Labeling of bovine heart cytochrome c oxidase with analogues of phospholipids. Synthesis and reactivity of a new cardiolipin benzaldehyde probe

The syntheses of two new radioactive probes derived from cardiolipin and phosphatidylcholine are reported. These probes are derivatives of natural lipids and contain an amine-specific benzaldehyde in the head-group region. This functional group allows a choice of timing of the reaction (e.g., after equilibration and detergent removal) because an irreversible covalent bond is formed only upon the addition of reducing agent. These probes, as well as a benzaldehyde analogue of phosphatidic acid, and a water-soluble benzaldehyde reagent were covalently attached to bovine heart cytochrome c oxidase. After reconstitution into vesicles, the lipid-benzaldehyde probes selectively incorporated into the smaller polypeptides of the enzyme, while the remaining subunits (I-IV) exhibited little incorporation of label. The accessibility of amine groups labeled under the conditions used here was independent of the structural and charge differences between the benzaldehyde probes. This suggests that all three lipid probes react with polypeptides of the cytochrome c oxidase complex at general contact sites for membrane phospholipids. A water-soluble benzaldehyde reagent predominantly labeled subunits IV, Va, and Vb and polypeptides of VII-VIII. A comparison of these results facilitates a more refined view of the disposition of polypeptides of cytochrome c oxidase in respect to the lipid and aqueous phases.     

Adduct formation between the cupric site of phenylalanine hydroxylase from Chromobacterium violaceum and 6,7-dimethyltetrahydropterin

The interaction of pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum with the cofactor analogue 5-deaza-6-methyltetrahydropterin and the cofactor 6,7-dimethyltetrahydropterin (DMPH4) has been investigated by multifrequency electron spin resonance (ESR) spectroscopy. 5-Deaza-6-methyltetrahydropterin, which lacks the N-5 nitrogen present in the pyrazine ring of DMPH4, binds tightly to the cupric form of the enzyme; however, no changes are observed in the ESR parameters of the copper center. In contrast, the binding of DMPH4 (or 6-methyltetrahydropterin) shifts the ESR parameters (g and A) associated with the cupric enzyme. In addition, superhyperfine transitions were resolved and assigned to hyperfine splitting from nitrogen ligands. ESR spectra of the enzyme recorded in the presence of [5-14N]DMPH4 or [5-15N]DMPH4 were computer simulated and found to be consistent with pterin serving as a direct donor ligand to the copper center through the N-5 position.     

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.     

Mechanism and base specificity of DNA Breakage in intact cells by neocarzinostatin

When electrophoresed on an agarose gel, the DNA isolated from neocarzinostatin- (NCS-) treated HeLa cells migrates in a ladder of discrete bands indicative of preferential breakage in the linker region of the nucleosomes. The 5'-termini of the drug-induced DNA strand breaks were characterized by reduction of the nucleoside 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase and treatment of the DNA with hot alkali and alkaline phosphatase prior to the kinase assay to give the total 5'-termini. In DNA isolated from NCS-treated cells, nucleoside aldehyde accounts for 30-45% of the drug-generated 5' ends; the remainder have PO4 termini. By contrast, 5'-terminal nucleoside aldehyde in DNA cut with the drug in vitro exceeds 80% of the total 5' ends. Of the 32P representing nucleoside aldehyde in DNA from NCS-exposed cells, 77% is in TMP; the rest is in AMP much greater than CMP greater than GMP, a distribution in excellent agreement with that obtained for in vitro drug-treated DNA. DNA sequencing experiments, using the 340 base pair alphoid DNA fragment isolated from drug-treated cells, show that the pattern of breakage produced by NCS within a defined sequence of DNA in intact cells is similar to that in the in vitro reaction, with a preferential attack at thymidylate residues, but a much higher concentration of the drug was required to cause comparable breakage in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epsilon subunit of Escherichia coli F1-ATPase: effects on affinity for aurovertin and inhibition of product release in unisite ATP hydrolysis

The epsilon subunit of Escherichia coli F1-ATPase is a tightly bound but dissociable partial inhibitor of ATPase activity. The effects of epsilon on the enzyme were investigated by comparing the ATPase activity and aurovertin binding properties of the epsilon-depleted F1-ATPase and the epsilon-replete complex. Kinetic data of multisite ATP hydrolysis were analyzed to give the best fit for one, two, or three kinetic components. Each form of F1-ATPase contained a high-affinity component, with a Km near 20 microM and a velocity of approximately 1 unit/mg. Each also exhibited a component with a Km in the range of 0.2 mM. The velocity of this component was 25 units/mg for epsilon-depleted ATPase but only 4 units/mg for epsilon-replete enzyme. The epsilon-depleted enzyme also contained a very low affinity component not present in the epsilon-replete enzyme. In unisite hydrolysis studies, epsilon had no effect on the equilibrium between substrate ATP and product ADP.P1 at the active site but reduced the rate of product release 15-fold. These results suggest that epsilon subunit slows a conformational change that is required to reduce the affinity at the active site, allowing dissociation of product. It is suggested that inhibition of multisite hydrolysis by epsilon is also due to a reduced rate of product release. epsilon-depleted F1-ATPase showed little of no modulation of aurovertin fluorescence by added ADP and ATP. Aurovertin fluorescence titrations in buffer containing ethylenediaminetetraacetic acid (EDTA) revealed that epsilon-depleted enzyme had high affinity for aurovertin (Kd less than 0.1 microM) regardless of the presence of nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Endocrine responses in relation to compensatory testicular growth after neonatal hemicastration in boars

Mass (TM) and relative mass (organ mass/body mass; RTM) of the right testis and epididymis (EM and REM, respectively) were determined every 14 days from 10 to 122 days of age for intact boars (I) and boars hemicastrated on Day 10 (HC) in two crossbred herds (Trial 1 and Trial 2). Plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, growth hormone (GH), and testosterone were determined in four blood samples from each pig, three collected 24 h prior to castration and one immediately prior to castration. Values for TM and RTM of HC boars were approximately double (p less than 0.0001) those of I boars by 38 days of age, and these differences were maintained through Day 122. Both EM and REM were greater (p less than 0.05) in HC than in I boars from Day 52 to Day 122. The TM, RTM, EM and REM were greater (p less than 0.05) in Trial 1 than in Trial 2 for both I and HC boars from Day 80 to Day 122, indicating an earlier onset of pubertal testicular growth in the Trial-1 boars. Plasma GH concentration was greater (p less than 0.05) in HC than in I boars from Day 16 to Day 38. A transient increase in plasma FSH (p less than 0.05) was observed from Day 24 to Day 38. After Day 38, there was no difference (p greater than 0.05) in FSH or GH between HC and I boars, or between trials. Plasma LH, prolactin, and testosterone concentrations were also similar in HC and I boars.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proof of safety vs proof of hazard

This paper refines and extends the work of Bross (1985, Biometrics 41, 785-793). One method to determine whether an environment is safe or hazardous is to frame the problem in the context of hypothesis testing. Any "proof" of safety or hazard will depend on the size of both the Type I and Type II errors associated with the test. Many past environmental monitoring programs, however, have ignored the power of the design, regardless of whether the objective was proof of safety or hazard.