Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Interaction of poly(L-lysine) and copolymers of lysine with immobilized DNA.

Sonicated DNA has been covalently attached to Sepharose 4B by a carbodiimide method [Rickwood, P. (1972) Biochim. Biophys. Acta269, 47–50] which minimizes modification of the DNA and matrix. Columns of this material have been used to study the interaction between cationic polypeptides and DNA. When poly(l-lysine) is bound to such columns at low ionic strength and then eluted with a linear salt gradient the polypeptide elutes over a broad range of salt concentration, presumably reflecting different strengths of interaction with various sites on the DNA. The broadness of the elution profile cannot be attributed to heterogeneity in the poly(l-lysine) sample but rather to the $\text{AT}\text{GC}$ content of various DNA sites. Studies with other polypeptides, poly(l-Lys⁷⁹, l-Leu²¹) and poly-(l-Lys-l-Ala-Gly), as well as studies at different temperatures, have helped to clarify the possible roles of conformational mobility, polypeptide hydrophobicity, and the presence of contiguous lysines in determining the strength of interaction of polypeptides and proteins with DNA sites.     

Purification and properties of malate dehydrogenase from the extreme thermophileBacillus caldolyticus

The enzyme malate dehydrogenase (EC 1.1.1.37) from an extreme thermophileB. Caldolyticus was purified to about 91% homogeneity. The molar mass of the enzyme was determined as 73 000 daltons and it is composed of two subunits, each with a molar mass of 37 000. Initial velocity studies with oxaloacetic acid and NADH as substrates at pH 8.1, over a range of temperatures, indicate that the enzyme operates via a sequential type mechanism. Van't Hoff plots of the kinetic parameters displayed sharp changes in slope at characteristic temperatures, whereas the Arrhenius plot exhibited no such breaks over the temperature interval investigated. The enzyme was found to be stable at 41°C and lower temperatures. At 51°C and 59°C an almost immediate 20% reduction in activity was obtained, but no further inactivation occurred during the 60 min of incubation. At 59°C the enzyme lost 50% of its initial activity in about 38 s. High concentration of NADH was observed to greatly stabilize the enzyme at that temperature.It is suggested that the slope changes in the Van't Hoff plots and the stability profies at 51°C and 59°C are representative of a temperature induced conformational change in the enzyme.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.     

Partial purification and characterization of nitrous oxide reductase fromParacoccus denitrificans

We report the first partial purification of nitrous oxide reductase, a unique and labile enzyme of denitrifying bacteria. The procedure, which required anaerobic conditions throughout, resulted in a 60-fold purification relative to crude lysate in the case ofParococcus denitrificans. The molecular weight was estimated by gel exclusion chromatography to be about 85,000. The partially purified enzyme is inactivated rapidly by O₂, dithionite, and mercaptoethanol and is reversibly inhibited by moderate concentrations of common salts. Up to 80% of the original activity can be reconstituted following O₂ inactivation by incubating the enzyme with reduced benzyl viologen for 2 to 3 h. TheV _max pH profile shows a broad maximum at pH 8. The enzyme is irreversibly retained by common anion exchangers in the range pH 7 to 8 but can be eluted in acceptable yield as one of the last components from an imidazole-based anion exchange material by means of a pH gradient. This behavior implies that nitrous oxide reductase is very acidic. Among the several peptides observed by sodium dodecyl sulfate slab electrophoresis, only two, with apparent molecular weights of 58,000 and 25,000, correlated closely with the activity of fractions eluted from the imidazole column. These two peptides together comprised about 30% of the total protein in the fractions with highest specific activity.     

Isolation and characterization of a mixotrophic sulfur-oxidizing Thermus scotoductus

Thermophilic, faculatatively mixotrophic sulfur-oxidizing bacteria were isolated from a sulfide-rich, neutral hot spring in Iceland. The strain, IT-7254, used thiosulfate and elemental sulfur as electron donors, oxygen and nitrate as electron acceptors, and acetate and other organic compounds as carbon sources. After a few days of growth in the presence of thiosulfate, this strain formed sulfur globules. Comparison of intracellular enzymes and heme proteins of heterotrophically and mixotrophically grown cells showed some differences. The new isolate belonged to Thermus scotoductus because the small subunit (SSU) rRNA gene sequence analysis showed 98.6% sequence similarity and 84% DNA:DNA reassociation to Thermus scotoductus NMX2 A. 1. It is also close to Thermus antranikianii HN3-7, with 98.3% and 79% SSU rRNA sequence similarity and DNA:DNA reassociation, respectively. It was also found that both Thermus NMX2 A.1 and T. antranikianii HN3-7 were able to oxidize thiosulfate but that the T. scotoductus type strain SE-1 was not. This is the first report of Thermus strains that are capable of mixotrophic growth with sulfur oxidation.     

Inhibition of cGMP-dependent protein kinase II by its own splice isoform

cGMP- and cAMP-dependent protein kinases (cGK I, cGK II, and cAK) are important mediators of many signaling pathways that increase cyclic nucleotide concentrations and ultimately phosphorylation of substrates vital to cellular functions. Here we demonstrate a novel mRNA splice isoform of cGK II arising from alternative 5' splicing within exon 11. The novel splice variant encodes a protein (cGK II Delta(441-469)) lacking 29 amino acids of the cGK II Mg-ATP-binding/catalytic domain, including the conserved glycine-rich loop consensus motif Gly-x-Gly-x-x-Gly-x-Val which interacts with ATP in the protein kinase family of enzymes. cGK II Delta(441-469) has no intrinsic enzymatic activity itself, however, it antagonizes cGK II and cGK I, but not cAK. Thus, the activation and cellular functions of cGK II may be determined not only by intracellular cGMP levels but also by alternative splicing which may regulate the balance of expression of cGK II versus its own inhibitor, cGK II Delta(441-469).     

Sulfolobicins, specific proteinaceous toxins produced by strains of the extremely thermophilic archaeal genus Sulfolobus

Several novel strains of "Sulfolobus islandicus" produced proteinaceous toxins, termed sulfolobicins, which killed cells of other strains of the same species, as well as of Sulfolobus solfataricus P1 and Sulfolobus shibatae B12, but not of the producer strains and of Sulfolobus acidocaldarius DSM639. The sulfolobicin purified from the strain HEN2/2 had a molecular mass of about 20 kDa. It was found to be associated with the producer cells as well as with cell-derived S-layer-coated spherical membrane vesicles 90 to 180 nm in diameter and was not released from the cells in soluble form.     

Bacteria in cancer therapy: a novel experimental strategy

Resistance to conventional anticancer therapies in patients with advanced solid tumors has prompted the need of alternative cancer therapies. Moreover, the success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity to normal tissues. Several decades after Coley's work a variety of natural and genetically modified non-pathogenic bacterial species are being explored as potential antitumor agents, either to provide direct tumoricidal effects or to deliver tumoricidal molecules. Live, attenuated or genetically modified non-pathogenic bacterial species are capable of multiplying selectively in tumors and inhibiting their growth. Due to their selectivity for tumor tissues, these bacteria and their spores also serve as ideal vectors for delivering therapeutic proteins to tumors. Bacterial toxins too have emerged as promising cancer treatment strategy. The most potential and promising strategy is bacteria based gene-directed enzyme prodrug therapy. Although it has shown successful results in vivo yet further investigation about the targeting mechanisms of the bacteria are required to make it a complete therapeutic approach in cancer treatment.