Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including alpha-(2----8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis alpha-(2----8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3200 pmol of [3H]TTX-binding sites/mg of protein and a single polypeptide of approximately 285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.     

The immunocytochemical localization of superoxide dismutase in the enterocytes of the avian intestine: The effect of vitamin D3

Summary Both light microscopical and electron microscopical immunocytochemical techniques were utilized to localize CuZnsuperoxide dismutase (SOD) in the duodenum of normal, rachitic and vitamin-D₃-replete chicks. This enzyme catalyses the dismutation of the superoxide anion, a toxic free radical generated during the normal aerobic metabolism of most respiring cells. Light microscopy showed no SOD activity associated with the duodenal enterocytes of normal and rachitic chicks. However, in rachitic animals subsequently treated with vitamin D, i.e. vitamin-D-replete chicks, intense immunoreactivity for the enzyme was seen in association with the apical border of the duodenal absorptive cells. Immunostaining for SOD was not seen in goblet cells. With electron microscopy, immunostaining for SOD activity was identified in association with the apical microvilli and, to a lesser degree, with the terminal web, a well as in association with both lysosomes and peroxisomes. From this report it appears that there is a physiological relationship between vitamin D, SOD and the intestinal absorptive cell. However, the precise relationship must await further clarification.     

The combined effects of chlorine and ammonia on litter breakdown in outdoor experimental streams

The response of Potamogeton crispus L. breakdown to controlled doses of different levels of chlorine and chlorine + ammonia was investigated over two years in outdoor experimental streams. In 1985, downstream riffles of 2 streams were dosed (observed in-stream concentrations) at ca. 10 μg/L Total Residual Chlorine (TRC), one stream at 64 μg/L TRC and one stream at 230 μg/L TRC. Two control streams were not dosed and the upstream riffles of each stream served as within stream controls. In 1986, the downstream riffle of one stream was dosed at 70 μg/L TRC and a second stream was dosed at 200 μg/L TRC. Four streams were also dosed with 2.5 mg/L NH₃-N: one stream with no chlorine, one stream with ca. 10 μg/L TRC, one with 56 μg/L TRC, and one with 150 μg/L TRC. A seventh stream was dosed for 2 h at 2000 μg/L TRC and 2.5 mg/L ammonia and then allowed to recover (recovery stream). Each year, litter decomposition (degree day k values) was measured during two 35 day trials (Jun–Jul and Aug–Sep). In 1985, when streams were dosed with chlorine alone, decomposition was significantly reduced with the high (230 μg/L TRC) chlorine dose. Downstream decomposition was 27% (Jun–Jul) and 59% (Aug–Sep) of the upstream (control) rate. No other chlorine effects were found during this period. In Jun–Jul 1986, there was significantly lower decomposition in the downstream dosed sites of the 200 μg/L TRC alone stream, the 146 μg/L TRC + ammonia stream and the recovery stream; downstream decay rates were (respectively) 56%, 42% and 64% of the upstream control sites. No other up-down pairs were different in July 1986. In Aug–Sep, all three streams with chlorine + ammonia (6, 56 and 146 μg/L TRC + 2,5 mg/L ammonia) and the 70 μg/L TRC alone stream had significantly lower decomposition rates in the downstream dosed sites. For these streams, downstream decay rates ranged from 46% (high chlorine + ammonia) to 73% (low chlorine + ammonia) of the upstream control rates. No other up-down pairs were different during this trial. Up and downstream sites of the stream dosed with 2.5 mg/L ammonia alone were nearly identical for both trials (< 3% difference). These results indicate that TRC at less than 250 μg/L can significantly reduce litter decomposition and strongly suggest that addition of ammonia to chlorinated water can increase the toxic effect of chlorine. currently at the Department of Fisheries and Wildlife currently at the Department of Fisheries and Wildlife     

Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure

The predicted conformation and position of the central transforming region (residues 55–67) of the p21 protein are compared with the conformation and position of this segment in a recently determined X-ray crystal structure of residues 1–166 of this protein in the activated state bound to a nonhydrolyzable GTP derivative. We previously predicted that this segment of the protein would adopt a roughly extended conformation from Ile 55-Thr 58, a reverse turn at Ala 59-Gln 61, followed by an -helix from Glu 62-Met 67. We further predicted that this region of the activated protein occupies a position that is virtually identical to corresponding regions in the homologous purine nucleotide-binding proteins, bacterial elongation factor (EF-tu), and adenylate kinase (ADK). We find that there is a close correspondence between the conformation and position of our predicted structure and those found in the X-ray crystal structure. A mechanism for activation of the protein is proposed and is corroborated by X-ray crystallographic data.     

Cardiac alpha-crystallin. I. Isolation and identification

A water soluble protein, a major component of the cytosolic fraction of rat heart cells, was purified using either reverse phase HPLC or antibodies affinity chromatography procedures and characterized. The protein has an apparent Mr of 24 k, as judged by SDS-gel electrophoresis. Under non-denaturing conditions, however, the protein occurs as a homomultimer (Mr between 400 and 650 k) of the monomeric 24 kDa species and could be selectively enriched by fractionation of the cytosolic fraction on 10 to 40% sucrose gradients. Polyclonal antibodies, raised against the denatured 24 kDa protein, were used to investigate its tissue distribution. Besides the heart, where it is very abundant, the 24 kDa protein is expressed also in other red muscles and in kidneys, but was not detectable in stomach, thymus, liver, and brain. The amino acid composition of the protein and the partial amino acid sequence of various proteolytic fragments was determined. A search for homologies of the primary structure of known proteins has shown that the 24 kDa protein is strikingly similar, if not identical to alpha-B-crystallin. In fact, the two proteins were found to be indistinguishable also by immunological criteria. This study demonstrates that the lens protein alpha B-crystallin is a major cytosolic component of heart cells.     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

Strain DCB-1 conserves energy for growth from reductive dechlorination coupled to formate oxidation

Strain DCB-1 is a strict anaerobe capable of the reductive dechlorination of chlorobenzoates. The effect of dechlorination on the yield of pure cultures of DCB-1 was tested. Cultures were incubated with formate or H₂ as electron donors and CO₂ as a putative carbon source. Relative to control cultures with benzoate, cultures which dechlorinated 3-chlorobenzoate and 3,5-dichlorobenzoate had higher yields measured both as protein and cell density. On the media tested the apparent growth yield was 1.7 to 3.4 g cell protein per mole Cl^- removed. Dechlorination also stimulated formate oxidation by growing cultures. Resuspended cells required an electron donor for dechlorination activity, with either formate or elemental iron serving this function. Resuspended cells did not require an electron acceptor for formate consumption, but reductive dechlorination of 3CB to benzoate stoichiometrically stimulated oxidation of formate to CO₂. These results indicate that DCB-1 conserves energy for growth by coupling formate, and probably, H₂ oxidation to reductive dechlorination.Non-standard abbreviations 3CB 3-chlorobenzoate - 35DCB 3,5-dichlorobenzoate - PCF Propionibacterium sp. culture fluid