substrate of the pPORA, and a tyrosine amino transferase presumably involved in conferring photoprotection onto the PIM complex. Two other constituents were found to be related to previously characterized outer envelope membrane proteins: a 33K protein to TOC33 which is a GTP binding protein, and a 16K protein to a voltage-dependent, amino acid-sective channel protein in the outer plastid envelope.

Shedding of the 67-kD laminin receptor by human cancer cells.

The 67-kD laminin receptor (67LR) is a cell membrane-associated molecule exhibiting high affinity for the basement membrane glycoprotein, laminin. While export of the 67LR toward the extracellular matrix has been recently suggested by electron microscopy studies, there is to date no evidence of shedding of the 67LR from cells. Using two monoclonal antibodies directed against the 67LR, we developed a double-determinant radioimmunoassay that demonstrates that the 67LR is released from cancer cells into the culture medium. The shed molecule exhibited the same apparent molecular weight as that of the membrane-associated 67LR, suggesting that no proteolytic cleavage is involved in the process. Furthermore, we demonstrate that the 67LR is not anchored to the membrane through a glycolsyl-phosphatidylinositol bridge. However, the observation that lactose increased the release of 67LR suggests that a lectin-type interaction is involved in the cell membrane association of this laminin binding protein and the cell surface. Interestingly, the released 67LR recovered after HPLC gel filtration was found free as well as associated to high molecular weight complexes. The free 67LR retained its ability to bind to the cell surface. Our study is the first demonstration that the 67LR is effectively shed by cancer cells. The released free 67LR could play an important role in modulating interactions between cancer cells and laminin during tumor invasion and metastasis.

Hoechst 33342 Induces Apoptosis and Alters Tata Box Binding Protein/DNA Complexes in Nuclei from BC3H-1 Myocytes

Hoechst 33342 and Hoechst 33258 bind to adenine-thymine rich regions of the minor groove of DNA. Hoechst 33342, but not Hoechst 33258, induces BC3H-1 myocyte cell death and DNA fragmentation into an internucleosomal pattern characteristic of apoptosis. Hoechst 33342 has been shown to inhibit endogenous nuclear topoisomerase I activity. Another enzymatic activity utilizing the minor groove of DNA, the initiation of RNA polymerase II activity by formation of a TATA box binding protein/TATA box promoter complex, is shown to be altered using a gel mobility shift assay. A [32P]-labeled 24-oligonucleotide containing a TATA box element formed one molecular weight complex in control and Hoechst 33258 treated cells. The presence of Hoechst 33342 (26.7 μM) decreased the amount of the control complex and increased the presence of lower molecular weight species suggesting degradation of nuclear TBP and/or release of other transcription factors from the complex creating a smaller sized molecular complex which retains TATA box binding capacity. These results suggest that the pathway utilized to induce apoptosis in BC3H-1 myocytes may also involve the alteration of normal TBP/DNA complex formation and reduction in the initiation of new transcription.

Hepatic removal of 125I-DLT gelatin after burn injury: a model of soluble collagenous debris that interacts with plasma fibronectin

The decline of plasma fibronectin after surgery, trauma, and burn, as well as during severe sepsis after injury, appears to limit hepatic Kupffer cell phagocytic activity. Intravenous infusion of gelatin-coated particles to simulate blood-borne particulate collagenous tissue debris in the circulation after injury also depletes plasma fibronectin. We used soluble gelatin conjugated with 125I-labeled dilactitol tyramine (DLT-gelatin) as a model of soluble collagenous tissue debris. We studied its blood clearance as well as organ localization in normal and postburn rats. Fibronectin-deficient plasma harvested early after burn exhibited limited ability to support in vitro phagocytic uptake of the gelatinized microparticles by Kupffer cells in liver tissue from normal rats. However, Kupffer cells in liver tissue from normal and postburn rats phagocytized the test particles at a normal rate when incubated in normal plasma. The DLT-gelatin ligand bound to fibronectin in a dose-dependent manner as verified by its capture with anti-fibronectin coated plastic wells when coincubated with purified fibronectin. By gel filtration chromatography, the binding of fibronectin with the DLT-gelatin ligand was readily detected, resulting in the formation of a high-molecular-weight complex. In normal animals the plasma clearance and liver localization of 125I-DLT-gelatin was competitively inhibited by infusion of excess nonradioactive gelatin. The blood clearance and liver localization of the soluble gelatin ligand were also impaired after burn injury during periods of fibronectin deficiency similarly to the pattern observed with gelatin-coated microparticles. By autoradiography, the cellular site for the uptake of the 125I-DLT-gelatin was primarily but not exclusively hepatic Kupffer cells; 125I-DLT-asialofetuin and 125I-DLT-ovalbumin were removed by hepatocytes and sinusoidal endothelial cells, respectively. Thus, gelatin conjugated with 125I-DLT can be used to simulate blood-borne soluble collagenous tissue debris after burn. It rapidly binds to plasma fibronectin before its hepatic Kupffer cell removal, and its blood clearance is markedly delayed after burn injury during periods of plasma fibronectin deficiency.

Hypothalamic binding sites for pituitary adenylate cyclase activating polypeptide: characterization and molecular identification 1

The goal of these experiments was to identify and characterize binding sites in the rat hypothalamus for the peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The 27 amino acid form of PACAP (PACAP27) was used as the radiolabeled ligand in these experiments. Binding of [125I]PACAP27 to hypothalamic membrane preparations was rapid, reversible on addition of unlabeled peptide, and at least partially regulated by GTP. Nonhydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), guanosine-5'-(2-thiodiphosphate) (GDP beta S), and guanylylimidophosphate (GppNHp) also displaced [125I]PACAP27 binding to hypothalamic membrane preparations in a dose-dependent manner. The order of potency for the three analogs was GTP gamma S greater than GDP beta S greater than GppNHp. Both forms of the peptide, PACAP27 and PACAP38, were highly potent in displacing bound [125I]PACAP27, whereas VIP or PACAP(1-23) were unable to displace binding at concentrations of up to 500 nM. Scatchard analysis of the PACAP27 and PACAP38 displacement curves revealed that the fit of both curves was consistent with a single class of high-affinity binding sites, although the site exhibited a greater affinity for PACAP38 compared with PACAP27 (PACAP27 Kd = 1452 +/- 59 pM; PACAP38 Kd = 175 +/- 13 pM; Bmax 23.2 +/- 1.1 pmol/mg protein). The possibility of the existence of a class of binding sites with extremely low affinity cannot be discounted. After covalent cross-linking of [125I]PACAP27 with its receptor, the molecular weights of the complexes were estimated by electrophoresis and autoradiography. A major band of 60 Kd was evident when membranes were incubated with VIP or PACAP(1-23). Previous incubation with unlabeled PACAP27 or PACAP38 eliminated visualization of this band. These results suggest that a specific, high-affinity binding site for PACAP27 is present in rat hypothalamus, and that this site shows a greater affinity for PACAP38 compared with PACAP27. The molecular weight of the peptide-receptor complex is 60,000 kDa, and therefore the receptor itself has an apparent molecular weight 57,000.

Expression of A-type PDGF receptor in cytoplasm of tumor cell lines synthesizing PDGF

Two tumor cell lines, WM 266-4 melanoma and SW 707 colorectal carcinoma, both of which synthesize platelet-derived growth factor (PDGF) but do not express cell surface PDGF receptor, were tested for the presence of intracytoplasmic PDGF receptor. Using a novel method of intracytoplasmic (newly synthesized), receptor precipitation by the growth factor, we identified a 125,000 molecular weight protein crosslinked with 125I-labeled PDGF in a 140,000 molecular weight complex. In vitro translation of the corresponding mRNA revealed a 125,000 molecular weight product which presumably represents the A-type PDGF receptor. Exogenous PDGF was found to activate synthesis of RNA and cell proliferation.

Uracil-DNA Glycosylase Inhibitor Gene of Bacteriophage PBS2 Encodes a Binding Protein Specific for Uracil-DNA Glycosylase

The uracil-DNA glycosylase inhibitor gene (ugi) of the Bacillus subtilis bacteriophage PBS2 has been subcloned to a 720-base pair DNA fragment contained in pZW2-0.7 and its nucleotide sequence determined. Using nucleotide deletion analysis, we have located the cloned ugi gene along with potential regulatory elements. A promoter-like region (-10 and -35 consensus sequences) similar to other B. subtilis genes and the Shine-Dalgarno sequence characteristic of Gram-positive bacteria were both identified upstream from the initiator AUG codon. A 17-nucleotide exact inverted repeat followed by runs of adenine and thymine residues was positioned almost immediately downstream of the ochre codon. The ugi gene product was identified on sodium dodecyl sulfate-polyacrylamide gels using Escherichia coli minicells containing pZW2-0.7 and by recovering uracil-DNA glycosylase inhibitor activity following electrophoresis. The ugi gene codes for an acidic polypeptide of 9,477 molecular weight (84 amino acids) whose electrophoretic mobility was greater than predicted for a protein of this size. The mode of inhibition did not appear to involve a catalytic process nor did it directly involve inhibitor-DNA interaction. Rather, the inhibitor protein was shown to bind physically to the E. coli uracil-DNA glycosylase, forming a 36,000 molecular weight complex. This complex seems to be reversible, since inhibitor activity was recovered after heat treatment of the complex. In addition, we demonstrated that the inhibitor protein is active against uracil-DNA glycosylases isolated from several diverse biological sources but inactive against E. coli deoxyuridine triphosphatase, DNA polymerase I, and DNA polymerase alpha, beta, and gamma.

Radiolabeling of the interferon-α receptor

The action of alpha interferon (IFN-alpha) is initiated by its binding to a specific cell-surface glycoprotein, the IFN-alpha receptor, which is not well characterized. IFN-alpha A was reacted with an 125I-labeled, cleavable, heterobifunctional reagent. The derivatized IFN-alpha A was bound to human Daudi cells and photoactivated, forming a covalent IFN/receptor complex of apparent molecular weight 130,000-140,000 by SDS-polyacrylamide gel electrophoresis. Cleavage of the complex produced a new 125I-labeled 110 kDa band, representing the 125I-labeled IFN-alpha receptor free of IFN-alpha. This result provides a better estimate of the apparent molecular weight of the IFN-alpha receptor, and also provides a tool for tracking the migration of the free receptor in SDS-polyacrylamide gel electrophoresis.