Photoaffinity labeling of the head-activator receptor from hydra.

A photoaffinity ligand for the head-activator (HA) receptor from hydra was synthesized using solid-phase peptide synthesis and coupling of two HA peptides over their epsilon-amino groups of Lys7 with succinimidyl esters. The new ligand, Bpa-HA-HA bipeptide, contains one normal HA peptide and another where p-benzoylphenylalanine (Bpa) was added at the amino terminus to allow ultraviolet activation and Tyr11 instead of Phe11 for radioiodination. The 125I-Bpa-HA-HA bipeptide bound with nanomolar affinity to the HA receptor from the multiheaded mutant of Chlorohydra viridissima as measured in a filter assay. After photoaffinity labeling of the hydra membrane fraction, a 200-kDa band was detected using reducing or non-reducing SDS/PAGE and autoradiography. Unlabeled HA derivatives, but no other neuropeptides, inhibited the labeling. Competition experiments with HA-HA homobipeptide in the nanomolar range indicate that predominantly the low-affinity and not the high-affinity HA receptor was photolabeled. Further evidence that the labeled molecule is the HA receptor comes from specific photoaffinity labeling with a second ultraviolet-activatable ligand containing p-nitrophenylalanine. The HA receptor could be functionally solubilized with Triton X-100 or Chaps. In the solubilizate the 200-kDa HA receptor was photolabeled specifically by both ligands. Liquid-phase isoelectric focussing of the solubilizate indicated a pI of about 5.4 of the photolabeled molecule. After chemical deglycosylation with trifluoromethanesulfonic acid, the apparent molecular mass of the labeled molecule was decreased to 180 kDa, indicating that the receptor is glycosylated.

Accumulation of transcripts encoding a lipid transfer-like protein during deformation of nodulation-competent Vigna unguiculata root hairs.

A cDNA library was constructed from RNA of Vigna unguiculata root hairs harvested 1 day and 4 days after inoculation with Rhizobium sp. NGR234. A heterologous probe was used to identify a cDNA clone, the predicted 99-amino-acid sequence of which shares homology with a nonspecific lipid transfer protein (LTP) of Hordeum vulgare. Other characteristics, including an estimated molecular weight of 10.4 kD, an isoelectric point of 8.6, and a signal peptide with a hydrophobic region at the amino-terminal end, are shared by most LTPs. A transcript of 630 nt was found in all tissues tested, except nodules. Levels of mRNA increased in root hairs 24 hr after treatment with Rhizobium sp. NGR234, with different hormones, or with Nod factors. Amounts of transcripts were dependent on the concentration of Nod factors. Accumulation of transcripts during nodule development correlated with root hair deformation, the first visible step in the Rhizobium-legume symbiosis.

Anaerobic degradation of malonate via malonyl-CoA by Sporomusa malonica, Klebsiella oxytoca, and Rhodobacter capsulatus.

Anaerobic decarboxylation of malonate to acetate was studied with Sporomusa malonica, Klebsiella oxytoca, and Rhodobacter capsulatus. Whereas S. malonica could grow with malonate as sole substrate (Y = 2.0 g.mol-1), malonate decarboxylation by K. oxytoca was coupled with anaerobic growth only in the presence of a cosubstrate, e.g. sucrose or yeast extract (Ys = 1.1-1.8 g.mol malonate-1). R. capsulatus used malonate anaerobically only in the light, and growth yields with acetate and malonate were identical. Malonate decarboxylation in cell-free extracts of all three bacteria was stimulated by catalytic amounts of malonyl-CoA, acetyl-CoA, or Coenzyme A plus ATP, indicating that actually malonyl-CoA was the substrate of decarboxylation. Less than 5% of malonyl-CoA decarboxylase activity was found associated with the cytoplasmic membrane. Avidin (except for K. oxytoca) and hydroxylamine inhibited the enzyme completely, EDTA inhibited partially. In S. malonica and K. oxytoca, malonyl-CoA decarboxylase was active only after growth with malonate; malonyl-CoA: acetate CoA transferase was found as well. These results indicate that malonate fermentation by these bacteria proceeds via malonyl-CoA mediated by a CoA transferase and that subsequent decarboxylation to acetyl-CoA is catalyzed, at least with S. malonica and R. capsulatus, by a biotin enzyme.

Malonate decarboxylase of Malonomonas rubra, a novel type of biotin-containing acetyl enzyme.

Cell suspensions or crude extracts of Malonomonas rubra grown anaerobically on malonate catalyze the decarboxylation of this substrate at a rate of 1.7-2.5 mumol.min-1.mg protein-1 which is consistent with the malonate degradation rate during growth. After fractionation of the cell extract by ultracentrifugation, neither the soluble nor the particulate fraction alone catalyzed the decarboxylation of malonate, but on recombination of the two fractions 87% of the activity of the unfractionated extract was restored. The decarboxylation pathway did not involve the intermediate formation of malonyl-CoA, but decarboxylation proceeded directly with free malonate. The catalytic activity of the enzyme was completely abolished on incubation with hydroxylamine or NaSCN. Approximately 50-65% of the original decarboxylase activity was restored by incubation of the extract with ATP in the presence of acetate, and the extent of reactivation increased after incubation with dithioerythritol. Reactivation of the enzyme was also obtained by chemical acetylation with acetic anhydride. These results indicate modification of the decarboxylase by deacetylation leading to inactivation and by acetylation of the inactivated enzyme specimens leading to reactivation. It is suggested that the catalytic mechanism involves exchange of the enzyme-bound acetyl residues by malonyl residues and subsequent decarboxylation releasing CO2 and regenerating the acetyl-enzyme. The decarboxylase was inhibited by avidin but not by an avidin-biotin complex indicating that biotin is involved in catalysis. A single biotin-containing 120-kDa polypeptide was present in the extract and is a likely component of malonate decarboxylase.