Repeating structure of chick tropoelastin revealed by complementary DNA cloning

A cDNA library was constructed from chick aorta poly(adenylic acid)-containing RNA in the expression vector pEX1. Several clones were identified by screening the library with a polyclonal antiserum raised against chick tropoelastin and confirmed by DNA sequencing. Analysis of the deduced amino acid sequence, corresponding to the mature tropoelastin and most of the signal peptide, revealed that the molecule is composed of at least 8, and possibly 13, repeating units. The common features of each unit include an N-terminal region composed largely of alanines and lysines and ending with an aromatic amino acid, followed by a GAG span and then a C-terminal region consisting mostly of valines, prolines, and glycines often present in several copies of the sequence (VPGV). This structure is discussed in terms of the functional properties of the molecule.     

Kinetic mechanism of DNA polymerase I (Klenow)

The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A., & Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.     

Covalent binding of 4-carbamoylbenzenediazonium chloride to deoxyguanine bases of DNA resulting in apparent irreversible inhibition of poly(adenosine diphosphoribose) polymerase at the nicotinamide binding site

The poly(adenosine diphosphoribose) polymerase activity of isolated liver nuclei was inhibited by 4-carbamoylbenzenediazonium chloride, referred to as 4-diazoniobenzamide, an effect that was dependent on the time of incubation and the concentration of the diazonium compound, with inhibition following first-order kinetics. The inhibition was not reversed by reisolation of nuclei and centrifugal washing, whereas the inhibition by benzamide or 4-aminobenzamide was completely reversible under these conditions. Simultaneous incubation of 4-diazoniobenzamide with benzamide prevented enzyme inhibition. The 4-diazoniobenzoic acid analogue was not inhibitory. The mechanism of action of 4-diazoniobenzamide was traced to a specific covalent binding to dGMP of DNA to form N2-[(4-carbamoylphenyl)azo]-2'-deoxyguanosine 5'-monophosphate. Coenzymic DNA, by tight association with the polymerase protein, fixes the -C(O)NH2 moiety of the adduct at the nicotinamide-binding site of the enzyme.     

Virtual and solution conformations of oligosaccharides

The possibility that observed nuclear Overhauser enhancements and bulk longitudinal relaxation times, parameters measured by 1H NMR and often employed in determining the preferred solution conformation of biologically important molecules, are the result of averaging over many conformational states is quantitatively evaluated. Of particular interest was to ascertain whether certain 1H NMR determined conformations are "virtual" in nature; i.e., the fraction of the population of molecules actually found at any time within the subset of conformational space defined as the "solution conformation" is vanishingly small. A statistical mechanics approach was utilized to calculate an ensemble average relaxation matrix from which (NOE)'s and (T1)'s are calculated. Model glycosidic linkages in four oligosaccharides were studied. The solution conformation at any glycosidic linkage is properly represented by a normalized, Boltzmann distribution of conformers generated from an appropriate potential energy surface. The nature of the resultant population distributions is such that 50% of the molecular population is found within 1% of available microstates, while 99% of the molecular population occupies about 10% of the ensemble microstates, a number roughly equal to that sterically allowed. From this analysis we conclude that in many cases quantitative interpretation of NMR relaxation data, which attempts to define a single set of allowable torsion angle values consistent with the observed data, will lead to solution conformations that are either virtual or reflect torsion angle values possessed by a minority of the molecular population. On the other hand, calculation of ensemble average NMR relaxation data yields values in agreement with experimental results. Observed values of NMR relaxation data are the result of the complex interdependence of the population distribution and NOE (or T1) surfaces in conformational space. In conformational analyses, NMR data can therefore be used to test different population distributions calculated from empirical potential energy functions.     

Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with nitrogen-15

A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts [see Delihas, N., Anderson, J., & Singhal, R. P. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190] but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a "normal" chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for our understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed.     

Multiparameter study of denaturation of 20 beta-hydroxysteroid dehydrogenase by urea in the presence of stabilizing agents

We investigated the denaturation of tetrameric 20 beta-hydroxysteroid dehydrogenase (20R)-17 beta,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) to find out whether intermediate states are formed during the process. The denaturation process was studied in the presence and absence of stabilizers, both specific, such as NADH, and non-specific, such as the salting-out anion phosphate. Changes in enzymatic activity, intrinsic protein fluorescence and far-ultraviolet circular dichroism were monitored. When NADH was present, denaturation of the enzyme by urea was a one-step transition between the native and the completely denatured state. In dilute phosphate, and even more so in concentrated phosphate, the existence of intermediate states with different stability is evidenced by the noncoincidence of the transition curves that probe for different functional and conformational aspects of the enzyme. Therefore, for 20 beta-hydroxysteroid dehydrogenase the formation of intermediates can be prevented by adding NADH, or enhanced by adding concentrated phosphate.     

Laser-Raman and infrared spectroscopic studies of protein conformation in the eggshell of the fish Salmo gairdneri

Laser-Raman and infrared spectroscopic studies reveal abundant beta-pleated sheet conformation in the eggshell proteins of the fish Salmo gairdneri. This secondary structure is the underlying molecular conformation, dictating the formation of the helicoidal architecture of the eggshell. Disulphide bonds crosslink the eggshell proteins of the fertilized eggs and are apparently found in g-g-g (gauche-gauche-gauche), g-g-t (gauche-gauche-trans) and t-g-t (trans-gauche-trans) conformation. There is no evidence for the existence of free sulphydryls. The tyrosines appear to act as hydrogen-bond acceptors, whereas the aromatic residues phenylalanine and tryptophan are also eggshell protein constituents.     

Structural and physico-chemical properties of amino acids of functionally important regions of proteins

A correlation of the localization of functionally important regions with places having low and high values on twelve profiles built on a basis of amino acid sequences was analysed using a broad set of proteins. The profiles of hydrophilicity, resemblance to the sequences of human proteins, flexibility, mutability and others were considered. The resemblance profile was plotted by the program fixing short similar fragments in the testing protein and 92 human ones. The active centres were shown to be located in the primary structure regions having relatively low values on the resemblance profiles. Similar effect was observed in the mutability and alpha-helicity profiles. The potential functionally important sites of the human leukocyte interferon and interleukin-2 isolated on the basis of the analysis of this profiles were in accord with the available literary data.     

Androgenic regulation of luteinizing hormone secretion: relationship to androgen binding in sheep pituitary

Castrated ram lambs (wethers) were investigated for sensitivity to androgen feedback and to determine whether this feedback inhibition of luteinizing hormone (LH) was associated with changes in pituitary androgen receptors. Administration of Silastic capsules containing either dihydrotestosterone or testosterone was found to produce dose-dependent inhibitory effects on serum LH levels in wethers. Physiological dosages of these androgens (i.e., those that produce serum levels of dihydrotestosterone [0.24 ng/ml] or testosterone [2.1 ng/ml] similar to those of intact rams) resulted in differential inhibition of serum LH and LH content of the anterior pituitary. Whereas the inhibitory effect of dihydrotestosterone on pituitary LH content was much more dramatic than that seen with testosterone, the high dosage of testosterone also produced a substantial decrease in pituitary LH content. Responses of the pituitary to changes in serum androgen were compared to responses of the seminal vesicle, which served as a control androgen target organ. Androgen levels were positively correlated with seminal vesicle weights, but pituitary weights were unaffected by castration and/or androgen replacement. Treatments with dihydrotestosterone were associated with decreased cytosol androgen binding activity (i.e., receptors) in pituitary and seminal vesicle, suggesting that both of these tissues were sites of androgen action. Although testosterone inhibited serum LH levels, pituitary cytosol androgen receptors were not affected by changes in serum testosterone. We conclude from these data that dihydrotestosterone is a physiological regulator of pituitary LH secretion in the ram and that further study is needed to investigate the complex actions of testosterone and its metabolites on pituitary function.