Effect of Plant Species and Environmental Conditions on Ice Nucleation Activity of Pseudomonas syringae on Leaves.

Selected plant species and environmental conditions were investigated for their influences on expression of ice nucleation activity by 15 Pseudomonas syringae strains grown on plants in constant-temperature growth chamber studies. Ice nucleation frequencies (INFs), the fraction of cells that expressed ice nucleation at -5 or -9 degrees C, of individual strains varied greatly, both on plants and in culture. This suggests that the probability of frost injury, which is proportional to the number of ice nuclei on leaf surfaces, is strongly determined by the particular bacterial strains that are present on a leaf surface. The INFs of strains were generally higher when they were grown on plants than when they were grown in culture. In addition, INFs in culture did not correlate closely with INFs on plants, suggesting that frost injury prediction should be based on INF measurements of cells grown on plants rather than in culture. The relative INFs of individual strains varied with plant host and environment. However, none of seven plant species tested optimized the INFs of all 15 strains. Similarly, incubation for 48 h at near 100% relative humidity with short photoperiods did not always decrease the INF when compared with a 72 h, 40% relative humidity, long-photoperiod incubation. Pathogenic strains on susceptible hosts were not associated with higher or lower INFs relative to their INFs on nonsusceptible plant species. The ice nucleation activity of individual bacterial strains on plants therefore appears to be controlled by complex and interacting factors such as strain genotype, environment, and host plant species.

Alpha-bungarotoxin binding in house fly heads and Torpedo electroplax.

House fly heads contain a site that binds alpha-bungarotoxin with high affinity. It is present at about 23 pmol/g of heads and binds alpha-bungarotoxin (labeled with [3H]pyridoxamine phosphate) reversibly with a Kd of 6 nM. The effects of 48 drugs have been compared on the alpha-bungarotoxin binding sites of house fly and Torpedo. The pharmacology of the house fly sites is similar to that previously reported for neuronal alpha-bungarotoxin binding sites in both vertebrates and invertebrates and is distinguishable from that of the classic nicotinic neuromuscular acetylcholine receptor, as exemplified by that of Torpedo electroplax. Differences between the house fly site and Torpedo include higher affinities of the Torpedo receptor for decamethonium, hexamethonium, carbamylcholine, and acetyl-beta-methylcholine, but lower affinities for nicotine, atropine, and dihydro-beta-erythroidine.

Properties and affinity purification of the mixed-type putative acetylcholine receptor from wild and a mutant strain of hose flies.

Binding of decamethonium to a soluble preparation from house fly head (either wild or a mutant strain) showed a single kind of binding with values for wild strain of Kd = 0.095 micrometers and Bmax = 0.22 nmol/mg protein. The mutant had a four-fold greater affinity and a seven-fold lesser amount. The binding was blocked by both nicotinic and muscarinic drugs. The decamethonium binding migrated in sucrose gradients as a single peak, with sedimentation coefficient s20,w = 12.5 S and therefore a molecular weight of 342 000. Purification by affinity chromatography was achieved with only partial loss of activity, andthe purified material demonstrated a single band on analytical disc gel electrophoresis. Electrophoresis in sodium dodecyl sulphate gels showed two subunits of molecular wegiths 94 000 and 64 000. Both subunits had an isoelectric point of 4.8.

Substrate preferences of wild and a mutant house fly acetylcholinesterase and a comparison with the bovine erythrocyte enzyme.

Comparisons were made of purified acetylcholinesterase from the heads of wild type house flies with a mutant form (which bound organophosphates and carbamates less tightly). Using 12 substrates and 6 quaternary inhibitors, the only substantial difference was that the Km for butyrylcholine was 25 times greater for the mutant enzyme, suggesting that butyrylcholine and the organophosphates and carbamates shared a common binding site. The pure enzyme from the wild type house fly was also compared with bovine erythrocyte acetylcholinesterase. The major difference was again with butyrylcholine as substrate: the ability to acylate or deacylate was 30 times greater in the fly enzyme (the Km values differed by a factor of 4).

Molecular and structural characteristics of house fly brain acetylcholinesterase.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from the brains of house flies (Musca domestica L., tetrachlorvinphos-resistant strain) was examined for molecular and structural features, including molecular weight, Stokes radii, partial specific volumes, sedimentation coefficients and frictional ratios. Acetylcholinesterase purified by affininity chromatography was examined in the electron microscope by negative staining and three molecular forms were clearly observed (monomers, dimers and tetramers). Several tetrameric configurations were observed as well as structures of similar size showing tails. In the preparations of acetylcholinesterase so far examined, no globular structures having more than four monomeric units were observed.

Purification of acetylcholinesterase from house fly brain by affinity chromatography.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from the heads of house flies (Musca domestica L.) was purified by affinity chromatography. The enzyme was adsorbed from the crude extracts on an affinity column containing trimethyl(p-aminophenyl) ammonium chloride hydrochloride (Ki approximately 1.7 - 10(-4) M), covalently linked to Sepharose 4B, then eluted with a solution of a selective reversible inhibitor, 1,5-bis (4-allyl dimethyl ammoniumphenyl)-pentan-3-one dibromide (BW 284C51; Ki approximately 1 - 10(-7) M). The enzyme was purified 1223 times in one step and had a specific activity of 752 units/mg protein. Disc gel electrophoresis in polyacrylamide gel revealed five protein bands, four corresponding to the enzyme activity bands and one devoid of enzyme activity. On the basis of periodic acid-Schiff stain intensity, the slower moving isozyme I and the contaminating band appear to be rich in carbohydrate. The purity of the enzyme estimated by disc gel electrophoresis was 94%. Density gradient centrifugation in sucrose showed two major species each of which ran as a single band on disc gel electrophoresis. The average molecular weights were 306000 (+/- 11 150) for heavy (s20,w = 11.5 S) form and 143000 (+/- 4700) for light (s20,w = 6.9 S) form.

Acetylcholine receptor oligomers from electroplax of Torpedo species.

Sedimentation in sucrose gradients of alpha-bungarotoxin-labeled crude and pure acetycholine receptor preparations from Torpedo californica showed two major oligomers. The molecular weights, corrected for the bound Triton X-100 by comparing sedimentation in H2O and in D2O, were 330 000 for the heavy (H) oligomer and 190 000 for the light (L) oligomer. Lesser peaks found in preparations of T. marmorata and purified preparations of T. californica with molecular weights of 500 000 (HH) and 80 000 (LL). These molecular weights are based upon the assumption of globularity, and may require adjustment if the assumption is wrong. The H and L peaks have similar drug sensitivities, but at pH 10 the L peak was stable whereas the H peak dissociated to components sedimenting as LL. Treatments with p-chloromercuribenzoate, which blocks acetylcholine binding partially without affecting alpha-bungarotoxin binding, had no effect upon the pattern of sedimentation. This and other evidence suggested that the heterogeneity of oligomers was unrelated to the heterogeneity of site affinities for acetylcholine and nicotinic drugs.

Evidence that alpha-dihydrograyanotoxin II does not bind to the sodium gate.

The basis for the ability of alpha-dihydrograyanotoxin II (alpha-2HG-II) to promote Na+ conductance in axons was sought. The apparent binding of tritiated alpha-2HG-II to neural and other preparations was studied, using equilibrium dialysis, with lobster axon membranes, Torpedo electroplax, housefly head, and rat brain, liver and kidney. In every case the "binding" was nonsaturating and was suggested to involve nonspecific partitioning into the tissue. Supporting evidence was the similarity of extent of "binding" in all tissues and its relative insensitivity to neuropharmacological agents. Alpha-2HG-II did not affect the Na+ conductance of phospholipid bilayers, nor did it permit transport of 22Na into a bulk organic phase. It was concluded that alpha-2HG-II did not bind to the sodium gate, but possibly to a sodium permease present at a frequency of less than one per mu2 of cell membrane.