Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers.

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas putida KT2442 during growth on carbohydrates was studied. PHAs isolated from P. putida cultivated on glucose, fructose, and glycerol were found to have a very similar monomer composition. In addition to the major constituent 3-hydroxydecanoate, six other monomers were found to be present: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate. The identity of all seven 3-hydroxy fatty acids was established by gas chromatography-mass spectrometry, one-dimensional 1H-nuclear magnetic resonance, and two-dimensional double-quantum filtered correlation spectroscopy 1H-nuclear magnetic resonance. The chemical structures of the monomer units are identical to the structure of the acyl moiety of the 3-hydroxyacyl-acyl carrier protein intermediates of de novo fatty acid biosynthesis. Furthermore, the degree of unsaturation of PHA and membrane lipids is similarly influenced by shifts in the cultivation temperature. These results strongly indicate that, during growth on nonrelated substrates, PHA monomers are derived from intermediates of de novo fatty acid biosynthesis. Analysis of a P. putida pha mutant and complementation of this mutant with the cloned pha locus revealed that the PHA polymerase genes necessary for PHA synthesis from octanoate are also responsible for PHA formation from glucose.     

Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium.

The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules.     

Influence of benzo[a]pyrene diol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-trans-anti-[BP]G.C adduct structure and comparison with the (+)-trans-anti-[BP]G.C enantiomer.

Benzo[a]pyrene (BP) is an environmental genotoxin, which, following metabolic activation to 7,8-diol 9,10-epoxide (BPDE) derivatives, forms covalent adducts with cellular DNA. A major fraction of adducts are derived from the binding of N2 of guanine to the C10 position of BPDE. The mutagenic and carcinogenic potentials of these adducts are strongly dependent on the chirality at the four asymmetric benzylic carbon atoms. We report below on the combined NMR-energy minimization refinement characterization of the solution conformation of (-)-trans-anti-[BP]G positioned opposite C and flanked by G.C base pairs in the d(C1-C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17- G18-A19-T20-G21-G22) duplex. Two-dimensional NMR techniques were applied to assign the exchangeable and non-exchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid in the modified duplex. These results establish Watson-Crick base pair alignment at the [BP]G6.C17 modification site, as well as the flanking C5.G18 and C7.G16 pairs within a regular right-handed helix. The solution structure of the (-)-trans-anti-[BP]G.C 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOE buildup curves as constraints in energy minimization computations. The BP ring spans both strands of the duplex in the minor groove and is directed toward the 3'-end of the modified strand in the refined structure. One face of the BP ring of [BP]G6 stacks over the C17 residue across from it on the partner strand while the other face is exposed to solvent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.

The cytochrome o complex is a bo-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature. This paper presents a relatively simple preparation of the enzyme starting with a strain of Escherichia coli which overproduces the oxidase. The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively. A slight modification of the purification protocol yields an oxidase preparation that contains a possible fifth subunit which may be the cyoE gene product. The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper. There is no electron paramagnetic resonance detectable copper in the purified enzyme. Hence, the equivalent of CuA of the aa3-type cytochrome c oxidases is absent in this quinol oxidase. There is also no zinc in the purified quinol oxidase. Finally, monoclonal antibodies are reported that interact with subunit II. One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase. Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in the bo-type ubiquinol oxidase.     

Potent slow-binding inhibition of cathepsin B by its propeptide.

A peptide (PCB1) corresponding to the proregion of the rat cysteine protease cathepsin B was synthesized and its ability to inhibit cathepsin B activity investigated. PCB1 was found to be a potent inhibitor of mature cathepsin B at pH 6.0, yielding a Ki = 0.4 nM. This inhibition obeyed slow-binding kinetics and occurred as a one-step process with a k1 = 5.2 x 10(5) M-1 s-1 and a k2 = 2.2 x 10(-4) s-1. On dropping from pH 6.0 to 4.7, Ki increased markedly, and whereas k1 remained essentially unchanged, k2 increased to 4.5 x 10(-3) s-1. Thus, the increase in Ki at lower pH is due primarily to an increased dissociation rate for the cathepsin B/PCB1 complex. At pH 4.0, the inhibition was 160-fold weaker (Ki = 64 nM) than at pH 6.0, and the propeptide appeared to behave as a classical competitive inhibitor rather than a slow-binding inhibitor. Incubation of cathepsin B with a 10-fold excess of PCB1 overnight at pH 4.0 resulted in extensive cleavage of the propetide whereas no cleavage occurred at pH 6.0, consistent with the formation of a tight complex between cathepsin B and PCB1 at the higher pH. The synthetic propeptide of cathepsin B was found to be a much weaker inhibitor of papain, a structurally similar cysteine protease, and no pH dependence was observed. Inhibition constants of 2.8 and 5.6 microM were obtained for papain inhibition by PCB1 at pH 4.0 and 6.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Topological mapping of the active sites of cytochromes P4502B1 and P4502B2 by in situ rearrangement of aryl-iron complexes.

Cytochrome P4502B1 reacts with phenylhydrazine or phenyldiazene to give an iron-phenyl complex that oxidatively rearranges in situ to the two N-phenylprotoporphyrin IX regioisomers with the phenyl group on pyrrole rings A (NA) and D (ND) [Swanson, B. A., Dutton, D. R., Lunetta, J. M., Yang, C. S., & Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19258-19264]. The conclusion that the active site of cytochrome P4502B1 is open above pyrrole rings A and D but not B and C is extended here by studies with larger arylhydrazines. The N-arylprotoporphyrin IX standards required for product identification were obtained by reaction of the arylhydrazines with equine myoglobin. Cytochrome P4502B1 aryl-iron complex formation followed by oxidative shift of the aryl group produces the following N-aryl-protoporphyrin IX NA:ND regioisomer ratios: phenylhydrazine (39:61), 3,5-dimethylphenylhydrazine (29:71), 4-tert-butylhydrazine (25:75), 2-naphthylhydrazine (less than 2:greater than 98), and 4-(phenyl)phenylhydrazine (87:13). Electron-withdrawing substituents (as in 3,5-dichlorophenyl) prevent the aryl group shift. The increase in the proportion of the ND regioisomer with increasing bulk of the aryl group suggests that the region over pyrrole ring A is more sterically encumbered than that over pyrrole ring D. The regiospecificity is reversed, however, with 4-(phenyl)phenylhydrazine, which primarily gives the NA regioisomer. This reversal suggests that the active site has a sloping roof that is higher over pyrrole ring A than pyrrole ring D and that provides a larger steric barrier to the shift of tall aryl moieties than the barrier over pyrrole ring A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.     

The concentration dependence of the depolarization of yeast by monovalent cations.

Monovalent cations decrease the initial rate of uptake of the membrane potential probe 2-(dimethylaminostyryl)-1-ethyl-pyridinium (DMP) into metabolizing cells, showing that the cells are depolarized. A steep decrease in this rate was found even at low cation concentrations, reaching 62%, 42%, 58%, 40% and 40% at high concentrations of K+, Rb+, Cs+, Na+ and Li+, respectively. The corresponding concentrations at which half-maximum decrease was found were 0.22, 0.36, 1.2, 17 and 17 mM. These values are of the same order of magnitude as the half-saturation concentrations for monovalent cation uptake by the yeast.