Nuclear magnetic resonance studies of amino acids and proteins. Rotational correlation times of proteins by deuterium nuclear magnetic resonance spectroscopy

We show that measurement of the spin-lattice (T1) and spin-spin (T2) relaxation times (or line widths) of irrotationally bound 2H nuclei in macromolecules undergoing isotropic rotational motion outside of the extreme narrowing limit (i.e., for the case omega 02 tau R2 much greater than 1) permits determination of both the rotational correlation time (tau R) of the macromolecule and the electric quadrupole coupling constant (e2qQ/h) of the 2H label. The technique has the advantage over 13C nuclear magnetic resonance (NMR) that no assumptions about bond lengths (which appear to the sixth power in 13C relaxation studies) or relaxation mechanisms need to be made, since relaxation will always be quadrupolar, even for aromatic residues at high field. Asymmetry parameter (eta) uncertainties are shown to cause negligible effects on tau R determinations, and in any case it is shown that both e2qQ/h and eta may readily be determined in separate solid-state experiments. By way of example, we report 2H NMR results on aqueous lysozyme (EC 3.2.1.17) at 5.2 and 8.5 T (corresponding to 2H-resonance frequencies of 34 and 55 MHz). Interpretation of the results in terms of the isotropic rigid-rotor model yields e2qQ/h values of approximately equal to 170 or approximately equal to 190 kHz, respectively, for the imidazolium and free-base forms of [epsilon 1-2H] His-15 lysozyme in solution, in excellent agreement with e2qQ/h values of approximately 167 and approximately 190 kHz obtained for the free amino acids in the solid state. In principle, the method may in suitable cases permit comparison between the dynamic structures of proteins in solution and in the crystalline solid state.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

Crystallographic quaternary structural analysis of AMP nucleosidases from Escherichia coli and Azotobacter vinelandii

Adenosine monophosphate nucleosidases from Azotobacter vinelandii and Escherichia coli have been studied crystallographically to determine their quarternary structures. Preliminary characterization of the A. vinelandii enzyme shows that the crystals are monoclinic, C2 with a = 347 A, b = 204 A, c = 114 A, and beta = 91.7 degrees. The asymmetric unit contains 12 or 9 subunits of Mr 54,000. Self-rotation functions with data from the AMP nucleosidases from A. vinelandii and from E. coli (Giranda, V. L., Berman, H. M., and Schramm, V. L. (1986) J. Biol. Chem. 261, 15307-15309) are consistent with the monomers arranged as hexamers with point symmetry 32. The hexamers are arranged in the unit cells so that crystallographic 2-fold axes are coincident with the local 2-folds of the point group 32.     

Enzymes Involved in Anaerobic Polyethylene Glycol Degradation by Pelobacter venetianus and Bacteroides Strain PG1

In extracts of polyethylene glycol (PEG)-grown cells of the strictly anaerobically fermenting bacterium Pelobacter venetianus, two different enzyme activities were detected, a diol dehydratase and a PEG-degrading enzyme which was characterized as a PEG acetaldehyde lyase. Both enzymes were oxygen sensitive and depended on a reductant, such as titanium citrate or sulfhydryl compounds, for optimal activity. The diol dehydratase was inhibited by various corrinoids (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, and methylcobalamin) by up to 37% at a concentration of 100 μM. Changes in ionic strength and the K⁺ ion concentration had only limited effects on this enzyme activity; glycerol inhibited the enzyme by 95%. The PEG-degrading enzyme activity was stimulated by the same corrinoids by up to 80%, exhibited optimal activity in 0.75 M potassium phosphate buffer or in the presence of 4 M KCI, and was only slightly affected by glycerol. Both enzymes were located in the cytoplasmic space. Also, another PEG-degrading bacterium, Bacteroides strain PG1, contained a PEG acetaldehyde lyase activity analogous to the corresponding enzyme of P. venetianus but no diol dehydratase. Our results confirm that corrinoid-influenced PEG degradation analogous to a diol dehydratase reaction is a common strategy among several different strictly anaerobic PEG-degrading bacteria.     

Regulation of glycogen synthetase and phosphorylase phosphatase activities in rat adipose tissue.

Incubation of a rat adipose tissue homogenate causes a time and temperature dependent activation of glycogen synthetase (UDP glucose:glycogen 4-alpha-glucosyltransferase) and simultaneous inactivation of phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-glucosyltransferase, EC 2.4.1.1). Activation of glycogen synthetase at 15 and 23 degrees C was preceded by a lag period. The duration of the lag period could not be correlated with significant changes in phosphorylase activity. Addition of glucose and methylxanthines caused an increase in the rates of glycogen synthetase activation and phosphorylase inactivation. The effect on glycogen synthetase activation was mainly on the linear phase. Addition of AMP inhibited phosphorylase inactivation and accelerated glycogen synthetase activation. Addition of muscle phosphorylase alpha caused a prolongation of the lag period which lasted until phosphorylase alpha activity had decreased to the level originally present in the preparation. It is concluded that in adipose tissue activation of glycogen synthetase is not dependent on prior inactivation of phosphorylase and that other factors should be looked for to explain the lag period preceding glycogen synthetase activation.     

Structural variability in the genome of phage ?H of Halobacterium halobium

Summary The partially circularly permuted, terminally redundant structure of the DNA of phage H has been confirmed by a cleavage map for the restriction enzymes PstI, ClaI, BglII, HindIII, and, partially, BamHI.Six variants of phage H have been isolated from 71 single plaques. Their genomes differ by several insertions, a deletion, and an inversion of a DNA segment with a minimal length of 11 kb. The inversion occurs with high frequency in variants carrying at the flanks of the invertible DNA in verted repeats of a 1.8 kb DNA element which shares sequence homology with the DNA of H. halobium and may be involved in the extreme variability of its genome.     

Irreversible enzyme-shuttle immunoassay

The concept of a competitive enzyme immunoassay that utilizes simultaneously the bound and the free analyte-enzyme conjugate (heterobifunctional conjugate) for signal generation in response to varying analyte concentrations in samples has been investigated. Two antigenic sites of the heterobifunctional conjugate are used in the assay for binding to immunoglobulins: the analyte derivative binds to an immobilized antibody, Ab(1), and the enzyme component binds to a spatially separated immobilized antibody, Ab(2). The analytical system is set up such that in the absence of analyte, the conjugate is predominantly bound in the compartment that contains Ab(1). With increasing concentration of native analyte in samples, an increasing concentration of native analyte in samples, an increasing amount of conjugate migrates to the second compartment that contains Ab(2). The enzyme bound in each compartment is used for signal generation. Mathematical models have been developed to determine the optimal conditions and to predict the performance of such dual-antibody systems. The theoretical predictions are supported by experimental results. The dual-antibody system has been compared with a conventional competitive enzyme immunoassay using the same reagents.     

Isotope trapping and positional isotope exchange with rat and chicken liver phosphoenolpyruvate carboxykinases

Isotope-trapping studies of the enzyme.MgGTP complex were carried out with rat liver cytosolic and chicken liver mitochondrial phosphoenolpyruvate carboxykinases. For the rat liver enzyme, MgGTP was partially trapped from both E.MgGTP and E.MgGTP.OAA complexes, consistent with a steady-state random mechanism. For the chicken liver enzyme, MgGTP was 100% trapped from the E.MgGTP.OAA complex, consistent with a steady-state ordered mechanism. The rate constants for the interaction of MgGTP with the free enzymes are approximately 10(7) M-1 S-1, somewhat lower than the diffusion limit for association. The dissociation rate for the enzyme.MgGTP complexes is 26-92 s-1, reflecting a tightly bound complex with high commitment to catalysis in the presence of oxaloacetate. Positional isotope-exchange studies were also carried out with phosphoenolpyruvate carboxykinases from rat and chicken. No exchange if the beta gamma-18O in [beta gamma-18O, gamma-18O3]GTP to form [beta-18O, gamma-18O3]GTP was detected in the absence of oxaloacetate. In the presence of oxaloacetate, no positional isotope exchange of [beta gamma-18O, gamma-18O3]GTP was detected during initial rate conditions. The results indicate that at least one of the products dissociates rapidly from the E.MgGDP.PEP.CO2 complex relative to the net rate of MgGTP formation from the E.MgGDP.PEP.CO2 complex. A rapid equilibrium between the central complexes in which the beta-phosphoryl of GDP is restricted with respect to torsional rotation cannot be excluded but is unlikely on the basis of the relative rates of catalysis and torsional rotation. The addition of Mn2+, an activator of phosphoenolpyruvate carboxykinase, did not influence the positional isotope-exchange results.(ABSTRACT TRUNCATED AT 250 WORDS)