Function of the delipidated beta-adrenergic receptor appears to require a fatty acid or a neutral lipid in addition to phospholipids

Detergent-solubilized preparations of the beta-adrenergic receptor (R) and of the guanyl nucleotide binding proteins (Gs) were extensively treated to remove phospholipids and cholesterol. Reconstitution of an R-Gs system was subsequently performed in the presence of a mixture of natural phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine or the synthetic dioleoyl derivatives of the same phospholipids. In both cases, an additional lipid was required for the agonist-dependent activation of Gs. The requirement could be fulfilled by alpha-tocopherol, or by unsaturated fatty acids such as oleic acid. Inclusion of this non-phosphorylated lipid in the reconstituted system enhanced the isoproterenol-dependent activation of Gs by guanosine 5'-O-[gamma-thio]triphosphate 16-33-fold. The rate of activation was largely dependent on the addition of the agonist. Efficient functional reconstitution of R-Gs was thus achieved in a totally defined lipid system. Additional studies of the reconstituted system and of the native membrane led to the notion that the non-phosphorylated lipid plays a role in the function of the hormone-R complex.     

Indole-3-ethanol oxidase in Phycomyces blakesleeanus. Is indole-3-ethanol a “storage pool” for IAA?

Indole-3-ethanol (IEt) was extracted from Phycomyces blakesleeanus Bgff. and purified by TLC and HPLC. Identification was performed by mass spectrum. The HPLC-purified compound showed an UV-spectrum typical for indoles, with absorption maxima at 220 and 281 nm. The IEt content varied between 1.5 nmol (g fresh weight)⁻¹ and 5.6 nmol (g fresh weight)⁻¹. The observed variations were strongly correlated with certain developmental stages of the fungus. Furthermore, the decrease of IEt between 60 and 84 h of fungal development coincides with a high IEt oxidase activity. The product of the enzyme reaction was indole-3-acetaldehyde, which was identified by co-chromatography with an authentic standard in several TLC and HPLC systems and by chemical conversion to indole-3-acetaldoxime.     

Spontaneous epimerization of (S)-deoxycoformycin and interaction of (R)-deoxycoformycin, (S)-deoxycoformycin, and 8-ketodeoxycoformycin with adenosine deaminase

(R)-Deoxycoformycin (pentostatin), (S)-deoxycoformycin, and 8-ketodeoxycoformycin were compared as inhibitors of calf intestine adenosine deaminase. In contrast to (R)-deoxycoformycin, which had been demonstrated as a tight-binding inhibitor with a dissociation constant of 2.5 X 10(-12) M [Agarwal, R. P., Spector, T., & Parks, R. E., Jr. (1977) Biochem. Pharmacol. 26, 359-367], (S)-deoxycoformycin and 8-ketodeoxycoformycin are slope-linear competitive inhibitors with respect to adenosine. The kinetic constants are 33 microM for inhibition by (S)-deoxycoformycin, 43 microM for 8-ketodeoxycoformycin, and 16 microM for the Km for adenosine. The stereochemistry of carbon 8 of the diazepine ring therefore causes a (1.3 X 10(7]-fold change in the affinity for the enzyme which is specific for the R configuration. This difference is attributed to an induced conformational change which cannot be initiated by the S isomer or the 8-keto analogue of (R)-deoxycoformycin. The studies were complicated by the need to remove traces of tight-binding inhibitor(s) from (S)-deoxycoformycin, since as little as 0.001% of the R isomer causes significant inhibition. The R and S isomers of deoxycoformycin are unstable in neutral or mildly acidic aqueous solutions. Isomerization of the secondary hydroxyl at carbon 8 of the diazepine ring is one of the reactions, resulting in S to R and R to S conversions for deoxycoformycins. Opening of the aglycon is also a major reaction. The tight-binding inhibitor generated from (S)-deoxycoformycin was identified as (R)-deoxycoformycin by high-pressure liquid chromatography, spectroscopy, circular dichroism, and chemical criteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional electron microscopic analysis of the chalice form of phosphorylase kinase

Phosphorylase kinase (Mr 1.3 X 10(6], a Ca2+-calmodulin-dependent protein kinase, plays a key role in the initiation of glycogenolysis. After purification on hydroxylapatite, the negatively stained enzyme was used for electron microscopy. In electron micrographs, phosphorylase kinase shows two major molecular forms: a butterfly form (approx. 60%) and a chalice form (approx. 40%). Images of the chalice form of the enzyme were computer-averaged by the method of single particle averaging. The following apparent molecular dimensions were obtained from the averages: total height, 20 nm; maximal width, 18 nm. The chalice form of phosphorylase kinase consists of a major structure termed the cup (11 nm X 18 nm), containing a large accessible cleft, and a minor structure termed the stem (8 nm X 9 nm). A closer examination of the images by averaging of molecular parts revealed two subpopulations of the cup part: a flexed (closed) type and an extended (open) type. The orifice, which can be closed partly by two protrusions (I, I'), is about 6 nm wide when the protrusions are flexed and 9 nm wide when they are extended. It is suggested that the substrates, e.g. phosphorylase b, may be accommodated in the large cleft of the enzyme. While the orientation of the protrusions (I, I') is the most obvious difference between the two types, more structural differences can be detected, suggesting a concerted movement of the protein domains against each other.     

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.     

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.     

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.