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.     

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.     

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.     

Fibrinogen-sepharose interaction with prothrombin, prethrombin 1, prethrombin 2 and thrombin

Binding of prothrombin, prethrombin 1, prethrombin 2 and thrombin to fibrinogen-Sepharose was studied. Thrombin and prethrombin 2 bound to fibrinogen-Sepharose, while prethrombin 1 and prothrombin did not. Bound thrombin and prethrombin 2 were recovered from the column by eluting with 0.1 M NaCl/0.05 M Tris-HCl buffer (pH 7.4). The affinity of thrombin and prethrombin 2 to fibrinogen-Sepharose depended on ionic strength and reached a maximum at 50 mm concentration. Prethrombin 2 interacts with fibrinogen as well as thrombin; and prothrombin fragment 1.2 is not important in the formation of this complex. Thus, prethrombin 2, which is a precursor of thrombin without measurable enzymatic activity and which lacks the single cleavage at Arg-322-Ile-323 present in thrombin, has the same or very similar structural conformation as thrombin and has the same macromolecular substrate recognition site. These results confirm the earlier results that active center is not necessary in fibrinogen-thrombin interaction.     

The dependence of glyceroglycolipid orientation and dynamics on head-group structure

The head-group orientations and molecular dynamics of three glyceroglycolipids in aqueous dispersions, as determined by 2H-NMR, are compared. 1,2-Di-O-tetradecyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerol (alpha-DTGL) and 1,2-di-O-tetradecyl-3-O-(alpha-D-mannopyranosyl)-sn-glycerol (alpha-DTML), selectively 2H-labelled on the pyranose ring, at the exocyclic hydroxymethyl group, and at C3 of glycerol, have been studied by 2H-NMR and the results compared with those reported earlier for 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL). The alpha-glucolipid exhibits a gel-to-liquid crystal phase transition and a lamellar to hexagonal mesophase transition at temperatures which are similar to those of the beta-anomer, beta-DTGL. However, alpha-DTGL exhibits head group orientations and molecular ordering in the lamellar and hexagonal phases which differ strikingly with those reported for the corresponding beta-glucolipid. Whereas the head group of beta-DTGL is extended away from the bilayer surface into the aqueous phase, that of alpha-DTGL is almost parallel to the bilayer surface. alpha-DTGL exhibits a molecular order parameter of 0.56 which is substantially greater than that of its anomer, beta-DTGL, 0.45. The latter indicates that the head group region of the alpha-glyceroglucolipid is characterized by smaller angular fluctuations than that of beta-DTGL. On entering the hexagonal mesophase the pyranose ring of the beta-glucolipid undergoes a large reorientation relative to the motional axis of the head group, whereas the alpha-anomer exhibits only a small orientational change. 1,2-Di-O-tetradecyl-3-O-(alpha-D-mannopyranosyl)-sn-glycerol (alpha-DTML) undergoes a phase transition at 47 degrees C, attributed to the unusual lamellar gel to hexagonal phase transition. The pyranose ring of alpha-DTML, in a mixture with dimyristoylphosphatidylcholine (1:9 mol ratio) to give a lamellar liquid crystalline phase, is oriented away from the bilayer surface into the aqueous environment and has an Smol of 0.75. The results for alpha-DTML, 2H-labelled at the C3 position of glycerol, suggest that this segment also has high molecular ordering. alpha-DTML in a lamellar environment has the least flexible membrane surface of the glyceroglycolipids investigated to date. 2H-NMR spin lattice relaxation times have been used to probe the head group motions of the glycolipids. The results indicate that the rate of head group motion increases in the order alpha-DTML less than alpha-DTGL less than beta-DTGL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunogenicity and antigenicity of immunoglobulins: detection of human immunoglobulin light-chain carbohydrate, using concanavalin A

Concanavalin A binding to glycoprotein bands on nitrocellulose blots was used to detect mannose, sorbose, N-acetylgalactosamine and/or glucose residues on 100% (31/31) of human Bence Jones protein light chains, following sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All (20/20) light chains form IgG myeloma proteins and light chains from a preparation of normal polyclonal human IgG were also bound by concanavalin A. The specificity of concanavalin A for glycoproteins was demonstrated by its binding to human Fc fragments and a human monoclonal anti-Rhesus D antibody (REG-A), but not to human albumin pFc' fragments and aglycosylated REG-A derived from cells grown in the presence of the glycosylation inhibitor tunicamycin. These results suggest that all Bence Jones proteins and light chains from myeloma IgG proteins contain mono- or oligosaccharides linked O-glycosidically to serine or threonine residues.     

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)