A monoclonal antibody specific for the red-absorbing form of phytochrome

Characterisation of a new monoclonal antibody (mAb), designated LAS 41, directed against 124-kilodalton (kDa) etiolated-oat (Avena sativa L.) phytochrome, indicates that it recognises an epitope unique to the red-light-absorbing form, Pr. In a solid-phase enzyme-linked immunosorbent assay (ELISA), LAS 41 exhibits a seven- to eight-fold higher affinity for Pr than for the far-red-light-absorbing form of phytochrome, Pfr. In addition, in immunoprecipitation assays LAS 41 effectively precipitates 100% of phytochrome presented as Pr but only precipitates a maximum of 24.5% of phytochrome presented as Pfr. These values are indicative of binding exclusively to Pr. Peptide-mapping studies show that LAS 41 recognises and epitope located within a region 6–10 kDa from the aminoterminus of the phytochrome molecule. Since binding of LAS 41 to Pr induces alterations in the spectral properties of Pr, this indicates that at least part of the 4 kDa domain to which the antibody binds is essential for protein-chromophore interaction. Subsequent photoconversion of LAS 41-Pr complexes produces native Pfr spectra, with concomitant production of free antibody and antigen, as shown by a modified ELISA. The specificity of LAS 41 for Pr has facilitated the purification of Pfr which is free of contaminating Pr. This has enabled direct determination of the mole fraction of Pfr established by red light to be 0.874.Abbreviations ELISA enzyme-linked immunsorbent assay - kDa kilodalton - mAb monoclonal antibody - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - (A) difference in absorbance (A ₆₆₅ ^Pr –A ₇₃₀ ^Pr )-(A ₆₆₅ ^Pfr –A ₇₃₀ ^Pfr ) - Ar/Afr spectral change ratio (SCR) - max mole fraction of Pfr following saturating red light     

Metabolic switching patterns provide definitive evidence of substrate-specificity and delineate subtle differences in positioning at the aromatase active site [Poster 06]

We found that extensive metabolic switching can be triggered in aromatization. Up to 20–35% of (19-³H₃)-labeled substrate is diverted to non-estrogenic product, namely 1β- and 2β-hydroxy androgens, negating usefulness for [³H]water assays. The strikingly substrate-specific metabolite patterns observed reflect positioning at the active site, and the large kinetic isotope effects involved. This switching is unusual in that it involves: 1) tritium labeling, 2) a multistep enzymic process, and 3) the potential unraveling of an enzymic mechanism.     

High-Efficiency Conversion of Pyruvate to Acetoin by Lactobacillus plantarum during pH-Controlled and Fed-Batch Fermentations

The influence of pH on the type and concentration of metabolites produced from pyruvate by Lactobacillus plantarum ATCC 8014 was examined in pH-controlled fermentors at pH values of 4.5 to 6.5. Specific growth rates, cell dry weights, and diacetyl concentrations were highest at pH 5.5, with values of 0.78 h⁻¹, 190 mg/liter, and 1.2 mM, respectively. While the conversion efficiency (millimoles of acetoin formed per millimoles of pyruvate utilized) was highest (94.6%) at pH 4.5, acetoin levels were similar (20 mM) between pH 4.5 and 5.5. Feeding stationary-phase cells exogenous pyruvate increased acetoin levels to 78 mM.     

Plant Cell Wall Carbohydrates as Substrates for Azospirillum brasiliense

Carbohydrate components (simple sugars and polysaccharides) of cell walls of pearl millet (Pennisetum americanum L., cv. Gahi) were studied as potential substrates for the root-associated diazotroph Azospirillum brasiliense Sp. 7. Simple sugars were utilized, but no evidence was obtained to support the suggestion that the polysaccharide components tested might serve as substrates for growth following hydrolysis by the associated azospirilla.     

Factor XIII binds to the A alpha- and B beta- chains in the D-domain of fibrinogen: an immunoblotting study

The binding sites in fibrinogen for Factor XIII were localized using an immunoblotting technique. Platelet Factor XIII bound to fibrinogen and to plasmin degradation products of fibrin(ogen) including Fragments: X, D1-D3, and D-dimer, but did not bind to Fragment E. Binding of Platelet Factor XIII was independent of calcium ions but could be inhibited by the presence of 0.5 M NaCl. Binding could also be inhibited by preincubating Factor XIII with a 100-fold molar excess of fibrinogen but not by 100-fold molar excess of Fragment E. Binding of Factor XIII to fibrinogen was specific, since several other proteins tested (ovalbumin, bovine serum albumin, alpha 2-macroglobulin, beta-galactosidase, fructose kinase, lactic dehydrogenase, triose phosphate isomerase, fumarase and pyruvate kinase) did not bind Factor XIII. Furthermore, binding was not observed either when Factor XIII was left out or when antiFactor XIII antiserum was substituted with nonimmune serum. When fibrinogen was reduced prior to electrophoresis, Factor XIII bound to the A alpha and B beta chains of fibrinogen and des A,B fibrinogen, the B beta-chain of Fragment X, but not the gamma-chains. Localization of the Factor XIII binding sites to the carboxy terminal segments of the A alpha and B beta chains in the Fragment D-domain of fibrinogen could have important physiological consequences.     

Organothallium(III) reagents for modification of biomacromolecules: irreversible labelling of phosphoglycerate kinase from rabbit muscle

Organothallium(III) reagents, by analogy with organomercurials, have been found to rapidly label phosphoglycerate kinase from rabbit muscle. By use of a radio-labelled version of p-methylphenylthallium(III) bis-trifluoroacetate (MPT) the inhibition was shown to be irreversible by the criterion of gel filtration desalting. The rate of labelling was shown to depend on the temperature, enzyme and thallium reagent concentrations, and the presence or absence of the various substrates of the enzyme. The structure and oxidation state of the thallium reagent used affected the extent of modification by the compounds MPT, o-carboxyphenylthallium(III) bis-trifluoroacetate, thallic trifluoroacetate and thallous acetate. A number of pieces of evidence implicate cysteine residues in the labelling, including changes in the free thiol titre of the enzyme on thalliation, model studies on the interaction of thiols (e.g. glutathione) with thallium(III) and thallous materials, the lack of inactivation of phosphoglycerate kinase from yeast (which has only one thiol residue distant from the active site), and the partial restoration of enzymic activity by treatment of thalliated enzyme with sulphydryl reducing agents. Substrate protection studies showed that modification of rabbit muscle phosphoglycerate kinase by MPT was fully prevented by 3-phosphoglycerate and partially by MgATP. The latter protected only against the fast phase of thallic modification, the slower phase being unaffected. The presence of MgADP potentiated the labelling by MPT. No evidence of an MgADP-induced conformational change in the enzyme could be obtained from fluorescence or circular dichroic spectroscopies, although changes of the native spectra were noted on thalliation by MPT alone. The cross-linking potential of these arylthallium(III) reagents is discussed along with conformational changes required to trigger the hinge-movement between the N- and C-domains of the protein.     

Chromosome organisation in polyploid mouse trophoblast nuclei

At least one-third of mouse trophoblast cells undergo endoreduplication during the first half of gestation. It has been suggested that the endoreduplicated chromosomes may be polytenised. Here it is shown, using in situ hybridisation to the -1 antitrypsin genes, which map at a unique site, that while there is a tendency for duplicated chromosomes to cluster, this does not involve the complete fusion of replicated chromatids found in fully polytene chromosomes, and in a substantial proportion of homologues the sites on the chromosome arms corresponding to these genes are widely separated. The centromeres do not fuse into a single chromocentre but the possibility is not ruled out that individual chromosomes may be polytenised in the centromeric region. Evidence is also presented showing that endoreduplication in trophoblast nuclei is not accompanied by the formation of new prekinetochore structures, in contrast to the situation in polyploid mouse liver and C127 cells.