Symmetry laws for interaction between helical macromolecules.

The power of symmetry laws is applied in many scientific areas from elementary particle physics to structural biology. The structures of many biological helices, including DNA, were resolved with the use of pertinent symmetry constraints. It was not recognized, however, that similar constraints determine cardinal features of helix-helix interactions vital for many recognition and assembly reactions in living cells. We now formulate such symmetry-determined interaction laws and apply them to explain DNA "over-winding" from 10.5 base pairs per turn in solution to 10 in hydrated fibers, counterion specificity in DNA condensation, and forces observed over the last 15 A of separation between DNA, collagen, and four-stranded guanosine helices.     

Reversible inhibition of protein synthesis in HeLa

Protein synthesis in suspended HeLa S₃ cells is inhibited by more than 50% immediately after addition of 100 μg pronase/ml or 500 μg trypsin/ml. Polyribosome profiles are not altered by exposure of cells to 1 or 2 mg trypsin/ml suggesting that the inhibition affects peptide chain elongation. Protein synthesis resumes after removal of proteases by sedimentation and resuspension of the cells.     

Reversible inhibition of protein splicing by zinc ion

Protein splicing involves the self-catalyzed excision of a protein-splicing element, the intein, from flanking polypeptides, the exteins, which are concomitantly joined by a peptide bond. Taking advantage of recently developed in vitro systems in which protein splicing occurs in trans to assay for protein-splicing inhibitors, we discovered that low concentrations of Zn(2+) inhibited splicing mediated both by the RecA intein from Mycobacterium tuberculosis and by the naturally split DnaE intein from Synechocystis sp. PCC6803. Inhibition by Zn(2+) was also observed with a cis-splicing system involving the RecA intein. In all experimental systems used, inhibition by Zn(2+) could be completely reversed by the addition of EDTA. Zinc ion also inhibited hydroxylamine-dependent N-terminal cleavage of the RecA intein. All other divalent transition metal ions tested were less effective as inhibitors than Zn(2+). The reversible inhibition by Zn(2+) should be useful in studies of the mechanism of protein splicing and allow structural studies of unmodified protein-splicing precursors.     

Comparison of rabbit androgen binding protein with testosterone estradiol binding globulin--I. Physical and chemical properties

Rabbit epididymal androgen binding protein (rbABP) and serum testosterone estradiol binding globulin (rbTeBG) were purified and their physicochemical properties compared. Both proteins bound dihydrotestosterone (DHT) with high affinity. Both contained two components, Heavy (H) and Light (L), and their molecular weights and pI values were comparable. rbABP and rbTeBG were different with regard to their ConA-Sepharose binding property. rbABP was not bound by ConA-Sepharose while rbTeBG was found and retained by this lectin; thus, rbABP and rbTeBG differed in their carbohydrate structure. Peptide mapping on SDS-PAGE indicated that the H components of rbABP and rbTeBG were distinct even though they showed a high degree of homology. By contrast, the L components of these two proteins appeared to be identical. The structure of the steroid binding sites of these two proteins was analyzed by peptide mapping of [1,2(3)H]17 beta hydroxy-androsta-4,6-dien-3-one photoaffinity labeled protein. The size distribution of radioactive peptide fragments generated appeared to be identical for these two proteins. However, the distribution of labeled peptides was slightly different when examined by high pressure liquid chromatography (HPLC). The observations suggest that the differences between rbABP and rbTeBG might reside not only in carbohydrate moieties but also in their amino acid sequences.     

Comparison of rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin--II. Immunological properties

Purified rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin have been immunologically compared. A comparison using the steady state gel method of Ritzen et al. indicated immunological cross-reactivity. In order to further compare their immunological properties we developed a radioimmunoassay for both rbABP and rbTeBG using specific antisera directed against each. When these assays were compared, the extent or completeness of displacement proved to be the only parameter that was significantly different. This data obtained with homologous and heterologous radioimmunoassays is consistent with the idea that these two proteins contain minor antigenic determinants which are distinct.     

The 56 kDa androgen binding protein is an aldehyde dehydrogenase.

We have described a 56 kDa protein from genital skin fibroblasts that specifically binds androgen and that is generally not expressed in genital skin fibroblasts from patients with androgen insensitivity due to genetic defects of the androgen receptor. We have isolated a partial cDNA clone for the 56 kDa protein from an expression library of genital skin fibroblasts. In vitro translation of message selected with this clone faithfully produces the 56 kDa protein which can be immuneprecipitated with an anti-56 kDa antiserum. Northern blots probed with this clone show a 2.2 kb message, which parallels the expression of the 56 kDa protein. The sequence of this 998bp clone is identical to human liver aldehyde dehydrogenase 1, the cytoplasmic isoenzyme. On activity gels of genital skin fibroblast cytosol covalently labelled with androgen, aldehyde dehydrogenase activity comigrates with the single band labelled specifically with androgen. Thus, the 56 kDa androgen binding protein is an aldehyde dehydrogenase, which is prominently expressed in normal genital skin fibroblasts, but not in non-genital skin fibroblasts.     

Reversible inhibition of cyclic AMP-dependent protein kinase by insulin

Summary Extracts of fasted rat diaphragms, previously treated with or without insulin were assayed for glycogen synthase, protein kinase and cyclic [³H]-AMP binding. Treatment with insulin produced an elevation in the % of glycogen synthase I and a concurrent decrease in cyclic AMP-dependent protein kinase activity and cyclic [³H]-AMP binding. Analysis of extracts by disc gel electrophoresis demonstrated the inhibition of cyclic [3H]-AMP binding to involve the Type I protein kinase holoenzyme. Inhibition of protein kinase activity was most apparent in the presence of 0.2 µM cyclic AMP, with enzymatic activity of the insulin-treated extracts typically 60–65% of control. Higher assay concentrations diminished the difference between control and insulin-treated extracts and concentrations greater than 20 µm abolished it.The inhibition of cyclic AMP-dependent protein kinase activity after insulin was a transient and labile phenomenon. The effect was independent of ATP concentration in the assay, but was sensitive to the pH of tissue extraction, requiring a pH of 7.0 to 8.4 to be observed.Insulin-mediated inhibition of protein kinase activity was reversed upon preincubation of extracts at 0–2°. Relatively concentrated homogenates (<4 µl buffer/mg tissue) yielded extracts which exhibited little or no inhibition of protein kinase activity compared to extracts prepared from more dilute (6–10 µl/mg) homogenates. A model for the inhibition of the cyclic-AMP dependent protein kinase by an insulin-generated inhibitor which becomes directly associated with the Type 1 holoenzyme is proposed.Abbreviations cyclic AMP (cAMP) Adenosine 3,5-monophosphate - Tricine N-Tris (Hydroxy-methyl) methyl glycine - G-6-P glucose-6-phosphate - MES 2-[N-morpholino]ethane sulfonic acid A preliminary report was communicated to the 61st meeting of the F.A.S.E.B., April, 1977.     

Reversible inhibition of protein synthesis in lung by halothane

Alterations in the synthesis and degradation of proteins were investigated in intact lungs exposed to the volatile anaesthetic halothane. In rat lungs perfused in situ with Krebs-Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6 mM-glucose, plasma concentrations of 19 amino acids and 690 microM-[U-14C]-phenylalanine and equilibrated with O2/N2/CO2 (4:15:1), protein synthesis, calculated based on the specific radioactivity of aminoacyl-tRNA, was inhibited by halothane. The anaesthetic did not affect degradation of lung proteins. The inhibition of protein synthesis was rapid in onset, dose-dependent, and quickly reversible. It did not appear to be associated with overall energy depletion, with non-specific changes in cellular permeability, or with decreased availability of amino acids as substrates for protein synthesis.     

Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.

Reversible binding among components of the cellular submembrane cytoskeleton and reversible binding of some of these components with the plasma membrane likely play a role in nonelastic morphological changes and mechanoplastic properties of cells. However, relatively few studies have been devoted to investigating directly the kinetic aspects of the interactions of individual components of the membrane skeleton with the membrane. The experiments described here investigated whether one component of the erythrocyte membrane cytoskeleton, protein 4.1, binds to its sites on the membrane reversibly and if so, whether the different 4.1-binding sites display distinct kinetic behavior. Protein 4.1 is known to stabilize the membrane and to mediate the attachment of spectrin filaments to the membrane. Protein 4.1 previously has been shown to bind to integral membrane proteins band 3, glycophorin C, and to negatively charged phospholipids. To examine the kinetic rates of dissociation of carboxymethyl fluorescein-labeled 4.1 (CF-4.1) to the cytofacial surface of erythrocyte membrane, a special preparation of hemolyzed erythrocyte ghosts was used, in which the ghosts became flattened on a glass surface and exposed their cytofacial surfaces to the solution through a membrane rip in a distinctive characteristic pattern. This preparation was examined by the microscopy technique of total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP). Four different treatments were employed to help identify which membrane binding sites gave rise to the multiplicity of observed kinetic rates. The first treatment, the control, stripped off the native spectrin, actin, 4.1, and ankyrin. About 60% of the CF-4.1 bound to this control binded irreversibly (dissociation time > 20 min), but the remaining approximately 40% binded reversibly with a range of residency times averaging approximately 3 s. The second treatment subjected these stripped membranes to trypsin, which presumably removed most of the band 3. CF-4.1 binded significantly less to these trypsinized membranes and most of the decrease was a loss of the irreversibly binding sites. The third treatment simply preserved the native 4.1 and ankyrin. CF-4.1 binded less to this sample too, and the loss involved both the irreversible and reversible sites. The fourth treatment blocked the gycophorin C sites on the native 4.1-stripped membranes with an antibody. CF-4.1 again binded less to this sample than to a nonimmune serum control, and almost all of the decrease is a loss of irreversible sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rabbit red cell cyclic AMP-dependent protein kinase. I. Reversible subunit interaction

Cyclic AMP-dependent protein kinase I consists of two dissimilar functional subunits: a catalytic subunit and a cyclic AMP binding subunit. The interaction of the two subunits appears to be reversible.     

Determination of strontium binding to macromolecules.

An equilibrium dialysis technique for determining the binding of strontium to macromolecules is described. The major difficulty to be overcome is that 90Sr has a decay product, 90Y, which is also a beta-emitter. The described protocol is used to determine the Sr binding isotherm to bovine prothrombin fragment 1. The binding is found to be cooperative, somewhat weaker than Ca binding, and to involve approximately nine strontium sites. The stoichiometric equilibrium constants are determined by nonlinear regression. The procedure should be of great utility for many macromolecules that show strontium affinity.     

Physical studies of the interaction between the Escherichia coli DNA binding protein and nucleic acids.

The interaction of nucleic acid with the Escherichia coli DNA-binding protein has been studied by fluorescence emission spectroscopy and sedimentation velocity analysis. The protein binds to single-strand DNA with an apparent equilibrium dissociation constant of 2 X 10(-9). It binds to the homopolymers poly (dA) and poly (dT) slightly more tightly, but has a larger apparent equilibrium dissociation constant to poly (dC). The protein also binds tightly to ribohomopolymers and to tRNA, but not to duplex DNA. By the use of defined-length oligonucleotides, it has been shown that the protein binds to DNA in a highly cooperative manner. The extent of cooperativity is seen as the difference in binding between an isolated monomeric protein molecule bound to DNA and two or more molecules binding to contiguous sites.     

Reversible membrane interaction of BAD requires two C-terminal lipid binding domains in conjunction with 14-3-3 protein binding

BAD is a Bcl-2 homology domain 3 (BH3)-only proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Binding of BAD to mitochondria is thought to be exclusively mediated by its BH3 domain. We show here that BAD binds to lipids with high affinities, predominantly to negatively charged phospholipids, such as phosphatidylserine, phosphatidic acid, and cardiolipin, as well as to cholesterol-rich liposomes. Two lipid binding domains (LBD1 and LBD2) with different binding preferences were identified, both located in the C-terminal part of the BAD protein. BAD facilitates membrane translocation of Bcl-XL in a process that requires LBD2. Integrity of LBD1 and LBD2 is also required for proapoptotic activity in vivo. Phosphorylation of BAD does not affect membrane binding but renders BAD susceptible to membrane extraction by 14-3-3 proteins. BAD can be removed efficiently by 14-3-3zeta, -eta, -tau and lesxs efficiently by other 14-3-3 isoforms. The assembled BAD.14-3-3 complex exhibited high affinity for cholesterol-rich liposomes but low affinity for mitochondrial membranes. We conclude that BAD is a membrane-associated protein that has the hallmarks of a receptor rather than a ligand. Lipid binding is essential for the proapoptotic function of BAD in vivo. The data support a model in which BAD shuttles in a phosphorylation-dependent manner between mitochondria and other membranes and where 14-3-3 is a key regulator of this relocation. The dynamic interaction of BAD with membranes is tied to activation and membrane translocation of Bcl-XL.     

Reversible inhibition by diamide of cyclic AMP-dependent protein kinases from bovine thyroid.

The effects of diamide on protein kinases isolated from bovine thyroid were studied. Cyclic AMP-dependent protein kinase activity was directly, rapidly, and reversibly inhibited by diamide. This inhibition was non-competitive with respect to ATP or histone and could be prevented by thiol-reducing agents. However, a cyclic nucleotide-independent thyroid protein kinase was not affected. Our data indicate that diamide specifically inhibits protein kinases which are cyclic AMP-dependent.